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Characterization of glycerol metabolism and related metabolic pathways in the haloarchaeon Haloferax volcanii

Permanent Link: http://ufdc.ufl.edu/UFE0042555/00001

Material Information

Title: Characterization of glycerol metabolism and related metabolic pathways in the haloarchaeon Haloferax volcanii
Physical Description: 1 online resource (258 p.)
Language: english
Creator: Rawls, Katherine
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2010

Subjects

Subjects / Keywords: deor, genetic, glpr, glycerol, haloarchaea, haloferax, metabolism, pts, regulation, sugar
Microbiology and Cell Science -- Dissertations, Academic -- UF
Genre: Microbiology and Cell Science thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: The molecular mechanisms surrounding carbon utilization and its regulation are not well characterized in haloarchaea. Glycerol is a readily-abundant energy source for halophilic, heterotrophic communities as a result of its large-scale production by the halotolerant green algae Dunaliella sp. This study sought to characterize glycerol catabolism in the model haloarchaeon Haloferax volcanii. This work provides evidence that glycerol is a preferred carbon source over glucose and that the former is metabolized through chromosomally-encoded glycerol kinase (glpK) and glycerol-3-phosphate dehydrogenase (gpdA1B1C1). Both glpK and gpdA1 transcripts were glycerol-inducible, and the enzymatic activity of their gene products was not inhibited by glucose. Furthermore, glpK and gpdA1B1C1 are under the control of a strong, glycerol-inducible promoter. The glycerol metabolic operon also includes a putative glycerol facilitator as well as a homolog of the bacterial phosphotransferase system (PTS) protein Hpr. Additional bacterial PTS homologs EI and EII are encoded in the H. volcanii genome and preliminary evidence suggests that these EI and EIIBFru homologs may be involved in H. volcanii fructose metabolism. This work also examines the regulation of haloarchaeal carbon metabolism in H. volcanii, including the characterization of a DeoR/GlpR-type transcriptional repressor of glucose and fructose metabolic enzymes. The putative DeoR/GlpR-type protein encoded by glpR is transcriptionally-linked to pfkB encoding phosphofructokinase (PFK). Based on qRT-PCR, enzyme activity, and transcriptional reporter analyses, GlpR is likely a transcriptional repressor of genes encoding PFK and 2-keto-3-deoxygluconate kinase, two key enzymes of haloarchaeal fructose and glucose metabolism, respectively. This GlpR protein purified as a tetramer by gel filtration chromatography under both high and low salt and is postulated to use a phosphorylated intermediate of either glucose and/or fructose metabolism as its ligand. A putative repressor binding motif consisting of an inverted hexameric repeat was identified adjacent to characterized archaeal promoter consensus motifs upstream of the kdgK1 and glpR-pfkB genes. Taken together, our results provide insight into the carbon metabolic pathways of various investigated heterotrophic haloarchaea.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Katherine Rawls.
Thesis: Thesis (Ph.D.)--University of Florida, 2010.
Local: Adviser: Maupin, Julie A.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2012-12-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2010
System ID: UFE0042555:00001

Permanent Link: http://ufdc.ufl.edu/UFE0042555/00001

Material Information

Title: Characterization of glycerol metabolism and related metabolic pathways in the haloarchaeon Haloferax volcanii
Physical Description: 1 online resource (258 p.)
Language: english
Creator: Rawls, Katherine
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2010

Subjects

Subjects / Keywords: deor, genetic, glpr, glycerol, haloarchaea, haloferax, metabolism, pts, regulation, sugar
Microbiology and Cell Science -- Dissertations, Academic -- UF
Genre: Microbiology and Cell Science thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: The molecular mechanisms surrounding carbon utilization and its regulation are not well characterized in haloarchaea. Glycerol is a readily-abundant energy source for halophilic, heterotrophic communities as a result of its large-scale production by the halotolerant green algae Dunaliella sp. This study sought to characterize glycerol catabolism in the model haloarchaeon Haloferax volcanii. This work provides evidence that glycerol is a preferred carbon source over glucose and that the former is metabolized through chromosomally-encoded glycerol kinase (glpK) and glycerol-3-phosphate dehydrogenase (gpdA1B1C1). Both glpK and gpdA1 transcripts were glycerol-inducible, and the enzymatic activity of their gene products was not inhibited by glucose. Furthermore, glpK and gpdA1B1C1 are under the control of a strong, glycerol-inducible promoter. The glycerol metabolic operon also includes a putative glycerol facilitator as well as a homolog of the bacterial phosphotransferase system (PTS) protein Hpr. Additional bacterial PTS homologs EI and EII are encoded in the H. volcanii genome and preliminary evidence suggests that these EI and EIIBFru homologs may be involved in H. volcanii fructose metabolism. This work also examines the regulation of haloarchaeal carbon metabolism in H. volcanii, including the characterization of a DeoR/GlpR-type transcriptional repressor of glucose and fructose metabolic enzymes. The putative DeoR/GlpR-type protein encoded by glpR is transcriptionally-linked to pfkB encoding phosphofructokinase (PFK). Based on qRT-PCR, enzyme activity, and transcriptional reporter analyses, GlpR is likely a transcriptional repressor of genes encoding PFK and 2-keto-3-deoxygluconate kinase, two key enzymes of haloarchaeal fructose and glucose metabolism, respectively. This GlpR protein purified as a tetramer by gel filtration chromatography under both high and low salt and is postulated to use a phosphorylated intermediate of either glucose and/or fructose metabolism as its ligand. A putative repressor binding motif consisting of an inverted hexameric repeat was identified adjacent to characterized archaeal promoter consensus motifs upstream of the kdgK1 and glpR-pfkB genes. Taken together, our results provide insight into the carbon metabolic pathways of various investigated heterotrophic haloarchaea.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Katherine Rawls.
Thesis: Thesis (Ph.D.)--University of Florida, 2010.
Local: Adviser: Maupin, Julie A.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2012-12-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2010
System ID: UFE0042555:00001


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1 CHARACTERIZATION OF GLYCEROL METABOLISM AND RELATED METABOLIC PATHWAYS IN THE HALOARCHAEAON H aloferax volcanii By KATHERINE SHERWOOD RAWLS A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2010

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2 2010 Katherine Sherwood Rawls

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3 To my husband, Colin Rawls, who has been my guiding light, and to my parents, Steve and Francy Sherwood, for their unconditional love and support

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4 ACKNOWLEDGMENTS I express my deepest gratitude to my academic advisor, Dr. Julie MaupinFurlow, for challenging my intellectual capabilities and for expanding my scientific curiosity. Her guidance during my g raduate career has been crucial toward successful completion of my degree. I also wish to thank the additional members of my graduate supervisory committee, Drs. Linda Bloom, Graciela Lorca, Lonnie Ingram and K.T. Shanmugam for their project insight and fo r generous use of their equipment. Special thanks are extended to current and former graduate students, post doctoral students, laboratory technicians and undergraduate researchers of the MaupinFurlow research laboratory for creating a stimulating, pleasant environment in which to work. Specifically, I would like to acknowledge David Cano for his help in generating the glycerol kinas e mutant; Shalane Yacovone and Mario Corro for t he i r assistance in generating several knockout strains including the phosphotransferase system mutants, the dihydroxyacetone kinase mutants and the GlpR repressor mutant ; and Mayra Souza for her additional assistance in generating the GlpR repressor mutant as well as the transcriptional promoter reporter fusion constructs Also, I wish to thank Gosia GilRamadas for her guidance as my undergraduate research mentor. Special thanks are also extended to Dr. Sivakumar Uthandi and Nikita Nembhard for their insightful advice and support, especially during my last semester of graduate s chool. Thank you to all graduate funding agencies including the University of Florida Alumni Foundation, the Doris Lowe and Verna and Earl Lowe Scholarship Fund, the National Institute of Health, and the Department of Energy for their financial support of my dissertation project

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5 I finally wish to acknowledge my immediate family members: my parents, Francy and Steve Sherwood; my sister, Kelley Sherwood; my niece, Reagan Timmons; my grandmothers Johnnie Sherwood and Helen Glenn; and my late grandfathers Jim Sherwood and Bill Glenn, who have always graciously provided their moral and financial support throughout my academic career. I most importantly thank my husband, Colin Raw ls, for providi ng unwavering love and encouragement throughout this challenging process

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6 TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................. 4 LIST OF ABBREVIATIONS ........................................................................................... 14 ABSTRACT ................................................................................................................... 20 CHAPTER 1 LITERATURE REVIEW .......................................................................................... 22 Introduction ............................................................................................................. 22 An Overview of Glycerol Metabolism ...................................................................... 22 Biosynthesis of Glycerol ................................................................................... 23 Glycerol 3 phosphate dehydrogenase ....................................................... 24 Glycerol 3 phosphate phosphatase ........................................................... 25 Glycerol Dissimilation to Dihydroxyacetone Phosphate ................................... 25 Glycerol kinase .......................................................................................... 27 Glycerol 3 phosphate dehydrogenase ....................................................... 28 Glycerol dehydrogenase ............................................................................ 30 Dihydroxyacetone kinase ........................................................................... 30 Anaerobic Glycerol Dissimilation Pathways ...................................................... 32 Glycerol dehydratase ................................................................................. 33 1,3 Propanediol dehydrogenase ................................................................ 35 Glycerol Transport Across a Biological Membrane ................................................. 35 Intrinsic Permeability ........................................................................................ 36 Aquaglyceroporins ............................................................................................ 37 Glycerol facilitator protein GlpF .................................................................. 38 Fps1p ......................................................................................................... 39 Additional Aquaglyceroporins ........................................................................... 41 Active Transport of Glycerol ............................................................................. 44 Tran scriptional and Post Trans lati onal Control of Glycerol Metabolism .................. 45 Transcriptional Regulators of Glycerol Metabolism .......................................... 45 cAMP cAMP receptor protein ..................................................................... 46 FNR ........................................................................................................... 48 CcpA .......................................................................................................... 50 GlpR ........................................................................................................... 51 DhaR .......................................................................................................... 54 ArcA/ArcB .................................................................................................. 55 Additional transcriptional regulators of glycerol metabolism ...................... 57 Allosteric and Post trans lationa l Regulation of Glycerol Kinase Activity ........... 58 Hpr and EI dependent phosphorylation of glycerol kinase ........................ 59 Fructose 1,6bisphosphate ......................................................................... 60

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7 EIIAGlc ........................................................................................................ 61 Industrial and Biological Significance of Glycerol .................................................... 63 Glycerol as a Feedstock for Bioconversion ...................................................... 63 Therapeutic and Diagnostic Uses of Glycerol ................................................... 65 Biological Relevance of Glycerol to Halophilic Communities ............................ 66 Osmoprotectant properties of glycerol ....................................................... 66 Glycerol production by the halotolerant green algae Dunaliella ................. 68 Glycerol metabolism in halophilic bacteria ................................................. 70 Glycerol metabolism in haloarchaea .......................................................... 71 An Overview of Glucose and Fructose Metabolism in Haloarchaea ....................... 72 Transport of Fructose and Glucose Across a Biological Membrane ....................... 73 Overview of the Phosphotransferase System ................................................... 73 General carrier protein EI ........................................................................... 75 General carrier protein Hpr ........................................................................ 76 Sugar specific component EII .................................................................... 76 ATP Binding Cassette Transporters ................................................................. 77 Secondary Transporters ................................................................................... 79 Fructose and Glucose Metabolism and Their Regulation in Haloarchaea .............. 79 Glucose Degradation through a Modified Entner Doudoroff Pathway .............. 80 Fructose Degradation through a Modified EmbdenMeyerhof Parnas Pathway ........................................................................................................ 82 Regulation of Glucose and Fructose Metabolism in Haloarchaea .................... 83 TrmB .......................................................................................................... 8 3 General transcription factors TATA binding protein and transcription factor B ................................................................................................... 84 Project Rationale and Design ................................................................................. 86 2 METHODS AND MATERIALS ................................................................................ 91 Chemical s, Media, and Strains ............................................................................... 91 Chemicals and Reagents ................................................................................. 91 Strains, Plasmids, and Culture Conditions ....................................................... 92 High Performance Liquid Chromatography ...................................................... 93 DNA Procedures ..................................................................................................... 94 DNA Isolation and Analysis .............................................................................. 94 Polymerase Chain Reactions ........................................................................... 95 Cloning ............................................................................................................. 95 Construction of H. volcanii Deletion Strains ...................................................... 95 Southern Blot Analysis ..................................................................................... 96 Construction of Promoter Reporter Fusion Constructs ..................................... 97 RNA procedures ..................................................................................................... 98 RNA Isolation and Analysis .............................................................................. 98 (q)RT PCRs ...................................................................................................... 98 Protein Procedures ............................................................................................... 100 Protein Isol ation and Analysis ........................................................................ 100 StrepTactin Chromatography ......................................................................... 100 Gel Filtration Chromatography ....................................................................... 101

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8 Protein Quantification ..................................................................................... 102 Glycerol Kinase Activity Assay ....................................................................... 102 PFK and KDGK Activity Assays ..................................................................... 103 Galactosidase Assay ................................................................................... 104 Genome Analysis and Construction of Phylogenetic Trees ............................ 105 3 DISTRIBUTION OF PHOSPHOENOLPYRUVATE LINKED PHOSPHOTRANSFERASE SYSTEM HOMOLOGS IN ARCHAEA AND IMPLICATIONS AS TO THEIR BIOLOGICAL FUNCTION ................................... 130 Introduction ........................................................................................................... 130 Results and Discussion ......................................................................................... 132 Distribution of PTS Components in Archaea .................................................. 132 Distribution of DHAK Homologs in Archaea ................................................... 133 PTS Components and H aloarchaeal Glycerol Kinases .................................. 134 Preliminary Evidence for the Involvement of H. volcanii EI and EIIBFru in Fructose Metabolism ................................................................................... 136 Conclusion ............................................................................................................ 137 4 GLYCEROL KINASE AS THE SOLE ROUTE OF GLYCEROL CATABOLISM IN THE HALOARCHAEON Haloferax volcanii ...................................................... 146 Introduction ........................................................................................................... 146 Results and Discussion ......................................................................................... 147 Glycerol is Metabolized through Glycerol Kinase ........................................... 147 Glycerol Metabolism is not Reduced in the Presence of Glucose .................. 150 Levels of Glycerol 3 Phosphate Dehydrogenase and Glycerol Kinase Transcripts are Upregulated by the Addition of Glycerol ............................. 152 Glycerol Kinase and Glycerol 3 Phosphate Dehydrogenase Genes are under the Control of a Common Promoter .................................................. 153 Conclusion ............................................................................................................ 154 5 MOLECULAR CHARACTERIZATION OF THE PRIMARY GLYCEROL METABOLIC OPERON IN THE HALOARCHAEON Haloferax volcanii ................ 166 Introduction ........................................................................................................... 166 Results and Discus sion ......................................................................................... 166 H. volcanii Glycerol Metabolic Operon is under the Control of an Inducible Promoter ..................................................................................................... 166 Glycerol Metabolism Proceeds Primarily through GpdA1 ............................... 169 Distribution of Archaeal GpdA Homologs ....................................................... 170 Distribution of Archaeal Hpr Homologs and Putative Glycerol Facilitator Proteins ....................................................................................................... 171 Conclusion ............................................................................................................ 172

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9 6 GlpR REPRESSES FRUCTOSE AND GLUCOSE METABOLIC ENZYMES AT THE LEVEL OF TRANSCRIPTION IN THE HALOARCHAEON Haloferax volcanii .................................................................................................................. 185 Introduction ........................................................................................................... 185 Results and Di scussion ......................................................................................... 186 Identification of GlpR as a Putative Repressor of Metabolic Enzymes ........... 186 Transcripts Encoding GlpR and PFK are under the Control of a Common Promoter and are Repressed in the Absence of Fructose .......................... 187 GlpR Represses PFK Transcription during Growth in the Absence of Fructose ...................................................................................................... 189 GlpR Represses KDGK Transcription during Growth in the Ab sence of Glucose ....................................................................................................... 191 GlpR and Sugar Metabolism .......................................................................... 194 Promot er Regions for kdgK1 and glpR pfkB Include a Putative GlpR Binding Motif ............................................................................................... 195 GlpR Purifies as a Tetramer from PeptideRich Media un der Both High and Low Salt ...................................................................................................... 196 Conclusion ............................................................................................................ 196 7 SUMMARY AND CONCLUSIONS ........................................................................ 218 Summary of Findings ............................................................................................ 218 Future Directions .................................................................................................. 219 LIST OF REFERENCES ............................................................................................. 222 BIOGRAPHICAL SKETCH .......................................................................................... 257

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10 LIST OF TABLES Table page 2 1 Strains and plasmids used in C hapter 3. .......................................................... 112 2 2 Primers used in C hapter 3. ............................................................................... 113 2 3 Strains and plasmids used in C hapter 4. .......................................................... 115 2 4 Primers used in C hapter 4. ............................................................................... 116 2 5 Strains and plasmids used in C hapter 5. .......................................................... 118 2 6 Primers used in C hapter 5. ............................................................................... 120 2 7 Strains and plasmids used in C hapter 6. .......................................................... 124 2 8 Primers used in C hapter 6. ............................................................................... 126 3 1 Distribution of PTS homologs in archaea ......................................................... 139 5 1 Transcription of a bgaH galactosidase reporter gene from gpdA1, glpK and trpA promoters .................................................................................................. 174 5 2 Glycerol 3 phosphate dehydrogenase specific activity in various glycerol metabolic mutants. ........................................................................................... 175 5 3 Predicted locations of transmembrane domains of H. volcanii GlpX. ............... 176 6 1 Transcription of a bgaH galactosidase reporter gene from glpR pfkB kdgK1 and kdgK2 promoters ........................................................................... 198

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11 LIST OF FIGURES Figure page 1 1 Glycerol synthesis, release, and catabolism in halophilic ecosystems. .............. 88 1 2 Metabolic conversion of glucose to pyruvic acid in haloarchaea. ....................... 89 1 3 Potential routes for the metabolic conversion of fructose to pyruvic acid by haloarchaea. ....................................................................................................... 90 3 1 Genomic organization of complete phosphoenolpyruvate:phosphotransferase system utilization operons in archaea ............................................................... 140 3 2 Organization of glycerol utilization operons from haloarchaea whose genom e sequences have been completed ..................................................................... 141 3 3 Alignment of haloarchaeal glycerol kinases and biochemically characterized bacterial glycerol kinases ................................................................................. 142 3 4 PCR and Southern blot confirmation of H. volcanii mutant strain KS3 (H26 fruB ptsI ). ....................................................................................................... 143 3 5 H. volcanii mutant strain KS3 deficien t in PTS components EI and EIIBFru (H26 fruB ptsI ) is unable to metabolize fructose ........................................... 144 3 6 PTS components EI and EIIBFru are not required for glucose metabolism in H. volcanii. ........................................................................................................ 145 4 1 Halophilic microorganisms assimilate glycerol into DHAP by one of two catabolic routes. ............................................................................................... 156 4 2 Phylogenetic distribution of glycerol kinases .................................................... 157 4 3 PCR and Southern blot confirmation of H. volcanii glycerol kinase mutant strain glpK ) .................................................................................... 158 4 4 H. volcanii metabolizes glycerol through glycerol kinase. ................................. 159 4 5 H. volcanii glycerol kinase activity is dependent on glpK stimulat ed by glycerol, and uninhibited by glucose ................................................................. 160 4 6 Parent H26 and glycerol kinase mutant strain glpK ) exhibit similar growth phenotypes on glucose minimal media ................................................ 161 4 7 Glycerol and glucose are differentially metabolized by H. volcanii ................... 162 4 8 Genomic organization and transcript analysis of glpK and glycerol 3 phosphate dehydrogenaserelated operons of H. volcanii. ............................... 164

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12 5 1 Genomic organization of the primary glycerol metabolic operon in H. volcanii. 177 5 2 PCR and Southern blot confirmation of H. volcanii gpdA1 and gpdA2 mutant strains KS12 (H26 gpdA1 gpdA2). .................................... 178 5 3 Glycerol is primarily catabolized through GpdA1 and not GpdA2 in H. volcanii 180 5 4 Phylogenetic distribution and genomic organization of GpdA homologs in Archaea and E. coli .......................................................................................... 181 5 5 Many haloarchaeal genomes encode a full length GpdA1 and a C terminally truncated GpdA2. ............................................................................................. 182 5 6 B. subtilis Hpr phosphorylative residues histidine15 and serine46 are conserved in haloarchaeal Hpr homologs ....................................................... 183 5 7 GlpX is a putative glycerol facilitator protein based on the presence of predicted transmembrane domains .................................................................. 184 6 1 H. volcanii glpR (HVO_1501) is linked on the chromosome with genes of sugar metabolism ............................................................................................. 199 6 2 Genomic organization and transcript analysis of pfkB and glpR in H. volcanii 202 6 3 PCR and Southern blot confirmation of H. volcanii glpR mutant strain KS8 (H26 glpR ). ..................................................................................................... 204 6 4 PFK activity increases when cells are grown on glycerol minimal medium after deletion of glpR ........................................................................................ 205 6 5 GlpR is not required for reduction of PFK activity on peptide media in the absence of fructose or glucose ......................................................................... 206 6 6 Genomic organization and transcript analysis of KDGK genes located on the chromosome and megaplasmid pHV4 of H. volcanii ........................................ 207 6 7 Phylogenetic distribution of PFK and KDGK in Bacteria and Archaea.............. 209 6 8 KDGK activity is increased by deletion of glpR in cells grown on glycerol minimal medium. .............................................................................................. 210 6 9 GlpR is not required for reduction of KDGK activity on peptide media in the absence of fructose or glucose. ........................................................................ 211 6 10 Deletion of Glp R does not impact glycerol or glucose consumption in H. volcanii ............................................................................................................ 212 6 11 Glycerol and fructose are cometabolized in H. volcanii. .................................. 213

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13 6 12 Genomic regions upstream of glpR pfkB and kdgK1 include a conserved inverted repeat that may serve in GlpR binding ................................................ 215 6 13 H. volcanii GlpR purifies as a tetramer by gel filtration ..................................... 216 6 14 H. volcanii GlpR purifies to homogeneity after tandem StrepTactin and gel filtration chromatography ................................................................................. 217 7 1 H. volcanii grows aerobically on biodiesel waste as a sole carbon source. ...... 221

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14 LIST OF ABBREVIATION S Angstrom A260 Absorbance at 260 nm A280 Absorbance at 280 nm A340 Absorbance at 340 nm A405 Absorbance at 405 nm A595 A bsorbance at 595 nm AAA+ ATPase associate d with diverse cellular activities ABC ATP binding cassette AC Adenylate cyclase ADP Adenosine5 diphosphate Amp Ampicillin AP Antarctic phosphatase AR Activating region ATP Adenosine5 triphosphate ATCC American type culture collection BFD B acterioferri tin associated ferredoxin BLAST Basic local alignment search tool C Degrees Celsius C Carboxyl CA Casamino acids cAMP Cyclic AMP cAMP CRP cAMP complexed with CRP CFU Colony forming units cm Centimeter

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15 COG Clusters of orthologous groups cre Catabolite responsive element Crh Catabolite repression protein Hpr CRP cAMP receptor protein CSPD Disodium 3 (4 methoxyspiro{1,2dioxetane3,2' (5' chloro)tricyclo[3.3.1.13,7]decan} 4 yl) phenyl phosphate CT Threshold count Da Dalton DBD DNA binding domain DEPC Diethylpyrocarbonate DHA Dihydroxyacetone DHAK Dihydroxyacetone kinase DHAP Dihydroxyacetone phosphate DIG 11dUTP 2' d eoxyuridine5' triphosphate coupled by an 11atom spacer to digoxigenin DMSO Dimethyl sulfoxide DNA Deoxyribonucleic acid dNTP Deoxyribonucleotide triphosphate EI Enzyme I of the PEP:PTS EII Enyzme II of the PEP:PTS EIIAGlc Glucose specific phosphocarrier EII protein of the PEP:PTS Ea Energy of activation (kcal 1) ED Entner Doudoroff EMP Embden Meyerhof Parnas EDTA Ethylenediaminetetr aacetic acid F1P Fructose 1 phosphate

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16 FAD Oxidized Flavin adenine dinucleotide FADH R educed Flavin adenine dinucleotide FBP Fructose 1,6 bisphosphate fg Femtogram FOA 5 Fluoroorotic acid Fru Fructose g Gravitational force G1P Glycerol 1 phosphate G3P Glycerol 3 phosphate G3PDH Glycerol 3 phosphate dehydrogenase G3PP Glycerol 3 phosphate phosphatase GD Glycerol dehydratase GDH Glycerol dehydrogenase GK Glycerol kinase Glu Glucose Gly Glycerol HPA 3 Hydroxypropionaldehyde HPLC High performance liquid chr omatography Hpr Histidine containing phosphorylatable protein of the PTS Hpt Histidine containing phosphotransfer domain HTH Helix turn helix in Inch Kav Gel phase distribution coefficient kcal Kilocalorie Kd Binding dissociation constant

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17 kDa Kilodalton KDG 2 keto 3 deoxygluconate KDGK KDG kinase KDPG 2 keto 3 deoxy 6 phosphogluconate Km Michaelis Menten constant (mol 1) LB LuriaBertani medium lb Pound LDH Lactate dehydrogenase M Molar (mol iter1) MFS Major facilitator superfamily mg Milligram g Microgram M Micromolar ( mol iter1) min Minutes MIP Major intrinsic protein ml Milliliter MM Minimal medium mM Millimolar (mmol1) MOPS 3 (N Morpholino)propanesulfonic acid, 4Morpholinepropanesulfonic acid N Amino NAD+ Oxidized Nicotinamide adenine dinucleotide NADH Reduced Nicotinamide adenine dinucleotide n.d. Not determined nm Nanometer NMR Nuclear magnetic resonance

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18 Nv Novobiocin OD600 Optical density at 600 nm ONPG o nitrophenyl D galactopyranoside OR Oxidoreductase ORF Open reading frame PAGE Polyacrylamide gel electrophoresis PCR Polymerase chain reaction PDH 1,3 Propanediol dehydrogenase PEP Phosphoenolpyruvate PFK Phosphofructokinase PFL Pyruvate formate lyase PMSF P henylmethylsulfonyl fluoride PNK Polynucleotide kinase PRD PEP carbohydrate PTS regulatory domain PTS Phosphotransferase system qRT PCR Quantitative reversetranscriptase polymerase chain reaction R2 C oefficient of determination rRNA Ribosomal RNA RNA Ribonucleic acid RPM Revolutions per minute RT Reverse transcriptas e s Seconds SAM S adenosyl methionine SD Standard deviation SDS Sodium dodecyl sulfate

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19 SSC Saline sodium citrate TBP TATA binding protein TFB Transcription factor B TIM Triosephosphate isomerase TM Transmembrane Tris N tris(hydroxyl methyl) aminomethane U Enzyme activity unit [ 1 ( mg protein)1] UV Ultraviolet V Volt v Volume (in ml) Vo Void volume of the column ( ml) VC Geometric bed volume ( ml) Vmax Maximal velocity (mol 1s1) VR Retention (elution) volume of the protein ( ml ) w Weight ( grams) YPC Yeast peptone casamino acids medium

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20 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy CHARACTERIZATION OF GLYCEROL MET ABOLISM AND RELATED METABOLIC PATHWAYS IN THE HALOARCHAEAON H aloferax volcanii By Katherine Sherwood Rawls December 2010 Chair: Julie MaupinFurlow Major: Microbiology and Cell Science The molec ular mechanisms surrounding car bon utilization and its regulation are not well characterized in hal oarchaea. G lycerol is a readily abundant energy source for halophilic, heterotrophic communities as a result of its large scale production by the halotolerant green algae Dunaliella sp. T h is study sought to characterize glycerol catabolism in the model haloarchaeon Haloferax volcanii. This work provides evidence that glycerol is a prefer r ed carbon source over glucose and that the former is metabolized through chromosomally encoded glycerol kinase ( glpK ) and glycerol 3 phosphate dehydrogenase ( gpdA1B1C 1 ). Both glpK and gpdA1 transcripts were glycerol inducible, and the enzymatic activity of their gene products was not inhibited by glucose. Furthermore, glpK and gpdA1B1C1 are under the control of a strong, glycerol inducible promoter. The glycerol metabolic operon also includes a putative glycerol facilitator as well as a homolog of the bacterial ph osphotransferase system (PTS) protein Hpr A dditional bacterial PTS homologs EI and EII are encod ed in the H. volcanii genome and preliminary evidence suggests that these EI and EIIBFru homologs may be involved in H. volcanii fructose metabolism. This work also examines the regulation of haloarchaeal carbon metabolism in H. volcanii, including the characterization of a

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21 DeoR/GlpRtype transcriptional repressor of glucose and fructose metabolic enzymes The putative DeoR/GlpRtype protein encoded by glpR is transcriptionally linked to pfkB encoding phosphofructokinase (PFK). Based on qRT PCR, enzyme acti vity, and transcriptional reporter analyses, GlpR is likely a transcriptional repressor of genes encoding PFK and 2keto 3 deoxygluconate kinase, two key enzymes of haloarchaeal fructose and glucose metabolism, respectively. T his GlpR protein purified as a tetramer by gel filtration chromatography under both high and low salt and is postulated to use a phosphorylated intermediate of either glucose and/or fructose metabolism as its ligand. A putative repressor binding motif consisting of an inverted hexameri c repeat was identified adjacent to characterized archaeal promoter consensus motifs upstream of the kdgK1 and glpR pfkB genes Taken together, o ur results provide insight into the carbon metabolic pathways of various investigated heterotrophic haloarchaea.

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22 CHAPTER 1 LITERATURE REVIEW Introduction This literature review is designed to present the most relevant, primary scientific literature concerning glycerol, glucose, and fructose metabolism and their regul ation in the three domains of life. The review will highlight characterized glycerol metabolic enzymes, regulation of gl ycerol metabolism, glycerol transport across the biological membrane, and the biological relevance of glycerol including its industrial and medical applications. This review will additionally focus on glucose and fructose metabolism in haloarchaea with regards to metabolic enzymes, regulation, and sugar transport across the biological membrane. An Overview of Glycerol Metabolism Glycerol is a ubiquitous molecule that serves as an important biosynthetic precursor. Glycerol is the structural component of many lipids, and glycerol derivatives glycerol 1 phosphate (G1P) or its enantiomer glycerol 3 phosphate (G3P) are used as the backbone for archaeal or bacterial and eukaryotic biological membrane lipids respectively (Kates, 1978) Glycerol is also the principal compatible solute produced in response to decreased extracellular water activity by yeast (Brown and Simpson, 1972; Blomberg and Adler, 1989) as well as algae (Craigie and McLachlan, 1964; Ben Amotz and Avron, 1973b; Borowitzka and Brown, 1974; Ben Amotz and Avron, 1979) Glycerol also serves as a cryoprotective agent and contributes to inorganic phosphate recycling as well as energy production, neoglucogenesis, and redox balance (Brisson et al., 2001) In fungi, glycerol is primarily produced from dihydroxyacetone phosphate (DH AP) through G3P dehydrogenase (G3PDH encoded by gpd ABC or glp ABC ) and G3P

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23 phosphatase (G 3P P). There are two characterized routes of aerobic glycerol ca tabolism: i) through glycerol kinase (GK encoded by glpK ) and G3PDH or ii) throug h glycerol dehydrogenase (GDH) and dihydroxyacetone kinase (DHAK encoded by d haKLM ) (either phosphoenolpyruvate:phosphotransferase system (PEP:PTS) linke d or ATP dependent) Glycerol can also be ferment ed to 1,3 propanediol as a major product through the action of glycerol dehydratase (GD) and 1,3propanediol dehy drogenase (1,3 PDH) and for this reason has been widely used as a feedstock in bioconversion. Glycerol can pass through the cell membrane by f acilitated diffusion, simple diffusion, or through active transport and r egulation of glycerol metabolism occurs both transcriptionally and post translationally and at different steps in the metabolic pathway Each of these aspects of glycerol metabolism is discussed. Biosynthesis of Glycerol Glycerol is produced by e ukaryotes and has osmoregulatory, biosynthetic, and/ or redox balancing functions. Fungal glycerol production has been best characterized with regards to biochemistry and cellular physiology where glycerol is involve d in many important cellular functions including osmoregulation during low water activity (Blomberg and Adler, 1992) and maintenance of redox balance during fermentati on of glucose to ethanol (Ansell et al., 1997; Bjrkqvist et al., 1997; Nissen et al., 2000) Glycerol is cytosolically synthesized by Saccharomyces cerevisiae from DHAP as catalyzed by GPD1 and GPD 2 encoded G3PDH and the GPP1P and GPP 2P encoded G 3P P. Both GPD1P and G P P2P are osmotically induced, whereas their isoenzymes ( GPD2P a nd GPP1P) are induced during anaerobic metabolism (Albertyn et al., 1994; Ohmiya et al., 1995; Norbeck et al., 1996; Ansell et al., 1997; Bjrkqvist et al., 1997) Dunaliella tertiolecta also contains multiple isoenzymes for glycerol synthesis that are

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24 differentially induced. The mode of induction depends on the ultimate function of the glycerol molecule, specifically whether it will be further modifi ed for lipid synthesis or will be used directly as an osmoprotectant. Glycerol 3 p hosphate d ehydrogenase G3PDH [ sn G3P : nicotinamide adenine dinucleotide (NAD+) 2 oxidoreductase (OR), EC 1.1.1.8] catalyzes the reversible reduction of DHAP to G3P and is di stributed throughout all domains of life. I n higher plants and algae, G 3 PDH is often referred to as DHAP reductase because at physiological pH and substrate concentrations the enzyme is essentially inactive as a dehydrogenase (Gee et al., 1988a; Gee et al., 1988b; Gee et al., 1989) Interestingly, certain yeast s including S. cerevis i a e and S chizosaccharomyces pombe encode two isoenzymes of G3 PDH which modulate different cellular activities ( GPD1P and GPD2P ) While GPD1P is osmotically induced and appears to increase glycerol synthesis in response to osmotic stress GPD2P is induced under anoxic conditions ( Albertyn et al., 1994; Ohmiya et al., 1995; Norbeck et al., 1996; Ansell et al., 1997; Bjrkqvist et al., 1997) Overproduction of either GPD1 or GPD2 in S. cerevisiae increases glycerol production, indicating that DHAP reduction may be a ratelimiting step in glycerol synthesis (Nevoigt and Stahl, 1996; Remize et al. 2001) Similarly, the microalgae D tertiolecta also has multiple isoforms of G3PDH which are differentially located and differentially induced (Gee et al., 1989; Gee et al., 1993; Ghoshal et al., 2002) Two major G3PDH isoenzymes are located in the chloroplast, and a m inor form is located in the cytoplasm. One of the major isoenzymes is thought to be involved in lipid synthesis on the basis of its constitutive enzymatic activity and its sensitiv ity toward detergents, lipids, and longchain acyl CoA derivates. The other major species is thought to be involved in osmotic stress response based on

PAGE 25

25 its stimulation during growth on high salt. The minor form of the enzyme is believed to contribute to lipid synthesis based on its constitutive nature. Glycerol 3 phosphate p hosphatase G3PP ( G3P phosphohydrolase, EC 3.1.3.21) catalyzes the hydrolysis of G3P to glycerol and phosphate. S. cerevisiae encodes two isoenzymes of G3PP which are differentially expressed under either anaerobiosis or salt stress Although both G3PP isoenzymes (Gpp1 and Gpp2) are induced during hyperosmotic conditions (Phlman et al., 2001) only the expression of GPP 1 is induced under anaerobic conditions (Phlman et al., 2001) Mutants lacking both GPP1 and GPP2 are devoid of G3PP activity and produce only small amount s of glycerol, confirming the essential role of G3PP in glycerol biosynthesis (Phlman et al., 2001) Unlike G3PDH, overproduction of either Gpp1 or Gpp2 does not enhance glycerol production in yeast, indicating that G3P hydrolysis is not a rate limiting step in glycerol sy nthesis (Phlman et al., 2001) Glycerol Dissimilation to Dihydroxyacetone Phosphate There are two characterized routes of gly cerol dissimilation to DHAP: i) through GK and G3PDH [ flavin adenine dinucleotide (FAD) dependent or NAD(P)+dependent ] or ii) through NAD(P)+dependent GDH and DHAK ( ATP or PEP:PTS dependent ). A number of yeasts including S. cerevisiae (Sprague and Cronan, 1977) Debar yomyces hansen ii (Blomberg and Adler, 1989) and Candida glycerinogenes (Chen et al., 2008) metabolize glycerol primarily through the former route. In this route, g lycerol is first converted to G3P by GK. G3P can either serve as a precursor for lipid biosynthesis or can be subsequently converted into the glycolytic intermediate DHAP by G3PDH. S cerevisiae cells deficient in either GUT1 encoded GK or GUT2 encoded G3PDH are unable to utilize glycerol as a sole carbon source (Sprague and Cronan, 1977) In

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26 contrast, some yeasts such as S pombe solely metabolize glycerol through GDH (Gancedo et al., 1986; Matsuzawa et al., 2010) and DHAK (Gancedo et al., 1986; Kimura et al., 1998) Interestingly, the yeasts S. cerevisiae (Norbeck and Blomberg, 1997) D hanseii (Blomberg and Adler, 1989) and Zygosaccharomyces rouxii (Wang et al., 2002) metaboliz e glycerol through either route; however, the lat ter route ( through GDH and DHAK) is more commonly activated during osmotic stress. Higher e ukaryotes also encode GK and G3PDH enzymes of glycerol dissimilation that are highly conserv ed in amino acid sequence (Ayala et al., 1996; Brisson et al., 2001) GDHs and DHAKs are also found in higher eukaryotes, although the latter are based on sequence similarity to bacterial DHAKs. GK and G3PDH homologs are widely distributed in archaea with the exception of autotrophic methanogenic archaea which are unable to utilize glycerol as an energy source (Nishihara et al., 1999) GDH and DHA K homologs are not as common as GK and G3PDH homologs in archaea, with distribution limited to haloarchaea. Specifically GDH e nzyme activity has only been detected in Halobacterium salinarium and Halobacterium cutirubrum which are not known to metabolize glycerol (Rawal et al., 1988) and DHAK has only recently been identified in archaea based on genome sequenc es (Bolhuis et al., 2006; Hartman et al., 2010; Benson et al., 2010) GK and G3PDH are widely distributed in bacteria, although several bacterial GDH s and DHAK s hav e also been characterized, especially in Enterobacteriaceae (Bouvet et al., 1995) Some bacteria including certain strains of Enterococcus faecalis can metabolize glycerol to DHAP using either route (Bizzini et al., 2010)

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27 Glycerol k inase GK (ATP : glycerol phosphotransferase, EC 2.7.1.30) catalyzes the transfer of the gamma phosphoryl group of a denosine5 triphosphate (ATP) to glycerol to form G3P GK is a ubiquitous member of the ribonuclease H like family of kinases, a protein superfamily whose members are composed of two domains with an intervening cleft containing the ATPase catalytic site (Cheek et al., 2005) The crystal structure of Escherichia coli GK reveals that adenosine5 diphosphate (ADP) binds in the intercleft site and that glycerol binds directly below ADP in the interdomain cleft (Hurley et al., 1993) Glycerol binding is stabilized by arginine83, glutamate184, tyrosine135, and aspartate245 r esidues which form hydrogen bonds with the hydroxyl groups of glycerol, and through van der Waals interactions between typtophan103, phenylalanine270, and the glycerol carbon backbone. The aspargaine10 and aspartate245 residues are thought to serve as the Mg2+ binding site s, the former of which is also proposed to be involved in ATP hydrolysis. Kinetic and biochemical evidence suggests that E. coli GK exists in equilibrium between functional dimeric and tetrameric forms (de Riel and Paulus, 1978a) the lat t er of which can be inactivated by allosteric regulators. GK is known to be a ratelimiting and regulated step of glycerol metabolism in many bacteria. GK is primarily regulated to limit glycerol metabolism when more preferable carbon sources such as glucose are available. In E. coli GK regulation also prevents toxic accumulation of methylgloxyl during growth on glycerol (Freedberg et al., 1971) GK catalytic activity is allosterically inhibited by the small molecule fructose 1,6bisphosphate (FBP) in both Gram negative and Gram positive bacteria. Additionally, the glucose specific phosphocarrier protein of the PEP:PTS (EIIAGlc) inhibits GK activity

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28 solel y in Gram n egative bacteria (Grke and Stlke, 2008) and GK activity is activated through an enzyme I (EI) dependent phosphorylation by heat stable histidine phosphorylatable protein (Hpr ) exclusively in Gram positive bact eria (Grke and S tlke, 2008) Furthermore, the E. coli glycerol facilitator protei n GlpF may increase GK activity as evidenced by the significant increase in the Michaelis Menten constant (Km) and decrease in maximal velocity (Vmax) of GK upon deletion of glp F (Thorner and Paulus, 1973; Voegele et al., 1993) GK is also regulated at the transcriptional level through a variety of proteins including members of the CRP/FNR super family (specifically, CRP and Ers), CcpA ( a LacI/GalR superfamily member) and GlpR ( a DeoR/GlpR superfamily member) Glycerol 3 phosphate dehydrogenase G3PDHs are either NAD+( sn G3P : NAD+ 2 OR, EC 1.1.1.8), FAD ( sn G3P: cytochrome OR, EC 1.1.5.3 ) or NADP+dependent ( sn G3P : NADP+ 1 OR, EC 1.1.1.177) enzymes which catalyze the reversible conversion of G3P to DHAP. Despite cofactor differences, G3PDHs are conserved with greater than 45% protein sequence identity across all do mains of life (Yeh et al., 2008) While NAD+dependent G3PDHs are cytosolically located, FAD dependent G3PDHs are localized to the cytoplasmic membrane in bacteria (Walz et al., 2002) and are tightly bound to the outer surface of the inner mitochondrial membrane in eukaryotes (Lin, 1977) In eukaryotes, FAD dependent G3PDH, together with its cytosolic counterpart, form the G3P shuttle. This shuttle is responsible for the reoxidation of NADH by the mitochondrial electron transport chain (Ansell et al., 1997; Larsson et al., 1998) NADP+dependent G3PD Hs are more limited in distribution compared to either NAD+or FAD dependent G3PDHs

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29 with the only biochemically characterized representative in the hyperthermophilic archaeon Archaeoglobus fulgidus (Sakasegawa et al., 2004) T he E. coli genome encodes two G3PDH which are differentially expressed depending up on oxygen availability. Under oxygenrich conditions, a membrane associated homodimeric protein encoded by glpD is produced (Schryvers et al., 1978) U nder anaerobic conditions, the glpABC operon is preferentially expressed. This membrane associated, anaerobic G3PDH consists of a tri heteromeric complex which utilizes fumarate or nitrate as the external electron acceptor (Cole et al., 1988; Varga and Weiner, 1995) Catalytic activity is carried by the soluble GlpAC dimer and membrane associated GlpB mediates electron transfer from the GlpAC dimer to the terminal electron acceptor fumarate through the membraneboun d menaquinone pool (Cole et al., 1988; Varga and Weiner, 1995). GlpA binds FAD cofactor noncovalently, GlpC binds flavin mononucleotide, and GlpB contains two ironsulfur clusters (Cole et al., 1988) Seven fully active structures of E. coli GlpD, both native and substrate analoguebound, have been solved to 1.75 resolution (Yeh et al., 2008) GlpD is composed of both a soluble, extramembranous C terminal cap domain as well as a cytoplasmic membraneassociated N terminal domain. Binding of the N terminus of the protein to the membrane is mediated by the exposure of a basic amphipathic helix that inserts into the hydrophobic core regions of membrane lipids (Walz et al., 2002) In addition to serving as the anchor for GlpD, the N terminus of the protein contains the substrate as well as the cofactor binding domains, and is thought to contain a ubiquinonedockin g site (Yeh et al., 2008)

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30 Glycerol d ehydrogenase GDHs are either NAD+( glycerol : NAD+ 2 OR, EC 1.1.1.6) or NADP+dependent ( glycerol : NADP+ 2 OR, EC 1.1.1.72) enzymes which catalyze the pH dependent, reversible oxidation of glycerol to DHA. While NAD+dependent GDHs are widespread, NADP+dependent GDHs are primarily found in fungi (Jennings, 1984) halophilic algae (Borowitzka et al., 1977) higher eukaryotes (Toews, 1967 ; Kormann et al., 1972) and the bacterium Gluconobacter sp. (Adachi et al., 2008; Richter et al., 2009) The directionality of the redox reaction is pH dependent; the oxidation of glycerol occurs under basic conditions (optimum at pH 10) and the reduction of DHA occurs under slightly acidic conditions (optimum at pH 6) Although glycerol dissimilation to DHA P usually occurs through GK in E. coli, GDH may be alternatively used upon GK inactivation (St Martin et al., 1977) The E. coli GDH exhibits broad substrate specificity, act ing on 1,2propanediol and its analogs, and is activated by ammonium potassium, and rubidium ions and is strongly inhibited by N ethylmalemide, 8hydroxyquinoline, 1,10phenanthroline, and cupric or calcium ions (Tang et al., 1979) Dihydroxyacetone k inase DHAKs (glycerone kinases, EC 2.7.1.29) are a family of amino acid sequenceconserved enzymes which utilize either ATP (eukaryotes and bacteria) or PEP (bacteria) as the source of the highenergy phosphoryl group (Bchler et al., 2005a) DHAKs display wide distribution in eukaryotes and bacteria, and the recent sequencing of haloarchaeal genomes have led to the identification of DHAK homologs in archaea (Bolhuis et al., 2006) The DHAK family can be divided into four distinct groups: i) single subunit ATP dependent DHAKs (found in Citrobacter freundii yeasts, plants, and animals), ii) PEP dependent DHAKs consisting of DhaK DhaL, and a singledomain

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31 DhaM subunit (distributed throughout bacteria), iii) PEP dependent DHAKs consisting of DhaK, DhaL, and a multi domain DhaM subunit (found in Coryn e bacterium di phtheriae E. coli, Desulfovibrio vulgaris and other Proteobacteria as well as some Actinobacteria), and iv) incomplete DHAKs including two subunit DHAKs encoded by discistronic operons without DhaM and DHAKs whose function resembles ATP dependent DHAKs, but whose sequence is more PEP:PTS dependent like (often found in bac terial species such as Yersinia pestis Burkholderia pseudomallei and Sinorhizobium meliloti which encode a reduced PTS that often do es not contain carbohydrate transporters) (Erni et al., 2006) DHAK crystal structures have been solved for the PEP:PTS dependent DHAKs of E. coli (Siebold et al., 2003b; Oberholzer et al., 2006) and Lactococcus lactis (Zurbriggen et al., 2008) and for the ATP dependent DHAK of C. freundii (Siebold et al., 2003a) While ATP dependent DHAKs consist of a single polypeptide with twodomains (DhaK and DhaL), PEP:PTS dependent DHAKs consist of three subunits (DhaK, DhaL, and DhaM). The DhaK subunit s from ATP and PEP:PTS dependent DHAKs have similar topology, consisting of an N terminal domain resembling the mannose family of transporters and a C terminal domain which is structurally similar to FtsZ, a cell division protein. DhaK bin ds DHA covalently between the carbonyl carbon of DHA and the imidazole ring (N 2) on the active site histidine residue which is stabiliz ed through hydrogen bonding. This hemiaminal linkage between the substrate and Dh aK is not involved in catalysis but instead allows for chemical discrimination between short chain carbonyl compounds which are capable of forming such linkages (suc h as DHA) and polyols which cannot form the covalent interaction (such as glycerol). This chemical

PAGE 32

32 discrimination allows DHAK s to retain activity even in the presence of molar quantities of glycerol (Erni et al., 2006) DhaL topology is conserved between ATP and PEP:PTS dependent DHAKs, although the surface potential of these proteins differs significantly (Oberholzer et al., 2006) DhaL contains a novel fold consisting of an eight helix barrel of regular updown topology, a hydrophobic core, and a capped nucleotidebinding site in a shallow depression at the narrow end of the barrel (Bchler et al., 2005a; Oberholzer et al., 2006) ADP is coordinated by two magnesium (Mg2+) ions which are in turn complexed by three gamma carboxyl groups from invariant aspartate residues. Water molecules help to complete the octahedral coordination sphere. Although the DhaL topology between ATP and PEP:PTSdependent DHAKs is conserved, the bound nucleotide assumes a different role in each case. In ATP dependent DHAKs, ADP serves a catalytic substrate of the reaction, similar to the nucleotide role in other kinases. In contrast, the nucleotide in PEP:PTS dependent DHAK s serves as a coenzyme which is not exchanged for ATP, but is instead rephosphorylated in situ by DhaM (Bchler et al., 2005a) Treatment of E. coli DhaL with a chelating agent destabilizes nucleotide binding, thus decreasing the thermal unfolding temperature of DhaL significantly (Bchler et al., 2005a) In most bacterial PEP:PTS dependent DHAKs DhaM consists of a single domain which is homologous to the PTS mannosespecific transporter, IIABMan (Erni et al., 2006; Zurbriggen et al ., 2008) Occasionally, DhaM contains of additional domains which are homologous to the general carrier PTS proteins, Hpr and EI (Gutknecht et al., 2001) Anaerobic Glycerol Dissimilation Pathways Several members of the Enterobacteriaceae including Klebsiella pneumoniae, E. coli, and Enterobacter sp. and many Firmicute s such as Clostridium pasteurianum

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33 Clostridium butyricum C. freundii and Lactobacillus reuteri are capable of anaerobic growth on glycerol as the sole source of carbon and energy (Bouvet et al., 1995) As a result, their fermentative pathways have been more extensively studied and these m icroorganisms have often been used as microbial powerhouses for the anaerobic conversion of glycerol to more valuable chemical products In the absence of an external oxidant, glycerol is converted by Enterobacteriaceae members to 1,3 propanediol through t he action of GD and 1,3 PDH. 1,3 propanediol is the major anaerobic conversion product, accounting for 5060% of t he glycerol consumed, although minor products including ethanol, 2,3 propanediol, acetic acid, and lactic acid are also formed (Daniel et al., 1998) Glycerol d ehydratase GD ( glycerol hydrolyase, EC 4.2.1.30) catalyzes the coenzyme B12(most GDs ) or S andenosyl methionine (SAM)dependent ( C. butyricum GD ) de hydration of glycerol to form 3hydroxypropionaldehyde (3 HPA) Coenzyme B12 dependent GDs are widespread in Firmicute s and Enterobacteriaceae, and have been biochemically characterized from many representatives including Citrobacter Klebsiella Clostridium and Prop ionibacter (Toraya et al., 1980) SAM dependent GD has only more recently been described in C. butyricum (Raynaud et al., 2003; O'Brien et al., 2004) Coenzyme B12dependent GD s have similar biochemical properties to diol dehydratases; however their substrate specificity, subunit composition, monovalent cation selection, and cofactor affinity differ (Toraya, 2000) Both diol dehydratases and coenzyme B12dependent GDs proceed through a radical mechanism in which an adenosyl radical forms in the active site through homolytic cleavage of the cofactor cobalamin (Co) carbon (C) bond which triggers substrate activation by abstraction of a

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34 hydrogen atom (Toraya, 2003) Due to the nature of the biochemical reaction, GDs are easily inactivated by both substrate and cofactor analogs (Bachovchin et al., 1977) ; thus, the genomes of GD containing members often encode reactivase enzymes. Upon inactivation of GD holoenzyme in K. pneumoniae, the enzymebound coenzyme loses the adenine moiety from its upper axial ligand through irreversible cleavage of the CoC bond (Mori and Toraya, 1999) The reactivating factor (encoded by gdrAB ) mediates the ATP dependent exchange of the enzymebound, modified cobalamin for coenzyme containing the adenine moiety. The modified coenzyme is released from the active site and is converted back to its adenylated form through reductive adenosylation. I n C. freundii the structural genes of GD encoded by dhaBCE are part of the dha regulon which also encodes genes for reactivation of inactivated GD ( dhaFG ) as well as additional genes of the pathway ( dhaDKT ) (Seifert et al., 2001) This regulon is under the control of the transcriptional activator DhaR which induces expression under anaerobic conditions when either glycerol or DHA are present. SAM dependent GD is similar in tertiary structure to SAM dependent pyruvate formate lyase (PFL) and, also like E. coli PFL, crystallizes as a dimer (Becker et a l., 1999; O'Brien et al., 2004) As a result of the structural homology between SAM dependent GD and PFL, SAM dependent GD is thought to proceed through a radical mechanism similar to SAM dependent PFL in which r eductive cleavage of SAM results in the t ransient formation of a 5 deoxyadenosyl radical (Frey et al., 1994; Wagner et al., 1999) While this mechanism is reminiscent of the coenzyme B12 dependent GD mechanism, the exact chemical details are no t well understood due to the highly transient nature of the SAM radical (O'Brien et al., 2004)

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35 1,3 Propanediol dehydrogenase 1,3 PD H ( 1,3 propanediol oxidoreductase, EC 1.1.1.202) catalyzes the NADH dependent dehydrogenation of 3 HPA to 1,3 propanediol. Although 1,3propanediol cannot be metabolized further by glycerol fermenting microorganisms without genetic manipulation (Zhu et al., 2002) the NAD+ generated during the reduction of 3HPA can be used by GDH to s upport oxidative glycerol metabolism. The biochemical properties of 1,3PDHs from Lactobacilli sp. and K. pneumoniae have been elucidated. The tetrameric 1 ,3 PDH from L. reuteri reduces acetol and DHA in addition to 3HPA and oxidizes glycerol and propan ediol (Talarico et al., 1990) These properties are in contrast to the 1,3PDH enzymes from K. pneumoniae, Lactobacillus brevis and Lactobacillus buch n eri which purify as either an octomer or a hexamer and are unable to utilize glycerol as an oxidative substrate (Johnson and Lin, 1987; VeigadaCunha and Foster, 1992) Many 1,3 PDHs are inactivated by both metal chelaters and oxygen (Johnson and Lin, 1987; Daniel et al., 1995) In C. butyricum the dhaT gene encodi ng 1,3PDH is under the control of a twocomponent signal transduction system, DhaAS/DhaA, which activates gene expression in response to cellular physiological parameters such as redox potential, similar to the DhaR protein found in other 1,3propandiol generating bacteria (Seo et al., 2009) Glycerol Transport Across a Biological Membrane Two types of membrane transport systems have been distinguished for small molecules, active and passive transport. Active transport systems are characterized as those requiring metabolic energy to move a s ubstance across a biological membrane against an electrochemical gradient. By contrast, passive transport systems are energy independent. Passive transport can occur with (facilitated diffusion) or without (simple

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36 diffusion) the help of facilitator proteins. Bacteria and eukaryotes utilize passive transport with respect to glycerol transport while fungi can also employ active glycerol transport mechanisms. Glycerol transport in archaea has not been characterized at the molecular level, although bioinformat ic analysis predicts the presence of at least GlpF like proteins in some archaea based on primary amino acid sequence identity. Due to the absence of information on archaeal glycerol transport this section will only focus on glycerol transport in bacteria and eukaryotes Intrinsic Permeability The small size and uncharged nature of glycerol allows cells to be intrinsically permeable to this molecule Shigella flexneri and E. coli mutants deficient in glycerol facilitator protein GlpF are able to grow on gl ycerol as a sole carbon source (Richey and Lin, 1972) Both mutants exhibit a calculated maximal glycerol equilibration half life of 20 to 40 s compared to the 2 s equilibration half life of their parental strains. Thus, although glycerol equilibration can proceed through simple diffusion, facilitated diffusion enables much more rapid uptake of glycerol. Glycerol is much less permeable t hrough the membranes of yeast and algae which accumulate the molecule as an osmoprotectant (Brown et al ., 1982; Karlgren et al., 2005) G ated glyc erol export channels allow yeast cells to rapidly control and finetune their glycerol content (Van Aelst et al., 1991) Despite limited glycerol permeability under standard conditions, Dunaliella sp. display increased glycerol permeability in response to environmental fa ctors such as increased temperature (Wegmann et al., 1980; Elevi Bardavid et al., 2008) and osmotic s tress (Fujii and Hellebust, 1992)

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37 Aquaglyceroporins The major intrinsic protein (MIP) family of integral membrane proteins is a ubiquitous family whose members serve as transmembrane (TM) channels that conduct water as well as linear polyalcohols by energy independent mechanisms (Reizer et al., 1993) Members of this family include aquaporins which facilitate water flux across the membrane This family also includes aquaglyceroporins which are highly selective for small molecules such as glycerol and other aldols, and not ions and charged solutes which would otherwise dissipate the electrochemical potential across the cell membrane. Given the specificity of aquaglyceroporins for small molecules, it is without quest ion that they serve important roles in nutrient uptake, osmoregulation, and probably other cellular processes. Aquaglyceroporins are distributed throughout bacteria, filamentous fungi, yeast, and some archaea (Reizer et al., 1993) Representatives from all but the archaeal domain have been characterized at the biochemical level. The structures of various members of the MIP family have been determined (Fu et al., 2000; Sui et al., 2000; Froger et al., 2001; Andrews et al., 2008) revealing a conserved right handed bundle of six TM helices. Symmetry in the N and C terminal halves of the protein structure suggests that the aquaglyceroporins originated from a tandem, intragenic duplication event of an ancestral protein containing three TM domains (Wistow et al., 1991) GlpF and Fpsp1 are the best characterized aquaglyceropori ns in bacteria and eukaryotes, respectively. Additional aquaglyceroporins AQP3, AQP7, and AQP9 in mammals Yfl054 like in yeast and others are also discussed.

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38 Glycerol facilitator p rotein GlpF Glycerol facilitator protein ( GlpF ) is an MIP that catalyzes the equilibration of glycerol between the intracellular and extracellular spac e s in bacteria (Duchesne et al., 2001) GlpF has been best characterized in E. coli where it was first des cribed as a channel protein due to i) its substrate specificity which i s dependent on molecular weight, not chemical structure, and ii) its temperatureinsensitivity with regards to diffusion (Heller et al., 1980) Like other genes of the glp regulon in E. coli, the gene encoding GlpF is induced by G3P (Sanno et al., 1968) and provides a strong growth advantage when extracellular glycerol is limiting (Richey and Lin, 1972) Upon internalization of extracellular glycerol, intracellular glycerol is subsequently converted into G3P by GK This metabolic conversion provides the chemical potential difference of gl ycerol uptake. Due to the inability of GlpF to use G3P as a substrate for transport, an imbalance of glycerol concentration across the membrane is created, and G3P remains trapped intracellularly where it is further metabolized (Heller et al., 1980) Although G3P can not be transported out of the cytoplasm by GlpF, G3P can be transported into the cell through the facilitator GlpT (Hayashi et al., 1964) a member of the major facilitator superfamily (MFS) of transporters. Upon internalization G3P is oxidized to DHAP by G3PDH. Similar to other aquaglyceroporins, GlpF is strictly sel ective for nonionic compounds and has reduced conductivity for water compared to aquaporins (Fu et al., 2000) In addition to glycerol, GlpF conducts linear polyalcohols (al ditols) and urea derivatives, for which it is stereoand enantioselective (Fu et al., 2000) The influx of glycerol as mediated by GlpF requires low energy of activation (Ea 4.5 kcal mol1) (Heller et al., 1980) and is non saturable at more than 200 mM glycerol (Fu et al., 2000)

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39 The crystal structure of an E. coli GlpF tetramer has been determined at 2.2 resolution with each channel containing three glycerol molecules (Fu et al., 2000) T he g lycerol molecules proceed through an amphipathic channel with a radius of 15 on the periplasmic side that constricts to a selectivity filter region with a radius of <3.5 extending 28 in length to the cytoplasmic surface. Due to the constriction s in the selectivity filter region, glycerol and related alditols are predicted to pass through this region in single file. Two conserved aspartic acidprolinealanine motifs comprise a key interface betw een the duplicated threeand one half membranespanning helices (M1 through M8) that form a right handed helical bundle around each channel. Two Mg2+ ions are located in the center of the tetramer near the periplasmic face of the protein; the outer Mg2+ ion is coordinated through the side chain of gluta mate 43 and water, and the inner Mg2+ ion is coordinated through tryptophan42 and water. Site directed mutagenesis suggests that glutamate43 but not tryptophan42 is critical for proper oligomerization, channel function, and in vivo stability of the GlpF protein (Cymer and Schneider, 2010) Fps1p In S cerevisiae the Fps1p aquaglyceroporin mediates glyc erol export and, therefore, plays a crucial role in osmoregulation (Van Aelst et al., 1991) Genome wide homology searches sugges t that Fps1plike proteins are restricted to y easts (Hohmann, 2002) During hyper osmotic conditions, yeast cells activate glycerol biosynthetic genes thr ough an elaborate signaling system involving a mitogenactivated protein kinase cascade and the highosmolarity glycerol pathway as a precondition for glycerol accumulation (Hohmann, 2002; Dihazi et al., 2004) In order for glycerol to accumulate as an organic osmotic solute, Fps1 p activity drastically reduces within seconds after

PAGE 40

40 exposure to the hyper osmotic stress. Fps1p is reactivated after sufficient glycerol accumulation or once the cells have been shifted to hypoosmotic conditions (Tams et al., 1999) R egulation and proper functioning of Fps1p are essential if cells are to maintain proper osmoregulation. Deletion of FPS1 renders cells sensitive to hypoosmotic shock due to the inability of these mutant cells to reduce excessive turgor pressure (Tams et al., 1999) Hyperactive Fps1p results in sensitivity to hyper osmotic stress due to the inability of cells to restore lost turgor pressure (Tams et al., 1999) To mediate proper glycerol export, Fps1p is regula ted by short C terminal (Hedfalk et al., 2004) and N terminal (Tams et al., 2003) domains within the protein, which are required for retention of cellular glycerol under hypert onic stress. These Fps1p regulatory domains were initially identified through truncation analysis (Tams et al., 2003; Hedfalk et al., 2004) and their importance has been confirmed through random geneti c screens for hyperactive Fps1pcontaining cells (Karlgren et al., 2004) The N terminal regulatory domain consists of a cytoplasmic extension near the first TM dom ain and the C terminal domain is located downstream of the sixth TM domain (Tam s et al., 2003; Hedfalk et al., 2004) The positioning of s everal critical amino acid residues of these domains are highly conserved in orthologs from other yeast species (Tams et al., 2003; Hedfalk et al., 2004) The N terminal regulatory domain has an amphiphilic character, and structural predictions indicate that it may fold into the membrane bilayer (Tams et al., 2003) The N terminal domain consists of a wellconserved motif (LYQNPQTPTVLP ) which is part of an approximately 25mer stretch of amino acids constituting a conserved sequenc e (Tams et al., 2003) The intervening, conserved 20 amino acid

PAGE 41

41 distance between the N terminal regulatory domain and the first TM domain is important for proper channel regulation (Tams et al., 2003) The dodecahedral motifs ( amino acid residues 535 546) which constitute the C terminal regulatory domain consist of a well conserved first half (HESPVN) and a variable latter half (Hedfalk et al., 2004) Fsp1p is also regulated by the paralogs Rgc1 and Rgc2 as evidenced by increased cell wall turgor pressure and e levated intracellular glycerol levels following gene deletion of both RGC1 and RGC2 (Be ese et al., 2009) The exact mechanism through which this regulation occurs is still unknown. The biological function of Fsp1p has been recently expanded to include arsenic flux as evidenced by modulation of Fps1p intracellular levels (MaciaszczykDziubinska et al ., 2010) Overexpression of Fps1p in S. cerevisiae increases arsenite tolerance while deletion of FPS1 increases arsenite sensitivity (MaciaszczykDziubinska et al., 2010) This study further determined that arsenite treatment increases the abundance of transcr ipt specific for FPS1 whose gene product localize s to the membrane, and transport experiments revealed that Fps1p in concert with the arsenite transporter Acr3p mediates arsenite efflux in S. cerevisiae Additional A quaglyceroporins In addition to the aqua glyceroporins GlpF and Fps1p which are well described, additional aquaglyceroporins which have not been characterized as extensively are discussed including : i) the fungal specific Yfl054 like aquaglyceroporins ii) a group of fungal aquaglyceroporins whos e members are neither Fps1pnor Yfl054like, iii) the mammalian aquaglyceroporins (AQP3, AQP7, AQP9, and AQP10) and iv) nodulin26 like aquaglyceroporins in plants.

PAGE 42

42 Yfl054 is a fungal specific aquaglyceroporin originally identified from the S cerevisiae genom e sequence (Hohmann et al., 2000) Additional homologs of Yfl054like proteins have been identified in other yeast as well as filamentous fungi. Yfl054like proteins are structurally distinguished from Fps1p as containing a long ( approximately 350 amino acids in length ) N terminal extension and a 50 amino acid C terminal extension. The core TM is highly sequenceconserved among Yfl054 like aquaglyceroporins while the long N terminal extension is variable, with the except ion of a h ighly conserved PVWSLNQPLPV motif (Pe ttersson et al., 2005) The biological function of Yfl054 has not been fully elucidated; however, one study has loosely implicated Yfl054 in glycerol flux based on slight differences in glycerol transport in parental and mutant strains (either FSP1 or FSP1 YFL054) in the presence of ethanol (Oliveira et al., 2003) Due to differences in ethanol sensitivity and glycerol permeability, the authors suggested that Yfl054 may serve a different cellular function from Fps1p. A third group of aquagly ceroporins has been solely identified in filamentous fungi as being neither Fsp1p like nor Yfl054like and whose members share limited sequence similarity and often differ in molecular size (Pettersson et al., 2005) Several members of this group have long N terminal extensions similar to Yfl054. H owever, these proteins differ in their primary sequence from Yfl054like proteins B iochemical or genetic characterizations of members from this group of proteins has not been performed. In mammals thirteen aquaporins have been characterized (AQP0 AQP12) (Ecelbarger et al., 1995; Lee et al., 1996; Lu et al., 1996; Ishibashi et al., 1998; Kuriyama et al., 2002; Mobasheri and Marples, 2004; Ishibashi, 2009) Of these, four

PAGE 43

43 have been c las sified as aquaglyceroporins: AQP3, AQP7, AQP9, and AQP10. These aquaglyceroporins describe a new class of water channels which are permeable to glycerol, but to a lesser degree than bacterial GlpF Phylogenetic analysis reveals that these proteins cluster more closely with bacterial GlpF than with additional eukaryotic aquaporins (Ishibashi, 2009) In huma ns, these aquaglyceroporins are localized in different tissues ; AQP3 and AQP9 i n the kidney (Kuriyama et al., 2002) AQP7 in adipose tissue (Kuri yama et al., 2002) and AQP10 in the gastrointestinal tract (Ishibashi, 2009) Although AQP10 is related to AQP7 based on a mino acid sequence identity (Zardoya, 2005) AQP10 is m ore closely related to AQP3 in function. N either AQP10 nor AQP3 are capable of transporting arsenite, whereas AQ P7 and AQP9 are known arsenite transporters (Liu et al., 2004) The nodulin 26like intrinsic protein subfamily is a highly conserved, plant specific protein family whose members transport a variety of uncharged solutes ranging from water to ammonia to glycerol. Two members in particular, nodulin26 and LIMP2, specifically transport glycerol as well as other uncharged polyols and have a low intrinsic water permeability (Wallace et al., 2002) The primary sequences of nodulinlike protein subfamily members consist of a hybrid of amino acid residues conserved in aquaglyceroporins as well as aquaporins and contain some residues unique to the nodulinlike family (Wallace et al., 2002) Subfamily members are often subject to post translational phosphorylation in response to environmental cues which can stimulate transport activity (Weaver and Roberts, 1992) Phosphorylation is common in both plant and animal MIPs, and this modification often occurs within the cytosolic termini and loop regions of the proteins resulting in modulation of their localization, regulation, or

PAGE 44

44 transport activity (Chaumont et al., 2005) Nodulin like proteins are generally expressed at low levels i n the plant compared to other MIPs, and several exhibit tissue specific localization and are spatially or temporally regulated (Wallace et al., 2006) The biological significance of the glycerol transport behavior by nodulin26 like family members is less well understood, since there is no apparent role for glycerol transport in metabol ism or osmoregulation (Wallace et al., 2006) Active Transport of Glycerol Although not yet described in bacteria, several yeasts and some fungi can accumulate glycerol through active, proton symport (Ferreira et al., 2005) In S. cerevisiae and Candida albicans Stl1p is responsible for mediating active glycerol / H+ symport ( Ferreira et al., 2005; Kayingo et al., 2009) Stl1p is a member of the sugar permease family of the MFS (Nelissen et al., 1997) and was implicated in glycerol / H+ symporter in S. cerevisiae based on the following arguments: i) STL1 mutants do not efficiently utilize or accumulate glycerol as a sole carbon and energy source, ii) active uptake of glycerol is absent in STL1 mutants iii) microarray and proteomic data correlate the expression of STL1 and the presence of its gene product directly with glycerol uptake activity iv) the localization of Stl1p in the plasma membrane and its glucose dependent inactivation are fully consistent with its function as a transporter v) heterologous expression of Stl1p in yeast cells without an active uptake mechanism enables the active uptake of glycerol and vi) glycerol accum ulation in the heterologous system is sensitive to the dissipation of proton motive force through the action of the protonophores and ionophores carbonyl cyanide 3chlorophenyl hydrazone and carbonyl cyanide 4(trifluoromethoxy)phenylhydrazone (Ferreira et al., 2005) Stl1p transport activity appears to be negligible when wildtype S. cerevisiae cells are actively growing

PAGE 45

45 in glucosebased complex media, regardless of salt concentration, but becomes measurable upon glucose exhaustion during salt stress (Lages and Lucas, 1997; Fer reira et al., 2005) or in response to increased temperature (Ferreira and Lucas, 2007) Transc riptional and Post Translational Control of Glycerol Metabolism Glycerol metabolism is controlled both transcriptionally and post trans latio nally and at different steps i n catabolism. Gram positive bacteria generally use Hpr as a central regulatory unit for carbon metabolism, whereas Gram negative bacteria, specifically enteric bacteria, utilize EIIAGlc as the key regulatory unit (Deutscher et al., 2006) In addition to regulation by PTS components, glycerol metabolism is also regulated by nonPTS proteins including FNR, GlpR, DhaR, ArcA and ArcB, cAMP CRP, CcpA, GlpP, Ers, and B, as well as small molecules such as FBP. Transcriptional Regulators of Glycerol Metabolism Gram positive and Gram negative bacterial genomes encode different tra nscriptional regulators of glycerol metabolism Gram negative bacteria use cAMP CRP to regul ate secondary carbon metabolism. The cAMP CRP levels are subject to regulation by adenylate cyclase (AC, 3,5 cyclic AMP synthetase, EC 4.61.1) encoded by cya which ca talyzes the synthesis of cAMP from ATP. In contrast, Firmicutes t ypically do not encode AC and the ability of these organisms to take up cAMP from the environment is unknown. I nstead, Firmicutes use cata bolite control protein A (CcpA) which binds to phosphorylated Hpr as the effector molecule to mediate catabolite repression. Most of the glycerol transcriptional regulators that have been characterized from Gram positive bacteria were done so in Bacillus subtilis whereas most of the Gram negative r egulators have been characterized in E. coli. As there are a large

PAGE 46

46 number of possible glycerol transcriptional regulators, only a handful will be addressed within the scope of this literature review. Regulation by FNR, GlpR, DhaR, Arc A and Arc B, cAMPCRP, CcpA, GlpP, Ers, and B will be discussed. cAMP cAMP receptor p rotein The cAMP CRP complex is a global regulator of transcription that is widespread in bacteria excluding Firmicute s where it is not predicted based on the absence of cya encoding AC (Deutscher et al., 2006) Many of regulatory targets of cAMP CRP are involved in secondary carbon catabolism (Zubay et al., 1970) H owever, cAMP CRP also controls other metabolic processes including but not limited to: osmoregulation (Landis et al., 1999) the stringent response (Johansson et al., 2000) nitrogen assimilation (Mao et al., 2007) bi ofilm formation (Jackson et al., 2002) iron uptake (Zhang et al., 2005) and multidrug resistance to antibiotics (Nishino et al., 2008) CRP is a member of the CRP FNR superfamily of transcription factors whose members are generally transcriptional activators or, less likely, repressors of metabolic genes in facultative or strictly anaerobic bacteria (Krner et al., 2003) Proteins belonging to the CRP FNR superfamily respond to a broad spectrum of both intracellular and extracellular signals including cAMP (Kolb et al., 1993) anoxia (Hutchings et al., 2002) redox state (Zeilstra Ryalls and Kaplan, 1996) nitric oxide (Van Spanning et al., 1999) carbon monoxide (Aono et al., 1996) temperature (Dramsi et al., 1993) and 2oxoglutarate (Muro Pastor et al., 2001) Th is response occurs either through i) intrinsic sensory molecules which allow for the binding of an allosteric effector molecule or through ii) prosthetic groups which interact with the signal molecule (Krner et al., 2003) CRP is activated by binding its allosteric effector, cAMP (Blaszczyk et al., 2001) and is transcriptionally repressed by Fis (Gonzlez Gil et al., 1998) a small DNA

PAGE 47

47 binding protein which regulates the expression of several genes needed for catabolism of sugars and nucleic acids. The crystal structures of cAMP CRP with (Passner and Steitz, 1997; Benoff et al., 2002) and without (Passner et al., 2000) bound DNA and apoCRP (Sharma et al., 2009) have been solved. CRP is a homodimer consisting of two domains : a C terminal helical DNA binding domain (DBD) and an N terminal dimerization domain. The N terminal sheets which contain a hydr ophobic pocket for cAMP binding that is connected by a linker region to the C terminal domain. In the apoform, the CRP Cterminal domain dimerizes with the DNA recognition helix F buried within its core. Upon binding cAMP, a reorientation occurs which liberates the DNA recognition helix (Sharma et al., 2009) Activated CRP then binds to a 22bp palindromic region of DNA which induces severe DNA bending upon binding (Ebright et al., 1989) This bending mechanism allows for two regions of CRP, activation regions 1 (Zhou et al., 1993) and 2 (Niu et al., 199 6) located in the C and N termini, respectively, to interact with RNA polymerase. cAMP CRP levels are subject to regulation by AC which is in turn regulated by EIIAGlc, and by both positive (Hanamura and Aiba, 1992) and negative (Aiba, 1983) autoregulatory mechanisms cAM P CRP activates promoters that are grouped as class I, class II, or class III, d epending on how many activation units ( molecules of cAMP CRP) are required and by the nature of the cAMP CRP interaction with RNA polymerase. In addition to activation, CRP represses transcription by promoter exclusion, exclusion of activator protein, interaction with repressor protein, or hindering promoter clearance.

PAGE 48

48 In E. coli several glycerol metabolic genes are subject to regulation by cAMP CRP complex. As evidenced by DNa seI footprint ing, the cAMP CRP dimer binds two regions centered at 60.5and 37.5bp upstream of the E. coli glpFK transcriptional start site, which also overlaps the GlpR binding site (Weissenborn et al., 1992) Furthermore, monomeric cAMP CRP bin ds in regions centered at 41.5, 91.5, and 131.5 bp upstream of the E. coli glpTQ transcriptional start sites and regions centered at 0.5, 40.5, and 90.5bp upstream of the E. coli glpABC transcriptional start site, each overlapping the GlpR a nd FNR binding sites (Larson et al., 1992) Additional cAMP CRP sites were centered 63.5bp upstream of the transcriptional start site of glpD and 58.5bp upstream of the transcriptional start site for glpEGR although these sites do not appear to overlap additional regulatory sites (Ye and Larson, 1988; Yang and Larson, 1998) The occurrence of tandem operators for cAMP CRP and GlpR as o pposed to overlapping operator regions may explain why glpD displays the greatest sensitivity to GlpRdependent repression. FNR FNR is a member of the CRP/FNR superfamily of transcription factors which mediates the transition from aerobic to anaerobic grow th in a number of bacteria (Krner et al., 2003) FNR activates genes involved in anaerobic metabolism acid resistance, chemotaxis, cell structure, and molecular biosynthesis and transport FNR also represses genes associated with aerobic metabolism (Salmon et al., 2003; Kang et al., 2005) FNR is composed of i) an N terminal sensory domain containing four essential cysteine residues which link the [4Fe4S]2+ cluster, ii) a C ter minal helix turn helix (HTH) DBD, and iii) a dimerization motif located between the N and C termini (Green et al., 1993; Bates et al., 2000; Krner et al., 2003) In the absence of ox ygen,

PAGE 49

49 FNR purifies as a homodimer and the oxygen sensitive [4Fe 4S]2+ cluste r promotes dimerization (Moore and Kiley, 2001) Oxygen destabilizes and inactivates the dimeric FNR complex through oxidation of the [4Fe4S]2+ cluster to two [2Fe 2S]2+containing monomers (Lazazzera et al., 1996; Khoroshilova et al., 1997) Prolonged oxygen exposure returns FNR to an apoprotein state through loss of the two [2Fe2S]2+ clusters (Sutton et al., 2004) Nitric oxide also inactivates FNR through nitrosylation of the [4Fe4S]2+ cluster (Cruz Ramos et al., 2002) Molecules of apoFNR are degraded by ClpXP protease which binds to motifs located in the N and C termini of FNR (Mettert and Kiley, 2005) In its activated form, FNR homodimer binds to a highly conserved, dyad consensus DNA sequence, TTGAT N4ATCAA (where N represents A, T, C, or G) (Eiglmeier et al., 1989) through contacts with serine212 and glutamate209 (Spiro et al., 1990) FNR activates transcription from both class I and class II promoters at locations 61, 71 82 or 92bp upstream and at an average distance of 41.5bp upstream of the transcriptional start site of the affected gene or operon, respectively (Wing et al., 1995) As a repressor, F NR often binds at sites located near the transcriptional start site of the affected gene or operon. Three activating regions (AR), AR1, AR2, and AR3, contact RNA polymerase through the 70 terminal domain of RNA polymerase, thus promoting transcription (Lee et al., 2000; Lamberg et al., 2002; Blake et al., 2002) E. coli glycerol metabolic genes whose transcription is activated by FNR during anaerobiosis include glpABC (Kuritzkes et al., 1984) and glpTQ (Yang et al., 1997) Although the FNR binding sites have not been directly demonstrated, FNR is predicted to bind 0.5and 40.5bp upstream of the

PAGE 50

50 transcriptional start site of E. coli glpABC and 91.5and 131.5bp upstream of the transcriptional start site of E. coli glpTQ based on sequence analysis (Eiglm eier et al., 1989) CcpA Many low G+ C Gram positive bacteria such as B. subtilis do not encode AC and were thus predicted to use a mode of regulation other than cAMP CRP for catabolite repression. In Firmicutes c arbon catabolite repression is mediated by CcpA and Hpr (Deutscher et al., 2006) CcpA is a pleiotropic transcription factor that is a member of the LacI/GalR family of repressors (Henkin et al., 1991) The phosphorylation status at serine46 of general PTS carrier protein Hpr and its homolog, catabolite repression protein Hpr (Crh), affect the ability of CcpA to regulate transcription of a variety of metabolic genes including glpK (Deutscher et al., 1994; Galinier et al., 1997) Thus, in Gram positive bacteria, Hpr participates both in sugar translocation and serves as a corepressor for met abolic enzymes. Although both Hpr and its homolog Crh contain the regulatory serine46 residue, the histidine15 residue is not conserved, indicating that Crh is not implicated in sugar transport. Hpr phosphorylation is catalyzed by homohexameric ATP dependent Hpr kinase ( HprK ) which is triggered by the availability of glycolytic intermediates such as FBP (Deutscher et al., 1995) and is inhibite d by inorganic phosphate (Reizer et al., 1998) Hp rK phosphorylates Hpr in an ATP dependent manner at the regulatory residue serine46 which inhibits EI dependent histidine15 phosphorylation up to 600fold (Stlke et al., 1998) In the presence of inorganic phosphate, HprK can also serve as a phosphorylase, catalyzing dephosphorylation at serine46 (Galinier et al., 1998) HprK is also present in Gram negative, nonenteric bacteria; however, due to the absence of

PAGE 51

51 CcpA in these or ganisms, HprK is thought to mediate different processes (Deutscher et al., 2005) Gram negative HprKs are suggested to mediate pathogenesis as HprKs often cluster with transcriptional regulators of virulence genes (Deutscher et al., 2005) and inactivation of Neis se ria meningentitis hprK leads to decreased cellular adhesion of the pathogen to human epithelial cells (Bol et al., 2003) Two molecules of phosphorylated Hpr serve as the effector f or the dimeric CcpA (Jones et al., 1997) Binding of these phosphorylated Hpr effector molecules to the CcpA C terminal domain triggers a slight rotational movement of the CcpA core which br ings the N terminal DBD into a position that is competent for DNA binding (Schu macher et al., 2004) The CcpA cofactor complex then binds to specific palindromic operator sequences in the promoter regions of catabolic operons known as catabolite responsive elements ( cre ) sites. In B. subtilis the cre site overlaps the 35 promote r region of glpFK (Miwa et al., 2000) GlpR GlpR is a member of the DeoR/GlpR protein family whose members are widespread among bacteria. A mino acid sequence similarity search results indicate that some archaea also encode DeoR /GlpR homologs. DeoR/GlpR type proteins often serve as transcriptional repressors (Munch Petersen and Jensen, 1990; Weissenborn et al ., 1992; Zeng and Saxild, 1999; Ray and Larson, 2004; Barrire et al., 2005; Haghjoo and Galn, 2007) or activators (Zhu and Lin, 1986; Gaurivaud et al., 2001) of either sugar or nucleoside metaboli sm. DeoR/GlpRtype regulators are comprised of approximately 250 amino acids, with a HTH D BD near the N terminus and a C terminal domain that often binds a sugar phosphate effector molecule of the relevant metabolic pathway (Zeng et al., 1996) In general, DeoR/GlpR family members display i) a conserved HTH motif with respect to the second helix, ii) a C terminal oligomerization domain, and iii) an inducer -

PAGE 52

52 binding domain. T he first two amino acid residues of the recognition helix (the first helix in the HTH motif) of DeoR/GlpRtype repressors are thought to confer specificity for binding to the DNA sequence of the operator site. DeoR /GlpR type repressors t ypically regulate transcription through operator binding and concomitant DNA looping (Amouyal et al., 1989; Larson et al., 1992) GlpR has been implicated as a repressor of glycerol metabolism in E. coli (Cozzarelli et al., 1968) as well as P. aeruginosa (Schweizer and Po, 1996) The GlpR proteins exhibit significant similarity (79.4% similarity and 49% identity at the primary amino acid level) (Schweizer and Po, 1996) ; however, only E. coli GlpR has been biochemically characterized. A study involving E. coli GlpR purified to near homogeneity revealed tha t under non denaturing conditions, active GlpR exists as a tetramer with a subunit molecular weight of 30 kDa (Larson et al., 1987) GlpR specifically binds : i) DNA harboring operator sites in a cooperative fashion and ii) G3P (Kd 20 50 M), the lat t er of which diminishes transcriptional repression of the glp operon (Hayashi and Lin, 1965; Larson et al., 1987) Flow dial ysis experiments indicated that each GlpR tetrameric subunit binds one molecule of G3P (Lar son et al., 1987) Binding specificity of G3P for GlpR was evidenced through competition assays involving the binding of unlabelled compounds ( 500 M) to radioactively labeled G3P (28 m) and GlpR (1.4 g) (Larson et al., 1987) Glycerol 2 phosphate, glycerol, PEP, and glucose6 phosphate have no apparent effect on G3P binding to Glp R. Compounds more clos ely resembling G3P, such as DHA P and glycerophosphodiesters have a small yet significant im pact on binding which increases significantly when competing compounds are included at concentrations greater than 1 mM. Addition of G3P (5 mM) dis sociates

PAGE 53

53 GlpR (300 nM) from the glpK operator (1.5 nM) This concentration of G3P (5 mM) was not suffici ent to dissociate GlpR from the promoter regions upstream of the gl pD glpABC or glpTQ regulons indicating that the affinities of the GlpR operator complexes for G3P may vary depending on the operator sequence. Higher concentrations (> 5 mM) of G3P were not examined for the additional operator complexes. For E. coli, the GlpR operator consensus sequence i s determined as 5 WATKYTCGWW 3 ( where W is A or T, K is G or T, Y is C or T, and the dot represents the center of symmetry ) (Zhao et al., 1994) In E. coli the glp regulon co m p rises five operons located at three different positions in the chromosome: i) glpTQ and glpABC operons are transcribed divergently from glpEGR located near 51 min of the linkage map, ii) glpD is located near the 77 min linkage map, and iii) glpFKX is located near the 89 min linkage map (Zeng et al., 1996) Each operon is negatively contr olled by G lpR to varying extent and repression is relieved by the inducer, G3P. G3P permease and glycerophosphodiesterase ( encoded by glpTQ ) and the subunits of G3PDH ( encoded by glpABC ) are divergently transcribed from a common control region containing five operator binding sites for GlpR (Larson et al., 1992) T ranscriptional repression is thought to occur through DNA looping similar to other characterized DeoR/GlpR regulators (Larson et al., 1992) The glpD operon encoding aerobic ally expressed G3PDH and the glpFKX operon encoding GlpF, GK, and fructose1,6 b isphosphatase II are tightly controlled by GlpR. GlpR binds both upstream of the glpFKX operon as well as internal to glpK (Weissenborn et al., 1992) and controls glpD expression through binding at one of four operator sites. Two operator sites are internal to the gene, one overlaps the 10 promoter element and one

PAGE 54

54 is located 30bp downstream of the transcriptional start site (Yang and Larson, 1996) The glpE and glpG genes encoding the uncharacterized proteins GlpE and GlpG display sequence homology to the minor thiosulfate sulfurtransferase and an in tramembrane serine protease cluster with g lpR and are transcribed divergently from the neighboring glpD gene. Multiple promoter elements control the transcription of the gl pEGR operon, allowing for differential expression of its gene products The glpEGR operon is not subject to autoregulation (Yang and Larson, 1998) The order of glycerol metabolic operon sensitivity to GlpR repression from greatest to least sensitive is glpD glpTQ an d glpFKX (Freedberg and Lin, 1973) One study has implicated P. aeruginosa GlpR as a transcriptional repressor of glycerol metabolic genes (Schweizer and Po, 1996) The P. aeruginosa glpR gene is located upstream of glpD encoding G3PDH. Inactivation of glpR results in constitutive expression of glycerol transport activity as evidenced by 14C glycerol uptake. GlpR also represses transcription from the glpT (encoding G3P permease) and glpD (encoding G3PDH) promoters as evidenced by transcriptional promoter reporter fusion assays DhaR DhaR is a member of the enhancer binding protein family and controls expression of the E. coli dha regulon through direct interaction with 70 of RNA polymerase and DNA looping (Rappas et al., 2005; Bchler et al., 2005b) DhaR consists of three domains: i) a C terminal DBD (5 kDa), ii) a central AAA+ ( A TPase a ssociated with diverse cellular a ctivities) ATPase domain (24 kDa), and iii) an N terminal receiver domain for ligands (37 kDa). The N terminal sensing domain of DhaR consists of both GAF ( cycli c G MP, a denylyl cyclase, F hlA) and PAS ( P er A RNT S im) domains which are the ligand and protein interaction domains of twocomponent system sensor kinases

PAGE 55

55 (Ponting and Aravind, 1997; Hurley, 2003) The central AAA+ domain consists of seven highly conserved sequence motifs which are shared among AAA+ proteins (Morett and Segovia, 1993) T he molecular mechanism concerning DhaR regulation of the dha operon has only been described by one study in E. coli (Bchler et al., 2005b) DhaR stimulates transcription of the dha regulon from a 70 promoter (the exact binding site of DhaR is unknown) and autorepresses transcription of dhaR DhaK and DhaL serve as co repressor and co activator of DhaR In the presence of DHA, a phosphoryl group is transferred from the ATP bound DhaL subunit to DHA allowing ADP bound DhaL to bind to DhaR in the receiver domain. This binding in turn activates expression of the dha regulon. DhaK directly compet es with ADP bound DhaL for DhaR. DHA binding to DhaK reduces the affinity of DhaK as a co repressor of DhaR binding Based on gel filtration chromatography following copurification, DhaR is proposed to bind as a dimer to either monomeric DhaL or dimeric DhaK. In the absence of DHA, DhaL is rephosphorylated in situ by DhaM, and is therefore incapable of binding DhaR. DhaR orthologs encoded near dha opero ns are found in other bacteria including C. freundii K. pneumoniae, and Vibrio parahaemolyticus although none have been characterized in detail. Interestingly, DhaR from C. freundii can compliment an E. coli dhaR mutant allowing for regulation of the dh a operon (Bchler et al., 2005b) ArcA/ArcB The global ArcA/ArcB two component regulatory system is used to reduce the expression of many aerobic genes under anaerobic conditions (Iuchi and Lin, 1988; Iuchi et al., 1989) Cytoplasmically located ArcA is a member of the OmpR/PhoB subfamily of response regulators and contains an N terminal rec eiver domain and a C -

PAGE 56

56 terminal DBD. ArcB is membrane associated tripartite protein consisting of i) an N terminal primary transmitter domain, ii) a central receiver domain, and iii) a C terminal histidine containing phosphotransfer domain (HPt). ArcB serves as the sensor histidine kinase which is autophosphorylated at histidine 292 under anaerobic conditions (Iuchi and Lin, 1992) Histidine 292 is predominantly in a phosphorylated state in response to fermentative, metabolic effectors such as D lactate, pyruvate, and acetate (Georgellis et al., 1999) During autophosphorylation, t he phosphoryl group is relayed from histidine 292 in the t ransmitter domain of ArcB, to the aspartate576 residue in the receiver domain of Ar cB, to th e histidine717 of ArcB Hpt and finally to aspartate54 in the receiver domain of ArcA, leading to the activation of ArcA as a DNA binding protein (Georgellis et al., 1997; Jeon et al., 2001) However, u nder aerobic conditions, this autophosphorylation reaction is inhibited (Georgellis et al., 2001) and dephosphorylation of ArcA by reverse phosphorelay is promoted (Georgellis et al., 1998) Physiological evidence h as implicated the ArcA/ArcB twocomponent regulatory system as a transcriptional repressor of aerobically expressed glpD encoding G3PDH in E. coli (Freedberg and Lin, 1973; Iuchi et al., 1990) G3PDH s pecific activity i s reduced in an E. co li strain deficient in both glpR and glpA mutants under anaerobic conditions compared to oxygenic conditions (Freedberg and Lin, 1973) This anoxic dependent reduction i n G3PDH activity is compensated by deletion of either arcA or arcB (Iuchi et al., 1990) Thus, in E. coli the global ArcA/ArcB two component regulatory system is proposed to reduce the expression of glpD encoding the aerobic G3PDH during anoxic conditions Ho wever, the molecular mechanisms surrounding repression i ncluding the ArcA/ArcB binding site are yet to be described.

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57 Additional transcriptional regulators of glycerol m etabolism Given that there are several transcriptional regulators of glyce rol metabolism, all of them can not be covered in the scope of this review. Therefore, only a few additional activators will be further discussed including GlpP, Ers, and B. GlpP is a member of the G3P responive antiterminator family which serves as an antiterminator for the glp regulon. In B. subtilis GlpP activates th e expression of the glpFK glpD and glpTQ transcriptional units through G3P activated antitermination (Holmberg and Rutberg, 1991; Beijer et al., 1993; Nilsson et al., 1994) Activation of GlpP by G3P (Holmberg and Rutberg, 1991) facilitate s binding to an inverted repeat element located in the leader region of glpD (Glatz et al., 1998) GlpP also presumably binds t o the glpFK and glpTQ leader regions based on sequence similarity. In addition to antitermination, GlpP also serves to stabilize glpD message stability (Gl atz et al., 1996) GlpP is widely distributed in Firmicutes; however, none of these additional proteins have been characterized. Ers is a member of the CRP/FNR family and serves as a pleiotropic activator of virulence as well as metabolism in E. faecalis (Giard et al., 2006) Ers shares common CRP/FNR family characteristics including a HTH motif, a cyclic nucleotide binding domain, and several conserved amino acid residues (Riboulet Bisson et al., 2009b) More recently, Ers was demonstrated to control glycerol metabolism of E faecalis through positive regulation of both ef0082 encoding a putative transporter protein and the glpKOF operon encoding GK, G3P oxidase, and GlpF (Riboulet Bisson et al., 2009a) Although the mechanism by which Ers serves as an activator is unknown, it is proposed to be indirect based on DNaseI footprint analysis (Riboulet Bisson et al., 2009a) Ers dist ribution appears to be limited to Firmicutes

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58 Lysteria monocytogenes transcriptionally regulates genes involved in stress related functions by employing an alternative sigma factor, B. Mutants lacking B display increased sensitivity to a wide range of stresses including osmotic pressure (Becker et al., 1998) acidic conditions (Wiedmann et al., 1998) temperature (Ferreira et al., 2001) and oxidative stress (Ferreira et al., 2001) More recently, B has been implicated as a regulator for virulence. Proteomic profiling of cells deficient in B identified proteins encoding putative DHAK subunits whose abundance was severely diminished compared to the parental strain (Abram et al., 2008) Growth analysis confirmed a reduction in the growth rate of a B mutant compared to the parental strain when grown on glycerol as the sole carbon source (Abram et al., 2008) Although proteomic and phenotypic evidence appears to suggest a role for B as a regulator of glycerol metabolism, molecular evidence is still lacking. Allosteric and Post trans l ational Regulation of Glycerol Kinase Activity In addition to its transcriptional regulation, bacterial GK activity is also regulated allosterically through the glycoly tic intermediate FBP as well as general and sugar speci fic PTS components and through post translational modification. Specifically, GK s from Firmicute s are activated through phosphorylation at a conserved histidine residue by Hpr in th e absence of preferred PTS substrates Although Gram positive and Gram negative GKs contain 50 to 60% sequence identity, this phosphorylatable histidine residue which is highly conserved in Firmicute s is noticeably absent from Gram negative bacterial proteins (Charrier et al., 1997) In contrast e nteric bacteria rely upon unphosphorylated EIIAGlc to mediate allosteric inhibit ion of GK activity (Novotny et al., 1985; de Boer et al., 1986) In addition to regu lation by PTS components, b oth Gram positive and Gram negative bacterial GKs are allosterically regulated by FBP (Novotny

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59 et al., 1985; de Boer et al., 1986) B oth FBP and EIIAGlc exhibit positive cooperativity with respect to bacterial GK s and optimally inhibit these enzymes at pH 6.5 (Novotny et al., 1985) Each of these aspects will be discussed within this section. Hpr and EI d epe ndent phosphorylation of glycerol k inase In Gram positive bacteria such as Enterococcus sp. ( Charrier et al., 1997) Hpr serves as an activator of GK activity through reversible phosphorylation (Deutscher and Sauerwald, 1986) In the presence of PTS substrates, EI phosphorylates Hpr which transfers the phosphate moiety to sugar specific permeases which, in turn, phosphorylate the incoming PTS substrate. However, in the absence of preferable PTS substrates such as glucose, a phosphoryl residue is transferred from the N3position of the histidine 15 residue of Hpr to GK allo wing for subsequent 1015 fold activation (Yeh et al., 2004) The site of GK phosphorylation in E faecalis was determined as a conserved histidine232 residue (Charrier et al., 1997) Although Hpr dependent phosphorylation is a dominant form of GK regulation for many Gram positive bacteria, not all members are subject to this type of r egulation. Glycerol is one of the few carbon sources that Mycoplasma pneumoniae is able to metabolize (Hames et al., 2009) As a result M. pneumoniae genes encoding glycerol metabolic enzymes are constitutively expressed, a nd GK enzyme activity is not subject to activation by Hpr dependent phosphorylation (Hames et al., 2009) In addition to transferring phosphoryl moieties to GK, Hpr proteins can also donate phosphoryl groups to transcription al regulators containing PEP carbohydrate PTS regu latory domains (PRDs) (Stlke and Hillen, 1998) Phosphorylation of PR Ds by Hpr is required for activation of these regulators Several operonspecific transcriptional regulators in both Gram positive and Gram negative bacteria, including antiterminators

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60 and activators, contain duplicate PRDs (Stlke et al., 1998) These PRD containing regulators often serve as activators of enzymes invol ved either in the catabolism or generation of sugars transported by the PTS (Stlke et al., 1998) Antiterminator containing PRDs have been demonstrated for regulators controlling glucosidase (AmsterChoder and Wright, 1992) sucrose (Crutz et al., 1990) glucose (Stlke et al., 1997) and lactose metabolism (Alpert and Siebers, 1997) Fructose 1,6b isphosphate FBP is a key intermediary metabolite of glycolysis that regulates the activit y of many metabolic enzymes including lactate dehydrogenase (Cameron et al., 1994) pyruvate kinase (Mellati et al., 1992) and GK (Thorner and Paulus, 1973) FBP allosterically binds to both tetrameric GK (Kd 0.79 0.63 mM) as well as dimeric GK (Kd 19 5 mM) (Yu and Pettigrew, 2003) and promotes tetramer ization by 2 to 4 orders of magnitude (de Riel and Paulus, 1978a; de Riel and Paulus, 1978b) FBP mediated inhibition of GK act ivity is dependent on GK concentration, with desensitization occurring with diluted GK (Yu and Pettigrew, 2003) FBP displays noncompetitive inhibition and negative cooperativity with respect to ATP concentration and uncompetitive inhibition with respect to glycerol (Thorner and Paulus, 1973; Orm et al., 1998) The crystal structure of FBP bound E. coli GK has been solved, and the allosteric binding site of FBP determined (Orm et al., 1998; Yu and Pettigrew, 2003) E. coli G K tetramer binds two molecules of FBP between two glycinearginine loops (residues 224236), where onehalf of the binding site is donated by each monomer at the regulatory interface. Ionic interactions occur between the phosphate moieties of FBP and the GK residue arginine236 (in the guanidinium loop) Hy drogen bonding between the GK

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61 residue glycine 234 (in the amide hydrogen) and the 6phosphate moiety of FBP stabilize s the structure of FBP bound to the allosteric site of GK Site directed m utagenesis of the E. coli G K argini ne236 residue to alanine drastically reduces FBP inhibition, but does not prevent FBP mediated G K tetramer association (Orm et al., 1998) In addition to sitedirected mutagenesis, hydroxylamine mutagenesis has been used to generate colonies which exhibited reduced catabolite repression of glycerol m etabolism (Liu et al., 1994; Pettigrew, 2009) Sequencing of the glpK gene of these E. coli colonies reve aled that an alanine65 to threonine mutation perturbs G K oligomerization and eliminates FBP inhibition while an aspartate72 to asparagine mutation decreases the affinity of FBP for GK (Liu et al., 1994; Pettigrew, 2009) Neither muta t ion however, showed significant change o f either G K catalytic activity or inhibition by EIIAGlc. EIIAGlc EIIAGlc (also called IIIGlc in older literature) is the central regulatory element of the PEP:PTS in enteric bacteria, serving as a signal for the availability of extracellular glucose EIIAGlc recognizes and binds in a phosphorylation status dependent manner to at least 10 different target proteins including lactose, melibiose, and maltose transport components as well as GK (Postma et al., 1993) Specifically, EIIAGlc modulates glp expression through both inducer exclusion and regulation of cAMP CRP. Both mechanisms of action are dependent on the phos phorylation status of EIIAGlc, especially at histidine90, which in turn is dependent on both the av ailability of PTS substrates as well as the ratio of PEP to pyruvate. EIIAGlc is preferentially dephosphorylated when cells are exposed to readily metabolized PTS substrates or when the ratio of PEP to pyruvate is low. In its non phosphorylated state, EIIAGlc directly binds to and inactivates

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62 metabolic enzymes and transporters of secondary carbon sources. Specifically, EIIAGlc binds to and inactivates G K, preventing glp inducer formation (Novotny et al., 1985) EIIAGlc can also modulate glp operon expression through regulation of AC In the absence of readily metabolized carbon sources (such as glucose), phosphorylated EIIAGlc activates AC As a result, levels of cAMP and cAMP CRP are elevated leading to the activation of second ary metabolic operons such as the glp regulon The inducer molecule (G3P) is subsequently produced which binds to and inactivates EIIAGlc, preventing inducer exclusion. I n its dephosphorylated state, EIIAGlc is unable to activate AC thus decreasing cAMP and cAMP CRP levels. The crystal structure of EIIAGlcbound to GK in the allosteric site has been solved for E. coli proteins (Hurley et al., 1993) The interaction of EIIAGlc with G K occurs in the C terminal domain about 30 from the glycerol binding site (Hurley et al., 1993) and is stimulated by the presence of Zn2+ ions (Pettigrew et al., 1998) Site directed mutagenesis of the EIIAGlc phosphoryl acceptor ( histidine 90 ) and the adjoining histidine 75 residues to glutamine results in a loss of EIIAGlc regulatory function and severely impairs EIIAGlc phosphorylation, respectively (Presper et al., 1989; Meadow and Roseman, 1996) X ray crystallography, differential scanning calorimetry, and nuclear magnetic resonance ( NMR ) reveal slight structural differences between the EIIAGlc mutant and parent protein structures (Pelton et al., 1996) Specifically, the h istidine 90 to glutamate mutant protein contained additional coordinated water molecules and the hist idine75 to glutamate mutant protein had a reduced phosphoEIIAGlc hydrogen bond network.

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63 Industrial and Biological Significance of Glycerol Glycerol has been considered a neglected variable in bi ological processes due to its importance as a biosynthetic precursor, as a compatible solute, and/or as an organic carbon and energy source in all domains of life (Brisson et al., 2001) Glycerol is of extreme biological significance to halophilic communities, where it serves as an osmoprotectant for the primary producer Dunaliella and as the primary energy source for heterotrophic com munity members. In addition to its neglect with respect to biological processes, glycerol has also been considered an underutilized feeds tock for bioconversion. Its low cost, ease of its production, and the highly reduced nature of its carbon atom s makes g lycerol an attractive feedstock for bioconversion. Natural glycerol fermenters as well as heterologous systems have recently gained attention for the conversion of glycerol rich waste streams generated from the biodiesel industry into more valuable product s. Glycerol has also gained importance for its properties in the treatment and diagnosis of disease. G iven the scope of this review, only the biological relevance of glycerol, in part icular to halophilic ecosystems and the biotechnological and medical appl ications of glycerol will be discussed. Glycerol as a Feedstock for Bioconversion Glycerol can be produced either by microbial fermentation, recovered as a by product of saponification, or can be chemically synthesized from petrochemical feedstocks. Glycerol has been of recent interest as a feedstock for bioconversion due to the low cos ts associated with its production as a result of the biofuels industry. Biodiesel is produced through base catalyzed esterification of vegetable oil and animal fats (triacylglycerols) with short chain alcohols such as methanol or ethanol. The principal by product [10% weight (w) / volume (v)] obtained through such reactions is glycerol

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64 (Papanikolaou et al., 2002; Gonzlez Pajuelo et al., 2004; Mu et al., 2006) Once considered a valuable coproduct from biodiesel production, crude glycerol is now considered a costly waste stream with high disposal costs (Yazdani and Gonzalez, 2007) As a result of the highly reduced nature of the carbon atom s and the low production cost, glycerol is gaining increasing interest as a feedstock for bioconversion. Purified glycerol as well as its conversion products are used in a variety of commercial products including but not limited to those produced by the cosmetics, paints, automotive, food, tobacco, pharmac eutical, pulp and paper, leather, and textile industries (Wang et al., 2001) F ermentative glycerol conversion pathways have been studied extensively in naturally fermentative species including Enterobacter sp. (Barbirato et al., 1997) C. freundii Clostridium sp (Forsberg, 1987) Lactobacillus sp. (Schtz and Radler, 1984) Bacillus welchii (Gonzlez Pajuelo et al., 2004) and K. pne umoniae (Biebl et al., 1998) and many of these organisms have been used for the conversion of g lycerol to the primary fermentative product, 1,3 propanediol. Conversion of a glycerol feedstock to products other than 1,3propanediol can be achieved through modulating culture conditions and/or genetic manipulation (Forsberg, 1987) A lternative gly cerol fermentation products include DHA (Hu et al., 2010) hydrogen (Ito et al., 2005; Sakai and Yagishita, 2007; SabourinProvost and Hallenbeck, 2009) ethanol (Ito et al., 20 05; Sakai and Yagishita, 2007) succinic acid (Lee et al., 2001) propionic acid (Liu et al., 2010) butanol (Biebl, 2001) 2,3 butanediol (Biebl et al., 1998; Petrov and Petrova, 2009) and biosurfactants such as rhamnolipids (Santa Anna et al., 2001) D ue to i) the pathogenicity, ii) the requirement of strict anaerobios is iii) the need for rich nutrient

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65 supplementation, and iv) the difficulty of genetic manipulation of the abovementioned glycerol fermenting species acidic glycerol fermentation by E. coli has also be e n recently investigated (Yazdani and Gonzalez, 2007) E. coli has been used for the anaerobic conversion of glycerol to 1,2propanediol, hydr ogen, lactate, formic acid, acetate, succinate, and ethanol (Dharmadi et al., 2006; Gonzalez et al., 2008; Murarka et al., 2008) Therapeutic and Diagnostic Uses of Glycerol In addition to its chemical and metabolic conversion, glycerol has also been widely used directly in a number of therapeutics, ranging from cancer treatment to rehydrating agents. The viscosity of glycerol enables its use a s a softener in cough syrups, emollients, and ointments (Brisson et al., 2001) Due to its osmoregulatory properties, glycerol is used as a purgative and for the treatment of cerebral edemas, glaucoma, intracranial hypertension, and acute strokes (McCurdy et al., 1966; Bayer et al., 1987; Brisson et al., 2001) Glycerol can also serve as a hydrating agent, facilitating water adsorption in the k idneys and intestines and has been used to treat acute gastrointestinal diseases and constipation (Brisson et al., 2001) Furthermore, glycerol has shown potential as an anti cancer agent since glycerol functions as a chemical chaperone and may help in correcting p53dependent apoptosis (Ohnishi et al., 1999) Glycerol also has implications in diagnostics. Increased median hemolytic time in 0.3 M glycerol solution serves as an indicator for various red blood cell disorders such as sickle cell anemia, sickle thalassemia (Gottfried and Robertson, 1974) Additionally, the levels of serum glucose following glycerol administration can serve as an indic ator for glucose intolerance (Senior and Loridan, 1968) and abnormal

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66 glycerol levels in the kidney may serve as an indicator of renal disease (Brisson et al., 2001) Biological Relevance of Glycerol to Halophilic Communities Aside from its bioin dustrial uses, glycerol is an im portant biological molecule for halophilic communities. High salt environments such as the Dead Sea, the Great Salt Lakes, and salterns are dominated by i) halophilic archaea, ii) halophilic, rod shaped bacteria of the genus Salinibacter and iii) the unicellular green alga Dunaliella sp. which serves as the primary producer for heterotrophic community members In order to withstand osmotic stresses, Dunaliella sp. produces molar quant ities of glycerol as an osmotic solute (Figure 1 1) Permeability studies indic ate that this glycerol is available to heterotrophic members, especially as a result of external stresses such as increased temperature or hypertonic conditions (Wegmann et al., 1980; Fujii and Hellebust, 1992; Elevi Bardavid et al., 2008) (Figure 1 1) Thus glycerol is often postulated to be one of the most important energy sourc es for heterotrophic prokaryotes in hypersaline ecosystems. This section will review the osmoprotectant nature of glycerol, the production and release of glycerol by Dunaliella sp. and glycerol metabolism by heterotrophic community members. The importance of DHA as a potentially key substrate in the saltern ecosystem will additionally be addressed. Osmoprotectant properties of glycerol Halophilic and halotolerant microorganisms have adapted different mechanisms to withstand the high osmotic pressure exerted by their surrounding hypersaline environment. Most h alophilic archaea as well as bacterial species from the anaerobic, fermentative order Halanaerobiales and the aerobic, halotolerant genus Salinibacter maintain a high intracellular cation concentration equal to that of the surrounding

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67 environment (Lanyi, 1974) In the case of Halanaerobiales the salt in strategy is the only feasible means of haloadaptation, since very little energy is obtained from fermentative pathways and the massive production of organic compatible solutes would leave insufficient energy for other cellular functions (Oren, 1999) Interestingly, Salinibacter sp. which coexist with Halobacteriaceae members in hypersaline environments, have archaeal like properties including an acidic proteome and similar ities in genomic composition, suggesting horizontal gene transfer occurred between the two heterotrophic community members (Oren, 2008; nton et al., 2008) In general, cells which employ a salt in strategy accumulate K+, Mg2+, and Clions and exclude Na+ ions (Christian and Waltho, 1962) This salt protects halophilic proteins that tend to contain an excess of acidic amino acids through charge shielding (Lanyi, 1974) As a result, most intracellular proteins require high salt to retain activity (Lanyi, 1974) M any halophilic bacteria, osmophilic yeast, and the halotolerant algae Dunaliella sp. exclude intracellular salts and instead accumulate compatible solutes. Interestingly, methanogenic archaea both accumulate salt and synthesize organic osmotic solutes such as glycine betaine and amino acids (Oren, 2008) The term compatible solute was invoked while observing that intracellular polyols accumulate in large quantities in osmophilic yeast and enable these cells to tolerate environments with low water activity (Brown and Simpson, 1972) Compatible solute is now generally used to describe a low molecular weight solute accumulating at high concentration which, by virtue of being a poor enzyme inhibitor, protects enzymes against inhibition which would otherwise occur under low water activity. A variety of compatible solutes including amino acids and their derivatives, sugar alcohols, and sugars have been described in halophilic and

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68 halotolerant microorganisms. Individual chemical classes of compatible solutes can be widely distributed or limited to members of particular a phylogenetic group. Glycine betaine is a widely distributed compatible solute used by both halophilic methanogens and a number of bacteria, although few prokaryotes are capable of de novo synthesis of this compound (Oren, 2008) More widespread in bacteria are cycli c amino acid derivatives ectoine and hydroxyectoine, the former of which is synthesized by a number of aerobic, heterotrophic bacteria (Oren, 2008) In contrast, the distribution of compatible solutes glucosylglycerol acetyl lysine is more limited; the former is found nearly exclusively in moderate to highly halophilic cyanobacteria while the lat t er is found only in aerobic Firmicute s (Oren, 2008) Polyols such as glycerol are employed as compatible solutes almost exclusively by eukaryotes, although Pseudomonas putida use s mannitol (Kets et al., 1996) Glycerol, in particular, is the principal compatible solute produced in response to decreased extracellular water activity by yeast (Brown and Simpson, 1972; Blomberg and Adler, 1989) as well as algae (Craigie and McLachlan, 1964; Ben Amotz and Avron, 1973b; Borowitz ka and Brown, 1974; Ben Amotz and Avron, 1979) Glycerol p roduction by the halotolerant green a lgae Dunaliella Glycerol accumulates in molar quantities as a compatible solute in the green, halotolerant algae Dunaliella sp. in response to increasing sal t concentrations (Craigie and McLachlan, 1964; Ben Amotz and Avron, 1973b; Borowitzka and Brown, 1974) The mechanisms surrounding glycerol synthesis have not been well elucidated. The available data sug gests that glycerol is primarily produced from starch reserves in the chloroplast which are converted to DHAP through glycolysis with consumption of reducing equivalents which are supplemented by the pentose phosphate pathway

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69 (Chitlaru and Pick, 1991) In Dunaliella salina it is proposed that G3P is produced from DHAP through an NAD+dependent G3PDH and is trans ported into the cytoplasm by a phosphate translocator in exchange for an inorganic phosphate molecule. Once in the cytoplasm, G3P is hydrolyzed by G3PP to form glycerol. Excess glycerol is returned to cellular metabolism by oxidation to DHA through an NADP+dependent GDH. Subsequent ph osphorylation of DHA by an ATP dependent DHAK generates DHAP which is transported back into the chloroplast. I n D. tertiolecta photosynthesis also contributes to glycerol synthesis as evidenced by 14CO2flux to glycerol ; however, its carbon contribution relative to starch degradation decreases with increasing osmotic stress (Goya l, 2007) Proteomic evidence for the involvement of both starch catabolism and photoassimilation of carbon dioxide in glycerol synthesis has also been reported in D. salin a (Liska et al., 2004) in which key enzymes involved in the Calvin cycle, starch mobilization, and energy production a re more abundant in a salt induced proteome as identified by mass spectrometry. Several factors contribute to the regulation of glycerol synthesis including i) intracellular pH, ii) PFK activity, and iii) cellular levels of ATP and inorganic phosphate. Cytoplasmic alkalinization following hyperosmotic shock of Dunaliella is proposed t o activate glycerol synthesis based on the pH dependence of starchcatabolizing enzymes in this alga (Goy al et al., 1987; Kuchitsu et al., 1989) PFK is implicated in regulating glycerol synthesis from starch, as it is a classical checkpoint for glycolysis. ATP and photoassimilation products such as phosphoglyceric acid inhibit PFK activity (and subsequent glycerol production), while inorganic phosphate activates PFK activity and glycerol synthesis (Chitlaru and Pick, 1991) NAD+dependent DHA reductase

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70 activity has been reported in Dunaliella parva (Ben Amotz and Avron, 1973a) as well as D. tertiolecta (G ee et al., 1989) with the former having very weak NAD+dependent G3PDH activity, suggesting that glycerol may also be synthesized from DHA. Due to the fact that glycerol is essential to the haloadaptibility of Dunaliella sp. glycerol is several order s of magnitude less permeable to the biological membrane of this type of microalgae when compared t o other organism s (Brown et al., 1982) Thus, glycerol release from lysed algal cells is an important means by which this organic carbon and energy source becomes available to halophilic, heterotrophic community members. A side from glycerol r elease from lysed cells, Dunaliella cells can leak glycerol, especially at temperatures above 45 C (Wegmann et al., 1980; Elevi Bardavid et al., 2008) Significant release of intracellular glycerol has also be observed in the marine alga D. tertiolecta as a result of hypotonic stress (Fujii and Hellebust, 1992) Thus, glycerol appears to be a significant organic carbon and energy source for members of halophilic communities. In addition to glycerol, DHA derived from the glycerol cycle is a substrate of potential interest to heterotrophic community members, although the extent to which DHA is permeable to the algal membrane is currently unknown (Elevi Bardavid and Oren, 2008) Glycerol m etab olism in halophilic bacteria Aside from the primary producer Dunaliella sp. the saltern community is also dominated by heterotrophic members including the halophilic bacterium Salinibacter sp. and various haloarchaea. As a result of its production and release in Dunaliella sp ., g lycerol has been r eported to be one of the most available and rapidly turned over organic substrates in saltern communities (Sher et al., 2004) Salinibacter is proposed to metabolize glycerol solely through a glycerol inducible GK based on enzymatic

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71 activity assa ys for which no signifi cant NAD+dependent GDH activity has been detected in cell lysate (Sher et al., 2004) Unlike haloarchaea, Salinibacter does not appear to produce organic acids following incomplete oxidation of glycerol (Sher et al., 2004) Instead, DHA has been detected as an overflow product of glycerol metabolism on the basis of a nonspecific colorimetric assay and a specific, enzyme activity assay (Elevi Bardavid and Oren, 2008) DHA production can account for up to 20% of the glycerol metabolized (Elevi Bardavid and Oren, 2008) Interestingly, in the absence of glycerol, Salinibacter consume s DHA (Elevi Bardavid and Oren, 2008) The molecular mechanisms surrounding this DHA production and metabolism are currently unknown. Glycerol metabolism in h aloarchaea Archaeal glycerol metabolism has been widely uncharacterized at the molecular level, especially in haloarchaea. Most of the current knowledge concerning haloarchaeal glycerol metabolism is based on enzyme activity analysis (Wassef et al., 1970; Rawal et al., 1988; Oren, 1994; Oren and Gurevich, 1994a) Similar to bacteria, archaea are believed to metabolize glycerol through either i) GK and G3PDH or ii) GD H and DHAK. GK enzymatic activity has been detected in many haloarchaea (Rawal et al., 1988) GDH has only been detected in H. salinari um and H. cutirubrum (Rawal et al., 1988) which are nonutilizers of carbohydrates GK is noticeably absent from those halophilic archaea that c annot use glycerol as an energy source, such as autotrophic methanogens (Nishihara et al., 1999) Thus, GK is not thought to be involved in the synthesis of the backbone of archaeal p hospholipids which is instead mediated by sn G1P dehydrogenase (Nishihara et al., 1999) Putative GKs ( glpK ) often cluster on the genome with putative G3PDH ( gpdABC ) as well as putative glycerol facilitators ( glpX glpF ) (Figure 3 1 ). Although most of the glycerol consumed by haloarchaea as an

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72 energy source is respired to carbon dioxide, some glycerol is incompletely oxidized to organic acids such as acetate, pyruvat e, and lactate in Haloferax and Haloarcula sp. possibly due to the low oxygen dissolution under high salt (Oren and Gurevich, 1994b) Aside from glycerol, DHA is also proposed to be a significant organic carbon source for members of the halophilic, heterotrophic community (Elevi Bardavid et al., 2008) Haloquadratum walsby i metabolizes DHA as evid enced by colorimetric and specific enzymatic activity assays when cells are grown in complex media with DHA supplemented at 5 mM (Elevi Bardavid and Oren, 2008) Along with H. walsbyi (Bolhuis et al., 2006) b ioinformatics predicts that Halobacterium lacusprofundi (Schneider et al., 2006) and H. volcanii (Hartman et al., 2010) encode homologs of each of the PEP:PTS DHAK components, although there is currently no evidence supporting the metabolism of DHA by the latter two organisms. Interestingly, DHA is produced both in Salinibacter and Dunaliella sp. and is consumed over time in saltern communities (Elevi Bardavid and Oren, 2008) However, the source of DHA turnover is complicated by the fact that : i) Dunaliella sp. encode a functional DHAK (Lerner and Avron, 1977; Lerner et al., 1980) and ii) DHA transporters and its permeability has not been characterized for any of halophilic community members. An Overview of Glucose and Fructose Metabolism in Haloarchaea Although glycerol is widely used by haloarchaea as a primary energy source, only a limited number of h aloarchaea metabolize sugars such as glucose and fructose. For instance, although H. volcanii utilize s both glucose and fructose as sole carbon and energy sources, Halobacterium sp. are not able to utilize either carbohydrate (Gochnauer and Kushner, 1969) Haloarchaeal fructose and glucose dissimilation pathways have been somewhat characterized; h owever, knowledge concerning the

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73 mechanisms through which these pathways are regulated and the means by which the sugar is t ransported into the cell remain limit ed. This section will focus on the transport of glucose and fructose, characterized pathways of glucose and fructose metabolism, and key regulators of sugar metabolic enzymes in haloarchaea. Transport of Fructose and Glucose A cross a Biological Membrane Da ta on sugar transport is limited in archaea, especially i n halophilic archaea. B ioinformatics predicts that PTS sugar specific permeases and general phosphocarrier components are encoded within archaeal genomes including various haloarchaea and Thermofilum pendens H owever, none of these PTS homologs have been characterized ABC transporters and secondary transport systems utilizing an electrochemical gradient of Na+ ions have been described in the translocation of fructose and glucose across the membrane. Each of these transport systems, including an overview of the bacterial PTS, will be discussed in detail within this section. O verview of the Phosphotransferase System The PTS catalyzes the concomitant phosphorylation and transport of sugar substrates in bacteria. The PTS consists of three essential catalytic entities which can be fused or encoded separately: cytoplasmic general energy coupling proteins EI and Hpr and membraneassociated sugar specific permease enzyme II (EII) (Postma et al., 1993) The EII complexes generally consist of three proteins or protein domains ( E IIA, E IIB and E IIC); however, the mannose permease family consists of an additional domain ( E II D). The EIIA and EIIB domains are involved in phosphorylation, while EIIC functions as a membranebound permease. Phosphoryl relay proceeds from p hosphoenolpyruvate (PEP) as generated by enolase, to the N3 position of histidine 189 of EI (Weigel et al., 1982a) to the N1 position of histidine15 of Hpr (Weigel et al.,

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74 1982b) to a phosphorylatable histidine residue in family specific E IIA, to a phosphorylatable histidine residue within permeasespecific E IIB and finally t o the sugar substrate transported by sugar specific permease E IIC. All phosphoryl transfer reactions between PTS protei ns are reversible, and the phosphorylation status of various PTS proteins is determined by both PTS transport activity and the PEP:pyruvate ratio, reflecting flux through glycolysis (Kotrba et al., 2001) The dynamic phosphorylation status of PTS proteins in response to nutritional conditions and the metabolic state of the cell serves as the basis for PTS mediated regulation of diverse metabolic processes These processes include the transport and metabolism of nonPTS carbon sources, cell division, chemoreception, carbon storage and metabolism, noncarbon compound transport, pathogenicity, cellular motility, cell physiology, gene expression, nitrogen metabolism, and switching between fermentative and respiratory metabolism (Barabote and Saier, Jr., 2005; Deutscher et al., 2006) PTS components are widely distributed in bacteria (Barabote and Saier, Jr., 2005) and uncharacterized homologs are additionally present in archaea. High G + C Gram positive bacteria generally have a partial PTS, although complete transport systems for glucose, fructose, and ascorbate have been described in some members including Corynebacterium Most Firmicute s contain the general carrier components Hpr and EI as well as the regulatory enzyme HprK and sugar specific permeases for glucose, fructose, and lactose. Spirochetes and proteobacteria often lack PTS component s or contain only the general carrier phosphoproteins; whereas, the and gammapr oteobacteria encode more complete PTS components. Furthermore, many gammaproteobacteria encode paralogs of EI, Hpr, and fructosespecific IIA proteins which

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75 function as a phosphorelay system, contributing to nitrogen regulation (Powell et al., 1995) Each of these PTS components will be discussed General carrier p rotein EI EI is a highly conserved, soluble protein which, in E. coli exists in equ ilibrium between a functional homodimer and a nonphosphorylatable monomer (subunit molecular mass 59 to 64 kDa) (Chauvin et al., 1994) O nly the dimeric EI accept s the phosphoryl moiety from PEP, and the relatively slow EI monomer to dimer transition appears to be the ratelimiting step during in vitro phosphotransfer (Chauvin et al., 1996) Transfer of the phosphate moiety from PEP to EI requires the presence of Mg2+ ions although subsequent phosphotransfer to Hpr does not require this additional cofactor (Weigel et al., 1982a) The amino acid sequence of EI exhibits 30% similarity with pyruvate phosphate dikinase and PEP synthase which autophosphorylate at their respective activesite histidine residues using ATP or PEP as a phosphoryl donor (Poc alyko et al., 1990; Kotrba et al., 2001) The EI monomer is composed of two functional domains of equal size as determined by highsensitivity differential scanning calorimetry in Salmonella typhimurium (LiCalsi et al. 1991) The N terminal domain contains both the active site histidine residue which is located within a conserved signature motif of PEP utilizing enzymes and the Hpr interaction site (Kotrba et al., 2001) The EI C terminus binds PEP and is necessary for self dimerization (Chauvin et al., 1996) Biophysical analysis of the Streptomyces coelico lo r EI with several effectors indicates that EI is partly unfolded under acidic conditions and that PEP induces structural changes in this protein (Hurtado Gomez et al., 2006)

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76 General carrier p rotein Hpr Hpr is a soluble, general carrier protein consisting of a single domain of approximately 9 kDa of vari able amino acid sequence (Postma et al., 1993) Despite sequence variability, the site of phosphorylation involved in sugar transport, histidine15, is well conserved among Hpr proteins and is within a common consensus motif. The tertiary structure of Hpr is highly conserved, consisting of an arrangement of three sheets (Klevit and Waygood, 1986) The histidine15 residue is located with in the amino terminus helix. The NMR solution structures of Hpr complexed with EI (Garrett et al., 1999) and EIIAGlc (W ang et al., 2000) do not exhibit significant changes in the chemical shift values, indicating that Hpr does not undergo large conformational changes with different binding partners. Most low G + C Gram positive bacterial and a few Gram negative bacterial Hpr proteins can also be phosphorylated at serine46 by an ATP dependent HprK. This regulatory phosphorylation does not participate in sugar transport, although it inhibit s EI dependent histidine 15 phosphorylation by up to 600fold (Stlke et al., 1998) Sugar specific component EII EII components are multi domain pr oteins or protein complexes consisting of a family specific phosphoryl donor EIIA, a permease specific phosphoryl donor EIIB, a sugar specific permease/receptor EIIC, and, in the case of mannose permeases, an auxiliary protein EIID (Barabote and Saier, Jr., 2005) The EIIAB components consist of two peripheral proteins or domains of similar size, while the EIICD are integral proteins or domains (Kotrba et al., 2001) The EIIABC can exist as separate proteins or can be fused. There are seven characterized PTS permease families including: i) the glucose family that transport s glucose, N acetylglucosami ne, maltose, glucosamine, glucosides,

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77 sucrose, trehalose, and N acetylmuramic ac id ii) the fructose family that transport s fructose, mannitol, mannose, and 2O mannosyl D glycerate, iii) the lactose family that transports glucosides, cellobiose, N,Ndiacetylchitobiose, and lichenan oligosaccharides iv) the glucitol family that transports glucitol and 2methyl D erythitol v) the galactitol family that transports galactitol and D arabinol vi) the mannose family that transports gluco se, mannose, sorbose, fructose, glucosomaine, galactosamine, N acetylglucosamine, and other related compounds and vii) the ascorbate family that transports Lascorbate (Barabote and Saier, Jr., 2005) A non transporting soluble EII complex (DhaMLK) phosphorylates DHA at the expense of PEP with the aid of Hpr and EI (Gutknecht et al., 2001) ATP Binding Cassette T ransporters ATP binding cassette (ABC) transporters, which appear to be widespread in archaea (Saier, Jr., 2000) have been biochemically characteri zed in all domains of life. ABC transporters are a ubiquitous class of proteins involved in varying cellular processes such as substrate uptake or export, osmosensing and osmoregulation, and antigen processing (Holland and Blight, 1999) ABC transporters consist of two integral membrane permease proteins and an ATPase, the latter of which drives the substrate translocation event through ATP hydrolysis. In eukaryotes, each of these functional components is contained within a single polypeptide, whereas in prokaryotes, single proteins constit ute these activities (Albers et al., 2004) ABC transporters often have a very high affinity for their substrate, and have even been reporte d as low as the subnanomolar range for archaea (Xavier et al., 1996; Woodson et al., 2005) This high affinity provides an advantage for organisms that inhabit nutrient deprived ecological niches. The ATPase components are generally conserved at several motifs including

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78 Walker A and B sites, a helical domain, a linker peptide, and a switch region. The permease components typically consist of five to six membrane spanning helices and a n EAA loop located 100 amino acids from the C terminus Prokaryotic ATP transporters function through extracellular substratebinding proteins which bind the substrate and deliver it to the permease domains. Upon docking to the permeases, the substrate is released from the substratebinding protein and is subsequently translocated across the membrane. Prokaryotic substratebinding proteins are structurally conserved and consist of two distinct globular domains which are dr awn nearer to each other in a hingebending mechanism upon substrate binding within the cleft region (Quiocho and Ledvina, 1996) Unlike their bacterial counterparts arc haeal substratebinding proteins are often glycosylated (Koning et al., 2001; Elferink et al., 2001; Koning et al., 2002) although this modification is not required for substrate binding based on heterologous protein production studies in E. coli (Horlacher et al., 1998; Koning et al., 2001; Koning et al., 2002) G lycosylati on is proposed to stabilize these extracellular substratebinding proteins against proteolysis, influence their interaction with the cell membrane, and/ or alter their thermostability (Albers et al., 2004) Many archa eal ABC transporters have been characterized from hyperthermophiles (Xavier et al., 1996; Horlacher et al., 1998; Albers et al., 1999; Koning et al., 2001; Koning et al., 2002; Bevers et al., 2006) although genetic evidence for ABC transporters in methanogenic (Jovell et al., 1996; Schmidt et al., 2007; Chan et al., 2010) and halophilic archaea (Wanner and Sopp a, 1999; Woodson et al., 2005) has been demonstrated.

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79 Secondary Transporters Secondary transporters depend on the electrochemical gradient of sodium ions or protons to translocate the substrate across the cytoplasmic membrane. This group of transporters is widely used in mammals for amino acid as well as glucose and fructose transport (MacDonald et al., 1977; Lerner, 1987) H. volcanii transport s both glucose (Tawara and Kamo, 1991; Severina et al., 1991) and fructose (Takano et al., 1995) through a sodium dependent electrochem ical gradient which is induced by the respective hexoses. Interestingly, inhibitors of mammalian glucose transport such as phloridzen and forskolin inhibit the H. volcanii glucose transporter (Tawara and Kamo, 1991) de spite the somewhat low (26% ) primary amino acid sequence identity Fructose and Glucose Metabolism and Their Regulation in Haloarchaea Various haloarchaeal species including those of the genera Halobacterium Haloarcula, Haloferax and Halococcus are able to utilize glucose and fructose as sole carbon and energy sources (Rawal et al., 1988) Some notable noncarbohydrate utilizers includ e H. salinar um and H. cutirubrum (Rawal et al., 1988) While basic carbon metabolic pathways such as the Entner Doudoroff (ED) pathway are conserved in eukaryotes and eubacteria, archaea have diverse central metabolic networks. These highly variable central metabolic pathways of carbohydrate include modifications to the ED pathway as well as the EmbdenMeyerhof Parnas pathway (EMP pathway, glycoly sis) (Siebers and S chnheit, 2005) Unlike bacteria which primarily degrade hexoses through a classical EMP pathway, haloarchaea such as Halococcus saccharolyticus Haloferax mediterrane i and Haloarcula vallismortis catabolize the isomeric hexoses fructose and glucose through functionally separated, inducible pathways (Altekar and Rangaswamy, 1990; Altekar and Rangaswamy, 1992;

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80 Rangaswamy and Altekar, 1994; Johnsen et al., 2001) 13C NMR during fermentative growth, enzymatic studies, and DNA microarray analyses revealed that glucose is degraded only by way of a modified semi phosphorylative ED pathway in Halobacterium sacchar ovorum H mediterranei H. saccharolyticus H. volcanii and H. vallismortis (Tomlinson and Hochstein, 1972; Rawal et al., 1988; Johnsen et al., 2001; Zaigler et al., 2003) (Figure 1 2 ) whereas fructose is almost completely metabolized by way of a modified EM P pathway in H. vallismortis and H. saccharolyticus (Altekar and Rangaswamy, 1990; Johnsen et al., 2001) (Figure 1 3 ) Although the isomeric hexoses are catabolized by separate pathways according to 13C NMR, enzymatic activities for proteins involved in both pathways were detectable in cells grown in either sugar, although not to the same extent as pathway specific enzymes (Johnsen et al., 2001) As in the case of glycerol, haloarchaea including Haloferax sp. and Haloarcula sp. incompletely oxidize sugars to organic acids such as lactate, pyruvate, and acetate (Tomlinson and Hochstein, 1972; Oren and Gurevich, 1994b; Brsen and Schnheit, 2001) To date, the kn owledge concerning regulators of archaeal sugar metabolism has been limited. However pairs of general transcription factors TATA binding protein (TBP) and transcription factor B (TFB) contribute toward the expression of many genes including those involved in hexose metabolism (Facciotti et al., 2007; Coker and DasSarma, 2007) Aspects of each pathway including regulation are discussed Glucose Degradation through a Modified Ent ner Doudoroff Pathway Glucose degradation to pyr uvate through a modified ED pathway by haloarchaea was first proposed for H sacchar ovorum based on detectable enzymatic activities for glucose dehydrogenase and gluconate dehydratase and the absence of detectable activity for glucose6 phosphate dehydrogenase and 6phosphogluconate dehydrase

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81 activities in crude cell extract (Hochstei n, 1974) Evidence for a modified ED in glucose metabolism was subsequently reported for H mediterranei and H. vallismortis based on a higher observed enzymatic activity of glucose dehydrogenase (8.3 to 29.8 mU) than glucose6 phosphate dehydrogenase (0.6 to 3.7 mU) in glucosegrown cells (Rawal et al., 1988) More recently, radioisotope labeling experiments coupled with 13C NMR and specific enzyme activity assays for glucose dehydrogenase, gluconate dehydratase, 2 keto 3 deoxygluconate kinase (KDGK), and 2keto 3 deoxy 6 phosphogluconate (KDPG) aldolase has confirmed the degradation of glucose through a semi phosphorylative ED pathway in H. saccharolyticus (Johnsen et al., 2001) and DNA microarray analysis of H. volcanii confirmed significant upregulation of gene transcripts encoding glucose dehydrogenase and KDGK during growth on glucosecontaining media (Zaigler et al., 2003) Unlike the classical ED pathway which begins with phosphorylation of either glucose or its oxidized derivative gluconate, the semi phosphorylative ED pathway does not contain phosphorylated intermediates prior to KDPG (Figure 1 2) Instead, glucose is first oxidized to gluconate through an NADP+dependent glucose dehydrogenase. Following oxidation, gluconate dehydratase catalyzes the conversion of gluconate to 2keto 3 deoxygluconate (KDG) which is then phosphorylated by a unique KDG kinase (KDGK) to KDPG. A subsequent cleavage of KDPG occurs by KDPG aldolase, yielding py ruvate and glyceraldehyde 3phosphate. The latter compound is oxidized to pyruvate through a conventional process involving glyceraldehyde3 phosphate dehydrogenase, phosphoglycerate kinase, phosphoglycerate mutase, enolase, and pyruvate kinase.

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82 Fructose Degradation through a Modified EmbdenMeyerhof Parnas Pathway Analysis of fructose degradation in H vallismortis revealed that fructose is degraded to pyruvate through a modified EMP pathway based on measurement of ketohexokinase and phosphofructokinase ( PFK ) enzyme activities in fructosegrown cells (Altekar and Rangaswamy, 1990) As with glucose metabolism, radioisotope labeling experiments coupled with 13C NMR and specific enzy me activity assays for ketohexokinase, PFK, as well as classical fructose catabolic enzymes have confirmed the degradation of fructose through a modified EMP pathway in H saccharolyticus (Johnsen et al., 2001) (Figure 1 3). This modified EMP pathway differs from the classical fructose degradation pathways of bacteria only in the mechanisms of fructose 1phosphate (F1P) formation. M ost bacteria phosphorylate fructose during tr ansport by the PEP:PTS. However, in H. vallismortis and H. saccharolyticus F1P is formed through ketohexokinasedependent phosphorylation of fructose (Rangaswamy and Altekar, 1994; Johnsen et al., 2001) F1P is phosphorylated to FBP through PFK and is subsequently converted to two pyruvate molecules through the conventional enzymes of the EM pathway (Altekar and Rangaswamy, 1990; Johnsen et al., 2001) Some haloarchaea including H. volcanii Haloterrigena turkmenica, and Haloarcula marismortui encode PTS general carrier components EI and Hpr as well as fructosespecific permeases, providing a possibility that fructose phosphorylation may proceed through the PTS, similar to many bacteria (Figure 13). However, functional characterization of these proteins has not been performed.

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83 Regulation of Glucose and Fructose Metabolism in Haloa rchaea Currently, very little data is available concerning the regulation of glucose and fructose metabolism in archaea, especially in haloarchaea. A lthough preferential utilization of glucose over fructose was demonstrated for H. saccharolyticus, the molecular basis of this catabolite repression has not described (Johnsen et al., 2001) The majority of characterized archaeal regulators of carbon metabolism are transcription factors from hyperthermophiles (Lee et al., 2003; Lee et al., 2005; Kanai et al., 2007) although a few of haloarchaeal regulators have also been characterized (Facciotti et al., 2007; Coker and DasSarma, 2007; Schmid et al., 2009) Specifically, in H. salinar um pairs of general transcriptio n factors TBP and TFB as well as the transcription factor TrmB control gene clusters (Facciotti et al., 2007; Coker and DasSarma, 2007) ; however this haloarchaeon is not able to metabolize either glucose or fructose (Rawal et al., 1988) No regulators of sugar metabolism have been characterized in the model haloarchaeon H. volcanii, although homologs of characterized transcription factors are predicted based on primary sequence similarity. TrmB TrmB is a transcriptional regulator that governs the expression of a wide array of genes in response to cellular redox and energy status. T rmB is conserved across many archaeal and some bacterial species (primarily Firmicutes) ; however TrmB orthologs have not been characterized to date in bacteria (Lee et al., 2008) TrmB like proteins regulate maltose and glucose usage in thermophilic archaea (Lee et al., 2003; Lee et al., 2005; Kanai et al., 2007; Lee et al., 2008) and in nutrient limitation in halophilic archaea (Schmid et al., 2009)

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84 TrmB was originally identified in Thermococcus litoralis (Lee et al., 2003) where TrmB was implicated in regulating trehalose and maltose metabolism i n vitro In the absence of trehalose or maltose, TrmB blocks transcription of the trehalose and maltose ABC transporter operon, which also encodes trmB through direct binding of its promoter region. Dissociation of TrmB from its operator sequence occurs upon binding of sugar ligands resulting in subsequent transcriptional initiation. A recent in vitro analysis of P. fur iosus TrmB suggested that the maltodextrinspecific ABC transporter is also under the control of TrmB (Lee et al ., 2005) Maltodextrin and sucrose were found to relieve the TrmB repression of maltodextrin transport, whereas glucose increased transcriptional repression of the ABC transporters. More recently, H. salinarium TrmB has been implicated in coordinating t he transcription of various genes including those involved in cofactor synthesis i n response to nutrient limitation (Schmid et al., 2009) Microarray analysis of both parent and trmB d eficient strains grown in complex medium in the presence or absence of sugars ( glucose or glycerol ) indicated the differential transcription of 182 or 113 genes respectively, whose gene products are linked to carbohy drate, amino acid, cofactor, vitamin, a nd purine biosynthetic pathways many of which are incomplete. TrmB homologs can be found in most haloarchaea, but whether these homologs control similar gene clusters is unclear. General transcription f actors TATA b inding p ro tein and transcription f actor B General transcription factors are used for global gene regulation in all domains of life. Bacteria generally accomplish largescale transcriptional regulation through factors which respond to environmental stimuli. On the other hand, eukaryotes utiliz e multisubunit general transcription complexes to initiate large scale changes in

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85 transcription from various promoters. Despite the fact that many archaeal regulatory proteins resemble bacterial regulators, the transcriptional machinery is more eukaryotic like (Geiduschek and Ouhammouch, 2005) In addition to a multi subunit RNA polymerase, archaeal genomes contain t he basal transcription factors TBP and TFB, which are orthologous to the eucaryal TBP and TFIIB proteins and are necessary and sufficient for initiating basal transcription (Zillig et al., 1979; S oppa, 1999; Bell and Jackson, 2001) In h aloarchaea, these general transcription factors are often present in multiple copies, with H salinar um encoding six TBP s and seven TFB proteins (Baliga et al., 2000) Due to the interaction of TBP and TFB to recruit RNA polymerase and given the large number of orthologs present, there are several combinations of TBPs and TFBs which may drive transcription from an equally diverse set of promoters Through gene knockout of general transcription factor orthologs and subsequent transcriptome analysis of mutant strains, a large regulatory network which inclu des genes encoding putative s ugar metabolic enzymes was discovered (Facciotti et al., 2007; Coker and DasSarma, 2007) Co immunoprecipitation analysis identified roughly 37 % of all promoters (over 1,000) as being bound by a single transcription factor, whereas the majority of promoters are associated with multiple general transcription factors (Fac ciotti et al., 2007) Gene knockout and subsequent transcri ptome analysis revealed that 20% of the total genes regulated by general transcription factors are regulated by TbpD and TfbA (Coker and DasSarma, 2007) Although target promoters of general transcription factors have been identified, the molecular mechanisms of the interactions are still unclear.

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86 Project Rationale and Design The primary objective of this study was to characterize haloarchaeal carbon met abolism, specifically focusing on glycerol, glucose, and fructose metabolism, using H. volcanii as a model system Although glycerol is thought to be a primary energy source for heterotrophic members of halophilic communities, the molecular mechanism surro unding it s degradation and regulation has not been characterized. Thus, this study sought to el ucidate the molecular mechanism surrounding glycerol metabolism in H. volcanii. Similarly data concerning the regulation of haloarchaeal glucose and fructose me tabolic enzymes is severely limited. Thus, this study also sought to characterize regulators of haloarchaeal glucose and fructose metabolism, specifically focusing on a DeoR/ GlpRtype transcriptional regulator from H. volcanii. H. volcanii was chosen as a model haloarchaeon within which to study these pathways and their regulation based on the relevance of these carbon sources to this organisms ecology, the limited knowledge concerning these metabol ic pathways and their regulation within the model organism the ease with which H. volcanii is genetically manipulated, the availability of the genome sequence, and the availability of proteomic tools. After identifying homologs of bacterial glycerol metabolic enzymes in H. volcanii, involvement of each gene in g lycerol catabolism was confirmed through gene deletion and subsequent phenotypic and carbon utilization studies. The biochemical properties, organization, and regulation of each of these genes and their gene products was determined through specific enzyme activity assays, (q)RT PCRs, and transcriptional promoter reporter fusion assays. A bioinformatic approach to identify regulators of glucose and fructose metabolism led to the discovery of a DeoR/ GlpRtype repressor of glucose and fructose metabolic enzyme s. T he glpR gene clustered chromosomally with genes encoding sugar

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87 metabolic enzymes. The physiological role of this regulatory protein was determined through gene deletion and subsequent qRT PCRs enzymatic activity assays and transcriptional promoter re porter fusion assays. A putative GlpR binding site for each target promoter region was identified by bioinformatic analysis and a p reliminary mode of regulation i s suggested.

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88 Figure 1-1. Glycerol synthesis, release, and catabolism in halophilic ecosystems. Glycerol is produced by Dunaliella sp as an organic osmotic solute in response to salt stress through both degr adation of starch reserves and by enhancement of photosynthesis. Glycerol is released into the environment either through algal cell lysis or by leakage which occurs in response to increased temperatures (above 45 C) or hypotonic stress where it becomes a readily-available organic carbon source for heterotrophic community members. Halophilic, heter otrophic microorganisms can then convert glycerol into DHAP through one of two pathways: i) through GK and G3PDH or ii) through GDH and ATPor PEP:PTS-dep endent DHAK. The portion of the figure displaying salt-activated carbon flux in Dunaliella was modified from (Liska et al., 2004). Abbreviations: PM, plasma membrane; CA, carbonic anhydrase; PEP:PTS, phosphoenolpyruvate-linked phosphotransferase system.

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89 Figure 1-2. Metabolic conversi on of glucose to pyruvic acid in haloarchaea. Glucose is metabolized by a modified Entner-D oudoroff Pathway in a variety of haloarchaea including H. volcanii H. saccharovorum H. mediterranei Haloarcula vallismortis and H. saccharolyticus as evidenced by proteomic data, 13C-NMR, and enzyme activity assays (Hochstein, 1974; Rawal et al., 1988; Johnsen et al., 2001; Zaigler et al., 2003). Dashed arrow indicates multiple conversion steps to pyruvic ac id including conversions by classical enzymes glyceraldehyde-3-phosphate dehydrogenase, 3-phosphoglycerate kinase, phosphoglycerate mutase, enolase, and pyruvate kinase. Abbreviations: KDGK, 2-keto-3-deoxyg luconate kinase; KDPG, 2-keto-3deoxy-6-phosphogluconate.

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90 Figure 1-3. Potential routes fo r the metabolic conversion of fructose to pyruvic acid by haloarchaea. Conversion of fructose to F1 P is catalyzed by ketohexokinase in H. vallismortis and H. saccharolyticus as evidenced by 13C-NMR and enzyme activity analysis (Rangaswamy and Alte kar, 1994; Johnsen et al., 2001). Homologs of PTS general carrier components and fructose-specific PTS permease components are found in H. volcanii H. turkmenica and H. marismortui although it is unclear whether these components are functional. The PTS portion of the figure was modified from (Madigan et al., 2006). Dashed arrow indicates multiple conversion steps to pyruvic acid including conversions by classical en zymes glyceraldehyde-3-phosphate dehydrogenase, 3-phosphoglycerate ki nase, phosphoglycerate mutase, enolase, and pyruvate kinase. Abbrev iations: PFK, phosphofructokinase; FBP, fructose-1,6-bisphosphate; and TI M, triosephosphate isomerase.

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91 CHAPTER 2 METHODS AND MATERIAL S Chemicals, Media, and Strains Chemicals and Reagents Biochemicals and commercial enzymes were purchased from Sigma Aldrich (St. Louis, MO). Other organic and inorganic analytical grade chemicals were purchased from Fisher Scientific (Atlanta, GA) and BioRad Laboratories (Hercules, CA). Desalted oligonucleot ides were from Integrated DNA Technologies (Coralville, I A ). 2' Deoxyuridine5' triphosphate coupled by an 11atom spacer to digoxigenin (DIG 11dUTP), alkaline phosphataseconjugated antibody raised against DIG, disodium 3(4 methoxyspiro{1,2dioxetane3, 2' (5' chloro)tricyclo[3.3.1.13,7]decan} 4 yl) phenyl phosphate (CSPD) DIG labeled RNA molecular weight standard I (0.3 6.9 kb) and other DIG related biochemicals were purchased from Roche A pplied Science (Indianapolis, IN). Positively charged nylon membranes (Bright Star Plus) used for Southern hybridizations were from Ambion, Inc. (Austin, TX ). Phusion and Taq DNA polymerases, restriction enzymes, deoxy ribonucleotide triphosphates (dNTPs) T4 polynucleotide kinase (PNK), T4 DNA ligase, Antarctic P hosphatase (AP) and Quick Load 100 bp molecular weight standards were purchased from New England Biolabs (Ipswich, MA). A garose, Precision Plus ProteinTM Kalei doscope molecular mass markers and iQTM SYBR Green Supermix and iS criptTM cD NA synthesis kit used for (quantitative) reverse transcriptasepolymerase chain reactions [ (q)RT PCRs ] were purchased from Bio Rad Laboratories HiLo DNA molecular weight standards were from Minnesota Molecular, Inc. (Minneapolis, MN) S trep Tactin Superflow resin was purc hased from Qiagen (Valenia, CA).

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92 Strains, Plasmids and Culture Conditions Strains, oligonucl eotide primers used for cloning and screening of mutant strains and plasmids are listed in Tables 2 1 through 26 Escherichia coli strains Top10 (Life Technologies Carlsbad, California) was used for routine recombinant DNA experiments E. coli strain GM2163 (New England Biolabs) was used for isolation of plasmids lacking methylation for transformation into H. volcanii strains (Cline et al., 1989) Liquid cultures were aerated with orbital shaking at 200 revolutions per minute ( RPM ) E. coli strains were grown at 37 C in Luria Bertani (LB) medi um (Bertani, 1951) su pplemented with ampicillin (100 mg l1) as needed. H. volcanii strains were grown at 42 C in various media including yeast extract peptonecasamino a cids (YPC) American Type Culture Collection ( ATCC ) 974, c asamino a cids (CA) YPC supplemented with glucose (Glu) or fructose (Fru) and minimal medium (MM) supplemented with glycerol (Gly), Glu, Fru, succinate (Suc) and various combinations of these carbon sources. Medium formulae were according to The Halohandbook (Dyall Smith, 2008) with the following exception: Suc, Gly, Fru and/or Glu were included at 20 mM each where indicated. Biodiesel waste was received from Douglas Renk at the University of Florida, Department of Chemical Engineering Glycerol content of the biodiesel waste was analyzed by high performance liquid chromatography ( HPLC) using a BioRad Aminex HPX 87H column (300 x 7.8 mm) and a refractive ind ex detector with 4 mM H2SO4 as eluent (flow rate was set to 0.4 ml1min1). Biodiesel waste was then provided as the sole carbon source in the minimal medium to a final concentration of 20 mM glycerol Media were supplemented as needed with novobiocin (0.1 g ml1), 5 fluoroorotic acid (5FOA) (50 g ml1) tryptophan (820 gml1) and uracil (10 and 50 g ml1 for growth in the presence and absence of 5FOA,

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93 respectively) Uracil and 5FOA were dissolved in 100% [ v olume (v ) /v for 5 FOA or weight ( w ) /v for uracil ] dimethyl sulfoxide (DMSO) at 50 mg ml1 prior to addition to the growth medium For growth assays, cells were grown in media as indicated within Chapter 3 through Chapter 7. Cells were inoculated from 80 C glycerol stocks onto appropriate solid media. Cells were thrice subcultured during logarithmic phase growth and used as an inoculum for final growth analysis under various conditions as described below. Each subculture was started at an initial optical density at 600 nm (OD600) of 0.0 3 to 0.0 4 For determination of growth rate, cell yield, and carbon utilization, cells were grown in 20 ml of medium in 250 ml baffled Erlenmeyer flasks. For GK enzyme activity assays, cells were grown in 100 ml of medium in 1000 ml flasks. For G3PDH, PFK and KDGK enzyme activity assays and for RNA preparation, cells were grown in 25 ml of medium in 250galactosidase activity measurements, cells were grown in 3 ml of medium in 13 100 m m culture tubes Cell gro wth was monitored by an increase in O D600 [ where 1 OD600 unit equals approximately 1 109 colony forming units ( CFU) ml1 for all strains used in this study] All experiments were performed at least in triplicate and the means standard deviations (SD) of the results were calculated. For purificatio n of C terminally labeled StrepIItagged pr oteins, cells were grown in 500 ml of YPC in 1 000 m l flasks and pooled prior to protein purification High Performance Liquid C hromatography At various time points during growth, 1 ml culture sam ples o f parent (H26), GK glpK mutant (KS4) transcriptional repressor glpR mutant (KS8) and a mutant deficient in both glpK and glpR (KS10) were withdrawn and centrifuged (10 min, 10 000 g 4 C). Supernatant fractions were filtered using a 0.2 m pore size filters (Nalge Nunc

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94 International, Rochester, NY) and subsequently analyzed by HPLC using a BioRad Aminex HPX 87H column (300 x 7.8 mm ) and a refractive index detector with 4 mM H2SO4 as eluent (flow rate was set to 0.4 ml1min1) All experiments were performed at least in triplicate and the means SD of the results were calculated. DNA Procedures DNA Isolation and A nalysis Plasmid DNA was isolated from E. coli strains by use of the QI Aprep spin miniprep kit (Qiagen ). PCR products were purified by MinElute (Qiagen) prior to modification by restriction enzymes (BamHI, HindIII, KpnI, BlpI, BspHI, XbaI, HpaI, or NdeI), T4 PNK or T4 DNA ligase When applicable, plasmids were purified from agarose slices by QIAquick gel extraction kit (Qiagen). For rap id PCR screening, template DNA was extracted from H. volcanii mutant and parent strains and recombinant E. coli strains as described previously (Zhou et al., 2008) For Southern blot s, H. volcanii genomic DNA was isolated from 5 ml cultures by DNA spooling (Dyall Smith, 2008) The fidelities of all cloned PCR amplified products as well as mutant strains were confirmed by Sanger automated DNA sequencing using an Applied Biosystems model 3130 genetic analyzer (DNA Sequencing Facilities, Interdisciplinary Center for Biotechnology Research, University of Florida). DNA was separated by gel electrophoresis using 0.8% ( w /v) or 2.0 % (w /v) agarose gel s in 1 TAE electrophoresis buffer [ 40 mM N tris(hydroxymethyl) aminomethane ( Tris ) acetate (pH 8.5) and 2 mM ethylenediaminetetraacetic acid ( EDTA ) ] (Aaij and Borst, 1972) HiLo (Minnesota Mol ecular Inc. ) or Quic kL oad 100 bp (New England Biolabs) molec ular weight standards were used for product size estimation Gels were photographed with a Mini visionary imaging system (FOTODYNE, Hartland, W I ) after st aining with ethidium bromide ( 0.5 g 1)

PAGE 95

95 Polymerase Chain Reactions H igh fidelity doublestranded DNA PCR products used for construction of the plasmids listed in the Tables 2 1, 2 3, 2 5, and 2 7 were amplified using Phusion DNA polymerase. Taq DNA po lymerase was used for screening transformants for the generation of DIG labeled doublestranded DNA probes that were used for Southern blotting and for (q)RT PCRs All PCRs were performed according to the S upplier s instructions with the following modifications: 3% (v/v) DMSO was inclu ded and 0.1 mM dNTP mix was added to the standard DIG labeling reaction mixture which included 1 DIG dNTP ( Roche Applied Science). Primer pairs and template DNA used for t he PCRs are outlined in the Tabl es 2 2, 2 4, 2 6, 2 8 PCR s w ere performed using an iCycler or GeneCycler (BioRad Laboratories ). Cloning Prod uct inserts vectors and genomic DNA were digested using restriction enzymes ( BamHI, HindIII, KpnI, BlpI, BspHI, XbaI, HpaI SphI, MluI, ClaI or NdeI ) accordi ng to M anufacturers specifications. Digested vector DNAs were treated with AP for 45 min at 37 ation at 65 purification and subsequent ligation. For blunt end cloning, Vent polymerase was used to resolve overhangs according to the M anufacture rs instructions For inverse PCRs, the resulting products were treated with T4 PNK according to the S uppliers recommendations (New England Biolab s). Ligation reactions were pe r formed using T4 DNA ligase at 16 16 h ours according to the S uppliers recommendations Construction of H. volcanii Deletion Strains Several genes w ere targeted for markerless deletion from the chromosome of H. volcanii H26 using the pyrE2 based "popin/popout" method (B itan Banin et al., 2003;

PAGE 96

96 Allers et al., 2004) Briefly, t he ope n reading frame (O RF ) of the targeted gene along with 500bp of flanking region was cloned into pTA131 digested with BamHI and HindIII. After ligation, the resulting plasmid w as transformed into E. coli Top10, and screened for the presence of the insert After confirming sequence fidelity, inverse PCR was performed to remove the coding region of the targeted gene, and the subsequent PCR product was treated with T4 PNK prior to ligation to generate the new p lasmid construct This plasm id was transformed into E. coli GM2163 before transformation into H26 pyrE2 which is a uracil auxotroph. H. volcanii t ran s formants were plated onto CA medium without uracil and growth was counter selected on media containing 5FOA, allowing for either deletion of the target gene or restoration of the wildtype gene copy. Colonies were screen ed for the absence of a readily generated PCR product by use of internal primers specific for the ORF of the target gene ('NegativeForward' and Negative Reverse primer pairs ) Colo nies that did not yield this PCR product were confirmed to be mutant strains by Southern blotting and PCR with Confirm Forward and Confirm Reverse primer pairs that annea l within the target flanking regions cloned in to the suicide plasmids. These latter PCR products were separated by gel electrophoresis and sequenced to confirm DNA fidelity. Southern B lot Analysis H. volcanii parent H26 and mutant strains were subject ed to Southern blotting to confi rm mutation as described previously (Zhou et al., 2008) Briefly H. volcanii genomic DNA (10 g) was isolated from 5 ml cultures by DNA spooling (Dyall Smith, 2008) and subjected to restriction digestion for 6 8 hours. DNA was separated on a 0.8% (w/v) gel (20V, 12 16 h) and transferred (12 16 h) to a positively charged nylon membrane through capillary action. DNA was then cross linked to the membrane using

PAGE 97

97 a UV Stratalinker 2400 (Stratagene) and hybridized to a DIG labeled probe specific for the region flanking the 5 or 3 end of the target coding region (65 C, 12 16 h) Primers used for the construction of DIGlabeled probes are included in Tables 2 2, 2 4, and 28 Hybridization species were detected by CSPD mediated chemilumi nescence as recommended by the S upplier (Roche Applied Science) with the following modifications: an increase in stringency from 0.5x saline sodium citrate ( SSC ) [ 1 x SSC is 0.15 M NaCl with 0.015 M sodium citrate (pH 7.0) ] supplemented with 0.1 % (w/v) sodium dodecyl sulfate ( SDS ) to 0.1 x SSC supplemented with 0.1 % (w/v) SDS was included in the washing of the membranes at 65 C after hybridizations as needed. The DNA f ragment sizes were estimated by methylene blue staining of the Hi Lo DNA molecular weight markers on the membrane. Construction of Promoter Reporter Fusion Constructs A plasmid based reporter system was used to analyze transcription (Delmas et al., 2009) in which promoter regions of haloarchaeal operons/genes were fused to the Haloferax alicantei derived bgaH galactosidase. The bgaH gene was amplified from pTA102 ( the primers used for cloning of the insert are provided in Table 2 6 and 28 ) and cloned into pJAM202 using NdeI and BlpI which fused the bgaH gene dow nstream of the strong rRNA P2 promoter of H cutirubrum to generate pJAM2678. The regions upstream of glpR pfkB kdgK1, kdgK2 glpK gpdA1, and trpA were amplified from H. volcanii genomic DS70 DNA using the primers listed in Table s 2 6 and 2 8 and were f used with bgaH using XbaI and NdeI digestion to generate pJAM2689 (188bp glpR pfkB promoter region), pJAM2705 (89bp kdgK1 promoter region), pJAM2706 (524bp kdgK1 promoter region), pJAM2702 (232bp kdgK2 promoter region), pJAM2703 (122bp HVO_A0327kdgK2 promoter region), pJAM2679 (354bp

PAGE 98

98 glpK promoter region), pJAM2780 (310bp gpdA1 promoter region) and pJAM2712 (321bp trpA promoter region) (where bp represent the region upstream of the translational start codon of the first gene listed for each construct). Plasmid constructs were purified from E. coli strains Top10 an d GM2163 prior to transformation of H. volcanii parent or glpR mutant strains. Promoter sequences were predicted upstream of glpR pfkB kdgK1, kdgK2 glpK and gpdA1 according to methods previously described (Schneider et al., 2006) Plasmid controls pJAM2714 and pJAM2715 were constructed using pJAM2678 by removal of the H. cutirubrum rRNA P2 prom oter and the ShineDalgarno sequence (using XbaI and NdeI) or removal of only the rRNA P2 promoter (using XbaI and BamHI), respectively. RNA procedures RNA Isolation and Analysis T otal RNA used for (q)RT PCRs was isolated from H. volcanii parent H26 and glpR mutant KS8 strains (exponential phase; OD600 0.3 0.5 ) using the RNeasy RNA purification columns (Qiagen) RNA was treated with am plification grade DNaseI according to the S upplier s recommendations (SigmaAldrich), with the following modifications : 3 units ( U ) of enzyme were added per RNA and the mixture was incubated for 4 5 min at room temperature. RNA integrity was determined by gel electrophoresis. RNA concentration was determined by measuring the absorbance at 260 nm ( A260) using a Nanovue Plus Spectrometer instrument ( GE Healthcare Life Sciences, Uppsala, Sweden) (q)RT PCRs (q)RT PCRs were performed using H. volcanii total RNA as a ), appropriate primers (listed in Tables 2 4, 2 6 or 2 8 ), iQTM SYBR Green Supermix

PAGE 99

99 (Bio Rad Laboratories ) (for qRT PCRs) a nd an iCycler (Bio Rad Laboratories ) according to the S uppliers instructions (BioRad Laboratories ) RNA was reverse transcribed into cDNA using iScriptTM (Bio Rad Laboratories ) according to the M anufacturer s instructions. After cDNA synthesis (25 C 5 min; 42 C 30 min; 85 C 5 min), qRT PCRs were preheated to 95 C (4 min) followed by 40 amplification cycles consisting of denaturation (95 C, 30 s), annealing (temperatures listed in Tables 24, 2 6, and 28 1 min) and elongation (72 C, 17 s). For RT PCRs, reactions were preheated to 95 C (4 min), followed by 35 amplification cycles consisting of denaturation (95 C, 30 s), annealing (temperatures listed in Tables 2 4, 2 6, and 2 8 1 min) and elongation (72 C, 41 s), after which a final extension was performed at 72 C (10 min). For each primer pair, negative and positive controls were included to exclude genomic DNA contamination and confirm primer pair function, respectively. For the controls, reactions were identical with the following excepti ons: the sample was maintained on ice during the reversetranscription step for the negative control, and H. volcanii genomic DNA prepared as previously described (Ng et al., 1995) was used as a template for the positive control reactions PCR products from RT PCR s were sequenced as described above to confirm specificity. For qRT PCRs a bsolute (Rutledge and Ct, 2003) and/or relative (Pfaffl, 2001) quantification was performed for each transcript according to methods previously described T ranscript specific for the H. volcanii ribosomal protein L10 gene ( ribL) was used as an internal control based on a previous study (Brenneis et al., 2007) and our confirmation by qRT PCR that the N fold induction of transcripts specific for ribL was close to 1.0 when parent H26 or glpR mutant KS8 was grown in minimal media. V arying

PAGE 100

100 dilutions of cDNA samples were subjected to qRT PCR, and the threshold counts (CT) which fell within the linear range of H. volcanii genomic DNA standards (R2 > 0.99 for the linear regressions of all standards tested) were used for transcript quantification Experiments were performed in triplicate and the means SD of the results were calculat ed Protein Procedures Protein Isolation and Analysis C terminally StrepII tagged (Trp Ser His Pro Gln Phe Glu Lys) proteins were purified by Strep Tactin chromatography followed by gel filtration chromatography as detailed below. Purified protein fractions and molecular weight standards were analyzed by r educing 12% SDS polyacrylamide gel elect r o phoresis ( PAGE) (200 V, 50 60 m in) (Laemmli, 1970) Precision Plus ProteinTM Kaleidoscope molecular mass marker ( diluted at a ratio of 1:20 according to the S uppliers recommendations ) were prepared for electrophoresis by boiling for 10 min in 20 l of SDS PAGE b uffer [100 mM Tris H Cl ( pH 6.8 ) with 10% (v/v) mercaptoethanol, 2% ( w /v ) SDS, 10% (v/v) glycerol and 0.6 mg ml1 bromophenol blue] Gels were stained with Coomassie Blue and imaged on a BioRad XR imager according to M anufacturers protocol (BioRad Laboratories) Strep T actin Chromatography Cells were harvested from two 500 ml cultures by centrifugation (20 min, 4,300 g 4 C), washed once with buffer W [ 100 mM Tris HCl (pH 8.0) with 2 M NaC l ] and resuspended in 20 ml of buffer W containing 10 mM phenylmethylsulfonyl fluoride ( PMSF ) Cells were passed four times through a chilled French pressure cell at 20,000 lb 2. Cell lysate was centrifuged twice to remove cell debris (14,000 g 15 min 4 C).

PAGE 101

101 The filtrate obtained with a 0.45 m poresize filter ( Nalge Nunc International ) was applied to a Strep Tactin column (Qiagen) (1 ml column volume and 3 mg protein ml1 binding capacity) equilibr ated with 10 ml of buffer W. The column w as loaded with lysate (440 mg protein) and washed with 40 ml of buffer W Protein was eluted with 10 ml of buffer E [ 100 mM Tris HCl (pH 8.0) containing 2 M NaCl and 12 .5 mM desthiobiotin] int o 1 ml fractions Protein f ractions (770 g) were pooled and further purified by gel filtration chromatography (details presented below) Strep T actin c olumn resin was regenerated by washing the column w ith 1 5 ml of buffer R [ 100 mM Tris HCl ( pH 8.0) containing 1 50 mM NaCl, 1 mM EDTA and 1 mM hydroxy azoph enyl benzoic acid] Gel Filtration Chromatography After Strep Tactin chromatography, s amples (230 g in 0.5 ml per run) were applied to a Superdex 200 HR 10/30 gel filtration column ( GE Healthcare Lif e Sciences ) equilibrated in buffer W or buffer L [ 100 mM Tris HCl (pH 8.0) with 150 mM NaCl ] at a flow rate of 0.1 0.3 ml1m in1. Molecular mass standards for gel filtration included cytochrome C (12 kDa), carbonic anhydrase (29 kDa), serum albumin (66 kDa), alcohol dehydrogenase (150 kDa), amylase (200 kDa), thyroglobulin (669 kDa) and Blue Dextran (2, 000 kDa) (Sigma). P rotein elution was monit ored by UV absorbance at 280 nm (A280) and quantified by Bradford assay (Bradford, 1976) M olecular mass was estimated from the linear regression (R2 > 0.99) generated from plotting the logarithmic values of molecular mass against Kav and the equation Kav=(VR Vo)/(Vc Vo) where Kav is the gel phase distribution coefficient, VR is the retention (elution) volume of the protein, Vc is the geometric bed volume, and Vo is the v oid volume of the column. Column volume was calculated based on the measured column height of 29.94 cm and

PAGE 102

102 the column radius of 10 cm. Experiments were performed at least in triplicate and the mean s SD of the results were calculated. Protein Quantification Protein concentrations were determined by the Bradford method (Bradford, 1976) with bovine serum albumin (Bio Rad La boratories ) as the standard w ith the following modifications: the r eaction volume for the colori metric and performed in a 96well microplate reader at room temperature. Briefly, Bradford reagent was added to the protein sample and incubated for 5 minutes at room temperature, after which the absorbance at 595 nm ( A595) was determined for 100 l of sample reaction. The assay was linear between 0 and 4 00 1 of protein. Glycerol Kinase Activity Assay Exponential growth phase cells (0.3 0.5 OD600) were harvested by centrifugation (15 min, 6 000 g 4 C), washed once with 20 ml buffer K [ 100 mM potassium phosphate buffer (pH 7.4) with 3 M KCl] resuspended in 1 ml of buffer K and broken by sonication (4 20 s at 140 W). Debris was removed by centrifugation (10 min, 12 000 g 4 C). Protein concentration was estim ated using the Bradford assay and cell extract was used as the crude enzyme preparation. GK (ATP : glycerol 3 phos photra nsferase, EC 2.7.1.30) activity was determined at 42 C in a coupled photometric reaction as described previously (Sher et al., 2004) with the following modifications: assay buffer was supplemented with 3 M KCl, negative controls lacked ATP, glycerol or were per formed using boiled parent cell lysate, and time points (after incubation at 42 C) for which samples were withdrawn included 10, 20, 30 35, 40, 45, 50, 60, and 90 min intervals Briefly, GK activity was coupled to the formation of glycerophosphate which was quantified in the presence of NAD+ and commercially available glycerophosphate

PAGE 103

103 dehydrogenase. The reaction mixture (5 ml) contained 1 ml of cell extract (0.9 1 mg protein ml1) 30 mol ATP, 100 mol L cysteine and buffer K, and was initiated wit h addition of 125 mol glycerol. Sample reactions were incubated at 42 C for varying amounts of time (described above) after which 1 ml samples were withdrawn and the reaction terminated with addition of an equal volume of 0.2 N H2PO4. Samples were centri fuged ( 10 min, 12, 000 g ) to remove precipitated proteins and the glycerophosphate con tent of 250 l portions were quantified in a total reaction volume of 2.27 ml containing 0.011 N NaOH, 1.1 mM NAD+, 0.66 M hydrazine sulfate, 1% (w/v) nicotinamide sodium carbonate buffer and 8 U of glycerophosphate dehydrogenase from rabbit muscle ( Sigma, EC 1.1.1.8) After 1 hour of incubation at 30 C, the absorbance at 340 nm (A340) was measured. Product formation was quantified by linear regressi on analysis (R2 > 0.99) using standards which w ere linear between 0 and 10 mM glycerophosphate. PFK and KDGK Activity A ssays Exponential growth phase cells (0.3 0.5 OD600) were harvested by centrifugation (20 min, 4,300 g 4 C), w ashed once with 20 ml buffer A [100 mM Tris HCl ( pH 7.5) with 2 M NaCl] resuspended in 1 ml of buffer A containing 1 mM PMSF and lysed by sonication (4 20 s at 140 W). Cell d ebris was removed by centrifugation (10 min, 12,000 g 4 C). Protein concentration was estimated using the Bradford assay. PFK and KDGK activity assays were carried out as previously detailed (Johnsen et al., 2001) with the following exceptions: all enzyme activit ies were carried out aerobically at 37 C in a 96well microplate reader filled with 0.1 ml of assay mixture ( the path length for a 100 l reaction was calculated as 0.2825 cm1) It was ensured that in coupled enzymatic assays, the auxiliary enzymes were not rate limiting. Background change in

PAGE 104

104 A340 for reactions containing no substrate was subtracted from reactions in which substrate was included to yield the overall change in absorbance. Reactions containing boiled enzyme and no NADH were also included as controls. All experiments were performed in triplicate, and the means SD of the results were calculated. One U of enzyme activity is defined as 1 mol substrate consumed or product formed per min with a molar extinction coefficient for NADH of 6,220 Mcm. PFK (EC.2.7.1.56) specific activity was determined at 37 C by measuring the ATP dependent formation of FBP from F1P, which was coupled to the oxidation of NADH by FBP aldolase, triosephosphate isomerase ( TIM ) and G3PDH The assay mixtu re contained 100 mM Tris HCl ( pH 8.5 ) with 30 mM MgCl2, 1 M KCl, 10 mM F1P sodium salt, 2 mM ATP, 0.3 mM NADH, 0.54 U FBP aldolase, 2 U TIM, 0.34 U G3PDH, and cell extract (1 to 3 g protein). KDGK (EC 2.7.1.45) specific activity was determined at 37 C by measuring t he ATP and gluconatedependent formation of pyruvate, which was coupled to the oxidation of NADH by lactate dehydrogenase (LDH). The assay mixture contained 100 mM Tris HCl ( pH 8.5 ) with 1 M KCl, 10 mM MgCl2, 2 mM ATP, 0.3 mM NADH, 10 mM sodium gluconate, 11 U LDH, and cell extract (5 g protein). G alactosidase A ssay Promoter activity for each transcriptional reporter fusion construct was assessed quantitatively by assaying galactosidase activity as previously described (Holmes and Dyall Smith, 2000) Briefly, cells were grown in 3 ml of appropriate media and harvested at exponential growth phase (0.3 to 0.5 OD600) by centrifugation (15 min, 6,000 g 4C). Cell pellet s were washed once in buffer B [50 mM Tris HCl ( pH 7.2 ) with 2.5 M NaCl and 10 M MnCl2] resuspended in 300 l of buffer C [ buffer B supplemented with

PAGE 105

105 0.1% (w mercaptoethanol] and lysed using 150 l of 2 % (v/v) Triton X 100. Debris was removed by centrifugation ( 10 min, 6,000 g 4C), and the protein concentration in the cell extract was determined using the Bradford assay C ell lysates were assayed at 25 C for galactosidase specific activity by measuring the i ncrease in absorbance at 405 nm (A405) due to the liberation of o nitrophenol from o nitrophenyl D galactopyranoside (ONPG). The assay mixture (100 l) contained 20 l of cell lysate (3 of buffer C and 2.66 mM ONPG (stock solution 8 mgml in 100 mM potass ium phosphate buffer, pH 7.2). Negative controls included cells carrying pJAM2714 and pJAM2715 (the bgaH reporter plasmids devoid of promoter elements). Background values in which no substrate (ONPG) was added to the reaction were subtracted from reactions containing substrate for each lysate tested. All experiments were performed in triplicate, and the means SD of the results were calculated. One galactosidase activity is defined as the amount of enzyme catalyzing the hydrolysis of 1 mol ONPG min with a molar extinction coefficient for o nitrophenol of 3 300 Mcm. Genome Analysis and Construction of Phylogenetic Trees Ninety two archaeal genomes (Benson et al., 2010) were searched for homologs of PTS general carrier and sugar specific proteins with the BLASTP (Altschul et al., 1997) and InterProScan (Hunter et al., 2009) search engines. Q ueries of characterized PTS general carrier proteins and sugar specific permeases from E. coli as well as B. subtilis were used for the identification of PTS components in archaea. Archaeal homologs of B. subtilis HprK were also analyzed. DHAK distribution in archaea was searched us ing E. coli or C. freundii DHAK as a query for BLASTP. For construction of phylogenetic trees and pr otein alignments, protein sequences were retrieved from the

PAGE 106

106 NCBI database (Benson et al., 2010) and were N and C terminally trimmed wi thin the BioEdit seq uence editor software v7.0.4.1(Hall, 1999) prior to p airwise and multiple sequence alignment s that were performed using CLUSTAL W (Thompson et al., 1994) Phylogenetic and molecular evolutionary analyses of t he primary amino acid sequences were conducted using MEGA v3.1 (Kumar et al., 2004) The mean e volutionary distances were estimated from the protein sequences using the pdistance substitution model. Consensus trees were generated using the neighbor joining method of construction (Saitou and Nei, 1987) and validated with the bootstrap phylogeny test (Felsenstein, 1985) (1 000 replicates; 64 238 seed) and pairwise gap deletion. Interior branch test values or bootstrap values greater than the 50% cutoff are indicated at the internal nodes Due to space constraints within the figure legends, accession numbers for the following figures are listed below: Figures 33, 4 2, 5 4, 6 1, and 67. Accession numbers for Figure 33 (alignment of haloarchaeal and bacterial GKs ) : are as follows: E coli (Eco) YP_543453; S. flexneri (Sfl) NP_838952; Bacillus cereus (Bce), ZP_03103890; Natrialba magadii (Nma), ZP_03694006; H salinarum (Hsa), NP_280665; H marismortui (Hma), YP_135274; H lacusprofundi (Hla) ZP_02016760; H walsbyi (Hwa) YP_657499; H volcanii (Hvo), YP_003535588.1; E faecalis (Efa), NP_815610.1; M pneumoniae (Mpn), NP_109738.1; and B subtilis (Bsu), NP_388810.2 Accessi on numbers for trimmed sequences for Figure 4 2 (phylogenetic distribution of GKs ) are as follows: Thermoanaerobacter sp X514, YP_001662655; Thermoanaerobacter pseudethanolicus YP_001664537; Thermoanaerobacter tengcongensis NP_6235761; Clostridium hiranonis ZP_032922601; Clostridium

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107 difficile YP_001087882; Thermotoga neapolitana YP_002534606; Thermotoga sp. RQ2, YP_001739349; Marinitoga piezophila, YP_002615530 ; Thermotoga petrophila YP_001244954; Petrotoga mobilis YP_001567349; Desulfotomaculum reducens YP_001114174; Carboxydothermus hydrogenoformans YP_360659; Mannheimia succiniciproducens YP_089180; Actinobacillus succinogenes YP_001344893; Haemophilus influenzae, NP_438851; Haemophilus parasuis ZP_02478129; Salmonella enterica, ZP_0234517 4; S typhimurium NP_462967; Citrobacter koseri YP_001454599; K pneumoniae, YP_002235969; Escherichia albertii ZP_02904065; Shigella boydii YP_001882621; E coli, YP_543453; S flexneri NP_838952; Oceanobacillus iheyensis NP_693396; Staphylococcus a ureus NP_646000; Exiguobacterium sibiricum YP_001813587; Exiguobacterium sp. ZP_02991677; Anoxybacillus flavithermus YP_002315832; Bacillus weihenstephanensis YP_001643829; B cereus ZP_03103890; Bacillus thuringiensis YP_035286; Bacillus anthraci s, NP_843527; Rubrobacter xylanophilus YP_643841; Nitrosococcus oceani YP_344681; Burkholderia xenovorans YP_560348; Burkholderia multivorans YP_001578811; Burkholderia ubonensis ZP_02377911; N magadii ZP_03694006; H salinarum NP_280665 ; H marismortui YP_135274; H lacusprofundi ZP_02016760; H walsbyi YP_657499; S cerevisiae CAA48791; and H volcanii YP_003535588.1 Accession numbers for protein sequences for Figure 54 (distribution of GpdA homologs in archaea and E. coli ) are as follows: i) m embers of Gene organization I include H alorhabdus utahensis Huta_1471 (YP_003130380.1) ; H alomicrobium mukohataei Hmuk_2516 (YP_003178331.1), H walsbyi HQ1734A (YP_657500.1), H marismortui rrnAC0554 (YP_135276.1), H salinar um VNG1969G (NP_280666.1), H

PAGE 108

108 lacusprofundi Hlac_1123 (YP_002565787.1) N magadii Nmag_0933 (YP_003479079.1) H alogeometricum borinquense HborDRAFT_0007 ( ZP_03997215.1) H. volcanii HVO_1538 (GpdA1) (YP_003535585.1) and HVO_A0269 (GpdA2) (YP_003533725.1) and E. coli EcGlpA (AP_002838.1) and (ii) members of Gene Organization II include H. utahensis Huta_0683 ( YP_003129602.1) H. mukohataei Hmuk_2572 ( YP_003178385.1), H borinquense HborDRAFT_3500 ( ZP_04000658.1), H. marismortui rrnAC1955 ( YP_136529.1), Sulfolobus islandicus LS215_0342 (YP_002831137.1), H. walsbyi HQ2675A (YP_658392.1), H. salinarum OE2553R (YP_001689097.1), T pendens Tpen_1127 (YP_920528.1), Metallosphaera sedula Msed_1177 (YP_001191262.1), Caldivirga maquilingensis Cmaq_1799 (YP_001541610.1), Sulfolo bus s o lfataricus SSO2526 (NP_343866.1), Sulfolobus acidocaldarius Saci_2032 (YP_256621.1) and Saci_1118 (YP_255763.1), Picrophilus torridus PTO1486 (YP_024264.1), Thermoplasma volcanium TVN0840 (NP_111359.1), and Thermoplasma acidophilum Ta0633 (NP_394105. 1). Genomic sequences for Fig. 61B ( genomic clustering of DeoR/GlpR transcriptional regulators and PFK genes in haloarchaea and Gram positive bacteria) are as follows: i) m embers of Group I include H volcanii (NC_013967.1) Halot e r rigena turkmenica (NC_013743.1) and Morella thermoacetic a (NC_007644.1); ii) members of Group II include T teng congensis (NC_003869.1) Symbiobacterium thermophilum (NC_006177.1) B cereus (NC_003909.8) Lactobacillus sakei (NC_007576.1) and E faecalis (NC_004668.1); and iii) members of Group III include H marismortui (NC_006396.1) and H mukohataei (NC_013202.1)

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109 A ccession numbers for trimmed protein sequences in Figure 61 (p hylogenetic distribution of repressor proteins of the DeoR /GlpR family from Bacteria and Archaea) are as follows: E coli SrlR ( NP_417187.1), UlaR (NP_290821.1), DeoT (NP_415800.1), SgcR (NP_418720.1), DeoR ( NP_286606.1), AgaR (N P_289702.1), GlpR ( NP_417881.1), b1770 ( NP_416284.1) and c4824 ( AAN83253.1) ; P aeruginosa GlmR ( NP_254237.1 ) ; L euconostoc citreum FruR ( YP_001728489.1) ; Staphylococcus aureus LacR ( NP_372720.1) ; L lactis LacR (NP_267113.1) and FruR ( YP_001032855.1) ; C orynebacterium glutamicum SugR ( NP_601141.1) and cgR_1764 ( YP_001138660.1) ; Lactobacillus casei IolR ( YP_001986205.1) and SorC ( YP_001986411.1) ; B subtilis IolR ( NP_391856.1) and DeoR ( NP_391822.1 ) ; S flexneri SFV_3430 ( YP_690777.1) and SFV_2884 (Y P_690265.1); H influenza HI1009 (NP_439170.1) and HI0615 (NP_438773.1); Streptococcus mutans LacR (NP_721845.1); K pneumoniae SorC (YP_002241037.1) ; Salmonella enterica Sty0448 (NP_455005.1) Arabidopsis thaliana F box protein ( NP_566421.1) ; Rhizobium legum ino sarum AraC ( YP_768464.1) ; T ten g congensis TTE2588 ( AAM25712.1) ; Heliobacterium modesticaldum HM1_2674 ( YP_001681214.1) ; S thermophilum STH793 ( YP_074622.1) ; Clostridium botulinum CBC_0871 ( ZP_02620452.1) ; H volcanii HVO_1501 ( YP_003535550.1) ; H turkmenica Htur_2761 ( YP_003404307.1) ; H borinquense HborDRAFT_0052 ( ZP_03997260.1) ; H marismortui rrnAC0341 ( YP_135095.1) ; and H mukohataei Hmuk_2660 (YP_003178473.1). Finally, gene name or locus tag and accession numbers for trimmed sequences used in Figure 67 (distribution of PFK and KDGK in Bacteria and Archaea) are as follows: H. volcanii HVO_05 49 ( YP_003534614.1), HVO_A0328 (YP_003533784.1)

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110 HVO_1500 ( YP_003535549.1) and HVO_2612 ( YP_003536628.1) ; H. borinquense HborDRAFT_0980 ( ZP_03998188.1), HborDRAFT_0358 ( ZP_03997566.1) and HborDRAFT_2235 ( ZP_03999442.1); H lacusprofundi Hlac_0463 (YP_002565135.1), Hlac_2870 (YP_002564316.1), Hlac_2162 (YP_002566809.1) and Hlac_2117 (YP_002566764.1); H. marismortui rrnAC0545 (YP_135269.1), rrnAC2551 (YP_137055.1) and rrnAC0342 (YP_135096.1); H. turkmenica Htur_3215 (YP_003404753.1), Htur_3911 (YP_003405439.1), Htur_1630 (YP_003403189.1), Htur_4085 (YP_003405592.1), Htur_0569 (YP_003402140.1) and Htur_2760 (YP_003404306.1); H. mukohataei Hmuk_2509 ( YP_003178324.1), Hmuk_0377 (YP_003176221.1), Hmuk_2764 (YP_003178576.1) and Hmuk_2661 (YP_003178474.1); Halalkalicoccus jeotgali HacjB3_14395 (ADJ16260.1), HacjB3_09545 (ADJ15292.1) and HacjB3_10180 (ADJ15419.1); N magadii Nmag_1292 ( YP_003479434.1), Nmag_3485 ( YP_003481597.1) and Nmag_2964 ( YP_003481078.1) ; H salinar um VNG0158G (NP_279296.1) and VNG1851G (NP_280577.1); H walsbyi HQ1455A (Y P_657227.1) ; H utahensis Huta_2283 (YP_003131183.1), Huta_0006 (YP_003128929.1), Huta_0501 (YP_003129421.1); Huta_0650 (YP_003129570.1) and Huta_1103 (YP_003130015.1) ; Natronomonas phar a o nis NP3184A ( YP_327240.1 ) ; S solfataricus KdgK (1 58431173) ; Thermotoga maritima KdgK ( 90108697) ; Sulfolobus tokodaii KdgK ( 88192770) ; E coli KdgK (N P_417983.2) and FruK (Y P_003500240.1) ; Thermus thermophilus KdgK ( 48425860) ; B subtilis KdgK ( NP_390093.1) ; Erwinia chrysanthemi KdgK ( CAA52961.1) ; Thermoproteus tenax KdgK ( CAF18464.1) ; C maquilingensis Cmaq_0369 ( YP_001540205.1) ; S. boydii SBO_3525 ( YP_409837.1) ; H. influenzae HI0049

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111 ( NP_438222.1) and HIAG_00717 ( ZP_05850080.1) ; Clostridium acetobutylicum CA_ C0395 ( NP_347035.1) ; L casei LCABL_28640 ( YP_001988772.1) ; S flexneri FruK ( NP_708065.1) ; L lactis FruB ( YP_003353441.1) ; Spiroplasma citr i FruK ( AAF08321.1) ; Borrelia burgdorferi PfkB ( EEF56173.1) ; Pseudomonas putida FruK ( YP_001751248.1) ; Homo sapiens PfkM ( AAA60068.1) ; Mus musculus PfkB ( AAA20076.1 ) ; and Bacillus licheniformis FruK ( YP_078830.1)

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112 Table 21 Strains and plasmids used in C hapter 3. Strain or Plasmid Description Source or Reference E. coli GM2163 F ara 14 leuB6 fhuA31 lacY1 tsx78 glnV44 galK2 galT22 mcrA dcm 6 hisG4 rfbD1 rpsL136 dam13::Tn 9 xylA5 mtl 1 thi 1 mcrB1 hsdR2 New England Biolabs H. volcanii DS70 Wild type isolate DS2 cured of plasmid pHV2 (Oren, 1994) H26 DS70 pyrE2 (Allers et al., 2004) KS3 H26 fruB ptsI (devoid of FruB start codon and EI ORF) This study Plasmids pTA131 Ap r ; pBluescript II containing P fdx pyrE2 (Allers et al., 2004) pJAM202 Ap r Nv r ; pBAP5010 containing P2 rrnA psmB his 6 His 6 expressed in H. volcanii (Kaczowka and Maupin Furlow, 2003) pJAM202c Ap r Nv r ; control plasmid derived from pJA M202 (Zhou et al., 2008) pJAM809 Ap r Nv r ; pJAM202 containing P2 rrnA hvo1862 strepII (KpnI site inserted upstream of StrepII coding sequence) (Humbard et al., 2009) pJAM2055 Ap r Nv r ; pJAM202c derived expression plasmid including HpaI site for C terminal fusion to StrepII tag This study pJAM2657 Ap r ; pTA131 containing ptsI with ~ 700 bp of genomic DNA flanking 5' and 3' of the ptsI coding region This study pJAM2660 Ap r ; pJAM2657 derived ptsI fruB suicide plasmid This study pJAM2663 Ap r Nv r ; pJAM2055 containing P2 rrnA ptsI fruB strepII This study pJAM2664 Ap r Nv r ; pJAM2055 containing P2 rrnA ptsI strepII This study pJAM2665 Ap r Nv r ; pJAM809 containing P2 rrnA fruB strepII This study The StrepII tag is a peptide that binds to the biotin binding site of streptavidin. Apr, ampicillin resistance; Nvr, novobiocin resistance.

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113 Table 22. Primers used in C hapter 3 Primer Names (paired as used) PCR/Pr oduct descriptions Primer Sequences fruB and ptsI (Hvo 1495; Hvo 1496) HindIII Forward ~500 bp of genomic DNA flanking 5 and 3 of ptsI generated using H. volcanii DS70 genomic DNA as a template; includes XbaI and HindIII sites for cloning into pTA131 to generate pJAM2657 5 TCATG AAGCTT ATCGAGTTCCTCCTCGACC 3 XbaI Reverse 5 GATG TCTAGA AATCCTTCGTCGAGC 3 Inverse Forward ptsI suicide plasmid pJAM2660 generated by inverse PCR using pJAM2657 as template to generate pJAM2660; the start codon of fruB was also deleted during construction of pJAM2660 5 AACTCGTCGCAGTCACATCCTGTCCGA 3 Inverse Reverse 5 AGCTTACTGTTTGGCTTCAGGCGTGGAAAG 3 Negative Forward ~1700 bp within ptsI coding region; ptsI mutants 5 ATGACCGAACGAACCCTCTC 3 Negative Reverse 5 GCGACTTCAGCCTTCGT 3 Positive Forward ~700 bp of genomic DNA flanking 5 and 3 of ptsI ; used to confirm ptsI mutation by PCR 5 TCCGACGACTGACCACACCGAA 3 Positive Reverse 5 CAGGAGGTCCGAGTCCATCCG 3 fruB Complimentary Forward Coding region of fruB generated using H. volcanii DS70 genomic DNA as a template; includes NdeI and KpnI sites for cloning into pJAM809 to generate pJAM2665 5 ATTACATC CATATG AAACTCGTCGCAGTCAC 3 fruB Complimentary Reverse 5 GTA GGTACC CGAGAACAGCTTCTTCAG 3 ptsI Complimentary Forward Coding region of ptsI generated using H. volcanii DS70 genomic DNA as a template; includes NdeI and HpaI sites for cloning into pJAM2055 to generate pJAM2664 5 AACAGTAA CATATG ACCGAACCGAACCCTCT 3 ptsI Complimentary Reverse 5 ATGGCGCT GTTAAC TTGGTCTAGTGTAAG 3

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114 Table 22. Continued Primer Names (paired as used) PCR/Product descriptions Primer Sequences ptsI fruB Complimentary Forward Coding region of ptsI fruB generated using H. volcanii DS70 genomic DNA as a template; includes NdeI and HpaI sites for cloning into pJAM2066 to generate pJAM2663 5 AACAGTAA CATATG ACCGAACCGAACCCTCT 3 ptsI fruB Complimentary Reverse 5 ATGGCGCT GTTAAC CGAGAACAGCTTCTTCAG 3 HindIII Forward ~700 bp probe generated using pJAM2658 as template, used to ptsI mutation by Southern blot 5 TCATG AAGCTT ATCGAGTTCCTCCTCGACC 3 Inverse Reverse 5 AGCTTACTGTTTGGCTTCAGGCGTGGAAAG 3 Restriction enzyme recognition sequences are underlined.

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115 Table 23 Strains and plasmids used in C hapter 4 Strain or Plasmid Description Source or Reference E. coli strains F recA1 endA1 hsdR17 (rK mK + ) supE44 thi 1 gyrA relA1 Life Technologies GM2163 F ara 14 leuB6 fhuA31 lacY1 tsx78 glnV44 galK2 galT22 mcrA dcm 6 hisG4 rfbD1 rpsL136 dam13 ::Tn 9 xylA5 mtl 1 thi 1 mcrB1 hsdR2 New England Biolabs H. volcanii strains DS70 Wild type isolate DS2 cured of plasmid pHV2 (Oren, 1994) H26 DS70 pyrE2 (Allers et al., 2004) KS4 H26 glpK (devoid of GlpK) This study Plasmids pTA131 Ap r ; pBluescript II containing P fdx pyrE2 (Allers et al., 2004) pJAM202c Ap r Nv r ; control plasmid derived from pBAP5010 (Zhou et al., 2008) pJAM2055 Ap r Nv r ; pJAM202c derived expression plas mid including HpaI site for C terminal fusion to StrepII tag This study pJAM2658 Ap r ; pTA131 derived presuicide plasmid containing glpK with ~500 bp genomic DNA sequences flanking 5 and 3 ends of glpK This study pJAM2675 Ap r ; pJAM 2658 derived g lpK suicide plasmid This study pJAM2666 Ap r Nv r ; pJAM2055 derived expression plasmid containing P2 rrn glpK StrepII tag This study The StrepII tag is a peptide that binds to the biotin binding site of streptavidin. Apr, ampicillin resistance; Nvr, novobiocin resistance.

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116 Table 2 4 Primers used in C hapter 4. Primer Names (paired as used) PCR/Product descriptions Primer Sequences glpK (Hvo 1541) XbaI Forward ~500 bp of genomic DNA flanking 5 and 3 of glpK generated using H. volcanii DS70 genomic DNA as a template; includes XbaI and HindIII sites for cloning into pTA131 to generate pJAM2658 5 GATC TTCTAGA TCGACGACCAGGCGT 3 HindIII Reverse 5 GACTGCT AAGCTT CGATGACAACGATGT 3 Inverse Forward glpK suicide plasmid pJAM2675 generated by inverse PCR using pJAM2658 as template 5 CACGTGTTTGAAGCATTCGCACTCCAGATTCC 3 Inverse Reverse 5 TTCTAACCAACCTCGATACGAACTCTCGGTGTGAGA 3 Negative Forward ~500 bp within glpK coding region; glpK mutants 5 CGACGCCGAGCAGTTAGAAGCCA 3 Negative Reverse 5 GGAGTTCGTCGAGCGTCTCCCAG 3 Positive Forward ~700 bp of genomic DNA flanking 5 and 3 of glpK ; used to confirm glpK mutation by PCR 5 CGTCGTGTACCTCCTGTTCGATG 3 Positive Reverse 5 GCGACGATGATGAGCGGTTC 3 Complimentary Forward Coding region of glpK generated using H. volcanii DS70 genomic DNA as a template; includes NdeI and HpaI sites for cloning into pJAM2055 to generate pJAM2666 5 TACGTTGG CATATG TCAGGAGAAACTTACGTCG 3 Complimentary Reverse 5 ATTGTT GTTAAC TTCCTCCCGTGCCCA 3 Inverse Forward ~500 bp probe generated using pJAM2658 as template, used to glpK mutation by Southern blot 5 CACGTGTTTGAAGCATTCGCACTCCAGATTCC 3 HindIII Reverse 5 GACTGC TAAGCTT CGATGACAACGATGT 3 (q)RT PCR Primers RT Forward ~200 bp probe for cDNA of 3 end of gpdA (Hvo 1540) and 5 end of glpK with intergenic region; used to determine if glpK and gpdA are transcriptionally linked 5 GGCTACGACATCAAGCACCC 3 RT Reverse 5 TGGTCGATGGCACCGAC 3

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117 Table 24. Continued Primer Names (paired as used) PCR/Product descriptions Primer Sequences ( q)RT PCR Primers qRT 1541 Forward ~200 bp probe within glpK coding region; used to quantify transcript levels of glpK by qRT PCR; AT=52.5C 5 GTACCATCGTCGGTATGAC 3 qRT 1541 Reverse 5 TCGACTGGAGCTGACA 3 qRT 1538 Forward ~200 bp probe within gpdA coding region; used to quantify transcript levels of chromosomally encoded gpdA (Hvo 1538) by qRT PCR AT=58.0C 5 CAGGTGGACACGGTCGTC 3 qRT 1538 Reverse 5 CGAGAGCGTCTCTATCATCAGGTC 3 qRT A0269 Forward ~200 bp probe within gpdA ; used to quantify transcript levels of megaplasmid encoded gpdA (Hvo A0269) by qRT PCR; AT=55.0C 5 GACTACGTTGTCAGTGCGAC 3 qRT A0269 Reverse 5 GGATGATGGTATCGCCCTC 3 qRT 0484 Forward ~200 bp probe within ribL coding region; used to quantify transcript levels of ribL (internal standard) by qRT PCR AT=57.5C 5 CCGGCGCCTGCTTGTTCTCGCG 3 qRT 0484 Reverse 5 CCGAGGACTACCCCGTCCAGATTAGC 3 Restriction enzyme recongnition sequences are underlined.

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118 Table 25 Strains and plasmids used in C hapter 5 Strain or Plasmid Description a Source or reference Strains E. coli Top10 F recA1 endA1 hsdR17 (r K m K + ) supE44 thi 1 gyrA relA1 Invitrogen GM2163 F ara 14 leuB6 fhuA31 lacY1 tsx78 glnV44 galK2 galT22 mcrA dcm 6 hisG4 rfbD1 rpsL136 dam13 ::Tn 9 xylA5 mtl 1 thi 1 mcrB1 hsdR2 New England Biolabs H. volcanii DS70 Wild type isolate DS2 cured of plasmid pHV2 (Oren, 1994) H26 DS70 pyrE2 (Allers et al., 2004) KS4 H26 glpK (devoid of GlpK) (Sherwood et al., 2 009) KS1 2 H26 gpdA1 (devoid of chromosomal GpdA1 ) This study KS11 H26 gpdA2 (devoid of pHV4 carried GpdA2 ) This study Plasmids pTA102 Ap r Nv r ; pGB70 containing H alicantei bgaH derived from pMLH32 (Delmas et al., 2009) pTA131 Ap r ; pBluescript II containing P fdx pyrE2 (Allers et al., 2004) pJAM202 Ap r Nv r ; pBAP5010 containing P2 rrnA psmB his 6 His 6 expressed in H V olcanii (Kaczowka and Maupin Furlow, 2003) pJAM202c Ap r Nv r ; control plasmid derived from pJAM202 (Zhou et al., 2008) pJAM809 Ap r Nv r ; pJAM202 containing P2 rrnA hvo1862 strepII ( KpnI site inserted upstream of StrepII coding sequence) (Humbard et al., 2009) pJAM2658 Ap r ; pTA131 derived presuicide plasmid containing glpK with ~ 500 bp genomic DNA sequences flanking 5' and 3' ends of glpK (Sherwood et al., 2009) pJAM2678 Ap r Nv r ; pJAM202 derived plasmid containing P2 rrnA bgaH from pTA102 (Rawls et al., 2010) pJAM2679 Ap r Nv r ; pJAM2678 containing P glpK 35 4 bp bgaH This study pJAM2680 Ap r Nv r ; pJAM2678 containing P gpdA1 310 bp bgaH This study pJAM2684 Ap r Nv r ; pJAM809 containing P2 rrnA ptsH2 strepII This study pJAM2693 Ap r Nv r ; pJAM809 containing P2 rrnA glpF strepII This study pJAM2694 Ap r ; pTA131 containing ~ 500 bp of genomic DNA flanking 5' and 3' of the g pdA1 coding region This study pJAM2695 Ap r ; pTA131 derived chromosomal gpdA1 suicide plasmid This study pJAM2696 Ap r Nv r ; pJAM809 containing P2 rrnA gpdA1 strepII This study

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119 Table 25. Continued Strain or Plasmid Description a Source or reference pJAM2697 Ap r ; pTA131 with ~ 500 bp of genomic DNA flanking 5' and 3' of the g pdA2 coding region This study pJAM2698 Ap r ; pTA131 derived pHV4 encoded gpdA2 suicide plasmid This study pJAM2711 Ap r Nv r ; pJAM809 containing P2 rrnA gpdA2 strepII This study pJAM2712 Ap r Nv r ; pJAM2678 containing P trpA 321 bp bgaH This study pJAM2715 Ap r Nv r ; pJAM2678 derived plasmid devoid of P2 rrnA from pTA102 (Rawls et al., 2010) Apr, ampicillin resistance; Nvr, novobiocin resistance; SD,ShineDalgarno s equence

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120 Table 2 6 Primers used in C hapter 5. Primer Name (paired as used) PCR/Product Description Primer Sequence (5 to 3) Knockout of gpdA1 (HVO_1538): BamHI Forward 0.5 kb of genomic DNA flanking 5 and 3 of gpdA1 generated using genomic DNA as a template; includes BamHI and HindIII sites for cloning into pTA131 to generate pJAM2694 5 A GGATCC GAACACCGGGTCGAGA 3 HindIII Reverse 5 TT AAGCTT CGCGTCGAAGTCCGTGAGA 3 Inverse Forward gpdA1 suicide plasmid pJAM2695 generat ed by inverse PCR using pJAM2694 as template 5 ATGGCGATAACTGACGA 3 Inverse Reverse 5 CTGTCTTTCGTGAGGTAG 3 Negative Forward 1.2 kb within gpdA1 coding region; used gpdA1 mutants 5 AAGGAGTGTATCGAAGAGAACCG 3 Negative Reverse 5 CCTGACAGTTGCCCATCG 3 Positive Forward 0.7 kb of genomic DNA flanking 5 and 3 of g pdA1 gpdA1 mutation by PCR 5 TCGACGTAGGCGAACGAGG 3 Positive Reverse 5 GGATGTCTTCGAGCTTGAGTCCG 3 Complimentary Forward c oding region of gpdA1 generated using genomic DNA as a template; includes NdeI and KpnI sites for cloning i nto pJAM809 to generate pJAM2696 5 GTACGA CATATG AAAAAATCGCCGAGCG 3 Complimentary Reverse 5 AT GGTACC GTTATCGCCATCTGC 3 Inverse Forward 0.45 kb probe generated using pJAM2694 as template, used to confirm gpdA1 mutation by Southern blot 5 ATGGCGATAACTGACGA 3 HindIII Reverse 5 TT AAGCTT CGCGTCGAAGTCCGTGAGA 3 Knockout of gpdA2 (HVO_A0269): BamHI Forward 0.5 kb of genomic DNA flanking 5 and 3 of gpdA2 generated using genomic DNA as a template; includes BamHI and HindIII sites for cloning into pTA131 to generate pJAM2697 5 T GGATCC CACTACCTCATCGCCTTTG 3 HindIII Reverse 5 TG AAGCTT TCCTCGGCGAACTCGATTTC 3

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121 Table 2 6 Continued Primer Name (paired as used) PCR/Product Description Primer Sequence (5 to 3) Inverse Forward gpdA2 suicide plasmid pJAM2698 generat ed by inverse PCR using pJAM2697 as template 5 ATGGCGATTGAGAGCGAC 3 Inverse Reverse 5 ATAA T T AT GAAGAACCATGGGTAGCG 3 Negative Forward 1.3 kb within gpdA2 coding region; used gpdA2 mutants 5 TCGTCCAGTTGGAGGGCGA 3 Negative Reverse 5 CGTGACGCTGACCCTTCCAG 3 Positive Forward 0.6 kb and 0.7 kb of genomic DNA flanking 5 and 3 of g pdA2 respectively, gpdA2 mutation by PCR 5 GGCGCGGTAGTCCACAATCACT 3 Positive Reverse 5 GCGGTCGACGTACGGCTTCA 3 Complimentary Forward coding region of gpdA2 generated using genomic DNA as a template; includes BspHI and KpnI sites for cloning (by generating blunt ends using Vent Polymerase) into pJAM809 cut with NdeI and KpnI to generate pJAM2711 5 CCGGCT TCATGA GCTACTCAGTCGTC 3 Complimentary Reverse 5 AT GGTACC ATCGCCATGGCTGCC 3 Inverse Forward 0.6 kb probe generated using pJAM2697 gpdA2 mutation by Southern blot 5 ATGGCGATTGAGAGCGAC 3 HindIII Reverse 5 TG AAGCTT TCCTCGGCGAACTCGATTTC 3 RT PCR Primers gpdAB RT Forward anneals to 3 end of gpdB and 5 end of gpdA coding regions; used to determine if gpdA and gpdB are transcriptionally linked (annealing at 56 C) 5 CGACGCCGACATCGACT 3 gpdAB RT Reverse 5 GCCGATGACGAGCACG 3 gpdBC RT Forward Anneals to 3 end of gpd C and 5 end of gpdB coding regions; used to determine if gpd B and gpdC are transcriptionally linked (annealing at 60.5 C) 5 ACTCCGACCGACCGA 3 gpdBC RT Reverse 5 AAGTCGGGTTGCCTGG 3

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122 T able 2 6 Continued Primer Name (paired as used) PCR/Product Description Primer Sequence (5 to 3) gpdCglpK RT Forward anneals to 3 end of g pdC and 5 end of glpK coding regions; used to determine if gpdC and g lpK are transcriptionally linked (annealing at 61 C) 5 GGCTACGACATCAAGCACCC 3 gpdCglpK RT Reverse 5 TGGTCGATGGCACCGAC 3 glpKX RT Forward anneals to 3 end of g lpX and 5 end of g lpK coding regions; used to determine if g lpK and glpX are transcriptionally linked (annealing at 66.4 C) 5 GAACTCCGCGAGAACTGGCAGGT 3 glpKX RT Reverse 5 CCTTCGAACTGTCGGATGACGATGCC 3 glpXptsH 2 RT Forward anneals to 3 end of ptsH2 and 5 end of g lpX coding regions; used to determine if g lpX and ptsH 2 are transcriptionally linked (annealing at 55 C) 5 GGGTCATCCCGAACTCG 3 glpXptsH 2 RT Reverse 5 CGTGCAGGCCGTCTT 3 Promoter Fusion Primers bgaH Forward bgaH encoding galactosidase from H. alicantei was amplified using pTA102 as template; includes NdeI and BlpI sites for cloning into pJAM202 to generate pJAM2678 5 CAGCGAC CATATG ACAGTTGGTGTCTGCT 3 bgaH Reverse 5 TATGTA GCTCAGC TCACTCGG ACGCGA 3 P gpdA1310 bp Forward Putative gpdA1 p romoter generated using genomic DNA template; has XbaI and NdeI sites for cloning into pJAM2678 to generate pJAM2 679 Promoter region i s 310 bp of genomic DNA upstream of the gpdA1 start codon 5 A TCTAGA CCGACCACTACCGA 3 P gpdA1310 bp Reverse 5 ACGAT CATATG TCTTTCGTGAGGT 3 P glpK 354 bp Forward P utative glpK promoter generated using genomic DNA template; has XbaI and NdeI sites for cloning into pJAM2678 to generate pJAM2680 Promoter region includes 354bp of genomic DNA upstream of the glpK start codon 5 A TCTAGA CGCACAACTGACGAACG 3 P glpK 354 bp Reverse 5 ACGGA CATATG TAACCAACCTCGATAC 3

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123 T able 2 6 Continued Primer Name (paired as used) PCR/Product Description Primer Sequence (5 to 3) P trpA 321 bp Forward P romoter of trpA generated using genomic DNA as a template; includes XbaI and NdeI sites for cloning into pJAM2678 to generate pJAM2712. Promoter region includes 321 bp of genomic DNA upstream of the start codon of trpA 5 GC TCTAGA ACGACGCCATCACCTCC 3 P trpA 321 bp Reverse 5 ACGATTT CATATG GCCGCCAATAGGTCCG 3 Restriction enzyme recognition sequences are underlined.

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124 Table 27 Strains and plasmids used in C hapter 6. Strain or Plasmid Description a Source or reference Strains E. coli Top10 F recA1 endA1 hsdR17 (r K m K + ) supE44 thi 1 gyrA relA1 Invitrogen GM2163 F ara 14 leuB6 fhuA31 lacY1 tsx78 glnV44 galK2 galT22 mcrA dcm 6 hisG4 rfbD1 rpsL136 dam13::Tn 9 xylA5 mtl 1 thi 1 mcrB1 hsdR2 New England Biolabs H. volcanii DS70 Wild type isolate DS2 cured of plasmid pHV2 (Oren, 1994) H26 DS70 pyrE2 (Allers et al., 2004) KS8 H26 glpR (devoid of GlpR) This study KS4 H26 glpK (devoid of GlpK) (Sherwood et al., 2009) KS10 H26 glpK glpR (devoid of GlpR and GlpK) This study Plasmids pTA102 Ap r Nv r ; pGB70 containing H alicantei bgaH derived from pMLH32 (Delmas et al., 2009) pTA131 Ap r ; pBluescript II containing P fdx pyrE2 (Allers et al., 2004) pJAM202 Ap r Nv r ; pBAP5010 containing P2 rrnA psmB his 6 His 6 expressed in H volcanii (Kaczowka and Maupin Furlow, 2003) pJAM202c Ap r Nv r ; control plasmid derived from pJAM202 (Zhou et al., 2008) pJAM809 Ap r Nv r ; pJAM202 containing P2 rrnA hvo1862 strepII ( KpnI site inserted upstream of StrepII coding sequence) (Humbard et al., 2009) pJAM2676 Ap r ; pTA131 containing glpR with ~ 700 bp of genomic DNA flanking 5' and 3' of the glpR coding region This study pJAM2677 Ap r ; pJAM2676 derived glpR suicide plasmid This study pJAM2678 Ap r Nv r ; pJAM202 derived plasmid containing P2 rrnA bgaH from pTA102 This study pJAM2682 Ap r Nv r ; pJAM809 containing P2 rrnA glpR strepII This study pJAM2689 Ap r Nv r ; pJAM2678 containing P glpR pfkB 188 bp bgaH This study

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125 Table 27. Continued Strain or Plasmid Description a Source or reference pJAM2702 Ap r Nv r ; pJAM2678 containing P kdgK2 2 32 bp bgaH This study pJAM2703 Ap r Nv r ; pJAM2678 containing P HVO_A0327 kdgK2 122 bp bgaH This study pJAM2705 Ap r Nv r ; pJAM2678 containing P kdgK1 89 bp bgaH This study pJAM2706 Ap r Nv r ; pJAM2678 containing P kdgK1 524 bp bgaH This study pJAM2714 Ap r Nv r ; pJAM2678 derived plasmid devoid of P2 rrnA from pTA102 and SD This study pJAM2715 Ap r Nv r ; pJAM2678 derived plasmid devoid of P2 rrnA from pTA102 This study Apr, ampicillin resistance; Nvr, novobiocin resistance; SD,ShineDalgarno s equence

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126 Table 28 Primers used in C hapter 6. Primer Name (paired as used) PCR/Product Description a Primer Sequence (5 to 3) Knockout of glpR (HVO_1501): BamHI Forward 0.7 kb of genomic DNA flanking 5 and 3 of glpR generated using genomic DNA as a template; includes BamHI and HindIII sites for cloning into pTA131 to generate pJAM2676 5 T GGATCC CACAAGGCGAACGTGAT 3 HindIII Reverse 5 TT AAGCTT GCACCTCGTCGTCGGTGA 3 Inverse Forward glpR suicide plasmid pJAM2677 generated by inverse PCR using pJAM2676 as template 5 CGGTGGCGATTCCTCGTTACGA 3 Inverse Reverse 5 CGGAGTCGCACGATGATTCTCACA 3 Negative Forward 0. 75 kb within glpR coding region; glpR mutants 5 TGTTACCAGCAGAGCGC 3 Negative Reverse 5 CATCGTGCGACTCCGT 3 Positive Forward 0.8 kb of genomic DNA flanking 5 and 3 of glp R used to glpR mutation by PCR 5 ACCTCTCGACGCTCACGC 3 Positive Reverse 5 GGCGCGGAGAGCACC 3 Complimentary Forward c oding region of glpR generated using genomic DNA as a template; includes NdeI and KpnI sites for cloning into pJAM809 to generate pJAM2682 5 ATGCGA CATATG TTACCAGCAGAGCGC 3 Complimentary Reverse 5 AT GGTACC TCGTGCGACTCCGTC 3 Inverse Reverse 0. 7 kb probe generated using pJAM2676 as template, used to glpR mutation by Southern blot 5 CGGAGTCGCACGATGATTCTCACA 3 HindIII Reverse 5 TT AAGCTT GCACCTCGTCGTCGGTGA 3 (q)RT PCR Primers

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127 Table 28. Continued Primer Name (paired as used) PCR/Product Description a Primer Sequence (5 to 3) RT Forward anneals to 3 end of glpR and 5 end of pfkB coding regions ; used to determine if glpR and pfkB are transcriptionally linked 5 GAGCTCTCGAAGCTC 3 RT Reverse 5 GGTTCGTC AAGTGA 3 qRT 1500 Forward 0. 2 kb probe within pfkB coding region; used to quantify transcript levels of pfkB by qRT PCR (annealing at 53 C ) 5 GCAAGGGTATCAACGTCG 3 qRT 1500 Reverse 5 GAGCACAGTCGTGTTCAG 3 qRT 1501 Forward 0. 2 kb probe within glpR coding region; used to quantify transcript levels of glpR by qRT PCR (annealing at 49 C ) 5 GTGACGCCGAGTATC 3 qRT 1501 Reverse 5 CGTAGACGCCCTGTTCG 3 qRT 0549 Forward 0.2 kb probe within kdgK1 coding region; used to quantify transcript levels of kdgK1 by qRT PCR (annealing at 6 3 C ) 5 ACCTGCTCGACTCGGT 3 qRT 0549 Reverse 5 CGTAGACGCCCTGTTCG 3 qRT A0328 Forward 0.2 kb probe within kdgK2 coding region; used to quantify transcript levels of kdgK2 by qRT PCR (annealing at 53 C ) 5 GCAGAACGAGACATCCG 3 qRT A0328 Reverse 5 GTTGCTCGTACACCGTTC 3 qRT 0484 Forward 0.2 kb probe within ribL coding region; used to quantify transcript levels of ribL (internal standard) by qRT PCR (annealing at 57.5 C ) 5 CCGGCGCCTGCTTGTTCTCGCG 3 qRT 0484 Reverse 5 CCGAGGACTACCCCGTCCAGATTAGC 3 Promoter Fusion Primers bgaH Forward H. alicantei bgaH galactosidase) amplified from pTA102 ; includes NdeI and BlpI sites for cloning into pJAM202 to generate pJAM2678 5 CAGCGAC CATATG ACAGTTGGTGTCTGCT 3 bgaH Reverse 5 TATGTA GCTCAGC TCACTCGGACGCGA 3

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128 Table 28. Continued Primer Name (paired as used) PCR/Product Description a Primer Sequence (5 to 3) P kdgK2 232 bp Forward Putative kdgK2 p romoter generated using genomic DNA as a template; includes XbaI and NdeI sites for cloning into pJAM2678 to generate pJAM2702. Promoter region includ e s 232bp of genomic DNA upstream of the kdgK2 start codon 5 CGCCGC TCTAGA ACACAATGATCAACGTGGTGA 3 P kdgK2 232 bp Reverse 5 AATAGT CATATG CGCCCCTCGGCGGCT 3 P HVO_A0327kdgK2122 bp Forward P utative HVO_A0327 kdgK2 promoter generated using genomic DNA template; includes XbaI and NdeI sites for cloning into pJAM2678 to generate pJAM2703. Promoter region includes 122bp of genomic DNA upstream of the HVO_A0327 start codon 5 GCGCCGC TCTAGA ACACAATGATCAACGTGGTGA 3 P HVO_A0327kdgK2122 bp Reverse 5 AATAGT CATATG TGCGGGCGGTGGGGC 3 P kdgK1 89 bp Forward Putative kdgK1 p romoter generated using genomic DNA template; includes XbaI and NdeI sites for cloning into pJAM2678 to generate pJAM2705. Promoter region includes 89bp of ge nomic DNA upstream of the kdgK1 start codon 5 A TCTAGA GCCGGCCGGAAGGGC 3 P kdgK1 89 bp Reverse 5 AATAGT CATATG CGGCCGTTCGCAGGC 3 P kdgK1 524 bp Forward Putative kdgK 1 p romoter generated from genomic DNA template; includes XbaI and NdeI sites for cloning into pJAM2678 to generate pJAM2706. Promoter region includes 524 bp of genomic DNA upstream of the kdgK1 start codon 5 A TCTAGA TCCGAGCGGGTCGCGT 3 P kdgK1 524 bp Reverse 5 AT GGTACC TCGTGCGACTCCGTC 3

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129 Table 28. Continued Primer Name (paired as used) PCR/Product Description a Primer Sequence (5 to 3) P glpR pfkB 188 bp Forward Putative p romoter region of glpR pfkB operon generated using genomic DNA as a template; includes XbaI and NdeI sites for cloning into pJAM2678 to generate pJAM2689. Promoter region includes 188 bp of genomic DNA upstream of the start codon of glpR 5 A TCTAGA CGAACCGG CGATTCG 3 P glpR pfkB 188 bp Reverse 5 ACGAT CATATG TGGCGATTCCTCG 3 Restriction enzyme recognition sequences are underlined.

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130 CHAPTER 3 DISTRIBUTION OF PHOS PHOENOLPYRUVATELINKED PHOSPHOTRANSF ERASE SYSTEM HOMOLOGS IN ARCHAEA AND IMPLICATI ONS AS TO THEIR BIOLOGICAL FUNCTION Introduction The PTS catalyzes the group translocation and concomitant phosphorylation of sugar substrates across the biological membrane in bacteria. PTS components include two soluble general energy coupling proteins, EI and Hpr, which lack sugar specificity and membraneassociated EII permease complexes which are sugar specific (Postma et al., 1993) The sugar specific EII complexes generally consist of three proteins or protein domains (EIIA, EIIB, and EIIC) ; however, the mannose permease family consists of an additional membranespanning domain (EIID). Phosphorelay proceeds from PEP to the N3 position of histidine189 of EI (Weigel et al., 1982a) to the N1 position of histidine15 of Hpr (Weigel et al., 1982b) to a phosphorylatable histidine residue in the family specific EIIA, to a phosphorylatable histidine residue within the permeas e specific EIIB, and finally to the sugar substrate transport ed by the sugar specific permease EIIC. All phosphoryl transfer reactions between PTS proteins are reversible and the phosphorylation status of various PTS proteins is determined by both PTS transport activity and the PEP to pyruvate ratio, reflecting flux through glycolysis (Kotrba et al., 2001) As a result, the dynamic phosphorylation status of PTS proteins in response to nutritional conditions and the metabolic state of the cell serves as the basis for PTS mediated regulation of diverse metabolic processes including the transport and metabolism of nonPTS carbon sources, cell division, chemoreception, carbon storage and metabolism, noncarbon compound transport, cellular motility, cell physiology, gene expression, and switching between fermentative and respiratory metabolism (Barabote

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131 and Saier, Jr., 2005; Deutscher et al., 2006) In addition to group translocation of sugars in bacteria, the PTS is also responsible for DHA metabolism ( but not transport) in many bacteria (Erni et al., 2006) Many Gram negative bacterial genomes also encode a nitrogen PTS which does not transport carbohydrates, but exerts regulatory functions implicated in metabolism of nitrogen and carbon, virul ence, and potassium homeostasis (Barabote and Saier, Jr., 2005; Deutscher et al., 2006) Until the recent examination of archaeal genomes (Comas et al., 2008) PTS proteins were believed to be exclusive to bacteria (Barabote and Saier, Jr., 2005; Lee et al., 2 007) Current analysis of the PTS distribution in archaea has been severely limited, as only 19 archaeal genomes have been analyzed for PTS homologs, and only one has been reported to encode bacterial PTS homologs (Comas et al., 2008) W it h the increasing availability of archaeal genome sequences t his study sought to critically re evaluate the presence of bacterial PTS ho mologs and their biological function in archaea. Ninety two archaeal genomes (Benson et al., 2010) were searched for homologs of PTS proteins with search engines such as BLASTP (Altschul et al., 1997) and InterP roScan (Hunter et al., 2009) using queries of characterized proteins from E. coli, B subtilis or C. freundii PTS homologs are found in many halophilic Euryarchaeota, some methanogenic Euryarchaeota, and at least one Crenarchaeon. Furthermore, six archaeons encode a complete PTS, consisting of homologs of general carrier proteins EI and Hpr as well as membraneassociated permeases specifi c for mannose, fructose, and galactitol. DHAK homologs which cluster near PTS homologs and may function in DHA utilization were also identified. Overall, this study is expected to provide insight into the function and evolution of sugar transport in archaea.

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132 Results and Discussion Distribution of PTS Components in Archaea Although the PTS is widely distributed in bacteria, its distribution in archaea is much more scattered. Ninety two archaeal genomes (Benson et al., 2010) were searched for homologs of PTS general carrier and sugar specific proteins using the search engines BLASTP (Altschul et al., 1997) and InterProScan (Hunter et al., 2009) Q ueries of characterized PTS proteins from E. coli as well as B. subtilis were used for BLASTP Archaeal homologs of HprK were analyzed using B. subtilis as a query for BLASTP. T he majority of archaeal genomes examined did not encode homologs of E. coli or B. subtilis PTS proteins. H owever, many halophilic and some methanogenic Euryarchaeota as well as at least one Crenarchaeon encoded PTS like components (Table 31) Homologs for Hpr and EI proteins were identified in six of the 92 archaeal genomes analyzed: T. pendens H. volcanii, H. lacusprofundi H marismortui H turkmenica, and H. walsbyi In each case, these general carrier componen ts were also accompanied with sugar specific permeases (Table 31, Figure 31) Interestingly the H. volcanii genome encodes three homologs of Hpr which cluster on the genome with additional PTS homolo gs (Table 31 Figure 3 1 ). In Gram positive bacteria, Hpr phosphorylation at serine46 is catalyzed by homohexameric ATP dependent HprK, which is triggered by the availability of glycolytic intermediates such as FBP (Deutscher et al., 1995) In the presence of inhibitory molecules such as inorganic phosphate (Reizer et al., 1998) HprK can also serve as a phosphorylase, catalyzing the dephosphorylation of serine46 (Galinier et al., 1998) Serine46 phosphorylation is not directly implicated in sugar translocation, however the modif ication can inhibit EI dependent histidine15 phosphorylation up to 600fold (Stlke et al., 1998) A lthough

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133 many haloarchaeal Hpr homologs contain a conserved serine46 residue, bioinformatics only predicts homologs of B. subtilis HprK in two haloarchaea, H. ma rismortui (at 27% to 39 % amino acid identity) and H borinquense (at 40 % amino acid identity) the latter of which does not contain any detectable PTS homologs (Table 31). H omologs of B. subtilis HprK are also found in Methanosarcina thermophila (at 37 % amino acid identity) and Methanoregula boonei (at 45% amino acid identity ) despite the absence of additional PTS homologs. H. volcanii possesses the most versatile PTS of all the archaeal genomes analyzed based on the presence of both fructoseand galactitol specific permeases ( Table 31, Figure 31) Suga r specific PTS permeases are additionally found in T. pendens (mannose type), H marismortui (fructose type), and H turkmenica (fructose type) (Table 31, Figure 31) D istribution of DHAK Homologs in Archaea DHAKs (glycerone kinases, EC 2.7.1.29) are a family of amino acid sequenceconserved enzymes which utilize either ATP (eukaryotes and bacteria) or PEP (bacteria) as the source of the highenergy phosphoryl group (Bchler et al., 2005a) PEPdependent DHAKs rely on the PTS general carrier components EI and Hpr as well as a tri partite DHAK consisting of DhaK, DhaL, and DhaM subunits to phosphorylate DHA for its subsequent use. Unlike tradit ional PTS substrates, however, DHA is not transported into the cell by DHA specific PTS permeases. T he PTS instead serves to phosphorylate intracellular DHA in a phosphorelay proceeding from PEP to EI to Hpr to DhaM to ADP bound DhaL and finally to DHA whi ch is bound by DhaK. Ninety two archaeal genomes (Benson et al., 2010) were searched for the presence of DHAK using BLASTP (Altschul et al., 1997) and InterProScan (Hunter et al., 2009) search engines. DHAK homologs were identified using E. coli and C. freundii

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134 DHAK s as a query for BLASTP. Out of the 92 available archaeal genome sequences, DHAK homologs were identified in only three haloarchaea. Specifically, only H. volcanii, H. walsbyi and H. lacusprofundi encoded complete DHAKs ( Table 31, Figure 31). The H. volcanii and H. lacusprofundi DHAK genes clustered chromosomally with genes encoding general carrier protein homologs Hpr (ptsH ) and/or EI ( ptsI ) (Figure 3 1). T he presence of DHAKs in haloarchaea may arise from the biologi cal significance of DHA as a n organic carbon source for haloarchaea. Although the permeability of DHA to the biological membrane i s currently unknown, DHA derived from the glycerol cycle of Dunaliella sp. or as an overflow product of glycerol metabolism in Salinibacter sp. may serve as a putative energy source for haloarchaea (Elevi Bardavid and Oren, 2008) PTS Components an d Haloa rchaeal Glycerol Kinases Glycerol is an important organic carbon and energy source for haloarchaea and other members of halophilic, heterotrophic communities as a result of its large scale production by the halotolerant green alga Dunaliella sp. (Elevi Bardavid et al., 2008) As a result, GKs are widely distributed in haloarchaea and often cluster chromosomally with additional glycerol utilization enzymes including G3PDH and a putative glycerol facilitator protein (Figure 32). In Gram positive bacteria such as Enterococcus s p. (Charrier et al., 1997) Hpr serves as an activator of GK activity through reversible phosphorylation (Deutscher and Sauerwald, 1986) In the presence of PTS substrates, EI phosphorylates Hpr which transfers the phosphate moiety to sugar specific permeases which, in turn, phosphorylate the incomi ng PTS substrate. However, in the absence of preferable PTS substrates such as glucose, a phosphoryl residue is transferred from the N3position of histidine15 of Hpr to GlpK, allowing for subsequent 1015 fold activation (Yeh et al., 2004) The site of GlpK phosphorylation in E faecalis

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135 was determined as a conserved histidine232 (Charrier et al., 1997) (Figure 3 3). This conserved histidineresidue is noticeably absent from GK s of Gram negative bacteria such as E. coli which employ a different mode of regulatio n of GK activity (Figure 33) and haloarchaeal GKs (Figure 3 3). The absen ce of the conserved histidine residue in haloarchaea may be due to the fact that glycerol is an important organic carbon sources for heterotrophic prokaryotes in hypersaline ecosystems as a result of its production and release by the halotolerant green alg a Dunaliella sp. (Wegmann et al., 1980; Fujii and Helle bust, 1992; Elevi Bardavid et al., 2008) Thus, haloarchaeal GK s may not be subject to activation as glycerol is likely a preferred carbon source for members of the halophilic, heterotrophic microbial community (Sherwood et al., 2009) Although Hpr dependent phosphorylation is a dominant form of GK regulati on for many Gram positive bacteria, not all members are subject to its regulation. Glycerol is one of the few carbon sources that M pneumoniae is able to metabolize (Hames et al., 2009) ; thus, M. pneumoniae GK enzyme activi ty is constitutive and is not subject to activation by Hpr dependent phosphorylation (Hames et al., 2009) EIIAGlc (also called IIIGlc in older literature) is the central regulatory element of the PEP:PTS in enteric bacteria, serving as a signal for the availability of extracellular glucose. EIIAGlc recognizes and binds in a phosphorylation status dependent manner to metabolic enzymes and transporters of secondary carbon sources, leading to their inactivation (Postma et al., 1993) Specifically in E. coli EIIAGlc binds to GK i nteract ing with several amino acid residues including arginine402 and glutamate479 (the primary site of interaction). Many of these binding residues including glutamate479 are conserved i n haloarchaeal GKs (Figure 33); however, bioinformatics does not predict

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136 any haloarchaeal genomes to encode a homolog of E. coli EIIAGl c (Figure 3 1). Previous studies have shown that when E. coli G K is modified at glycine 230 to aspartic acid by site directed mutagenesis, the enzymatic activity of GK increases and allosteric regulation by the glycolytic pathway intermediate FBP decreases significantly (Anderson et al., 2007) In all haloarchaeal G K homologues analyzed, this glycine 230 to aspartic acid point mutation has occurred, possibly allowing for increased GK activity (Figure 33). Preliminary Evidence for the Involvement of H. volcanii EI and EIIBFru in Fructose Metabolism Give n the versatility of the PTS components encoded by the H. volcanii genome, this haloarchaeon was selected as a model for preliminary examination of the biological function of PTS homologs. The open reading fram e of ptsI (EI) as wel l as the start codon of f ruB (EIIBFru) were deleted from the c hromosome of H26 using a marker less deletion method (Bitan Banin et al., 2003; Allers et al., 2004) The subsequent gene knockout was confirmed by DNA sequencing, Southern Blot, and PCR using primer s which annealed external to the recombinatory region (Figure 3 4). While parent H26 readily utilized both fructose and glycerol, KS3 cells deficient in ptsI and fruB were unable to metabolize fructose in minimal medium containing glycerol and fructose as sole carbon sources (Figure 35). A plasmid containing both fruB and ptsI under the control of the constitutive H salinar um rRNA P2 promoter fully compliment ed the mutation however the mutation was not complement ed by providing either gene alone in trans (data not shown). The PTS deletions did not appear to affect glycerol metabolism of the mutant strain KS3 based on HPLC analysis of glycerol utilization (Figure 3 5) These results are consistent with glycerol transpor t by a facilitator protein

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137 and not by the PTS as in E. coli (Sanno et al., 1968) Interestingly, when mutant strain KS3 was grown i n minimal medium containing glucose and glycerol, consumption of glucose and glycerol was not affected by the PTS mutations based on HPLC analysis of carbon utilization (Figure 3 6) In bacteria, glucose is transported and concomitantly phosphorylated by the PTS (Barabote and Saier, Jr., 2005) Given that H. volcanii lack s homologs for the PTS glucosespecific permease present in many bacteria such as E. coli and that a glucose specific sodium transporter has been characterized in H. volcanii (Tawara and Kamo, 1991) it is not surprising that glucose metabolism does not appear affected by deletion of PTS components. The data here demonstrate that both EI and EIIBFru are needed for H. volcanii f ructose metabolism, and suggest a role for these proteins in the concomitant transport and phosphorylation of fructose by bacterial PTS homologs in H. volcanii Conclusion In conclusion, this study examined 92 published, archaeal genomes for homologs of characterized PTS proteins from both Gram positive and Gram negative bacteria. PTS homologs were identified in halophilic Euryarchaeota, methanogenic Euryarchaeota, and one Crenarchaeon. From the genomes analyzed, six archaeal genomes encoded a complete PTS consisting of homologs of general carrier proteins EI and Hpr as well as mem braneassociated permeases specific for mannose, fructose, and galactitol. Preliminary examination of an H. volcanii mutant deficient in both EIIBFru and EI demonstrated that these PTS homologs are needed for fructose metabolism and suggest a biological fu nction of these proteins for group translocation of fructose in this haloarchaeon. Homologs of DHAKs which cluster near PTS homologs w ere also identified in three out of the 92 archaeal genomes analy zed. Overall, this study has

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138 expanded the knowledge concerning sugar transport in archaea and is expected to serve as a guideline for understanding the biological function and evolution of the PTS with in the third domain of life.

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139 Table 31. D istribution of PTS homologs in archaea Organism EI Hpr EIIA EIIB EIIC EIID DhaK DhaL DhaM HprK Crenarchaeota Thermofilum p endens Tpen_ 1092 Tpen_ 1091 Tpen_ 1098 (Man) Tpen_ 1097 (Man) Tpen_ 1100 (Man) Euryarcha e ota Haloferax volcanii HVO_ 1496 HVO_ 1497 HVO_ 1498 (Fru) HVO_ 1495 (Fru) HVO_ 1499 (Fru) HVO_ 2101 HVO_ 2102 (Gat) HVO_ 2104 (Gat) HVO_ 2103 (Gat) HVO_ 1543 HVO_ 1546 HVO_ 1545 HVO_ 1544 Halorub r um l acusprofundi Hlac_ 1461 Hlac_ 1462 Hlac_ 1458 Hlac_ 1459 Hlac_ 1460 Haloarcula marismortui pNG 7391 pNG 7389 pNG 7388 (Fru) pNG 7392 (Fru) pNG 7387 (Fru) rrnAC 0402 rrnAC0623, rrnAC2379 Halogeometricum borinquense DSM11551 HborDRAFT _2433 Haloterrigena turkmenica Htur_ 2756 Htur_ 2757 Htur_ 2758 (Fru) Htur_ 2755 (Fru) Htur_ 2759 (Fru) Haloquadratum w alsbyi HQ 2709A HQ 2708A HQ 2672A HQ 2673A HQ 2674A M ethanocaldococcus fervens AG86 Mefer_ 1365* Methanocaldococcus sp. FS406 22 MFS40622 __1559* Methanocaldococcus jannaschii DSM2661 MJ0581* Methanosarcina thermophila Mthe_1685 Methanoregula boonei Mboo_1155 Indicates a truncated EII protein in which an EIIB domain is contained within a hypothetical protein

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140 Figure 3-1. Genomic organization of complete phosphoenolpyruvate:phosphotransferase system utilization operons in archaea. Genes which are conserved across multiple species have been highlighted as follows: DeoR/GlpR-t ype transcriptional regulator ( glpR, purple), DHAK ( dhaKLM green), PTS general carrier protein Hpr ( ptsH red), PTS general carrier protein EI ( ptsI gold), sugar-specific mannose permease EIIMan (manABC, dark blue), PFK ( pfkB grey), leucine biosythesis genes ( leuBCD peach), sugar-specific fructose permease EIIFru (fruABC, bright blue), and sugar-specific galactitol permease EIIGat (gatABC, plum). Proposed Fructose Utilization Operons 2000Haloferax volcanii Hvo2100 ptsH3 gatA gatC gatB tpuI fbpA Hvo2107 arcR3 xylB arcR4 pdxAProposed Galactitol Utilization Operon fbpA fruB ptsI ptsH1 fruA fruC pfkB glpR leuB leuD leuC Hvo1505 pNG7385 pNG7386 fruC fruA ptsH ptsI fruB pNG7393 pNG7394 pNG7395 Htur2754 fruB ptsI fruA fruC pfkB glpR leuB ptsHHaloterrigena turkmenica Haloarcula marismortui Haloferax volcanii Hlac1457 dhaK dhaL dhaM ptsI ptsH Hlac1463 Halorubrumlacusprofundi glpX ptsH dhaM dhaL dhaK ileS Haloferax volcaniiProposed Dihydroxyacetone Utilization Operons Tpen1090 ptsH ptsI nagA glmS Tpen1095 Tpen1096 manA manB Tpen1099 manC Tpen1101Proposed Mannose Utilization OperonThermofilum pendens Haloquadratum walsbyi gpdA dhaM dhaL dhaK ileSProposed Fructose Utilization Operons 2000 2000Haloferax volcanii Hvo2100 ptsH3 gatA gatC gatB tpuI fbpA Hvo2107 arcR3 xylB arcR4 pdxAProposed Galactitol Utilization Operon fbpA fruB ptsI ptsH1 fruA fruC pfkB glpR leuB leuD leuC Hvo1505 pNG7385 pNG7386 fruC fruA ptsH ptsI fruB pNG7393 pNG7394 pNG7395 Htur2754 fruB ptsI fruA fruC pfkB glpR leuB ptsHHaloterrigena turkmenica Haloarcula marismortui Haloferax volcanii fbpA fruB ptsI ptsH1 fruA fruC pfkB glpR leuB leuD leuC Hvo1505 fbpA fruB ptsI ptsH1 fruA fruC pfkB glpR leuB leuD leuC Hvo1505 pNG7385 pNG7386 fruC fruA ptsH ptsI fruB pNG7393 pNG7394 pNG7395 pNG7385 pNG7386 fruC fruA ptsH ptsI fruB pNG7393 pNG7394 pNG7395 Htur2754 fruB ptsI fruA fruC pfkB glpR leuB ptsH Htur2754 fruB ptsI fruA fruC pfkB glpR leuB ptsHHaloterrigena turkmenica Haloarcula marismortui Haloferax volcanii Hlac1457 dhaK dhaL dhaM ptsI ptsH Hlac1463 Halorubrumlacusprofundi Hlac1457 dhaK dhaL dhaM ptsI ptsH Hlac1463 Halorubrumlacusprofundi glpX ptsH dhaM dhaL dhaK ileS glpX ptsH dhaM dhaL dhaK ileS Haloferax volcaniiProposed Dihydroxyacetone Utilization Operons Tpen1090 ptsH ptsI nagA glmS Tpen1095 Tpen1096 manA manB Tpen1099 manC Tpen1101 Tpen1090 ptsH ptsI nagA glmS Tpen1095 Tpen1096 manA manB Tpen1099 manC Tpen1101Proposed Mannose Utilization OperonThermofilum pendens Haloquadratum walsbyi gpdA dhaM dhaL dhaK ileS gpdA dhaM dhaL dhaK ileS

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141 Figure 3-2. Organization of glycerol utilization operons from haloarchaea whose genome sequences have been completed. Genes that are conserved across multiple species are as follows: G3PDH ( gpdABC green), GK ( glpK, blue), GlpF-like glycerol facilitator ( glpF yellow), and non-GlpF like glycerol facilitator ( glpX, red). Proposed Haloarchaeal Glyc erol Utilization OperonsHalorubrumlacusprofundi Hlac1126 gpdC gpdB gpdA glpK glpX cdc6 Hlac1119 Huta1468 gpdC gpdB gpdA glpK glpX nagC pykAHalorhabdusutahensis uspA gpdC gpdB gpdA ugpB glpK glpF cdc6 nagC pykA kdgKHalomicrobiummukohataei Htur2958 cdc6 gpdC gpdB gpdA glpK glpX Htur2965Haloterrigena turkmenica phoU gpdC gpdB gpdA glpK glpX cdc6 pgsAHaloquadratum walsbyi cdc6 gpdA gpdB gpdC glpK glpX ptsH2Haloferax volcanii 2000 rrnAC 0557 gpdC gpdB gpdA lysR glpK glpX cdc6 glcK pykAHaloarcula marismortuiProposed Haloarchaeal Glyc erol Utilization OperonsHalorubrumlacusprofundi Hlac1126 gpdC gpdB gpdA glpK glpX cdc6 Hlac1119 Hlac1126 gpdC gpdB gpdA glpK glpX cdc6 Hlac1119 Huta1468 gpdC gpdB gpdA glpK glpX nagC pykA Huta1468 gpdC gpdB gpdA glpK glpX nagC pykAHalorhabdusutahensis uspA gpdC gpdB gpdA ugpB glpK glpF cdc6 nagC pykA kdgK uspA gpdC gpdB gpdA ugpB glpK glpF cdc6 nagC pykA kdgKHalomicrobiummukohataei Htur2958 cdc6 gpdC gpdB gpdA glpK glpX Htur2965 Htur2958 cdc6 gpdC gpdB gpdA glpK glpX Htur2965Haloterrigena turkmenica phoU gpdC gpdB gpdA glpK glpX cdc6 pgsA phoU gpdC gpdB gpdA glpK glpX cdc6 pgsAHaloquadratum walsbyi cdc6 gpdA gpdB gpdC glpK glpX ptsH2 cdc6 gpdA gpdB gpdC glpK glpX ptsH2Haloferax volcanii 2000 2000 rrnAC 0557 gpdC gpdB gpdA lysR glpK glpX cdc6 glcK pykA rrnAC 0557 gpdC gpdB gpdA lysR glpK glpX cdc6 glcK pykAHaloarcula marismortui

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142 Figure 3-3. Alignment of hal oarchaeal glycerol kinases and biochemically characterized bacterial glycerol kinases. Protein s equences were retrieved using the NCBI database and subsequently Nand C-te rminally trimmed and aligned using CLUSTAL W (Thompson et al., 1994). Conserved regulatory sites are highlighted in yellow. The site of Hpr-dependent phosphorylation in Firmicutes is indicated by *. The primary site of EIIAGlc interaction is indicated as A point mutation (G230D) known to in crease enzymatic activity of E. coli GlpK is indicated by Accession numbers for the alignment can be found in Chapter 2. 10 20 30 40 50 60 70 80 9 0 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| Efa HVTDYSNASRTMLFNIHDL D WD Q E I LD L L N IPRV MLPK V VSNS E--VYG L T KN-YH F Y G S EVPIAG M AGDQQAALFGQ MA F EP GM V KNTY Eca HVTDYSNASRTML Y NIHK LEWD Q E I LD L L N IP SS MLPEV KSNS E--VYG H T RS-YH F Y G S EVPIAG M AGDQQAALFGQ MA F EK GM I KNTY Bsu HVTDYSNASRT LM FNIY DL K WDDELLD I L G VP KS MLPEV K PSSH--VY AE T VD-YH F F G KNIPIAGAAGDQQ S ALFGQ A CF E EGMG KNTY Bce HVTDYSNASRT LM FNIHDL Q WDDELLD M L T VP KS MLPEVRPSS E--VYG E T ID-YH F F G QN VPIAG V AGDQQAALFGQ A CF G EGMAKNTY Hvo H I TDVTNASRTMLFNIHD M EWDDELLD E F N VPREL LPEVRPSS DDDY YGT T DA-D GF L G A EVPV AGAL GDQQAALFGQ T CF DA G D AKNTY Hla H I TDVTNASRTML Y NI R DLEWDDELLE E F D VP KE M V PEVRPSS DEDY YGH T DA-D GF L G E EVPV AGAL GDQQAALFGQ T CF D EG D AKNTY Hma H I TDVSNASRTML Y NIHDM EWDDELLE E F G VP ES M V PEVRPSS DESL YGH T DA-D GF LKE EVPV AGAL GDQQAALFGQ T CF DK G D AKNTY Hwa H I TDVTNASRTML Y NI RELEWDDELLE E F R VPRS M V PEVRPSS DDEY YGH T DA-D GF L G A E I P V AGAL GDQQAA M FGQT CF D EG D AKNTY Hsa H I TDVSNASRTML Y NI T DLEWDD W LLEE F D IPRE MLPEVRPSS DEAVYG H T DP-D GF L G AA VP VTAA L GDQQAALFGQ T CF DA G D AKNTY Nma H I T E VTNASRTML Y NIHDLEWDD D LLEE F S IP EA MLPEVRPSS DDET YGT T DP-E GF LEA EVPV AGAL GDQQAALFGQ T CF DA G D AKNTY M pn HVTDVSNASRT L LF D I TTMTW SQ EL G D I F K VP LSILP K V M PS NAHFGDIVPSHWSTSATGM VPIR G V AGDQQAALFGQ L C VEPAM V KNTY Eco HVTDYTNASRTMLFNIH T L D WDDKM LE V L D IPRE MLPEVR R SS E--VYG Q T NI-G G KG G TRIPI S G I AGDQQAALFGQ L C VKEGMAKNTY Kpn HVTDYTNASRTMLFNIH E L D WDDKM LD A L D IPRA MLPEVR K SS E--VYG Q T NI-G G KG G TRIPIAG I AGDQQAALFGQ L C VKEGMAKNTY Sfl HVTDYTNASRTMLFNIH T L D WDDKM LE V L D IPRE MLPEVR R SS E--VYG Q T NI-G G KG G TRIPI S G I AGDQQAALFGQ L C VKEGMAKNTY 100 110 120 130 140 150 160 170 180 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| Efa GTGS F IVMNTGEE PQLS KNNLLTTIG Y G IN G K--V Y YALEGSIFVAG S AIQWLRD GLKMLQT AA E SE AV A KASTGHN-E VYVVPAFTGLG Eca GTGA F IVMNTGEE PQLS DNDLLTTIG Y G IN G K--V Y YALEGSIFVAG S AIQWLRD GLRM I ETSPQ SE E LA AKAKGDN-E VYVVPAFTGLG Bsu GTGC FMLMNTGE K A IK S E HGLLTTI AW G ID G K--V N YALEGSIFVAG S AIQWLRD GLRMFQDSSL SE SY A EK V DS TD GVYVVPAF V GLG Bce GTGC FMLMNTGE K AV A S E HGLLTTI AW G ID G K--V N YALEGSIFVAG S AIQWLRD GMRMFKD A SE SE VY A SR V ES TD GVYVVPAF V GLG Hvo GTGS FMLMNTG D EAVM S E HGLLTT V G F Q RS GE PV Q YALEGSIF IT GAAI E WL E D -MTLIDN AA E SE K LA RS V ES TD GVY F VPAFTGLG Hla GTGS F Y LMNTGN EAVK S D HGLLTTIG F Q MS GE PV Q YALEGSIF IT GAAI E WL E D -VDLINN AA QTAE LA RS V DS TD GVY M VPAFTGLG Hma GTGA F Y LMNTGS EAVA S DN GLLTT V G F Q MS GE PV Q YALEGSIF I AGAAI E WL E D -VDLINN AA QTAE LA RS V ES TD GVY M VPAFTGLG Hwa GTGS F Y LMNTGTD AV A S D HGLLTTIG F Q MS GE PV Q YALEGSIFV T GAAI EF L E D -VDLINN AA QTAE LA SS V DS TD GVY M VPAFTGLG Hsa GTGS F Y LMNTGE D AV S S E HGLLTTIG F Q LS GE PV Q YALEGSIFV T GAAI E WL E D -VDLINN AA QTAE LA SS V DT TD GVY M VPAFTGLG Nma GTGS F F LMNTGN EAVK S D HGLLTTIG F Q RS GE DV Q YALEGSIFV T GAAI E WL E D -MSLIDNPSETAE LA RS V DT TD GVYVVPAFTGLG M pn GTGC FMLMNI G N E LKYS Q H N LLTT VAWQ LENQK-PV YALEGS V FVAGAA LK WLRD SLKVMYS AA E S DFYA KLAQKEEQE V VFVPAFTGLG Eco GTGC FMLMNTGE K AV K S EN GLLTTI AC G PT GE -V N YALEG AV F M AGAS IQWLRD EMKLIND A YD SE YF A TK V QN T NGVYVVPAFTGLG Kpn GTGC FMLMNTGE K AV T S T HGLLTTI AC G PR GE -V N YALEG AV F M AGAS IQWLRD EMKLISD A FD SE YF A TK V KD T NGVYVVPAFTGLG Sfl GTGC FMLMNTGE K AV K S EN GLLTTI AC G PT GE -V N YALEG AV F M AGAS IQWLRD EMKLIND A YD SE YF A TK V QN T NGVYVVPAFTGLG 190 200 210 220 230 240 250 260 270 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| Efa APYWD SQARGA V FG L TRGT TR E DF V K ATL QAVAYQV RD IIDTM KE D T GIDIPV-------L K VDGGA AN N D FLMQFQ A DIL N T A V Q R AH Eca APYWD SEARGA V FG L TRGT T KE DF VRATL QAVAYQSK DV IDTM KK DSGIDIPL-------L K VDGGA A KNDL LMQFQ A DIL DIDV Q R AA Bsu T PYWD SDV RG SV FG L TRGT T KEHFI RATLES L AYQT K DV LD AMEADS N I SLKT-------LRVDGGAVKNNFLMQFQ G D L L NVPV E RP E Bce T PYWD SEV RGAM FG V TRGT T KEHFI RATLES L AYQT K DV LC AMEADSGI ELKT-------LRVDGGAVKNNFLM K FQSDIL DVPV E RP V Hvo AP H WD QRARGT I V G M TRGT RR EHIVRATLESIA F QTRDV A EAME S DS E I DLSS-------LRVDGGAVKNNFL C Q L QS N ILD T EIVRP Q Hla AP H WD GRARGT I V G M TRGT G KEHIVRATLESIAYQTRDV A EAMEADSG VETTS-------LRVDGGAVKNNFL C Q L QSDI IQ T EIARP E Hma AP H WD GRARGT I V G M TRGT R KEHIVRATLESIAYQTRD LAEAME E DSGVEMTT-------LRVDGGAVKNNFL C Q L QSDI IQ T DIARP Q Hwa AP H WD GRARGTLVG M TRGT E KEHIVRATLESI G YQTRDV A EAMEADSGI ETTS-------LRVDGGAVKNNFL C Q L QSDIL Q T DIVRP V Hsa AP H WD GRARGTLVG M TRGT R K A HIVRATLESIAYQTRD IAA AMEADSG VSTTT-------LRVDGGAVKNNFL C Q L QSDI IQ T DLARP E Nma AP H WD QRARGT I V G M TRGT R K G H V VRATLESIAYQTRDV A EAMEADSGI AMTT-------L K VDGGAVKNNFL C Q L QSDI IGSKIV RP V M pn APYWD ASARGAIFG IEANT KR EH L V K ATLE A IA F Q AN D LIK AM AS D LNSSIKK--------IKA DGGA CNS N Y LMQFQ A DI ANLE V II P K Eco APYWD PYARGAIFG L TRG VNAN HI I RATLESIAYQTRDV L EAMQ ADSGI RLHA-------LRVDGGAV A NNFLMQFQSDIL G T R V E RP E Kpn APYWD PYARGAIFG L TRG VNSN HI I RATLESIAYQTRDV L EAMQ ADSGI RLHA-------LRVDGGAV A NNFLMQFQSDIL G T R V E RP E Sfl APYWD PYARGAIFG L TRG VNAN HI I RATLESIAYQTRDV L EAMQ ADSGI RLHA-------LRVDGGAV A NNFLMQFQSDIL G T R V E RP E 280 290 300 310 320 330 340 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|... Efa NL ETTALGAA F LAGLAVG F W KD L E E IKA--FQEEGQQ F E P I M AEEER EDL Y E GW QQ AV AATQQFKRKNK---Eca NL ETTALGAAYLAGLAVG F W KD LDEL KS--MAEEGQM F T P E M PAEER DNL Y E GW KQ AV AATQTFKFKAKKEGE Bsu IN ETTALGAAYLAG I AVG F W KDRS E IAN--Q W NL D KR F E P ELEEEK R NEL Y K GW QK AV KAAMAFK-------Bce IN ETTALGAAYLAGLAVGYW KNQDE IKE--Q W HM D KR F E P T M EAEISEEL Y A GW KK A IEATKAFK-------Hvo V D ETTALGAAY A AGLAVGYW ET LDEL RE--NW QV DREF A P K-DPQNVEHR Y G RW KE AV D R S L D WA REE----Hla V D ETTALG S AY A AGLAVGYW DTVDELRD--NW QI DREF T P EKGQA E VDKL Y S RW DD AV E R S L N WA QDD----Hma V D ETTALG S AY A AGLAVGYW DTVDELRD--NW QV D E EF S P E M DAGKADKM Y A RW DD AV D R SRDWA QEE----Hwa V D ETTALG S AY A AGLAVGYW DTVDELRD--NW QV DREF ESE M DSADANTM Y D RW DD AV E R S L D WA QEE----Hsa V D ETTALGAAY A AGLAVGYW DS LD D L RE--NW RV DR S F E P E M DPSE ADSK Y G RW ED AV D R S L A WA TED----Nma V D ETTALG S AY A AGLAVGYW DDVD S L RD--NW QV D WT F DSR M DDAVADAQ Y D RW LD AV D R S L D WA RDG----M pn NV ETTTMGAAF LAGLAV N YW KDTKQ L EK--LTGIAKQ F KSQ M NQTV R EKKSK RW NE AV K R T L K WA SLD----Eco V R E V TALGAAYLAGLAVG F W QN LDEL QE--KAVIE REFR P GIETT ER NYR Y A GW KK AV K R AMAW EEHD----Kpn V R E V TALGAAYLAGLAVG F W QN LDEL QE--KAVIE REFR P GIETT ER NYR Y S GW KK AV K R A L A W EEHD----Sfl V R E V TALGAAYLAGLAVG F W QN LDEL QE--KAVIE REFR P GIETT ER NYR Y A GW KK AV K R AMAW EEHD----10 20 30 40 50 60 70 80 9 0 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| Efa HVTDYSNASRTMLFNIHDL D WD Q E I LD L L N IPRV MLPK V VSNS E--VYG L T KN-YH F Y G S EVPIAG M AGDQQAALFGQ MA F EP GM V KNTY Eca HVTDYSNASRTML Y NIHK LEWD Q E I LD L L N IP SS MLPEV KSNS E--VYG H T RS-YH F Y G S EVPIAG M AGDQQAALFGQ MA F EK GM I KNTY Bsu HVTDYSNASRT LM FNIY DL K WDDELLD I L G VP KS MLPEV K PSSH--VY AE T VD-YH F F G KNIPIAGAAGDQQ S ALFGQ A CF E EGMG KNTY Bce HVTDYSNASRT LM FNIHDL Q WDDELLD M L T VP KS MLPEVRPSS E--VYG E T ID-YH F F G QN VPIAG V AGDQQAALFGQ A CF G EGMAKNTY Hvo H I TDVTNASRTMLFNIHD M EWDDELLD E F N VPREL LPEVRPSS DDDY YGT T DA-D GF L G A EVPV AGAL GDQQAALFGQ T CF DA G D AKNTY Hla H I TDVTNASRTML Y NI R DLEWDDELLE E F D VP KE M V PEVRPSS DEDY YGH T DA-D GF L G E EVPV AGAL GDQQAALFGQ T CF D EG D AKNTY Hma H I TDVSNASRTML Y NIHDM EWDDELLE E F G VP ES M V PEVRPSS DESL YGH T DA-D GF LKE EVPV AGAL GDQQAALFGQ T CF DK G D AKNTY Hwa H I TDVTNASRTML Y NI RELEWDDELLE E F R VPRS M V PEVRPSS DDEY YGH T DA-D GF L G A E I P V AGAL GDQQAA M FGQT CF D EG D AKNTY Hsa H I TDVSNASRTML Y NI T DLEWDD W LLEE F D IPRE MLPEVRPSS DEAVYG H T DP-D GF L G AA VP VTAA L GDQQAALFGQ T CF DA G D AKNTY Nma H I T E VTNASRTML Y NIHDLEWDD D LLEE F S IP EA MLPEVRPSS DDET YGT T DP-E GF LEA EVPV AGAL GDQQAALFGQ T CF DA G D AKNTY M pn HVTDVSNASRT L LF D I TTMTW SQ EL G D I F K VP LSILP K V M PS NAHFGDIVPSHWSTSATGM VPIR G V AGDQQAALFGQ L C VEPAM V KNTY Eco HVTDYTNASRTMLFNIH T L D WDDKM LE V L D IPRE MLPEVR R SS E--VYG Q T NI-G G KG G TRIPI S G I AGDQQAALFGQ L C VKEGMAKNTY Kpn HVTDYTNASRTMLFNIH E L D WDDKM LD A L D IPRA MLPEVR K SS E--VYG Q T NI-G G KG G TRIPIAG I AGDQQAALFGQ L C VKEGMAKNTY Sfl HVTDYTNASRTMLFNIH T L D WDDKM LE V L D IPRE MLPEVR R SS E--VYG Q T NI-G G KG G TRIPI S G I AGDQQAALFGQ L C VKEGMAKNTY 100 110 120 130 140 150 160 170 180 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| Efa GTGS F IVMNTGEE PQLS KNNLLTTIG Y G IN G K--V Y YALEGSIFVAG S AIQWLRD GLKMLQT AA E SE AV A KASTGHN-E VYVVPAFTGLG Eca GTGA F IVMNTGEE PQLS DNDLLTTIG Y G IN G K--V Y YALEGSIFVAG S AIQWLRD GLRM I ETSPQ SE E LA AKAKGDN-E VYVVPAFTGLG Bsu GTGC FMLMNTGE K A IK S E HGLLTTI AW G ID G K--V N YALEGSIFVAG S AIQWLRD GLRMFQDSSL SE SY A EK V DS TD GVYVVPAF V GLG Bce GTGC FMLMNTGE K AV A S E HGLLTTI AW G ID G K--V N YALEGSIFVAG S AIQWLRD GMRMFKD A SE SE VY A SR V ES TD GVYVVPAF V GLG Hvo GTGS FMLMNTG D EAVM S E HGLLTT V G F Q RS GE PV Q YALEGSIF IT GAAI E WL E D -MTLIDN AA E SE K LA RS V ES TD GVY F VPAFTGLG Hla GTGS F Y LMNTGN EAVK S D HGLLTTIG F Q MS GE PV Q YALEGSIF IT GAAI E WL E D -VDLINN AA QTAE LA RS V DS TD GVY M VPAFTGLG Hma GTGA F Y LMNTGS EAVA S DN GLLTT V G F Q MS GE PV Q YALEGSIF I AGAAI E WL E D -VDLINN AA QTAE LA RS V ES TD GVY M VPAFTGLG Hwa GTGS F Y LMNTGTD AV A S D HGLLTTIG F Q MS GE PV Q YALEGSIFV T GAAI EF L E D -VDLINN AA QTAE LA SS V DS TD GVY M VPAFTGLG Hsa GTGS F Y LMNTGE D AV S S E HGLLTTIG F Q LS GE PV Q YALEGSIFV T GAAI E WL E D -VDLINN AA QTAE LA SS V DT TD GVY M VPAFTGLG Nma GTGS F F LMNTGN EAVK S D HGLLTTIG F Q RS GE DV Q YALEGSIFV T GAAI E WL E D -MSLIDNPSETAE LA RS V DT TD GVYVVPAFTGLG M pn GTGC FMLMNI G N E LKYS Q H N LLTT VAWQ LENQK-PV YALEGS V FVAGAA LK WLRD SLKVMYS AA E S DFYA KLAQKEEQE V VFVPAFTGLG Eco GTGC FMLMNTGE K AV K S EN GLLTTI AC G PT GE -V N YALEG AV F M AGAS IQWLRD EMKLIND A YD SE YF A TK V QN T NGVYVVPAFTGLG Kpn GTGC FMLMNTGE K AV T S T HGLLTTI AC G PR GE -V N YALEG AV F M AGAS IQWLRD EMKLISD A FD SE YF A TK V KD T NGVYVVPAFTGLG Sfl GTGC FMLMNTGE K AV K S EN GLLTTI AC G PT GE -V N YALEG AV F M AGAS IQWLRD EMKLIND A YD SE YF A TK V QN T NGVYVVPAFTGLG 190 200 210 220 230 240 250 260 270 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| Efa APYWD SQARGA V FG L TRGT TR E DF V K ATL QAVAYQV RD IIDTM KE D T GIDIPV-------L K VDGGA AN N D FLMQFQ A DIL N T A V Q R AH Eca APYWD SEARGA V FG L TRGT T KE DF VRATL QAVAYQSK DV IDTM KK DSGIDIPL-------L K VDGGA A KNDL LMQFQ A DIL DIDV Q R AA Bsu T PYWD SDV RG SV FG L TRGT T KEHFI RATLES L AYQT K DV LD AMEADS N I SLKT-------LRVDGGAVKNNFLMQFQ G D L L NVPV E RP E Bce T PYWD SEV RGAM FG V TRGT T KEHFI RATLES L AYQT K DV LC AMEADSGI ELKT-------LRVDGGAVKNNFLM K FQSDIL DVPV E RP V Hvo AP H WD QRARGT I V G M TRGT RR EHIVRATLESIA F QTRDV A EAME S DS E I DLSS-------LRVDGGAVKNNFL C Q L QS N ILD T EIVRP Q Hla AP H WD GRARGT I V G M TRGT G KEHIVRATLESIAYQTRDV A EAMEADSG VETTS-------LRVDGGAVKNNFL C Q L QSDI IQ T EIARP E Hma AP H WD GRARGT I V G M TRGT R KEHIVRATLESIAYQTRD LAEAME E DSGVEMTT-------LRVDGGAVKNNFL C Q L QSDI IQ T DIARP Q Hwa AP H WD GRARGTLVG M TRGT E KEHIVRATLESI G YQTRDV A EAMEADSGI ETTS-------LRVDGGAVKNNFL C Q L QSDIL Q T DIVRP V Hsa AP H WD GRARGTLVG M TRGT R K A HIVRATLESIAYQTRD IAA AMEADSG VSTTT-------LRVDGGAVKNNFL C Q L QSDI IQ T DLARP E Nma AP H WD QRARGT I V G M TRGT R K G H V VRATLESIAYQTRDV A EAMEADSGI AMTT-------L K VDGGAVKNNFL C Q L QSDI IGSKIV RP V M pn APYWD ASARGAIFG IEANT KR EH L V K ATLE A IA F Q AN D LIK AM AS D LNSSIKK--------IKA DGGA CNS N Y LMQFQ A DI ANLE V II P K Eco APYWD PYARGAIFG L TRG VNAN HI I RATLESIAYQTRDV L EAMQ ADSGI RLHA-------LRVDGGAV A NNFLMQFQSDIL G T R V E RP E Kpn APYWD PYARGAIFG L TRG VNSN HI I RATLESIAYQTRDV L EAMQ ADSGI RLHA-------LRVDGGAV A NNFLMQFQSDIL G T R V E RP E Sfl APYWD PYARGAIFG L TRG VNAN HI I RATLESIAYQTRDV L EAMQ ADSGI RLHA-------LRVDGGAV A NNFLMQFQSDIL G T R V E RP E 280 290 300 310 320 330 340 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|... Efa NL ETTALGAA F LAGLAVG F W KD L E E IKA--FQEEGQQ F E P I M AEEER EDL Y E GW QQ AV AATQQFKRKNK---Eca NL ETTALGAAYLAGLAVG F W KD LDEL KS--MAEEGQM F T P E M PAEER DNL Y E GW KQ AV AATQTFKFKAKKEGE Bsu IN ETTALGAAYLAG I AVG F W KDRS E IAN--Q W NL D KR F E P ELEEEK R NEL Y K GW QK AV KAAMAFK-------Bce IN ETTALGAAYLAGLAVGYW KNQDE IKE--Q W HM D KR F E P T M EAEISEEL Y A GW KK A IEATKAFK-------Hvo V D ETTALGAAY A AGLAVGYW ET LDEL RE--NW QV DREF A P K-DPQNVEHR Y G RW KE AV D R S L D WA REE----Hla V D ETTALG S AY A AGLAVGYW DTVDELRD--NW QI DREF T P EKGQA E VDKL Y S RW DD AV E R S L N WA QDD----Hma V D ETTALG S AY A AGLAVGYW DTVDELRD--NW QV D E EF S P E M DAGKADKM Y A RW DD AV D R SRDWA QEE----Hwa V D ETTALG S AY A AGLAVGYW DTVDELRD--NW QV DREF ESE M DSADANTM Y D RW DD AV E R S L D WA QEE----Hsa V D ETTALGAAY A AGLAVGYW DS LD D L RE--NW RV DR S F E P E M DPSE ADSK Y G RW ED AV D R S L A WA TED----Nma V D ETTALG S AY A AGLAVGYW DDVD S L RD--NW QV D WT F DSR M DDAVADAQ Y D RW LD AV D R S L D WA RDG----M pn NV ETTTMGAAF LAGLAV N YW KDTKQ L EK--LTGIAKQ F KSQ M NQTV R EKKSK RW NE AV K R T L K WA SLD----Eco V R E V TALGAAYLAGLAVG F W QN LDEL QE--KAVIE REFR P GIETT ER NYR Y A GW KK AV K R AMAW EEHD----Kpn V R E V TALGAAYLAGLAVG F W QN LDEL QE--KAVIE REFR P GIETT ER NYR Y S GW KK AV K R A L A W EEHD----Sfl V R E V TALGAAYLAGLAVG F W QN LDEL QE--KAVIE REFR P GIETT ER NYR Y A GW KK AV K R AMAW EEHD----

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143 A B Figure 34. PCR and Southern blot confirmation of H. volcanii fruB ptsI mutant strain KS3 (H26 fruB ptsI ). A) Confirmation of the fruB ptsI mutant strain KS3 by PCR. Primer pairs that annealed outside the genomic region cloned in suicide plasmid pJAM26 60 were used for confirmation of the fruB ptsI gene deletions by PCR. HiLo DNA markers and molecular masses are indicated on left. Genomic DNA from the followin g strains served as template: Lane 1. Parent strain H26, Lane 2. KS3 fruB ptsI ). B) Southern blot confirmation of the fruB ptsI mutant strain KS3 fruB ptsI ). Genomic DNA was digested with Sap I and Xho I and hybridized with a DIG labeled probe specific for ptsI The following strains served as the source of genomic DNA: Lane 1. Parent strain H26, Lane 2. KS3 fruB ptsI ) 6 1.5/1.4 2 3 4 5 8 1 kB 1 2 6 1.5/1.4 2 3 4 5 8 1 kB 6 1.5/1.4 2 3 4 5 8 1 kB 1 2 4 3 2 1.5SapI and XhoI with ptsI probe12 kB 4 3 2 1.5SapI and XhoI with ptsI probe12 kB

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144 Figure 35. H. volcanii strain KS3 deficient in both PTS components EI and EIIBFru (H26 fruB ptsI ) is unable to metabolize fructose. The growth rates of and levels of carbon utilization by parent strain H26 and mutant KS3 cells grown on Gly Fru MM are shown. Growth at 42C (200 RPM ) was monitored as an increase in OD600, where 1 U was equivalent to ap proximately 109 CFU per ml for both strains. At various time points, supernatant fractions were withdrawn from cultures and analyzed by HPLC for glycerol and fructose consumption. Experiments were performed in triplicate, and the means SD were calculated. 0 5 10 15 20 25 0 5 10 15 20 25 30 Time (h) Carbon Source Concentration (mM) 0 0.5 1 1.5 2 Cell Growth (OD600) 0 5 10 15 20 25 0 5 10 15 20 25 30 35 40 Time (h) Carbon Source Concentration (mM) 0 0.5 1 1.5 2 Cell growth (OD600) Fructose Glycerol Growth Fructose Glycerol Growth H26 Gly Fru MM KS3 ( fruB ptsI ) Gly Fru MM 0 5 10 15 20 25 0 5 10 15 20 25 30 Time (h) Carbon Source Concentration (mM) 0 0.5 1 1.5 2 Cell Growth (OD600) 0 5 10 15 20 25 0 5 10 15 20 25 30 35 40 Time (h) Carbon Source Concentration (mM) 0 0.5 1 1.5 2 Cell growth (OD600) Fructose Glycerol Growth Fructose Glycerol Growth Fructose Glycerol Growth H26 Gly Fru MM KS3 ( fruB ptsI ) Gly Fru MM

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145 Figure 36. PTS components EI and EIIBFru are not required for glucose metabolism in H. volcanii. The growth rates of and levels of carbon utilization by parent strain H26 and mutant KS3 cells grown on Gly Glu MM are shown. Growth at 42C (200 RPM ) was monitored by an increase in OD600, where 1 U was equivalent to approximately 109 CFU per ml for both strains. At various time points, supernatant fractions were withdrawn from cultures and analyzed by HPLC for glycerol and glucose consumption. Experiments were performed in triplicate, and the means SD were calculated. 0 5 10 15 20 25 0 10 20 30 40 50 60 70 Time (h) Carbon Source Concentration (mM) 0 0.5 1 1.5 2 2.5 Cell Growth (OD600) H26 Gly Glu MM 0 5 10 15 20 25 0 10 20 30 40 50 60 70 Time (h) Carbon Source Concentration (mM) 0 0.5 1 1.5 2 2.5 Cell Growth (OD600) KS3 ( fruB ptsI ) Gly Glu MM Glucose Glycerol Growth 0 5 10 15 20 25 0 10 20 30 40 50 60 70 Time (h) Carbon Source Concentration (mM) 0 0.5 1 1.5 2 2.5 Cell Growth (OD600) H26 Gly Glu MM 0 5 10 15 20 25 0 10 20 30 40 50 60 70 Time (h) Carbon Source Concentration (mM) 0 0.5 1 1.5 2 2.5 Cell Growth (OD600) KS3 ( fruB ptsI ) Gly Glu MM Glucose Glycerol Growth Glucose Glycerol Growth

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146 CHAPTER 4 GLYCEROL KINASE AS T HE SOLE ROUTE OF GLY CEROL CATABOLISM IN THE HALOARCHAEON Haloferax volcanii Introduction Halophilic and halotolerant microorganisms have adapted different methods for withstanding the high osmotic pressure exerted by their surrounding hypersaline environment. Halophilic archaea (Christian and Waltho, 1962; Lanyi, 1974) as well as the halophilic bacterium Salinibacter ruber (Oren et al., 2002) maintain a high intracellular salt concentration by accumulating K+ and Clions and excluding Na+ ions, thus requiring intracellular proteins to be active under highsalt conditio ns. Many halophilic bacteria (Ventosa et al., 1998) the halotolerant green alga Dunaliella sp. (Lanyi, 1974) and some haloarchaea (Lai et al., 1991) exclude cytoplasmic salts and rely on organic solutes such as ectoine, glycine betaine, and glycerol to provide osmotic balance. Glycerol, in particular, is accumulated in molar quantities by Dunaliella as an organic osmotic solute. Due to leakage from Dunaliella cells (Wegmann et al., 1980; Elevi Bardavid et al., 2008) and/or cellular lysis, glycerol is released into the surrounding environment, where it serves as a primary energy source for haloarchaea. Upon uptake, halophilic microorganisms assimilate glycerol into DHAP by one of two catabolic routes (Figure 4 1 ). In one route, glycerol is first phosphoryl ated by GK to form G3P which is subsequently oxidized by G3PDH to produce DHAP. Alternatively, glycerol can be first oxidized by GDH to form DHA, which is subsequently phosphorylated by an ATP or PEP: PTS dependen t DHAK to yield DHAP. Once generated from glycerol, DHAP can be channeled into p yruvate and other metabolic intermediates, including G1P used as a phospholipid backbone in archaea (Nish ihara et al., 1999)

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147 Although glycerol is an important carbon and energy source for members of halophilic, heterotrophic communities, little is known regarding glycerol metabolism, especially in haloarchaea. Halophilic archaea such as H. volcanii have been previously shown to metabolize glycerol (Rawal et al., 1988) and specific activities of glycerolmetabolizing enzymes in various halo archaea have been determined (Wassef et al., 1970; Rawal et al., 1988; Oren and Gurevich, 1994a) ; however the metabolic pathways surrounding glycerol utilization at the molecular level have not been described. This study provides genetic and biochemical evidence that H. volcanii metabolizes glycerol through GK (encoded by glpK ) and, most likely, a glpK linke d G3PDH (encoded by a gpdA 1 B 1 C 1 operon) These results provide insight into the central metabolic pathways of heterotrophic haloarchaea such as H. volcanii Results and Discussion Glycerol is M eta bolized through Glycerol K inase To analyze glycerol catabolism in H. volcanii, a gene encoding a G K homolog (HVO_1541; glpK ) was targeted for knockout in a pyrE2 mutant strain (H26). The deduced product of this gene, GlpK, was most closely related to (with 74 to 78% identity) and clustered in dendr ograms with other putative GKs of haloarchaea, including those of H lacusprofundi H marismortui H. walsbyi N magadii and H. salinarum with the notable absence of GlpK homologs in the haloalkaliphilic archaeon N pharaonis and other archaea ( Figure 4 2 ). The H. volcanii and other haloarchaeal GlpK proteins also clustered with the bacterial GKs with the greatest degrees of identity (up to 58%) to those of the Thermoanaerobacterales and Thermotogales ( Figure 42 ). The glpK gene was deleted from the chr omosome of H. volcanii by a markerless knockout strategy as described previously (Bitan Banin et al., 2003; Allers et al., 2004)

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148 Gene deletion was confirmed by PCR, Southern blotting, a nd sequencing analysis (Figure 4 3 ). The resultant GK mutant, H26 glpK (KS4), was incapable of growth either on glycerol minimal medium plates (Figure 4 4 ) or in liquid culture (data not shown). A pHV2 based self replicating plasmid containing the glpK gene under the control of a strong rRNA P2 promoter (pJAM2666) restored KS4 growth on glycerol while the plasmid vector alone (pJAM202c) did not complement this glpK muta t ion (Figure 4 4 ). These results indicate that the GK homolog encoded by glpK is required for the growth of H. volcanii on glycerol. Previous studies have detected GK but not GDH activity in the lysate of H. volcanii cells grown in the presence of glycerol (Rawal et al., 1988) These findings are in agreement with the present results that in H. volcanii, glycerol metabolism proceeds through the glpK encoded GK rather than through the conversion of glycerol to DHA by a GDH. These results also suggest that genes HVO_1546 to HVO_1544, which are predicted to enc ode putative DHAK subunits K, L, and M based on the H. volcanii genome sequence (Hartman et al., 2010) function in DHA metabolism and not glycerol metabolism. Consistent with this possibility, recent evidence suggests that S ruber mediates the incomplete oxidation of glycerol to y ield DHA as an overflow product which may then be taken up by heterotrophs present in hypersaline environments (Elevi Bardavid et al., 2008) To biochemically confirm that the glpK homolog HVO_1541 codes for GK the specific activities of GK in cell lysates from H26 and KS4 (H26 glpK ) were measured as described previously (Bublitz and Kennedy, 1954; Oren and Gurevich, 1994a) GK a ctivity was readily detected in cells of the parent strain H26 grown in medium containing glycerol (Gly MM or Gly Glu MM), regardless of th e presence of glucose

PAGE 149

149 (Figure 4 5 ). Significant levels of GK activity were also detected in H26 cells grown on medium with glucose alone (Glu MM), although the levels were twofold lower compared to those in H26 cells grown on media with glycerol (i.e., Gly MM and Gly Glu MM). GK activity was not detected in the GK mutant (KS4) or boiled cell lysate (the negative control) (Figure 4 5 ). The specific activity of GK in parent strain H26 grown in the presence of glycerol was 430 30 nmol 1mg protein1; although fivefold higher than previously reported specific activities of GK s in S ruber (90 nmol 1 protein1), H cutirubrum (14 nmol 1 protein1), and H. volcanii (31 nmol 1 protein1 for cells grown i n complex medium with peptides) determined in similar assays (Wassef et al., 1970; Oren and Gurevich, 1994a; Sher et al., 2004) this value was within a reasonable range of measurement for GK enzymes. These results demonstrate that the glpK homolog HVO_1541 encodes a GK and that this gene is required for the catabolism of glycerol in H. volcanii. It should also be n ote d that GK activity is not universal among the archaea, unlike bacteria, and is absent in those organisms that cannot us e glyce rol as an energy source [ e.g., autotrophic methanogens (Nishihara et al., 1999) ] Thus, GK is not thought to be involved in the synthesis of the backbone of archaeal phospholipids and is instead mediated by G1PDH, which generates G1P from DHAP. Therefore, glycerol does not appear to be channeled into DHA by a GDH for the growth of H. volcanii These results also reveal that GK activity in H. volcanii alth ough elevated by growth on gly cerol, is not reduced by the presence of glucose in the growth medium. The detection of comparable levels of GK activity in H. volcanii cells grown on glycerol regardless of glucose supplementation contrasts with results for the E. coli model, in

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150 which the glpK regulon encoding GK is subject to catabolite repression (Freedberg and Lin, 1973) Glycerol Metabolism is not Reduced in the Presence of Glucose To determine if the growth defect of the GK mutant (KS4) i s exclusive to glycerol and to further investigate the observed differences in catabolite repression between H. volcanii and E. coli the growth of and utilization of carbon by the H. volcanii H26 parent and KS4 mutant strains on minimal medium supplemented with either 20 mM glycerol and 20 mM glucose or 20 mM glucose alone (Gly Glu MM or Glu MM, respectively) were measured (Figure 4 6 A and Figure 47 A ). Although there are other carbon sources [ Tris buffer (30 mM), uracil (450 M), biotin (0.41 M), and thiamine (2.37 M ) ] in the minimal medium, these alone do not support the growth of H. volcanii (data not shown). Thus, any observed growth would be due to glucos e and/or glycerol in the medium The glpK mutant (KS4) and parent strain H26 grew and utilized glucose at nearly identical rates when cultured in minimal medium containing glucose as the sole carbon source (Glu MM) (Figure 4 6 A ). In both cases, roughly 85% of the glucose was metabolized within 60 h, contributing to an average overall OD600 of 1.27 and a growth rate of 0.11 doublings per h when media was supplemented with 20 mM glucose (Figure 4 6A) The addition of 20 mM glycerol to 20 mM glucose (Gly Glu MM) enhanced the growth rate and cell yield of the parent H26 approximately twofold to 0.22 doublings per h and a final OD600 of 2.0 ( Figure 47 A ). Consistent with these results, H26 metabolized both of these carbon/energy sources but displayed a preference for glycerol, with 79% of the glycerol and only 16% of the glucose in the Gl y Gl u MM being metabolized (Figure 47 A ). Thus, H26 metabolized glucose at a reduced rate when glycerol was included in the growth medium. In contrast to H26, the glpK

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151 mutant KS4 is unabl e to metabolize glycerol and does not induce glucose metabolism when grown in the pr esence of glycerol until approximately 30 h later than its parent, H26 (Figure 4 7 A ), and 20 h later than KS4 grown on medium with glucose alone (Figure 4 6 A ). Once KS4 initiated this delayed metabolism of glucose in glycerol supplemented medium (Gly Glu M M), the growth rate (0.095 doublings per h) and final OD600 (0.93) of these cells was only slightly lower compared to those of cells grown in medium with glucose alone. In addition to using 20 mM of each carbon source, a lower supplementation (5 mM) of gly cerol and glucose was also used to examine both growth rates and carbon utilization. Both parent H26 and GK mutant KS4 ( glpK ) metabolized glucose at similar rates (Figure 46B). For both strains, all of the glucose is metabolized within 35 hours and both cultures reached a maximum OD600 of 0.75 (Figure 46B). Similar to the carbon preference observed in the minimal medium supplemented with 20 mM glycerol and 20 mM glucose, pare nt H26 metabolized the majority of the glycerol prior to induction of glucose metabolism in minimal medi um supplemented wit h 5 mM glucose and 5 mM glycerol (Figure 4 7B) Based on these results H. volcanii glycerol metabolism is not reduced or delayed in the presence of glucose. The observed preference of H. volcanii for glycerol d irectly contrasts with that of E. coli which exhibits diauxic growth with glucose as the preferred carbon and energy source (Holtman et al., 2001) Preference for carbon compounds other than glucose is not novel. For example, members of the genus Pseudomonas exhibit organic acidinduced catabolite repression of glucose metabolism (Lynch and Franklin, 1978) However, preferential utilization of glycerol over glucose has not been reported previously.

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152 Levels of Glycerol 3 Phosphate Deh ydrogenase a nd G lycerol Kinas e Transcripts are U pregula ted by the Addition of G lycerol Based on the genome sequence (Hartman et al., 2010) H. volcanii has two putative NAD(P) +linked G3PDH operons: one ( gpdA2B2C2; HVO_A0269 to HVO_A0271) on the minichromosome pHV4 and one ( gpdA 1 B 1 C 1 ; HVO_1538 to HVO_1540) on the chromosome directly upstream of the GK gene (Figure 48 A). The protein paralogs encoded by t hese two operons are closely related in amino acid sequence (58 to 74% identical and 67 to 85% similar). The products of both operons are distinct from the enantiomeric glycerophosphate synthase (EgsA), an NAD(P)+linked G1PDH responsible for the formation of the G1P backbone of archaeal phospholipids (Nishihara et al., 1999) most likely encoded by HVO_0822 in H. volcanii. In order to determine whether either gpd operon is upregulated in the presence of glycerol, qRT PCR was performed using genespecific primers ( Table 24 ). Primers were designed to correspond to the first gene ( gpdA2 [HVO_A0269] and gpdA 1 [HVO_1538]) in each of the two operons in order to achieve the strongest signal fo r tr anscriptional analysis (Figure 48 A). In addition, qRT PCR primers for glpK were designed to determine if the transcription of this gene was induced by glycerol. A transcript specific to the ribosomal protein L10 gene ( ribL ) was used as an internal control based on data from a previous study (Brenneis et al., 2007) and confirmation by qRT PCR that the level of induction of transcripts specific for ribL in cells grown in the presence of glycerol and glucose was close to onefold compared to transcript levels in cells grown in the presence of glucose alone. By qRT PCR analysis, transcripts specific for glpK gpdA 1 and gpdA2 were detected at significant levels under all growth conditions examined (growth on Gly MM

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153 and Gly Glu MM) (Figure 4 8 B). In addition, transcripts specific for gpdA 1 and glpK were upregulated approximately 78and 9fold, respectively, in the presence of glycerol and glucose compared to those in the p re sence of glucose alone (Figure 48 B). In contrast, gpdA2 tr anscripts were not induced to significant levels (2.01.0 fold) by glycerol (Figure 4 8 B), indicating that the gpdA2 operon is not likely to be involved in glycerol metabolism. These results reveal that transcripts specific for glpK and its gene neighbor gpdA 1 are significantly induced by glycerol, supporting the argument that both genes and their encoded enzymes ( GK and G3PDH ) are involved in glycerol metabolism in H. volcanii Glyce rol Kinase and Glycerol 3 Phosphate D ehydrogenase G enes are u nder t he Control of a Common P romoter Due to the close proximity of glpK and gpdA 1 B 1 C 1 and the likely involvement of the encoded gene products in a common metabolic pathway (Lawrence, 1997; Overbeek et al., 1999) it was investigated whether these genes were cotranscribed in an operon. RT PCR was performed using primers designed such that the forward primer would anneal to the 3 coding region of gpdC 1 and the reverse primer would anneal to the 5 cod ing region of glpK amplifying a portion of each gene as well as the 364bp intergenic region ( Figure 48 A). A single PCR product of the expected size was detected using synthesized cDNA (Figure 4 8 C), and the sequence of the product was later confirmed. No product from the negative control reaction with RNA as a template was detected (Figure 4 8 C). Thus, glpK and gpdC 1 are linked at the transcriptional level. Although glpK and gpdC 1 of the putative gpdA 1 B 1 C 1 operon are transcriptionally linked, the reasons for the significant differences in the levels of induction of glpK and

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154 gpdA 1 specific transcripts in the presence of glycerol (Figure 4 8 B) remain to be determined. Multiple promoter elements may be involved in the transcription of this region of the ch romosome and account for these differences in induction. Consistent with the possibility that multiple promoters control the expression of the gpdA 1 B 1 C 1 glpK region, BRE and TATA box promoter consensus sequence elements were identified upstream of both gpd A 1 and glpK (Figure 48A, P1 and P2, respectively). Alternatively, the transcription of glpK (which is distal relative to gpdA 1 ) may be reduced by transcriptional polarity, which leads to the premature termination of polycistronic mRNA translation, resulting in the reduced transcription of genes locat ed distally from the operon (Wek et al., 1987) Another possibility is that the glpK specific transcripts are more susceptible to degradation than those specific for gpdA 1 Conclusion T his study demonstrates that glycerol metabolism in H. v olcanii requires GK encoded by the glpK gene (HVO_1541) and that this gene is transcriptionally linked to a putative G3PDH operon ( gpdA 1 B 1 C 1 ; HVO_1538 to HVO_1540) located upstream of glpK on the chromosome. The levels of both glpK and gpdA 1 specific tran scripts are significantly upregulated in the presence of glycerol, although not to the same extent, with the glycerol dependent induction of gpdA 1 specific transcripts being eight fold greater that of glpK specific transcripts. Promoter consensus elements upstream of both gpdA 1 and glpK suggest that, in addition to sharing a common promoter with gpdA 1 glpK may be regulated independently of gpdA 1 The present model is that glpK and gpdA 1 share a common P1 promoter immediately upstream of gpdA 1 that is tightly regulated in response to glycerol availability and that additional control of glpK transcription may be achieved through a gpdA 1 independent P2 promoter immediately

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155 upstream of glpK This study also provides evidence that H. volcanii displays differential utilization of glycerol and glucose. Overall, the results not only shed light on glycerol metabolism in H. volcanii, but also add to the understanding of central metabolic pathways of haloarchaea.

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156 Figure 4 1. Halophilic microorganisms assimilate glycerol into DHAP by one of two catabolic routes. In the route depicted on the right, glycerol is phosphorylated by GK and subsequently converted into DHAP through G3PDH In the route depicted on the left, glycero l is oxidized through GDH to form DHA, which is subsequently phosphorylated by an ATP or PEP:PTSdependent DHAK to yield DHAP. DHAP is channeled into pyruvate and other metabolic intermediates.

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157 Figure 4 2 Phylogenetic distribution of glycerol kinases. Protein sequences were retrieved from the NCBI database, N and C terminally trimmed and aligned using CLUSTAL W (Thompson et al., 1994) Pairwise comparisons were performed between sequences and mean genetic distance was evaluated using pdistance (gaps were analyzed using complete deletion). The best neighborhoodjoining tree was then constructed using MEGA 4.0. Bootstrap support values are indicated at the internal nodes and were obtained by per forming 1,000 replicates. Accession numbers for protein sequences are listed in Chapter 2.

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158 AB Figure 4-3. PCR and Souther n blot confirmation of H. volcanii glycerol kinase mutant strain KS4 (H26 glpK ). A) Confirmation of the GK mutant strain KS4 (H26 glpK) by PCR. Primer pairs that annealed outside of the recombinatory region were used for confirmation of the glpK gene knockout by PCR. Hi-Lo DNA markers and molecular masses are indicated on left. Genomic DNA from the following strains served as template: Lane 1. H26, Lane 2. H26 glpK. B) Southern blot confirmation of the glpK (GK) gene knockout KS4. Genomic DNA was digested with SphI and BspHI and hybridized with a DIGlabeled probe specific for glpK. The following strains served as the source of genomic DNA: Lane 1. H26, Lane 2. KS4 (H26 glpK).

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159 Figure 4 4 H. volcanii metabolizes glycerol through GlpK Both the H26 parent strain and the H26 glpK mutant KS4 t ransformed with a plas mid carrying glpK (pJAM2666) grew on Gly MM. In contrast, the glpK mutant strain (KS4) and KS4 transformed with vector alone (pJAM202c) were unable to grow on Gly MM. Cells were transferred with a loop from liquid Glu MM cultures onto plates of solid Gly MM, and the plates were incubated at 42C for 3.5 days.

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160 Figure 4 5 H. volcanii GK activity is dependent on glpK (HVO_1541), stimulated by growth on glycerol, and is detectable in the presence of glucose. H. volcanii H26 (parent) and KS4 ( glpK mutant) cells were grown in Gly Glu MM, Gly MM, or Glu MM. Measurements of GK activities were performed using lysates prepared from log phase cells as specified in Materials and Methods. indicates that enzyme activity was not detected. Experiments were performed in triplicate, a nd the means SD were calculated.

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161 A B Figure 4 6 P arent strain H26 and glycerol kinase mutant strain KS4 (H26 glpK ) exhibit similar growth rates, cell yields, and carbon utilizati on patterns when grown in glucose minimal medium Minimal medium was supplemented with either 20 mM Glu (A) or 5 mM Glu (B). Growth at 42C (200 RPM ) was monitored as an increase in OD600, where 1 U was equivalent to approximately 109 CFU per ml for all strains. At various time points, supernatant fractions were withdr awn from both parent H26 and KS4 cultures and analyzed by HPLC for glucose consumption. Experiments were performed in triplicate, and the means SD were calculated. Cell growth and glucose utilization levels are indicated. 0 5 10 15 20 25 0 10 20 30 40 50 6070 Time (h) Carbon Source Concentration (mM) 0 0.5 1 1.5 Cell growth (OD600) Glucose (H26) Glucose (KS4) H26 Growth KS4 Growth 0 1 2 3 4 5 6 0 5 10 15 20 25 30 35 40 Time (h) Carbon Source Concentration (mM) 0 0.2 0.4 0.6 0.8 1 Cell Growth (OD600) Glucose (KS4) Glucose (H26) Growth (H26) Growth (KS4) 0 1 2 3 4 5 6 0 5 10 15 20 25 30 35 40 Time (h) Carbon Source Concentration (mM) 0 0.2 0.4 0.6 0.8 1 Cell Growth (OD600) Glucose (KS4) Glucose (H26) Growth (H26) Growth (KS4) Glucose (KS4) Glucose (H26) Growth (H26) Growth (KS4)

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162 A Figure 4-7. Parent strain H26 and GK mutant KS4 (H26 glpK) exhibit differential utilization of glycerol and glucose. The growth rates of and levels of carbon utilization by parent strain H26 and mu tant KS4 cells grown on Gly Glu MM with carbon sources supplemented at either 20 mM each (A ) or 5 mM each (B) are shown. Growth at 42C (200 RPM) was monitored by an increase in OD600, where 1 U was equival ent to approximately 109 CFU per ml for all strains. At various time points, 1 ml culture volumes were withdrawn, and supernatant fractions were analyzed by HPLC for glycerol and glucose consumption. Experiments were performed in triplicate, and the means SD were calculated.

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163 B Figure 4-7. Continued. KS4 ( glpK ) Gly GluMM 0 1 2 3 4 5 6 05101520253035Time (h)Carbon Source Concentration (mM)0 0.2 0.4 0.6 0.8 1 1.2Cell Growth (OD600)H26 Gly GluMM 0 1 2 3 4 5 6 0510152025303540Time (h)Carbon Source Concentration (mM)0 0.2 0.4 0.6 0.8 1Cell Growth (OD600)Glucose Glycerol Growth KS4 ( glpK ) Gly GluMM 0 1 2 3 4 5 6 05101520253035Time (h)Carbon Source Concentration (mM)0 0.2 0.4 0.6 0.8 1 1.2Cell Growth (OD600)H26 Gly GluMM 0 1 2 3 4 5 6 0510152025303540Time (h)Carbon Source Concentration (mM)0 0.2 0.4 0.6 0.8 1Cell Growth (OD600)Glucose Glycerol Growth Glucose Glycerol Growth

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164 A Figure 4 8 Genomic organization and transcript analysis of the GK gene and G3PDH related operons of H. volcanii. (A) Schematic representations of the GK gene ( glpK ) and the G3PDH related operons ( gpdA 1 B 1 C 1 and gpdA2B2C2) of H. volcanii and location of annealing sites for ( q ) RT PCRs. Representations of the chromosomally located gl pK and gpdA 1 B 1 C 1 operon(s) and the pHV4based gpdA2B2C2 operon are presented. Vertical lines indicate annealing sit es of primers used for (q) RT PCR analyses. P1, P2, and P signify locations of BRE and TATA box archaeal promoter consensus elements. (B) Relative quantification of transcript levels specific for both the chromosomal gpdA 1 and pHV4based gpdA2 genes encoding G3PDH subunit A homologs (Hvo1538 and HvoA0269, respectively) and the GK glpK gene (Hvo1541). Transcripts for the chromosomal glpK and gpdA 1 genes are upregulated in the presence of glycerol. Transcript levels were derived by qRT PCR as described in Materials and Methods. Calcula tions are based on the N fold induction of transcription in the presence of Gly Glu MM compared to transcription in the presence of Glu MM. Results were normalized to the N fold induction of the internal control, ribL Experiments were performed in triplic ate, and the means SD were calculated. (C) Chromosomal GK ( glpK ) and G3PDH ( gpdA 1 B 1 C 1 ) genes are under the control of a common promoter. An RT PCR primer pair based on the 5' and 3' ends of glpK and gpdC 1 respectively, was designed. Hi Lo DNA markers an d molecular sizes are indicated to the left. Total RNA from parent H26 was extracted and reverse transcribed to generate cDNA, which was used as a template for PCR (lane 1). RNA which had not undergone reverse transcription was used as a negative control t emplate for PCR (lane 2).

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165 B C Figure 48. Continued. 1 1

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166 CHAPTER 5 CHARACTERIZATION OF THE PRIMARY GLYCEROL METABOLIC OPERON IN THE HALOARCHAEON H aloferax v olcanii Introduction Glycerol is a highly abundant energy source in hypersaline environments as a result of leakage from and lysis of Dunaliella sp. which accumulate glycerol in molar quantities as a organic, osmotic solute (Ben Amotz and Avron, 1973b; Borowitzka and Brown, 1974; Wegmann et al., 1980; Elevi Bardavid et al., 2008) Glycerol is aerobically catabolized by heterotrophic community members by one of two routes: i) through GK and G3PDH or ii) through GDH and DHAK. In H. volcanii, glycerol metabolism proceeds solely through glpK encoding GK (Sherwood et al., 2009) In this study the subsequent steps in glycerol metabolism was determined through biochemical and genetic characterization of two homologs of the bacterial, catalytic G3PDH subunit A Both chromosomal gpdA1 and pHV4carried gpdA2 of H. volcanii we re investigated for their role, if any, in conversion of G3P to DHAP This study confirms that G3P is metabolized primarily through gpdA1 and provides evidence that the H. volcanii primary glycerol metabolic operon is under the control of a strong, glycerol inducible gpdA1 promoter ( PgpdA1, P1) R esults and D iscussion H. volcanii Glyce rol Metabolic O peron is unde r the Control of an Inducible Promoter In H. volcanii, gpdC1 and glpK are linked at the transcriptional level (Sherwood et al., 2009) Due to the close chrom osomal proximity of genes e ncoding a GK ( glpK ) to a G3PDH homolog ( gpdA1B1C 1 ) a putative non GlpF glycerol facilitator protein ( glpX ) and bacterial PTS component Hpr homolog ( ptsH2 ) the putative operon(s) of this

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167 genomic region were investigated by performing a series of RT PCRs P rimers were designed to amplify a portion of the coding region of neighboring genes gpdA1 and gpdB 1 (13 bp overlap in coding sequence), gpdB1 and gpdC1 (3 bp overlap in coding sequence), glpK and glpF (4 bp intergenic region) and glpF and ptsH2 (2 bp overlap in coding sequence) (Figure 5 1A). In each case, a single PCR product of expected size was det ec ted (Figure 5 1B) and confirme d by DNA sequencing. No product was detected in the negative control reactions co ntai ning RNA as a template for PCR (Figure 5 1B). Thus, the genes encoded in the primary glycerol metabolic operon ( gpdA1B1C1glpKXptsH2) are linked at the level of transcription (Figure 51B ). Although the primary glycerol metabolic operon ( gpdA1B1C1glpKXptsH2) is transcriptionally linked, the reasons for the significant differences in the levels of induction of glpK and gpdA 1 specific transcripts in the presence of glycerol (Figure 3 8 A ) (Sherwood et al., 2009) remain to be determined. Possible explanations for the differences in abundance of glpK and gpdA1specific transcripts include: i) m ultiple promoter elements ii) reduced transcription of glpK (which is distal relative to gpdA 1 ) due to transcriptional polarity (Wek et al., 1987) and iii) differences in transcript susceptibility to degradation. To determine if the differences in transcript induction was the existence of multiple promoter elements, t ranscriptional promoter reporter fusion assays were performed using reporter constr ucts containing the 310bp gpdA1 and 354bp glpK promoter DNA upstream of their translational start codons These promoter regions were amplified, fused to the H. alicantei bgaH based transcriptional reporter and transformed into wildtype strain H26 Tra -

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168 galactosidase activity of cells grown on various carbon sources. Using this approach, significant levels of transcription were detected for the gpdA1 (38 to 310 mU protein1) promoter con struct compared to negative controls lacking promoter elements which were less than or equal to 8.1 mU protein1 for all conditions tested (Table 5 1 ). The gpdA1 promote r construct was highly inducible in glycerol medium [at least 7fold upregulated in cells grown on glycerol ( glucose) compared to glucose alone]. In contrast, transcription from the glpK promoter was weak and constitutive for all conditions tested (14 to 22 mU protein1) ( Table 5 1 ). The inducible gpdA1 promoter was very strong co mpared to the weaker, nonglycerol inducible glpK promoter for all conditions tested, except when cells were grown in glucose alone, for which it exhibited only slightly increased specific activity compared to the glpK promoter (38 mU protein1 for PgpdA 1 compared to 22 mU protein1 for PglpK) (Table 5 1 ). It should be noted that when cells were grown on glycerol the gpdA1 promoter was relatively robust at driving expression of the heterologous bgaH reporter with activities greater than that of the H cutirubrum rRNA P2 promoter (Table 51 ), used routinely for highlevel production of proteins in H. volcanii (Kaczowka et al., 2005; Uthandi et al., 2010) Although the glycerol responsive gpdA1 promoter was not as inducible as the tryptophanresponsive trpA promoter (7 fold induction for PgpdA1 versus 45fold induction for PtrpA) (Table 5 1 ), the gpdA1 promoter can serve as an alternative for experiments requiring differential gene regulation. In contrast the glpK promoter was relatively weak (14 22 mU protein1) at driving the expression of the heterologous bgaH reporter compared to the constitutive rRNA P2 promoter (2302 60 mU protein1) (Table 5 1 ). Overall, the data suggests that the H. volcanii glycerol metabolic operon is regulated by a tightly controlled,

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169 glycerol inducible gpdA1 promoter (P1) that drives the robust expression of the operon in the presence of glyce rol. Glycerol Metabolism Proceeds P rimarily through GpdA1 G lycerol is metabolized solely through GK in H. volcanii ( glpK HVO_1541) (Sherwood et al., 2009) To analyze the subsequent step in glycerol catabolism, two genes encoding homologs of bacterial G3PDH subunit A, gpdA1 (HVO_1538) and gpdA2 (HVO_A0269), were deleted from the H. volcanii H26 genome using a markerless deletion strategy as previously described (Bitan Banin et al., 2003; Allers et al., 2004) Subunit A of the G3PDH complex was targeted for gene knockout since this subunit is required for G3PDH activity in E. coli (Cole et al., 1988) The g pdA1 and gpdA2 gene deletions were confirmed by PCR, Southern Blot and sequencing analysis ( Figure 5 2 ). The resulting gpdA1 mutant (KS12) was incapable of growth of glycerol minimal medium (Fig ure 53 ). In contrast c ells deficient in gpdA2 (KS11 ) grew a t a similar rate to wild type H26 on glycerol as the sole carbon source (Fig ure 53 ). The glycerol dependent phenotype displayed by KS12 could be complimented by providing either gpdA1 or gpdA2 in trans under the control of the strong, constitutive H. cutirubrum rRNA P2 promoter (Figure 53 ). The ability of gpdA2 in trans to compliment cells deficient in gpdA1 suggests that GpdA2 may be a functional isoform of GpdA1 whose intracellular levels are insufficient to impact glycerol metabolism. To determ ine whether the difference in phenotype between the gpdA mutants was due to a difference in cellular levels G3PDH activity was assessed in various strains In parent H26, G3PDH activity was up regulat ed at least 2fold when cells were grown in the presence of glycerol ( glucose) when compared to glucose alone (Table 5 2 ). Upon deletion of gpdA1, G3PDH activity was no longer induced by growth in the presence of

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170 glycerol. This was evidenced by a 4fold reduction in G3PDH activity of the gpdA1 mutant KS12 com pared to parent H26 during growth on glycerol and gl ucose. G3PDH activity in the gpdA1 background was restored to parental levels by providing either gpdA1 or gpdA2 in trans A mutant deficient in glpK displayed a similar level of G3PDH activity compared t o gpdA1 when grown in minimal medi um containing glycerol and glucose which could be complemented by providing glpK in trans (Table 52 ). The loss of activity in the glpK mutant suggests that G3P serves as the inducer of the chromosomal glycerol regulon, similar to E. coli (Hayashi and Lin, 1965) Deletion of gpdA2 resulted in a slight, yet significant reduction in G3PDH activity compared to parent H26 when c ells were grown in glycerol ( glucose) which could be fully complimented by providing gpdA2 in trans (Table 52 ). No activity was detected for negative controls containing either no substrate or boiled cell lysate. It is worth noting that similar to E. co li (Cole et al., 1988) GpdA is required for catalytic activity of the H. volcanii G3PDH complex based on the fact that activity was greatly reduced in gpdA1deficient cells compared to parent H26 under all of the conditions tested (Table 5 2 ). Furthermore, gpdA2 encode s a functional subunit of G3PDH based on the observed reduction in activit y upon deletion of gpdA2 (Table 52 ) and the ability of gpdA2 in trans to restore both growth phenotype (Figure 5 3 ) and G3PDH specific activity to parental levels in a gpdA1deficient background (Table 52 ). Distribu tion of A rchaeal GpdA H omologs Interestingly, many haloarchaeal genomes encode two gpdA genes, although these homologs have marked differences from the H. volcanii gpdA genes Similar to H. volcanii other haloarchaeal gpdA1 genes often cluster chromosomally with additional glycerol met abolic genes including gpdBC glpK and a putative glycerol facilitator ( glpF

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171 or glpX ) (Figure 3 2 ). However, u nlike H. volcanii gpdA2 which is pHV4 carried and appears to be cotranscribed with gpdB2C2, gpdA2 from other haloarchaea is chromosomally located and does not cluster with additional gpdBC subunits (Figure 54 ). Furthermore, unlike H. volcanii GpdA1 and GpdA2 which are equal in amino acid length, other haloarchaeal GpdA2 s appear to have undergone a C terminal truncation event. Specifically, these truncated GpdA2s are missing the C terminal bacterioferritinassociated ferredoxin (BFD) like [2Fe 2S] center common to GpdA proteins (Figure 55 ). Although the physiological role of BFD [2Fe 2S] remains unclear it is thought to be a general redox and/or regulatory component involved in the iron storage or mobilization functions of bacterioferritin in bacteria (Garg et al., 1996) Despite differences with protein length and genomic organization, haloarchaeal GpdA homologs cluster phylogenetically (Figure 54 ). Within the haloarchaeal phylogenetic node, GpdA1 and GpdA2 are separate d into distinctive lineages for which the atypical H. volcanii GpdA2 is t he only exception to date (Figure 54 ). GpdA homologs are also found in other Euryarchaeota ( Thermoplasmata ) as well as Crenarchaeota ( Thermoprotei ) although additional GpdBC subunits are absent from these archaea (Figure 54 ). The absence of these additi onal subunits may be explained by the fact that glycerol is not commonly encountered as a primary carbon source by these microorganisms. Similar to GlpK, GpdA is noticeably absent from methanogenic archaea and other archaea who are unable to metabolize gly cerol as the primary carbon source. Distribution of A rchaeal Hpr Homologs and Putative G lycerol F acilitator Proteins Along with glpK and gpdA1B1C1, the glycerol metabolic operon of H. volcanii was also found to encode a homolog of the PTS general carrier protein Hpr ( ptsH2 ) as well as a putative glycerol facilitator protein ( glpX ) Interestingly, H. volcanii contains three

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172 homologs of Hpr, each of which clusters on the genome with additional PTS homol ogs (Figure 3 1 ) a nd has conserved histidine15 and serine46 residues (Figure 5 6 ). In B. subtilis, histidine 15 is the phosphorylation site of the phosphorelay reaction mediating sugar translocation and serine46 is the regulatory residue which serves as a corepressor for CcpA respectively In addition to Hpr, a putative glycerol facilitator ( glpX ) is also encoded within the glycerol metabolic regulon of H. volcanii. Although this putative glycerol facilitator does not exhibit sequence homology with characterized glycerol facilitator proteins, it is distributed throughout haloarchaea and often clusters chromosomally with glycer ol metabolic enzymes (Figure 32 ). GlpX may function as a glycerol facilitator based on genom ic clustering and the presence of seven transmembrane domains as predicted by the SOSUI server (Table 53, Figure 57 ) (Hirokawa et al., 1998) C onclusion T his study report s the organization and regulation of the primary g lycerol metabolic operon of H. volcanii. RT PCR analyses demonstrated that gpdA1B1C1 encoding G3PDH, glpK encoding GK glpF encoding a putative glycerol facilitator and ptsH2 encoding a homolog of bacterial PTS general carrier protein Hpr are transcription ally linked. Furthermore, transcription of this glycerol metabolic operon proceeds through a glycerol inducible gpdA1 promoter as evidenced by a t ranscriptional promoter reporter system The H. volcanii genome encodes two homologs of GpdA or GlpA, the cat alytic subunit of bacterial G3PDH P henotypic and biochemical characterization of mutants deficient in either chromosomal gpdA1 or pHV4carried gpdA2 has demonstrated that G3P is primarily catabolized through GpdA1 in H. volcanii. A lthough the gpdA1 mutant

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173 strain was unable to grow on glycerol as a sole carbon source, this phenotype could be restored by providing either gpdA1 or gpdA2 in trans suggesting that GpdA2 encodes a functional G3PDH isoform. Furthermore, G3 PDH activity is glycerol inducible, and this activity is reduced to basal levels in both glpK and gpdA1 mutant strains when grown in the presence of glycerol The dramatic loss of enzyme activity in gpdA1deficient cells could be restored to parental levels by providing either gpdA1 or gpdA 2 in trans confirming that the majority of G3PDH enzyme activity under glycerol rich conditions derives from GpdA1. These results also suggest that G3P is needed for induction of G3PDH based on the reduced level of G3PDH activity in the glpK mutant. The r esults presented here provide the first genetic and biochemical characterization of a G3PDH in haloarchaea. Overall, the results not only shed light on glycerol metabolism in H. volcanii but also add to the understanding of central metabolic pathways of h aloarchaea.

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174 Table 5 1 Transcription of a bgaH galactosidase reporter gene from the genomic region upstream of gpdA1, glpK and trpA Galactosidase Specific Activity (mUmg protein 1 ) a Carbon Source gpdA1 (310 bp) glpK (354 bp) trpA (321 bp) rrnA (551 bp) None (SD only) Gly 310 5 16 2 n.d. 260 10 8.1 0.1 Glu 38 0.001 22 0.7 n.d. 250 7 7.3 0.9 Gly Glu 280 8 14 2 n.d. 260 7 8.1 0.05 Suc Trp n.d. n.d. 1700 50 230 30 7.4 0.07 Suc n.d. n.d. 38 6 260 20 8.0 0.08 a galactosidase activities were determined from the lysate of cells carrying transcriptional fusion constructs of promoters grown to log phase in MM with Gly, Glu, Gly Glu, Suc Trp and Suc as indicated. Promoter fusions included the start codon and genomic region (indicated in bp) immediately upstream of each target gene. Experiments were performed in triplicate, and the means S D were calculated. Abbreviation: n.d. not determ ined

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175 Table 5 2. G3PDH activity in various glycerol m etabolic m utants. Glycerol 3 Phosphate Dehydrogenase Specific Activity (mUmg protein 1 ) a Carbon Source H26 gpdA1 gpdA2 glpK gpdA1 + gpdA1 gpdA1 + gpdA2 gpdA2 + gpdA2 glpK + glpK Gly 76 10 no growth 55 6 no growth 70 9 67 3 73 9 72 8 Glu 28 4 19 1 24 2 19 4 26 6 28 5 28 5 27 3 Gly Glu 67 10 18 1 47 6 21 1 68 9 67 4 72 7 67 5 a G3PDH activities were determined from the lysate of cells deficient in genes encoding glycerol metabolic enzymes GlpK, GpdA1 or G pdA2 as well as parent H26 and strains containing glpK gpdA1 or gpdA2 in trans Cells were grown to log phase in MM with Gly, Glu and Gly Glu as indicated. Experiments were performed in triplicate, and the means SD were calculated.

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176 Table 5 3. Predicted locations of transmembrane domains of H. volcanii GlpX. TM Helix Number N terminal Residue Primary Sequence C terminal Residue TM Helix Type Length (AA) 1 Ala 4 AFALQIPVIGVSTERFIVLLLAA Ala 26 1 23 2 Leu 26 LGALPSFIFTGFVVLLGETAGIV Val 58 1 23 3 Ile 123 ITYAFGTQPDILAVGGLFGVLGL Leu 145 2 23 4 Phe 157 PLDSVALSVMTTAFIARIAFGYP Phe 179 2 23 5 Ala 230 AGILGGWTWLITESFFLAYGISA Ala 252 1 23 6 Ala 282 AAPMVGGSEPLIIVAAAVGGLIG Gly 304 2 23 7 Met 328 MSITIYSLLIGVLFLLGVIPNSA Ala 350 1 23

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177 A B Figure 51. Genomic organization of the primary glycerol metabolic operon in H. volcanii A) Schematic representation of the chromosomally encoded gpdA1B1C1glpKXptsH2 glycerol metabolic operon and the related pHV4carried gpdA2B2C2 operon on the H. volcanii genome and location of annealing sites (represented as vertical lines) for ( q )RT PCR primers. B) RT PCR reveals genes within the gpdA1B1C1glpKXptsH2 are cotranscribed. Total RNA from parent H26 was extracted and reverse transcribed to generate cDNA, which was used as a template for PCR amplifying the 3 and 5 ends of: gpdA1 and g pdB1 (lane 1), gpdB1 and gpdC1 (lane 2), gpdC1 and glpK (lane 3), glpK and glpF (lane 4) and glpF and ptsH2 (lane 5), respectively. RNA which had not undergone reverse transcription was used as a negative control template for each PCR reaction (lanes 610) 100 bp Quick Load DNA markers and molecular sizes are indicated on left. 2000 bp gpdA1 gpdB1 gpdC1 glpK glpX ptsH2 RT PCR primers gpdC / glpK RT PCR primers gpdA1/ gpdB1 RT PCR primers gpdB1/ gpdC1 RT PCR primers glpK / glpX RT PCR primers glpX/ptsH2 2000 bp 2000 bp gpdA1 gpdB1 gpdC1 glpK glpX ptsH2 RT PCR primers gpdC / glpK RT PCR primers gpdA1/ gpdB1 RT PCR primers gpdB1/ gpdC1 RT PCR primers glpK / glpX RT PCR primers glpX/ptsH2 1 6 2 3 4 5 9 7 8 10 1 4 cDNA RNAkB 0.5 0.4 0.3 0.2 0.6 0.7 6 7 8 9 10 5 3 2 1 6 2 3 4 5 9 7 8 10 1 4 cDNA RNAkB 0.5 0.4 0.3 0.2 0.6 0.7 6 7 8 9 10 5 3 2

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178 AB Figure 5-2. PCR and Souther n blot confirmation of H. volcanii gpdA1 and gpdA2 mutant strains KS12 (H26 gpdA1 ) and KS11 (H26 gpdA2 ). (A) Confirmation of the gpdA1 mutant KS12 by PCR. Pr imer pairs that annealed outside the genomic region cloned in suicide plasmid pJAM2695 were used for confirmation of the gpdA1 gene knockout by PCR. Hi-Lo DNA markers and molecular masses are indicated on le ft. Genomic DNA from the following strains served as template: Lane 1. Pa rent strain H26, Lane 2. KS12 (H26 gpdA1 ). (B) Confirmation of the gpdA2 knockout KS11 by PCR. Primer pairs that annealed outside the genomic regi on cloned in suicide plasmid pJAM2697 were used for confirmation of the gpdA2 gene knockout by PCR. Hi-Lo DNA markers and molecular ma sses are indicated on left. Genomic DNA from the following strains served as template: Lane 1. Parent strain H26, Lane 2. KS11 (H26 gpdA2 ). (C) Southern blot confirmation of the gpdA1 knockout in strain KS12 (H26 gpdA1 ). Genomic DNA was digested with MluI and hybridized with a DIG-labeled probe specific for gpdA1 The following strains served as the source of genom ic DNA: Lane 1. Parent strain H26, Lane 2. KS12 (H26 gpdA1 ). (D) Southern blot confirmation of the gpdA2 knockout in strain KS11 (H26 gpdA2 ). Genomic DNA was digested with ClaI and BpuI and hybridized with a DIG-labeled probe specific for gpdA2 The following strains served as the source of genomic DNA: Lane 1. Parent strain H26, Lane 2. KS11 (H26 gpdA2 ).

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179 CD Figure 5-2. Continued

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180 Figure 5-3. Glycerol is primarily ca tabolized through GpdA1 and not GpdA2 in H. volcanii The parent strain H26 and GpdA 2-deficient strain KS10 exhibit similar growth rates and cell yields when grown in Gly MM. In contrast, GpdA1-deficient strain KS11 is unable to utilize glycerol as a sole carbon source. However, the KS11 phenotype could be restored to parental levels by providing either gpdA1 or gpdA2 in trans. Growth at 42C (200 RPM) was monitored by an increase in OD600, where 1 U was equivalent to approximately 109 CFU per ml for all strains. Experiments were performed in triplicate, and the means SD were calculated. Cell growth and carbon utilization levels are indicated. For simplicity, H26, KS10 and KS11 without vector control grown in Gly MM was not included in Figure 5-3, however, these strains exhibited identical growth rates and cell yields as those strains with vector control shown in Figure 5-3.

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181 Figure 54. Phylog enetic distribution and genomic organization of GpdA homologs in Archaea and E. coli Protein sequences were retrieved using the NCBI database and aligned using CLUSTAL W (Thompson et al., 1994) aft er N and C terminal trimming. Pairwise comparisons were performed between sequences and mean genetic distance was evaluated using pdistance (gaps were analyzed using pairwise deletion). The best neighborhoodjoining tree was then constructed using MEGA 4.0. Bootstrap values are indicated at the internal nodes and were obtained by performing 1,000 replicates. Biochemically characterized proteins are indicated by Genomic organizations of archaeal gpdA are indicated to the right. Two major gene neighborhood organizations for gpdA a re noted: i) Gene Organization I ( gpdA members belonging to this organization are highlighted in green) in which gpdA is encoded in the operon gpdABC and ii) Gene Organization II ( gpdA members belonging to this organization are highlighted in yellow) in which gpdA does not cluster on the genome with gpdBC Accession numbers for protein sequences are included in C hapter 2. Hmuk 2516 rrnAC1955 HQ2675A Huta 1471 Hlac 1123 OE2553R HVO 1538 (GpdA1) HborDRAFT 0007 Nmag 3076 HVO A0269 (GpdA2) VNG 1969G HQ1734A HborDRAFT 3506 Nmag 0933 Hmuk 2572 rrnAC0554 EcGlpA Tpen 1127 TNV0840 Ta0633 PTO1486 Cmaq 1799 Msed 1177 Saci 1118 Saci 2032 LS215 0342 SSO2526 Huta 0683 99 51 73 99 100 100 77 92 100 99 99 99 98 82 81 99 100 99 85 54 100 54 50 0.05gpdA gpdABC Gene organization 1 Gene organization 1 Gene organization 2 Gene organization 2 Euryarchaeota (Halobacteriaceae)Euryarchaeota (Thermoplasmata ) Crenarchaeota (Thermoprote i ) Hmuk 2516 rrnAC1955 HQ2675A Huta 1471 Hlac 1123 OE2553R HVO 1538 (GpdA1) HborDRAFT 0007 Nmag 3076 HVO A0269 (GpdA2) VNG 1969G HQ1734A HborDRAFT 3506 Nmag 0933 Hmuk 2572 rrnAC0554 EcGlpA Tpen 1127 TNV0840 Ta0633 PTO1486 Cmaq 1799 Msed 1177 Saci 1118 Saci 2032 LS215 0342 SSO2526 Huta 0683 99 51 73 99 100 100 77 92 100 99 99 99 98 82 81 99 100 99 85 54 100 54 50 0.05Hmuk 2516 rrnAC1955 HQ2675A Huta 1471 Hlac 1123 OE2553R HVO 1538 (GpdA1) HborDRAFT 0007 Nmag 3076 HVO A0269 (GpdA2) VNG 1969G HQ1734A HborDRAFT 3506 Nmag 0933 Hmuk 2572 rrnAC0554 EcGlpA Tpen 1127 TNV0840 Ta0633 PTO1486 Cmaq 1799 Msed 1177 Saci 1118 Saci 2032 LS215 0342 SSO2526 Huta 0683 99 51 73 99 100 100 77 92 100 99 99 99 98 82 81 99 100 99 85 54 100 54 50 0.05gpdA gpdABC Gene organization 1 Gene organization 1 Gene organization 2 Gene organization 2 Euryarchaeota (Halobacteriaceae)Euryarchaeota (Thermoplasmata ) Crenarchaeota (Thermoprote i )

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182 Figure 55 Many haloarchaeal genomes encode a full length GpdA1 and a C terminally truncated GpdA2. H. volcanii GpdA1 and GpdA2 were aligned with E. coli Ec GlpA along with additional haloarchaeal GpdA1 and GpdA2 proteins using CLUSTAL W (Thompson et al., 1994) The N terminal methionine was trimmed for alignment purposes. The conserved N terminal NAD+ binding domain typical of EcGlp A (GXGXXG) is indicated with above the binding motif as predicted by alignments with previously characterized GpdA proteins. The BFD like [2Fe 2S] motif (CXCX34CX4C) is indicated by placed above the sequence containing the binding motif. The [2Fe2S] coordinated cysteine residues within the binding motif are indicated as Accession numbers for protein sequences obtained are listed in C hapter 2. ****** 10 20 30 40 50 60 70 80 90 100 110 120 130 140 ....|....|....|....|....|....|....| ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| Ec GlpA KTRDSQ S SD V II IGGG A TG A GIARD C A L RG L R V I LVER H DI AT G A TGR N HGLLHSG A RYAV T D AE SA R ECI S EN QI L K R IA R HCVE P ---T N GLF I TL PED DLS F QATF I RA C E E A GI S A E A I DP QQ AR I I EP A V NP HVO_1538 (GpdA1) ---KK S PS V L VIGGGSTG T GIARDLAMRG L DVTLVE K G NLT H GTTGRMHGLLHSGGRYAVSDQ P SA K ECI E ENRVLR R IA G HCVE M ---TGGLFVQRPEDS DE YFE K KL E GC R ECGIP A EVLS A EEAR EI EP Y LA K HVO_A0269 (GpdA2) -----S YS V V VIGGG A TG T G T ARDLAMRG F DVTLVERG NLT E GTTGR T HG H LHSG A RYAVSD K E SA V D C M R E NRVL HR IA G HC I E D ---TGGLFVQ LEG DS DD YFE R KL A GC A EC D IP T EV I SGEEAR RR EP Y L TD Huta_1474 ---TS S PS V L VIGGGSTG V GIARD A AMRG F DVTLVE K G NLT H GTTGRMHGLLHSGGRYAVSDQ A SA R ECI D EN M I LR D IA T HCVE D ---TGGLFV K RPED TEE Y YQE KL D G LR EC S IP ATE L T A EEAR RK EP F LA R Hmuk_2516 ---AS T PR IV V V G GGSTG A G V ARDLAMRG A DVTLVE Q G NLT H GTTGRMHGLLHSGGRYAVSDQ K SA R ECI E ENRVLR D IA S HCVE M ---TGG M FV K RPEDS E S YFE E KL Q GC A D C D IP A EV I SGE R AR E R EP H LA T HQ1734A ---VSKPH IV VIGGGSTG T GI V RDLAMRG VE VTL L E Q G NLT H GTTGRMHGLLHSGGRYAV A DQ A SA K EC M L ENRVL QD IA T HCVE M ---TGGLFV K RPEDS EE YF QK KL Q GC H EC D IP A EVL T A EEAR A V EP H LA G rrnAC0554 ---GP T PH I A VIGGGSTG A GIARDLAMRG L DVTLVE Q G NLT H GTTGRMHGLLHSGGRYAVSDQ A SA T ECI E ENRVLR D IA S HCVE M ---TGGLFV K RPEDS EE YF QK KL N GC E ECGIP A EV V SGEEAR A M EP H LA K VNG1969G ---AQEPH V V V V GGGSTG A G V ARDLAMRG L G VTLVE Q G NLT H GTTGRMHG LLHSGGRYAVSDQ A SA R ECI I ENRVLR E IA T HCV DE ---TGGLFV K RPEDS E Q YF QE KL A GC EA C D IP T K VLSG A EAR QL EP H LA D Hlac_1123 ---DQQVD V V V V GGGSTG C G V V RDLA R RG V D AV LVE K G NLT H GTTGRMHGLLHSGGRYAVSDQ K SA R ECI E ENRVLR D IA G HCVE E ---TGGLFV K RPEDS EE YF QE KL E GC RA C D IP V E MI D GEEAR RR EP Y LA R Nmag_093 3 ---AHDTA V L V L GGGSTG C GIARDLAMRG V DVTLVERG T LT D GTTGRMHGLLHSGGRYAVSDQ A SA T ECI E EN E VLR E IA S HCVE E ---TGGLFVQRPEDS DE YF R E KL E GC R D C D IP AT VLSG R EAR EV EP Y LA D HborDRAFT_0007 ---TE T PS V L VIGGGSTG C GI V RDLAMRG L DVTLVE K G NLT H GTTGRMHGLLHSGGRYAVSDQ P SA T ECI E ENRVLR R IA S HCVE M --TGGLFVQRPEDS DD YFE E KL E GC R ECGIP A EVLS A EEAR EM EP Y LA E Hmuk_2572 ---TR S TT V L VIGGG A TG V GIARDLA L RG V DVTL A ERG G L AS GT S GR S HGLLHSG A RYA E A D PTG A E ECI E ENR I LR S IA GA C I R D ---TGGLFVQ L P A D DP D YFE R K RAA C E E L GI D T E L L DAT EAR DL V P D L S E HQ2675A ---TIQTD V A I IGGG A TG A GIARDLA L RG V DV S L F ERG G L GG GT S GR S HGLLHSGGRYA E SD PIG A E ECI R E S R I LR D IA EG C I R E ---TGGLFVQ LES D DPA YFE E KL EA C N E LD IP T E T I S A EEA H E RV P G L TE rrnAC1955 ---AIETD V L V V GGG A TG A G V ARDLA L RG I DVTLVER D G LT S GT S GR S HGLLHSG A RYA E A D R VG A E ECI T ENR I L KE IA GA C I R D ---TGGLFVQ LAG D DP D YFE T K RAA C E E I GIP V E T L DA D A AR E RV P D LA S OE2553R ---------L V V GGG A TG V G V ARDLA L RG V DVTLV D RG G L GS GTTGR S HGLLHSG A RYA D S TPAD A R ECI R EN E VLR D IA GA C I R D ---T D G F FVQ HAR D DPA YF D T K V AA C R D AD IP VS V I D G AT AR AR EP A L TP Nmag_3076 ---PI T TD V L VIGGG A TG T GIARDL T L RG V DVTL A ERG G L S A GTTGR S HGLLHSG A RYA E A D AEG A L EC LE EN QI LR E IA GE C I R E ---T R GLFVQ VAG D DSA YF DE K RAA C E E L GIP V EV V D G DD AR E AVSG LA A HborDRAFT_3506 ---ADDTD V V VIGGG A TG A GI V RDLA L RG V DVTLVERG G L S A GT S GR S HGLLHSG A RYA E A D E VG A R ECI A ENR I LR D IA GE CV R D ---TGGLF L Q LA ED DP E YFE A K RTA C E D I GIP I E T LSG D E V R NEISG L S E 150 160 170 180 190 200 210 220 230 240 250 260 270 280 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| ....|....|....|....|....|....|....|....|....|....| Ec GlpA A L IG A V K VPDG T VDPFRL TA AN ML DA K EHGA V I L T AH E VT G L I R E GAT V C GV R --------VR N HLT G ET Q A L H A PV VVNA A G I W GQH I A E Y A D L R I R M F P A KG ---S L L I M D H R INQH V I NRCR KPS DADI L VP G HVO_1538 (GpdA1) D IK RAI K VPDG AVDPFRL C VANAA S A V EHG ARIETH SE VTD V LVE G G E V V GVEV T H QTGTGPY VH GEP G EV E E I R AD Y VVNATGAWAG Q IG D F AGV N VEVRPSKG ---VMT I MN T RQVDTV V NRCRPKGDADI I VPH HVO_A0269 (GpdA2) A VERAI W VPDG AVDPFRL C VANAA S A V EHGARIETHA E V V DL V VE G G R V A GVEV KRQ GPNHHSEGAA G DT E TFE AD Y VV S ATGAWAG Q L AA MAGVD L E M AI SKG ---A M V V T N V RQ L DTV I NRC L PKG E G D T II PH Huta_1474 D IDK AI E VPD A A I DPFRL C VANA IS A AN HGARIET F SE VTDLLVE D G E I V G A EV T H ESGEGNR VH GVE G SV E E I RV D HI VNA A GAW T G E I AA MA D I D VEVRPSKG ---VMT I MN V RQVDTV I NRC Q PKGDADIVVPH Hmuk_2516 DV DK AI S VPDG AVDPFRLVVANAA S A Q EHGARIETH SK VTDLLVE S G E I V GVEV E H GAKSGDR VH GVQ SGR E T I R AD H VVNATGAWAG R IG D MA D L D V A VRPSKG ---VMT I MN V RQVDTV I NRCRPKGDADI I VPH HQ1734A D IDK AI S VPDG A I DPFRLVVANAADA K EH D ARIETH S T VTDLL I E D DA V V GVEV K H DSGTGER VH GMT G VT E R I Y AD H V I NATGAWAG H IG E MAGVD I A VRPSKG ---VMT V MN T RQVDTV I NRCRPKGDADIVVPH rrnAC0554 D IDK AI S VP DG A I DPFRLVVANAA S A Q EHGARIETH T K VTDLLVE S G E V V G I EV E H DSGPGKR VH GTE G GT E Q I R AD Y VVNATGAWAG R IG D MAG L D I EVRPSKG ---VMT I MN I RQVDTV I NRCRPKGDADIVVPH VNG1969G DVE K AI W VPD A A I DPFRLVVANAA S A Q EHGARIETH S T VTD VV VE D G A V V GVEV E H ATGAGKR VH GTE G GT E V I R AD H VVNA A GAWAG R L G E MAGVD VEVRPSKG ---VMT V MN T RQVDTV V NRCRPKGDADIVVPH Hlac_1123 DVE K AI A L PD A AVDPFRL C VANAADA R EHGARIETHA P VTD V LVE D G E I V GVE I E H ETGPGKR VH REP G TT E E I R A R H VVNATGAWAG N V G E MAGVD VEVRPSKG ---VMT V MN T RQVDTV I NRCRPKGDADI I VPH Nmag_0933 DV S RAI Q VPDG AVDPFRL C VANA L DA ER HGAR V ETHA E V I DLL R D G DD I Y GVEV R H DSGPGKRT H KAP G TT E E I T A EY VVNATGAWAG Q IG A MA D L D V A VRPSKG ---VMT I MN V RQVDTV I NRCRPKGDADI I VPH HborDRAFT_0007 D IK RAI R VPDG A I DPFRL C VANAADA I D HGAR V ETHA E V V D V L I E D G E V V GVEV E H GEADLGIAEGEP G TR E K L Y AD Y VVNATGAWAG Q IG D MAGVD V A VRPSKG ---VMT I MN V RQVDTV V NRCRPKGDADIVVPH Hmu k_2572 A VERA FR VPD A V I Y P S RLV A ANAADA A D Q GA S I HP N A P L ES V T VE D G R V T -----------SA H LGGTVS E T I EP D YL VNA A GAWAG E V AA MAG L D VE M A PS R G ---VM VA VD YD R V G S V L NRCR DPD D G DIVVPH HQ2675A DVERA FT VPDG V I Y P S RLVVANAADA R EHGA S I Y TH TP V ES V TTA N G E I T -----------E L H VGGEIN D T V E A TQ I VNATG AWA EV M G E DL GVD V S MQ P TR G ---VM IS V EYDG L D P V L NRCR DPD D G DI I VPH rrnAC1955 DVERA FE VPD A V I Y P S RLV A ANAADA RD HGA T I HP HA P V E D V LVE D G H V A -----------G V QVGGTVE D T I E AD Y VVNATGAWAG EFAA MA D L D VE MQ P TR G ---VM VS V EYDD L GP V L NRCR DPD D G DIVVPH OE2553R AA ER VAV VPDG V V L P S RLV A A T AA DA RD HGA T I RL H EP I T A L TT D A G R V T -----------GART -ADGTT L S A T H VVNA A GAWAG D L AAT AGVD V DM H P A S G ---VM VT V ETP E T DTV F NRCRP AA D G DIVVPH Nmag_3076 A VERA M W VPD A V V L P S RLV A A T AADA R EHGARI L THA P V ES MVL E GER I D -----------S V SLGGAAETV V EP E F VVNATG PH AG A V AA MAGV T VE M RP TR G ---VM VS VD HDG LE P V L NRCR DPA D G DI I VPH HborDRAFT_3506 A VERA M K VPD A V V S P S RLV A ANAADA RD HGA T I L T N A P V E DL H VE D G R I T -----------G V KVGGSVG E T I A A KQ VVNA A GAWA ED V G R MAGV E V A M RP T KG ---VM VS V QYDG L GP V L NR A R DPA D G DIVVPH 290 300 310 320 330 340 350 360 370 380 390 400 410 420 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| Ec Glp A D T I SLI GTT SLR I D YN E I DD NRVTAE EVD ILL R E GE K L A P VMA KT R I L R AY S GVRPL VASD D DP S -GR NVS R GIV LLDHAERDGL D G FI T I T GGKL M TYR L MAE WA T D A VC R K LG NTRP C T TAD LA LPGS Q E P A E V T HVO_1538 (GpdA1) ET T C ILGTTDEEV E -DP E DYPEE G WEVD L MI E T LSELVP M L A DAR T IRSFWGVRPLYEPP GT G TE DPTDITR E FFLLDHA D RD D L P GMTSIVGGKLTTYR M MAE Q IS DHVC EK LGV D A E CRTAD E PLPGS E D FTV L R D HVO_A0269 (GpdA2) ET T V L LG AN D D P VD -DPDDYPEE Q WEVD M MI D IA SE M VP VVA DAR M IR AY WGVRPLY D P NPKS T T DP G D V TR NY F V LDHAERDG V A G FA S V VGGKLTTYR E MAE S VS DHVC E V LGV E EP CRT D E V PLPGS A DP S A LD E Huta_1474 ET T A ILGTTDEEV G -DP E DYPEE E W EVD L MI E EL AK LVP I L D DAR T IRSFWGVRPLYEPP D T G TD DPTDITR E FFLLDH D ERD A L P GMT T IVGGKLTTYR L MAE Q L V DH L A EK FD V D TT CRTA EE PLPGS E D EGV I D D Hmuk_2516 ET T A ILGTTDEEV E -DP E DYPEE Q WEVD Q MI E T LSELVP M L A DAR S IRSFWGVRPLYEPP E V G SE DPTDITR DY FLLDH E ERDGL P GMT T IVGGK F TTYR M M G E E IA DHV VS K F G MD A D CRTA D V PLPGS EE F S V L R D HQ1734A ET T A ILGTTD V EVD -DP E DYPE KQ WEVD L MI D T LSELVP M L T DAR T IRSFWGVRPLYEPP E V G SD DPTDITR D FFLLDH Q ERD N L H G L TSIVGGKLTTYR M MAE Q IT DH I C ER F G IE EP CRTAD E PLPGS N D IRV I D D rrnAC0554 ET T A ILGTTDEEV E -DP E DYPEE Q WEVD M MI D T LSEL I P M L D E AR T IRSFWGVRPLYEPP D V G SD DPTDITR D FFLLDH D ERD D L P GMTSIVGGK F TTYR M M G E Q IA DHVC G K F G ID A D CRTAD E PLPGS E D F S V L R D VNG1969G ET T C ILGTTDEEV A -DP E DYPE QQ WEVD MV I D EL A EL I P M L D DAR TV RSFWGVRPLYEPP D VD SD DPTDITR DY FLL A H D D RD D L P GMT T IVGGKLTTYR M MAE E IT DHVC ER LGV T A D CRTAD VA LPGS A D NTA LD D Hlac_1123 ET AC ILGTTDEEVD -DP E DYPEE E WEVD L MI E T LSELVP M L K DAR TL RSFWGVRPLYEPP GT G TE DPTDITR DY FLLDH G D RD D L P GMT T IVGGKLTTYR M MAE S IS DHVC D A LG H D A T C D TAD A PLPGS EN P A R M D E Nmag_0933 ET T A ILGTTDEEV S -DPDDYPEE Q WEVD M MI D T L T ELVP I L E E AR T IRSFWGVRPLYEPP GT G T Q D S TDITR D FFLLDH D ERDG V S GM S SIVGGK F TTYR A MAE E IS DHVC A K LGV S A A C A TAD E PLPGS E D VRV L EE HborDRAFT_0007 ET T C ILGTTDEEVD -DP E DYPEE G WEVD L MI D T LSELVP M L SE AR T IRSFWGVRPLYEPP GT G TE DPTDITR D FFLLDHA D RD D L P GM S SIVGGKLTTYR M MAE K IS DHVC EKV GV E A E CRTA EE PLPGS E D F S V L R D Hmuk_2572 E R Q AV LGTT SVPAS -DPDDY ERAD WEV E TC I E E C A T ML P P V A DA EIE R TW WGVRPLY A P D E AE S E RRG I S R G F LR LDHAE DG V ENAV S V VGGKLTTYR Q MAE AT T D L L C DR LGV TTD C T TA EE PLPG AD DP GT LD E HQ2675A D G EV V LGTT SVA V E -DPDDY E E AD WEV E RS VD E C A EL I P D I T DA PE V R TW WGVRPLYEP D E DER D D GRG I S R G FF VIN HAER N EPAN L I S V VGGKLTTYR Q MAE AA S D Q I C A R LGV D A S C E T D E R PL HA S D DP A Q LD A rrnAC1955 E S E AV LGTT SVP V R -DPD E Y ET E Q WEV E ES I E E C A A ML P S VA DA PE V R TW WGVRPLY A P D E AE -RGRG I S R G FFLLDHA D DG V DN M A SIVGGKLTTYR Q MAE AT A D L VC D D LGV D A A CRTAD Q T LP SV D DP A Q LD A OE2553R G S T AV LGTT SV D VD -D A D EF ATPDAA V E R V I D E C S A L L P AA S DA PIRT TY WGVRPLY S P S D Y G D D ARA I S R G F YV LDH A D RD DTA G L T TV VGGKLTT H R L MAE AT A D L V A DR LGV TEPS RTA NE PLPG H D DP ER LD A Nmag_3076 D D EV V LGTT SVP VD -D L D EF ER DQ Q EV E RT I R E C A T ML P A VA DA PQ V R TW WG I RPLYEP D E AAR GGRG I S R G F VQ LDHAE DG V ANFV SIVGGKLTTYR R MAE S VS D L V A DR LGV GNSST TA GRE L I G A SSA S E LD A HborDRAFT_3506 D G EV V LGTT SI EVD -DPDD YPE D AQ EV E R M VE E C S KML P PAR E A ER V R TW WGVRPLY A P D E DSR E GARG I S R G FF C V DH G D DG V ENF TSIVGGKLTTYR Q MAE AT T D R VC EK LGV A A D CRTA A K R LPG AD DP EQ LD A 430 440 450 460 470 480 490 500 510 520 530 540 550 560 ....|....|....|....|....|.... |....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| Ec GlpA LRKVIS L P A P L RGS A V Y R H G D R TPAW L SEGRLHRSL V C E CE A VT AG E VQ Y A V EN LNVNS L LD L R R RTR VG MG T CQG EL C AC R A A GL L QRFNVTTSAQSIEQLSTFLN ERWKG V Q PIA WG DA L R ES EF TRW VY QGLCGL E HVO_1538 (GpdA1) Y MD D FGLRSPI G RRS AQ RLGSR AD E VL NSV D PNP V V C E CE A VTRAE IQ DA L DT A G T DLN S VRI Q TRA S MGNCQG AI C C HR M A N EL APE YD E KTV R A S L D D L Y QERWKG E RHA M WG T QLSQ TA L K H M LHA A T M NRD HVO_A0269 (GpdA2) Y MDEF D LRSPI A RRS GQ RLG D R AP E VL DID E PNP T L C E CE A VTRAE VR DAI DQ V G A DLN G VR L RT RA S MGNCQGGFC S HR L GA EL YPD HGAEVA R D A V D E L Y QERWKG Q RHALWG E QLSQA M L NAM LHA T T M N H D Huta_1474 A MD D FGLRSPI A K RS AE RLGSR TQ E VL DTN E PNP V V C G CE A VTRAE V N DAI EQ S G T DLN A VRIRTRA G MGNCQGGFC S HR L A N EL HPD FD E GRA R A A L D E L Y QERWKG Q RHALWG R QLSQA MI T Y T M HA L T M NRD Hmuk_2516 Y MDEFGLR SPI G RRSV E RLGSR AE E VL GTG E PNP V L C N CE G VTRAE VR DAI SQ A G A DLN GT RIRTRA S MGNCQGGFC I HR L A G EL HPE FD E ATV R D S W D D L L QERWKG Q RHALWG N QL Q QA MI N Y A LHA T T Q NRD HQ1734A Y MDEFGLRSPI G RRS AD RLGSR VD E VL DTD E PNP T I C E CE G VTRAE VQ DAI SQ S G S DLN A VRIRTRA T MGNCQGG I C S HR L A N EL HTT YD E SVV ID A W NEI L QERWKG Q RHALWG Q QLSQA M L N Y A LHA T T Q NRD rrnAC0554 Y MDEFGLRSPI G RRSV E RLGSR AD D VL KTDG PNP T V C A CE G VTRAE IQ DAI GG S G S DLN A VRIRTRA S MGNCQGGFC S HR M A S EL HSE YD E PVV R E A W D E L L QERWKG Q RHALWG E QLSQA A L N Y A LHA T T Q NRD VNG1969G Y M T EFGLRSP V A Q RSV D RLGSR AGS VL DTD D PNP T I C A CE A VTRA E IQ DAI EQ S G T DLN A VRIRTRA T MGNCQGGFC A HR L A N EL HTAGYD D ATV R A S W D E L LA ERW R G Q RHALWG D QLSQA M L S Y A LHA T T Q NR G Hlac_1123 L M E EFGLRSP V A RRS GQ RLGSR AA D VL DEY D PNP V V C E CE G VTRAE VQ DAI GE A G S DLN A VRIRTRA S MGNCQGGFC T HR I A A EL AQE YP E PVV R D A E D E L Y QERWKG Q RHALWG M QLSQA M L N H L LHA T T M NRD Nm ag_0933 G MD D FGLRSPI A RRS NQ RLGSR AA D VL ETN E PNP V V C E CE G VTRAE IQ DAI DQ S G S DLN A VRIRTRA S MGNCQGGFC CQN M A N EL HPR YD E ETV R E A L D E L F QERWKG E RHALWG E QLSQA M L N Y A LHA T T M NRD HborDRAFT_0007 W MDEFG I RSPI G RRS AQ RLGSR TD D VL GEW D G PNP V V C E CE G VTRAE I H DA M NH A G T DLN A VRIRTRA S MGNCQG A FC S HR M A N EL VED HS E LVV R D S L D E L Y QERWKG E RHALWG R QLSQA M L K H M LHA T T M NRD Hmuk_2572 FV AR F DG Q G P TDSDV V ASR ------------------------------------------------------------------------------------------------------------------------HQ2675A L I DEF DAV N P TD R DI V TTATPAES AAD ----------------------------------------------------------------------------------------------------------------rrnAC1955 L V A EF DG Q G P TDEDV V GSA -----------------------------------------------------------------------------------------------------------------------OE2553R L V D T F DAA SP ADAD A R ---------------------------------------------------------------------------------------------------------------------------Nmag_3076 FV DEF DG R G P TDADL V D Q D G ----------------------------------------------------------------------------------------------------------------------HborDRAFT_3506 YV T EFG GAG P TDEDV V RG -------------------------------------------------------------------------------------------------------------------------570 580 590 600 610 ....|....|....|....|....|....|....|....|....|....|....|.. Ec GlpA K E QKDAL -------------------------------------------------HVO_1538 (GpdA1) E DPA AADAD I D F AA FD D G --VASGGAVAD GG RERAADR --ADDDALGGADGDN HVO_A0269 (GpdA2) A N HVAGDEN IEY RA FD G G R ---------------TAVPEGSHGD -----------Huta_1474 R DPA GG GS L D F GA FD S G PSNTPATDDGQPADGPPGVATDGGRGDGRGD -------Hmuk_2516 H DPA T -QP V D F AA FD S G PG ----SDRRLGSDTVGEAADGD --------------HQ 1734A H DPA ADSS V D F TT FD S G E -----MQSDSGTTAVSVATDGGTQDGDRV -------rrnAC0554 Q DPA DG EP V D F SA FD S G RG ----QTGGS VDSADVAADGGTNGY ----------VNG1969G H DPA DADTE I D F TA FD A G ----------------PLAADGGPEGAHGN -------Hlac_1123 G DPA SLNSE V D F GA FD A G E -GGSEADAIDGGDAGTAATDGGIDGDR ---------Nmag_0933 N DPA RESTQ L D Y AQ FD G G ---A ---------------------------------HborDRAFT_0007 G DPA ASDAD V D F GA FD A G TG AAAGETSGTGGDTDRAATDGGAPTDGSLAEVNDADS Hmuk_2572 -------------------------------------------------------HQ2675A --------------------------------------------------------rrnAC1955 --------------------------------------------------------OE2553R --------------------------------------------------------Nmag_3076 --------------------------------------------------------HborDRAFT_3506 --------------------------------------------------------

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183 Figure 5-6. B. subtilis Hpr phosphorylative residues hi stidine-15 and serine-46 are conserved in haloarchaeal Hpr homologs Phosphorylative residues histidine15 and serine-46 are highlighted and additiona lly are indicated by *. Protein sequences were retrieved using t he NCBI database and aligned using CLUSTAL W (Thompson et al., 1994) a fter Nand C-terminal trimming. Accession numbers for protein sequences are as follows: B. subtilis Hpr (CA31317.1); H. jeotgali HacjB3_10165 (YP_003737208); H. lacusprofundi Hlac_1462 (YP_002566120.1); H. volcanii HVO_2101 (YP_003536125.1), HVO_1497 (YP_003535546.1), HV O_1543 (YP_003535590.1); H. marismortui pNG7389 (YP_134785.1); H. walsbyi HQ2708A (YP_658421.1); H. turkmenica Htur_2757 (YP_003404303.1); T. pendens Tpen_1091 (YP_920493.1); Aspergillus flavis AFLA_023720 (XP_ 002373883.1); and E. coli Hpr (NP_416910.1). 10 20 30 40 50 60 70 80 90 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|.... B. subtilis Hpr ---AQK T FK V TADS G I HARPATVLV Q TA SKY DADV NLEYNGK----T V NLK S IMGV MS LG IAKG AEITISA S G A D ENDAL NA LEETMKSEGLGE HacjB3_10165 SETH ERTVEI VPE A GLHARPA AT FV E TANDHDA T V KI G S A EDGDEDLID A G SM I AVT S LGV KQ GE HLRI VAEGE DAE E ALDA LERV L S TPEGES Hlac_1462 ---ERTVTVVPE A GLHARPAS KL V Q TANR FDADVSI G R A NDGDDGL V R A D SMLS V SG L N V GH GE SVRV VAEGAE A AD ALDAVCDLL TSEVEEG HVO_1497 (PtsH1) ---ERTVTVVPE D GLHARPAS K FV E TANK FDADVQL G R ADE--DDLV P A A SMLAVTG LGV GHDE SVRL VAEGD DAE A ALDA LEDI L S TPEAKQ HVO_1543 (PtsH2) ---ER I VTVVPKD GLHARPAS Q FV E TANS FDAD IQL G R ADE--DDLV P A A SMLAVTG LGV GH GE EIRL VAD G D DAE A ALDA LEAV L S TPEAGD HVO_2101 (PtsH3) -AAKSA TVV V EH E T GLHARPAS M FV Q TA SK F ET D ISVRK A GG--ETE V D A K S SI AVLS LGV GPDE EIVITA D G N D G E Q A VER L VELVRN-DFDL p NG7389 --TV ERTVT I VPE A GLHARPAS A FV QAVN DHE A E V SA G RP D D---DLV Q A A SM I A I T S LGV GQ G DDIKL VAD G S DAE SV LDE LERI L T TPEAEL HQ2708A ---P ER V VTVVPED GLHARPAS K FV E TANE FDAE V QV G HI D ---DNPV N A A SMLAVTG L A V TC G DDVQVRAEGP DADA ALDE L TRIL S TPEEDL Htur_2757 ---ERTVTVVPE D GLHARPA AK FV E TA TE FDADVRVAP ADG--DDPV D A A SMLAVTS LGV AS GE DVRLIAEGD DAE A ALDD LEEL L A TPETES Tpen_1091 ---KTLK V K V SNRS GLHARPA AV FV Q TA RK F KSRITVRKL D K----AADSKNI L QLLALGV DM G DEIEI VAEGP D E E E A IAE L GKLL TEVLPSI A FLA_023720 ---FSQEE T ITNPN G M H T R A A AQ FV KE AN I F TSNV TVSDGRK----T V NGKKLFPLQT L ALSQG NTLTITAEGE D EQNA VEH L CKLL AELY--*

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184 Figure 5 7 GlpX is a p utative glycero l facilitator protein based on the presence of predicted transmembrane domains. The SOSUI server predicts seven transmembrane helices for H. volcanii GlpX by examination of the amphilicity of the primary amino acid sequence. Membrane

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185 CHAPTER 6 G lp R REPRESSES FRUCTOSE AND GLUCOSE METABOLI C ENZYMES AT THE LEVEL OF TRANSCRIPTI ON IN THE HALOARCHAE ON H aloferax volcanii Introduction The archaeal basal tr anscriptional machinery closely resembles the euk aryotic RNA polymerase (RNAP) II apparatus. Along with a multi subunit RNAP (Zillig et al., 1979) archaea encode two basal transcription factors, TBP and TFB which are homologues of the euk aryotic TBP and general transcription factor TFIIB, respectively (Soppa, 1999; Bell and Jackson, 2001) Although archaeal transcriptional components are fundamentally eukaryotic like in nature (Reeve et al., 1997) the majority of candidate transcription al regulators are homologous to bacterial activators and repressors (Kyrpides and Ouzounis, 1999; Aravind and Koonin, 19 99) Only a few archaeal candidate regulators resemble eukaryotic genespecific transcription factors, one of the best characterized of which is GvpE, an activator of gas vesicle biosynthesis in haloarchaea which resembles the eukaryotic basic leucine z ipper proteins (Offner and Pfeifer, 1995; Krger et al., 1998) While b ioinformatics analysis predicts many candidate archaeal regulators, only a limited number have been characterized at the molecular level, most of which are from hyperthermophiles (Lee et al., 2003; Xie and Reeve, 2005; Bell, 2005; Kanai et al., 2007; Fiorentino et al., 2007; Keese et al., 2010) Molecular data pertaining to haloarchaeal transcriptional regulation, specifically regulators of carbon utilization, are severely limited. Only a few global regulators, namely transcription factors (Facciotti et al., 2007; Coker and DasSarma, 2007; Schmid et al., 2009) have been implicated in regulating sugar metabolism in haloarchaea. Specifically, in H salinarum pairs of general transcription factors TBP and TFB control gene clusters (Facciotti et al., 2007; Coker and DasSarma, 2007) and tr anscription

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186 factor TrmB regulates diverse metabolic pathways in response to nutrient limitation (Schmid et al., 2009) N o transcriptional regulators of sugar metabolism have been characterized in the model haloarchaeon H volcanii to date, although homologs of transcription factors can be predicted based on primary sequence. Furthermore, DeoR/GlpRtype regulators have yet to be characterized in archaea. In this study, biochemical and genetic approaches were used to character ize GlpR, a putative transcriptional regulator of glycerol and/or sugar metabolism in H volcanii This study present s evid ence that GlpR is not only autoregulatory, but also regulates transcription of the downstream pfkB gene encoding PFK as well as a dis tant chromosomal KDGK gene kdgK1. Taken together, these results provide the first example of a DeoR/GlpR type repressor protein that controls key enzymes of sugar metabolism in haloarchaea, and allow valuable insight into haloarchaeal metabolic transcriptional regulation. Results and Discussion Identification of GlpR as a Putative R epressor of Metabolic Enzymes In order to provide insight into the regulation of haloarchaeal central metabolism, t he H. volcanii genome (Hartman et al., 2010) was searched for ORFs encoding proteins with DNA binding domains that clustered near sugar metabolic operons. One such ORF (HVO_1501; designated glpR ), when searched against the NCBI protein database, clustered with the DeoR/GlpR family of transcriptional regulators of s ugar metabolism (COG1349) (Figure 61A) The DeoR/GlpR protein family is widespread among bacteria and its members often serve as transcriptional repressors (Munch Petersen and Jensen, 1990; Weissenborn et al., 1992; Zeng and Saxild, 1999; Ray and Larson, 2004; Barrire et al., 2005; Haghjoo and Galn, 2007) or activators (Zhu and

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187 Lin, 1986; Gaurivaud et al., 2001) of either sugar or nucleoside metabolism. DeoR/GlpRtype repressors are comprised of approximately 250 amino acids, possess a helix turn helix DNA binding motif near the N terminus and generally bind a sugar phosphate effector molecule of the relevant metabolic pathway in the C terminal portion of the protein which can also contain oligomerization domains (Zeng et al., 1996) In H. volcanii as wel l as in some haloarchaea and many G ram positive bacteria, glpR clusters on the genome with pfkB encoding PFK (Figure 61B ), a key enzyme of fructose metabolism in haloarchaea such as H. volcanii (Johnsen et al., 2001; Falb et al., 2008) Dendrogram analysis reveal ed that H. volcanii GlpR clusters to the DeoR/GlpR family of transcriptional regulators with closest relationship to uncharacterized proteins of haloarchaea and Firmicute s (Figure 6 1B and Figure 61C). This result suggested that GlpR may be involved in re gulating sugar metaboli c enzymes at the level of transcription in H. volcanii and was thus targeted for further analyses. Transcr ipts E ncoding GlpR and PFK are under the Control of a Common P romoter and are Reduced in the Absence of Fructose In haloarchaea PFK is involved in the metabolism of fructose through a modified Embden Meyerhof Parnas (EMP) pathway (Falb et al., 2008) Due to the close proximity of glpR and pfkB on the chromosome (4 bp overlap in coding sequence) (Fig ure 62A ) it was investigated whether these genes were cotranscribed in an operon. RT PCR was performed using primers designed to amplify a por tion of each coding region (Figure 62 A). A single PCR product of expected size (0.2 kb) was detec ted using cDNA as a template for PCR (Figure 62 B). The primer specificity was confirmed by DNA sequencing. No product was det ected in the negative control

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188 reaction containing RNA as a template for PCR (Figure 6 2 B). Thus, glpR and pfkB are linked at the level of transcription. Since glpR and pfkB are cotranscribed and PFK activity is largely fructoseinducible and to some extent glucoseinducible in haloarchaea (Johnsen et al., 2001) it was investigated whether this regulation was controlled at the level of pfkB transcript and whether glpR was also i nduced by sugars. RNA was extracted from parent H26 grown in minimal medium supplemented with different carbon sources including fructose, glucose, glycerol and various combinations thereof, and was subjected to qRT PCR using primers specific for the 200bp coding region of glpR and pfkB (Table 28 ). Q uantification was performed for each transcript The internal standard, ribosomal protein L10 gene ( ribL) transcript, was chosen based on previous use (Brenneis et al., 2007) and confirmation by qRT PCR that t he N fold induction of transcripts specific for ribL was close to 1.0 when parent H26 was grown in minimal medium Using this approach, transcripts were found to be upregulated for both glpR (> 20 7fold) a nd pfkB (> 10 3 fold) in the presence of fruc tose regardless of glycerol supplementation compar ed to glycerol alone (Figure 62 C). Transcripts for glpR were also upregulated (> 10 4 fold ) during growth on glucose regardless of glycerol supplementation compared to glycerol alone (Figure 6 2 C). Thus the data reveal that glpR and pfkB are co transcribed from a common promoter and that the transcript levels of this operon are in creased by fructose and, to a lesser extent, by glucose These results are consistent with the observed sugar dependent alterations in PFK activity for H saccharolyticus (Johnsen et al., 2001) in which the addition of fructose to peptide rich media stimulated

PAGE 189

189 the level of PFK activity, and suggest that PFK is regulated at the level of transcription in haloarchaea. GlpR R epresses PFK T ranscription in the Absence of Fructose To analyze the role of GlpR in regulating sugar metabolism, the glpR gene (HVO_1501) was targeted for knockout in H. volcanii to generate glpR mutant strain KS8. Gene deletion was confirmed by PCR, Southern blotti ng and sequencing analysis (Figure 6 3 ). qRT PCRs w ere used to compare transcript levels of KS8 to parent str ain H26 on various carbon sources Using this approach, a pfkB specific transcript was found to be significantly increased by the glpR knockout (10 to 12fold) compared to parent H26 when cells were grown in the absence of fructose (in Gly MM) (Figure 6 2D ). The abundance of pfkB specific transcript in the presence of glycerol was restored to wildtype levels at least in part by providing a copy of glpR in trans thus, ruling out polar effects of the markerless deletion of glpR on the glpR pfkB operon (Figure 62D ). In contrast to growth on glycerol alone, the glpR knockout had little if any impact on pfkB transcript levels when cells were grown in the presence of fructose w ith or without glycerol (Figure 62D ). Transcript levels remained high f or pfkB in fructose containing media f or all strains analyzed (Figure 62D ) and were not significantly altered by the glpR mutation (Figure 62D ). Thus, while GlpR was required for repression of pfkB specific transcripts in glycerol minimal medium GlpR wa s not needed for the highlevels of pfkB transcript present when cells were grown on fructose. Based on the qRT PCR findings showing that GlpR may serve as a repressor of pfkB specific transcript ion in the absence of fructose, PFK activity was tested in both parent and glpR deficient strains to determine if enzyme activity was altered. Consistent with the qRT PCR results PFK activity was increased in wildtype cells grown in media

PAGE 190

190 supplemented with fru ctose (versus glycerol alone) (Figure 6 4 ). Als o cons istent with the qRT PCR results, PFK specific activity was significantly increased by the glpR knockout (2 fold compared to parent and glpR complemented strains) when cells were grown on m edia with glycerol alone (Figure 64 ). Deletion of glpR also resulte d in a 1.5fold increase in PFK activity compared to parent and glpR complemented strains when cells were grown in the presence of both fructose and glycerol (Figure 64 ). In contrast to media with glycerol ( fructose), PFK activity was decreased during growth on fructose by deletion of glpR (Figure 6 4 ). T he reason for this latter finding remains to be determined but does not appear to be at the level of transcription based on the qRT PCR results The PFK activity of H. volcanii was also measured after gr owth on peptide rich YPC medium fructose or glucose. Similar to minimal media, PFK activity was reduced when YPC was not supplemented with hexose sugars (Figure 6 5 ). GlpR, however, was not required for this decrease, which is in contrast to the GlpR dep endent reduction in PFK activity observed when sugars were excluded fr om glycerol minimal medium (Figure 6 4 and Figure 65 ). It should be noted that the range of PFK activity values determined for the H. volcanii strains under the various conditions (140 to 650 mU protein1) was in agreement with PFK activities reported for other haloarchaea [ 22 mU protein1 for H. saccharolyticus (Johnsen et al., 2001) and 1,300 mU prot ein1 for H. vallismortis (Rangaswamy and Altekar, 1994) in peptide media with 25 28 mM fructose] To determine whether the glpR pfkB operon is regulated by GlpR at the level of transcription: i) the 188bp genomic region upstream of the start codon of glpR was

PAGE 191

191 fused to the coding region of the H. alicantei bgaH galactosidase a ctivity of this reporter was monitored in parent and glpR mutant strains grown on various carbon sources. Using this approach, significant and comparable levels of reporter activity were detected for both parent and glpR mutant strains when cells were grow n in the presence of fructose, regardless of g lycerol supplementation (Table 61 ). Under these conditions, galactosidase specific activity measured for the glpR pfkB promoter was 25 to 34 mU protein1 compared to the negative controls which lacked promoter elements that were less than or equal to 12 mU protein1 for all conditions tested (Table 6 1 ). In contrast, when cells were grown on glycerol alone, the promoter activity of the glpR pfkB operon was reduced in parent strain H26 to levels comp arable to the vector control, while deletion of glpR increased promoter activity to levels similar to growth on fructose (Table 6 1 ). Overall, the data obtained with the promoter fusions are consistent with the qRT PCR and PFK specific activity data, and r eveal that GlpR is required for transcriptional repression of the glpR pfkB operon during growth in the absence of fructose, possibly by interacting with promoter elements within the 188bp region upstream of this operon. The results also reveal that the g lpR pfkB promoter is relatively moderate at driving the expression of heterologous genes such as bgaH compared to the strong and constitutive H. cutirubrum rRNA P2 promoter which reached levels of up to 260 mU protein1 galactosidase activity for t he reporter fusion (Table 6 1 ). GlpR Represses KDGK T ranscription during Growth in the Absence of Glucose To address whether GlpR is involved in the repression of other sugar catabolic pathways, qRT PCR primers were designed for analysis of the transcript levels of genes encoding homologs of KDGK including chromosomally encoded kdgK1 (HVO_0549)

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192 and pHV4encode d kdgK2 (HVO_A0328) (Figure 66A ). In haloarchaea, KDGK is involved in the metabolism of glucose through a modified ED pathway (Falb et al., 2008) In H. volcanii, both kdgK1 and kdgK2 are predicted to encode active KDGK enzymes based on their conservation of active site residues and their close relationship to KDGKs from other microbes that have been characterized at the biochemical level (Figure 6 7 ). Since KdgK1 and KdgK2 are close homologs (62% identity and 74% similarity in amino acid sequence), DNA sequencing was used to confirm qRT PCR product specificity. Based on qRT PCRs, the t ranscripts of both kdgK genes were significantly up regulated (4 to 12fold) when parent strain H26 was grown on media containing glucose ( glycerol) com pared to glycerol alone (Figure 66B ). Furthermore, chromosomally encoded kdgK1 was significantly up regulated in the glpR mutant compared to parent H26 when cells were grown on glycerol minimal medium (14fold ) but was relatively unaltered when cells were grown on media with glucose [ i.e ., 0.38 0.02fold lower in the presence of gl ucose alone and 2.4 0.14fold higher on glucose plus glycerol] (Figure 66 C). Unlike kdgK1, the glpR mutation had little if any impact on kdgK2 transcript levels compared to parent H26 on all media examined ( i.e ., Glu MM, Gly Glu MM and Gly MM) (Figure 6 6 C). Based on the qRT PCR finding that GlpR may serve as a repressor of kdgK1 transcript levels during growth in the absence of glucose KDGK enzyme activity was monitored in both parent and glpR mutant strains to determine the role of GlpR at the protein level. Similar to transcript levels, KDGK activity was reduced when cells were grown on gl ycerol compared to glucose (Figure 68 ). Likewise, deletion of glpR resulted in significant increases in KDGK activity on glycerol, with the increase on glycerol and

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193 glucose modest (2fold) compared to glycerol alone (7fold) (Fig ure 6 8 ). These levels were reduced to parental levels by providing glpR in trans ( Figure 68 ). T he glpR knockout did not impact KDGK activity when cells were grown with glucose as the sole carbon source (Figure 68 ) or on peptiderich YPC media with or without glucose or fructose (Figure 6 9 ). Based on these results, the KDGK activity of H. volcanii was consistent with the qRT PCR data in which glpR was needed for repression of kdgK1 transcri pt levels on glycerol Furthermore, KDGK specific activity (7.1 to 45 mU protein1 ) although higher, was within a reasonable range observed for other haloarchaea ( i.e ., H. saccharolyticus at 2 to 5 mU protein1 ) (Johnsen et al., 2001) To further investigate t he role of GlpR at the level of KDGK transcription kdgK1 and kdgK2 promoter regions were fused to a bgaH based transcriptional reporter (as described above). R epor ter fusions of various lengths and start sites were generated to ensure that the entire promoter and potential regulatory elements were included for analysis, and transcription of the kdgK promoter constructs was monitored by assay of galactosidase activ ity galactosidase activities of the kdgK 1 and kdgK2 bgaH transcriptional fusions w ere somewhat high on glycerol alone (Gly MM) these activities increased significantly upon glucose supplementation (Table 61 ). Furthermore, galactosidase activity of both kdgK1bgaH fusions was higher in the glpR mutant compared to the parent when the cells were grown on glycerol alone (Tab le 6 1 ) In contrast to kdgK1, deletion of glpR did not impact transcription of the kdgK2promoter fusions as measured using the reporter constructs (Table 61 ). Thus, GlpR appears to reduce KDGK activity and kdgK1 transcript levels through transcriptional repression of kdgK1 when cells are grown in the absence of glucose ( on

PAGE 194

194 glycerol ) It should be noted that both the 89and 524bp kdgK1 and 122 bp HVO_A0327kdgK2 promoters are relatively robust at driving expression of the heterologous bgaH reporter galactosidase activit y for the kdgK promoters was greater than that of the H. cutirubrum rRNA P2 promoter (T able 6 1 ), used routinely for high level production of proteins in H. volcanii (Kaczowka et al., 2005; Uthandi et al., 2010) While the data reveal that GlpR is required for the transcriptional repression of kdgK1 and the reduction of KDGK enzyme activity when the cells are grown in the absence of glucose galactosidase activity are relatively high for both kdgK1 bgaH reporter fusions on glycerol compared to the nearly baseline level of transcripts detected for kdgK1 by qRT PCR during grow th on this same medi um. Post transcriptional mechanisms and/or insufficient levels of GlpR needed to repress transcription of the kdgK1bgaH reporter fusion on the multicopy plasmids, pJAM2705 and pJAM2706, may explain these findings. GlpR and Sugar Metabolism The data pres ented here indicate that GlpR represses transcription of the glpR pfkB operon and kdgK1 in the presence of glycerol, and that GlpR is no longer an active repressor of these operons when cells are grown on media with fructose and glucose. It is unclear, how ever, whether GlpR and/or these operons are responsible for the differential utilization of glucose and glycerol by H. volcanii (Sherwood et al., 2009) (Figure 6 10). To directly examine the role of GlpR in this phenomenon and determine whether glycerol and fructose are differentially metaboli zed the glpR deletion was introduced into a previously described glycerol kinase ( glpK ) mutant strain KS4 (Sherwood et al., 2009) for phenotypic analysis. Since both glpR and glpK deletions are

PAGE 195

195 in separate operons and markerless, this double mutant strain was readily generated. Parent and glpK mutant strains were analyzed as a replicate of previous experiments (Sherwood et al., 2009) along with single glpR and double glpR glpK mutant strains for growth and carbon utilization on minimal media including fructose, glucose and/or glycerol. Interestingly, glycerol and fructose are coutilized when provided at equimolar concentrations (Fig ure 611). Furthermore, GlpR did not appear to mediate the differential rate at which glycerol and glucose are metabolized (Figure 610). Promoter R egions for kdgK1 and glpR pfkB Include a Putative GlpR Binding M otif Based on the finding that GlpR likely controls pfkB and kdgK1 transcription within the 188bp and 89bp region upstream of the translational start codon for each gene, respectively, these regions were analyzed to determine putative promoter elements and Gl pR binding motif(s). ShineDalgarno sites and promoter elements including the TFB responsive element (BRE) and TATA box were predicted based on consensus sequences (Gregor and Pfeifer, 2005) Next, inverted repeats located near these elements were aligned to identify potential GlpR binding site( s). Using this approach, an inverted hexameric repeat TCS N C N(3 4)SSN GGA (where S is G or C and N is any nucleotide) was identified that overlaps the putative kdgK1 promoter and is downstream of the putative glpR pfkB promoter (Figure 612). This motif was not found within the kdgK2 prom oter region, consistent with the findings that GlpR regulates both kdgK1 and pfkB but not kdgK2. Future investigation is expected to provide insight as to whether GlpR binds this motif and represses transcription from the kdgK1 and glp R pfkB promoters when cells are grown on glycerol (in the absence of glucose or fructose, respectively)

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196 GlpR Purifies as a Tetramer from Peptide Rich M edia under Both H igh and Low S alt GlpR was C terminally StrepII tagged and the encoding gene was placed under the control of the constitutive rRNA P2 promoter from H. cutirubrum on plasmid pJAM 2682. GlpR was purified from H. volcanii cells grown in rich, peptide based media and harvested for purification using StrepT actin and Superdex 200 gel filtration chromatography GlpR StrepII was purified under both high (2 M NaCl) and low (150 mM NaCl) salt conditions Due to the fact that haloarchaea maintain a high intracellular cation concentration equal to that of the surrounding environment in order to reduce osmotic pressure, their proteins often require high concentrations of salt in order to retain activity (Lanyi, 1974) however, there are some notable exceptions (Uthandi et al., 2010) Under both high (data not shown) and low (Figure 613) salt, GlpR purified as a tetramer composed of monomeric subunits of approximately 30 kDa (Figure 614) Members from the DeoR/GlpR family of transcriptional repressors including E. coli GlpR are often tetramers (Larson et al., 1987) B oth before and after gel filtration chromatography the protein was confirmed to be homogenous by reducing 12% SDS PAGE and subsequent staining with Coomassie Blue (Figure 6 14). C onclusion T his stud y demonstrates that the DeoR /GlpR type GlpR represses the transcription of genes encoding both fructose and glucose metaboli c enzymes when H. volcanii cells are grown in the absence of either fructose or glucose Transcript levels and activities of key enzymes of fructose and glucose metabolism, PFK ( pfkB ) and KDGK ( kdgK1 ), were reduced in the presence of glycerol alone compared to fructose and/or glucose. Analysis of the transcriptional fusions of the pfkB and kdgK1 promoters

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197 in a glpR mutant strain revealed that GlpR was required for transcriptional repression of these genes during growth on g lycerol. In contrast, transcription of the megaplasmid pHV4 encoded kdgK 2, although reduced by glycerol, was not significantly altered by the glpR mutation, suggesting that an additional regulator is used for kdgK 2. The glpR and pfkB genes were co transcribed under a common promoter based on RT PCR analysis, suggesting that GlpR also serves as an autoregulator, and transcription of the glpR pfkB operon was reduced during growth on glycerol alone compared to growth on fructose or glucose. The results presented here provide the first genetic and biochemical evidence of a DeoR/GlpR type transcriptional repressor protein in haloarchaea. Future biochemical characterization of the GlpR regulon is expected to provide further insight into the transcriptional regulation of sugar metabolism in H. volcanii as well as other microorganisms with similar gene organizations. Further analysis will also provide new insight into how DeoR/GlpR family members can interact with and regulate archaeal basal transcriptional machineries composed of eukaryotic like RNAP and TBP and TFB proteins.

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198 Table 61 Transcription of a bgaH galactosidase reporter gene from the genomic regions upstream of glpR pfkB kdgK1 and kdgK2 in parent and glpR mutant strains grown on v arious carbon sources Galactosidase Specific Activity (mUmg protein1)a Promoter: Carbon glpR pfkB (188 bp) kdgK1 (524 bp) kdgK1 (89 bp) HvoA0327 kdgK2 (122 bp) kdgK2 (232 bp) rrnA (551 bp) none (SD only) Source Mutation Gly 9.2 2 160 3 180 3 85 0.01 14 0.01 260 10 8.1 0.1 glpR 32 3 270 10 250 8 99 0.8 14 0.01 250 1 8.2 0.2 Fru 27 1 n.d. n.d. n.d. n.d. 160 7 8.2 0.1 glpR 25 0.3 n.d. n.d. n.d. n.d. 150 5 7.9 0.2 Gly Fru 27 0.2 n.d. n.d. n.d. n.d. 190 10 7.3 0.9 glpR 34 0.1 n.d. n.d. n.d. n.d. 180 9 8.2 0.2 Glu n.d. 300 4 280 0.01 190 0.01 41 0.1 250 7 7.3 0.9 glpR n.d. 250 2 280 3 160 0.01 39 0.02 250 0.01 12.0 0.02 Gly Glu n.d. 230 5 200 0.01 150 4 33 0.01 260 7 8.1 0.05 glpR n.d. 300 4 280 1 160 0.01 34 0.01 260 0.01 8.2 0.2 a galactosidase activities were determined from the lysate of cells carrying transcriptional fusion constructs of promoters grown to log phase in minimal media ( MM) with Fru, Glu and Gly as indicated. Promoter fusions included the start codon and genomic region (indicated in bp) immediately upstream of each target gene. Note that glpR pfkB and HvoA0327kdgK2 are operons most likely transcribed from these promoters. galactosidase activities increased by glpR mutation are shaded. Experiments were performed in triplicate, and the means SD were calculated. Abbreviations: SD, ShineDalgarno site, n.d. not determined.

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199 A Figure 6-1. H. volcanii glpR (HVO_1501) encodes a homolog of DeoR/GlpR-type transcriptional regulators and is linked on the chromosome with genes of sugar metabolism. A) H. volcanii GlpR protein was searched against the NCBI protein database using BLASTP (Alt schul et al., 1997) and found to be significantly related to COG1349, the DeoR/GlpR family of transcriptional regulators of sugar and nucleoside metabolism. H. volcanii GlpR was aligned with DeoR/GlpR family members using CLUSTAL W (Thompson et al., 1994) after Nand C-terminal trimming. The conserved N-terminal DNA binding domain typical of DeoR/GlpR proteins is shown in yellow. (B) Genomic clustering of DeoR/GlpR-transcriptional regulator (red) and PFK (green) genes is conserved in some haloarchaea and many Gram-positive bacteria. Group I: glpR clusters with pfkB and genes encoding a complete fructose PTS; Group II: glpR clusters with pfkB and genes encoding a partial fructose PTS; Group III: glpR clusters with pfkB as well as fbpA encoding fructose 1,6bisphosphate aldolase. (C) Phylogenetic distribution of the DeoR/GlpR family from Bacteria and Archaea. Pairwise comparisons were performed between protein sequences and the mean genetic distance was evaluated using pdistance (gaps were analyzed using pairwise deletion). The best neighborhood-joining tree was then constr ucted using MEGA 4.0. Interior branch test values are indicated at t he internal nodes and were obtained by performing 1,000 replicates. Biochemical ly characterized proteins are indicated by Accession numbers for protein sequences are listed in Chapter 2. 10 20 30 40 50 60 70 80 90 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| HVO_1501 Haloferax volcanii ER KRR I VELVSDSDGR SV ES L SDHLGY S KATIRRDL RELE DRG LIER SHGGAVPVTS---------------VGREQTYGQ K EVQNLEG K GlpR Escherichia coli Q R HNG I IELVKQQ G YVS TEE L VEHFSV S PQTIRRDL NEL AEQNLIL R HHGGAALPSS----------------SVNT P WHDRKATQTEE K DeoR Escherichia coli ER IGQLL QE L KRSDKLHLKDAAALLGV S EMTIRRDL NNHSAPVVLL--GG YIVLEPR--------------SASHYLLSDQKSRLVEE K A gaR Escherichia coli ER REQ I IQR L RQQ G SVQ V ND L SALYGV S TVTIRN DL AFLE KQG IAVR AYGGALICDST-------------TPSVEPSVED K SALNTAM K SrlR Escherichia coli Q R QAA ILEY L QKQ G KCSV EE L AQYFDTTGT TIRK DL VILE HAG TVIR TYGG VVLNK----------------EESDP P IDHK TLINTHK K UlaR Escherichia coli Q R HQILL EM L AQL G FVT V EKVVERLGI S PAT A RRDINK L DES G KLKKVRN GA EAITQQ--------------RPRWT P MNLHQAQNHDE K SgcR Escherichia coli D R IKQML HY L WQHRHL S TQQAMELFGYAEA T V RRDFQYIVNQYPGMIRGH G CLDFDDS-------------TDDKEYVFDV K RTLQSVA K DeoR Bacillus subtilis QQLSIEAAR L YYQSDY S QQQIAEQLNI S RPT VS R L L QYAKEK G YVQIRVMDPFEDLDALGSILEEKYGLLEAHVVFS P TPDYAGITHDLS SugR Corynebacterium glutamicum ER QHA I ASL L APT G AVSV GD L AEHFHVTTE T V RRDL RIME SLG LLQR VHGGAISPEPMG-----------TSPPRLK P ALGK GMPPEPRV LacR Lactococcus lactis ER KRR I VDY L KLKRRATIEE L LTLMDC S IST L RRDL NELE KEKSLR R VHGGAELTQD---------------LSEELSISE K TSKNIQE K FruR Leuconostoc citreum ER KKQ ILKIIQKNQFI S LQE L KKVTKT S LST V RRDL DIL AAD G LVRR VHGG VEWIEP---------------SRGSL P VSERLSLYQAT K GlmR Psuedomonas aeruginosa Q R RHA ILAL L SEQ G EVSV DA L AKRFST S EVTIRK DL AAME SHG LLLR RYGGAVPMP----------------QEL---IGEAAQPVSPY K 100 110 120 130 140 150 160 170 180 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| HVO_1501 Haloferax volcanii RA IADR A VEELAE G Q-VVF F D A GTTTMEVA RKVPKDG---TILG VTNSPRL A IEL NEEDN--EVK L T GGTLRRRTKALV G PT A ES----GlpR Escherichia coli ER IARKVAEQ I PN G S-TLF I D I GTTPEAVA HA L LNHS---NLRI VTN NLNV ANT L MVKED-FRII L A GGELRSRDGGII G EATLD----DeoR Escherichia coli RRA A KL A ATLVEPDQ-TL F F D C GTTTPWIIEAIDNEI---PFTA V CY S LNTFLA L KEKPH-CRAF L C GGEFHASNAIFKPIDFQQ----A gaR Escherichia coli RSV A KA A VELI QP G H-RVILD S GTTTFEIA RLMRKHT---DVIAM TN GMNV ANA L LEAEG-VELLMT GGHLRRQSQ S FY G DQ A EQ----SrlR Escherichia coli EL IAEA A VSFI HD G D-SIILD A G S T VLQMVPL L SRFN---NIT V M TNSLHIVN A L SELDNEQTILMP GGTFRKKSA S FH G QL A EN----UlaR Escherichia coli VR IAKA A SQLVNP G E-SVVINC G S T AFLLGREMCGKP----VQII TN YLPL ANY L IDQEH-DSVIIM GGQYNK-SQ S ITLSPQGS----SgcR Escherichia coli RE IAAL A RTMI KD G D-CFFLD S G S T CLELA KC L ADAR----VK V ICN DIKI ANE L GCFPH-VESYII GGLIRPGYF S VGESL A LE----DeoR Bacillus subtilis RYG A EYMHETVKD G D-IVGVSW GTTMYQIA QNMQPKQ-VKGVE VVQLKGGISHSRVNTYSAETIQ L FAEAFQTMPRYLPLPVVFDNADVK SugR Corynebacterium glutamicum LEL A ET A VSLI TPLARSI FLD S G LACTAI A TV L GDPPEDARWT VVTS S PGAVIA L SATDATSTVV L H G -QVHGNCS S II G ST A VD----LacR Lactococcus lactis EE IAQK A LSKI KD G D-IIFLD A GTTTGILA ELINQSHL--YLTI VTNSVSHLAK L TDDRL--IVY L L GGRVKKVTDAII G SQ A LE----FruR Leuconostoc citreum QK IAQR A AQQLQG G E-IIFLD A GTTTGELIPY L ADKKP--AVT VVTNSVHH A AQL SDLMI--PVMII GGQVKQTTDAVI G AA A VN----GlmR Psuedomonas aeruginosa QA I GRAA AARI REHA-RIII D S G S TTAAMIPE L GHKP---GLV V M TNSLNVVN A L RELEHEPVLLMT GGTWDPHSE S FQ G QV A EQ----190 200 210 220 230 240 250 260 270 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| HVO_1501 Haloferax volcanii -----------------FMERTN FD LLF L G TNALD V E S G L TTPNEDE ARMK ELM VEK A AKVVLV AD----LSKL GRRSFVQFAS L --EEI GlpR Escherichia coli -----------------FISQFRL D FGILG IS G I D SDGSLLEFDYH E VRTK RAIIENSRHVMLVV D ----HSKF GRNAMVNMGSI--SMV DeoR Escherichia coli -----------------TLNNFCP D IAF YSAA G VHVSKG A T CFNLEE LPVK HWAMSM A QKHVLVV D ----HSKF G KVRPARMGD L --KRF A gaR Escherichia coli -----------------SLQNYH FD MLF L G V D AID L E R G VS T HNEDE ARLNRR M CEV A ERIIVVT D ----SSKFN R SSLHKIIDT--QRI SrlR Escherichia coli -----------------AFEHFT FD KLF M G T DG I D LNA G V TTFNEVYT-VSKA M CNA A REVILM AD----SSKF GRKSPNVVCS L --ESV UlaR Escherichia coli -----------------ENSLYAGHWM F TSGK G LTA E G-LYKTDMLTAMAEQK M LSVVGKLVVLV D ----SSKI G ERAGMLFSRA--DQI SgcR Escherichia coli -----------------MINAFSVERA F ISCD ALSL E T G I T NATMFE VGVK TRIIQRSREVILM AD----HSKFDAVEPHAVAT L --SCI DeoR Bacillus subtilis RMVEKDRHIERIIEMGKQANIAL F TVGTVRDEALLFRL G YFNEEEKALLK K QAVGDICSRFFDAKGNICSSAIND R TIGVELQD L RLKER SugR Corynebacterium glutamicum -----------------MISQLRA D IAF VEVD AIQSDTSLC T FFPETIPI K QAM IKN A AFTVAVLSPRSPQDQELQLLKHPFST L --ADF LacR Lactococcus lactis -----------------QLSAYQ F NSAF V G AN G F D N E H G AM T PDHEE AAIK GLAVKQSQNAFIL AD----SSKL G QMSFVKFANS--EEV FruR Leuconostoc citreum -----------------QINHMV F NVSF V G A DG LSV E F G L TTPDLEE AAIK KAVVERSQVSYVL AD----NSKI G TAAFAKAVD L --ERV GlmR Psuedomonas aeruginosa -----------------VLRSYD FD QLF I G A DG I D L E R G T TTFNELLG-LSRV M AEV A REVIVM A E----AD K V GRRIPNQELPW--SSI ....|... HVO_1501 Haloferax volcanii D LFITD-GlpR Escherichia coli D AVYTD -DeoR Escherichia coli D IVVS D -A gaR Escherichia coli D MII V D -SrlR Escherichia coli D KLITD-UlaR Escherichia coli D MLIT G-SgcR Escherichia coli KTI I S D -DeoR Bacillus subtilis SILVAG-SugR Corynebacterium glutamicum DALV TDDH LacR Lactococcus lactis ELITEN-FruR Leuconostoc citreum VLVTSS-GlmR Psuedomonas aeruginosa HTL ITD-10 20 30 40 50 60 70 80 90 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| HVO_1501 Haloferax volcanii ER KRR I VELVSDSDGR SV ES L SDHLGY S KATIRRDL RELE DRG LIER SHGGAVPVTS---------------VGREQTYGQ K EVQNLEG K GlpR Escherichia coli Q R HNG I IELVKQQ G YVS TEE L VEHFSV S PQTIRRDL NEL AEQNLIL R HHGGAALPSS----------------SVNT P WHDRKATQTEE K DeoR Escherichia coli ER IGQLL QE L KRSDKLHLKDAAALLGV S EMTIRRDL NNHSAPVVLL--GG YIVLEPR--------------SASHYLLSDQKSRLVEE K A gaR Escherichia coli ER REQ I IQR L RQQ G SVQ V ND L SALYGV S TVTIRN DL AFLE KQG IAVR AYGGALICDST-------------TPSVEPSVED K SALNTAM K SrlR Escherichia coli Q R QAA ILEY L QKQ G KCSV EE L AQYFDTTGT TIRK DL VILE HAG TVIR TYGG VVLNK----------------EESDP P IDHK TLINTHK K UlaR Escherichia coli Q R HQILL EM L AQL G FVT V EKVVERLGI S PAT A RRDINK L DES G KLKKVRN GA EAITQQ--------------RPRWT P MNLHQAQNHDE K SgcR Escherichia coli D R IKQML HY L WQHRHL S TQQAMELFGYAEA T V RRDFQYIVNQYPGMIRGH G CLDFDDS-------------TDDKEYVFDV K RTLQSVA K DeoR Bacillus subtilis QQLSIEAAR L YYQSDY S QQQIAEQLNI S RPT VS R L L QYAKEK G YVQIRVMDPFEDLDALGSILEEKYGLLEAHVVFS P TPDYAGITHDLS SugR Corynebacterium glutamicum ER QHA I ASL L APT G AVSV GD L AEHFHVTTE T V RRDL RIME SLG LLQR VHGGAISPEPMG-----------TSPPRLK P ALGK GMPPEPRV LacR Lactococcus lactis ER KRR I VDY L KLKRRATIEE L LTLMDC S IST L RRDL NELE KEKSLR R VHGGAELTQD---------------LSEELSISE K TSKNIQE K FruR Leuconostoc citreum ER KKQ ILKIIQKNQFI S LQE L KKVTKT S LST V RRDL DIL AAD G LVRR VHGG VEWIEP---------------SRGSL P VSERLSLYQAT K GlmR Psuedomonas aeruginosa Q R RHA ILAL L SEQ G EVSV DA L AKRFST S EVTIRK DL AAME SHG LLLR RYGGAVPMP----------------QEL---IGEAAQPVSPY K 100 110 120 130 140 150 160 170 180 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| HVO_1501 Haloferax volcanii RA IADR A VEELAE G Q-VVF F D A GTTTMEVA RKVPKDG---TILG VTNSPRL A IEL NEEDN--EVK L T GGTLRRRTKALV G PT A ES----GlpR Escherichia coli ER IARKVAEQ I PN G S-TLF I D I GTTPEAVA HA L LNHS---NLRI VTN NLNV ANT L MVKED-FRII L A GGELRSRDGGII G EATLD----DeoR Escherichia coli RRA A KL A ATLVEPDQ-TL F F D C GTTTPWIIEAIDNEI---PFTA V CY S LNTFLA L KEKPH-CRAF L C GGEFHASNAIFKPIDFQQ----A gaR Escherichia coli RSV A KA A VELI QP G H-RVILD S GTTTFEIA RLMRKHT---DVIAM TN GMNV ANA L LEAEG-VELLMT GGHLRRQSQ S FY G DQ A EQ----SrlR Escherichia coli EL IAEA A VSFI HD G D-SIILD A G S T VLQMVPL L SRFN---NIT V M TNSLHIVN A L SELDNEQTILMP GGTFRKKSA S FH G QL A EN----UlaR Escherichia coli VR IAKA A SQLVNP G E-SVVINC G S T AFLLGREMCGKP----VQII TN YLPL ANY L IDQEH-DSVIIM GGQYNK-SQ S ITLSPQGS----SgcR Escherichia coli RE IAAL A RTMI KD G D-CFFLD S G S T CLELA KC L ADAR----VK V ICN DIKI ANE L GCFPH-VESYII GGLIRPGYF S VGESL A LE----DeoR Bacillus subtilis RYG A EYMHETVKD G D-IVGVSW GTTMYQIA QNMQPKQ-VKGVE VVQLKGGISHSRVNTYSAETIQ L FAEAFQTMPRYLPLPVVFDNADVK SugR Corynebacterium glutamicum LEL A ET A VSLI TPLARSI FLD S G LACTAI A TV L GDPPEDARWT VVTS S PGAVIA L SATDATSTVV L H G -QVHGNCS S II G ST A VD----LacR Lactococcus lactis EE IAQK A LSKI KD G D-IIFLD A GTTTGILA ELINQSHL--YLTI VTNSVSHLAK L TDDRL--IVY L L GGRVKKVTDAII G SQ A LE----FruR Leuconostoc citreum QK IAQR A AQQLQG G E-IIFLD A GTTTGELIPY L ADKKP--AVT VVTNSVHH A AQL SDLMI--PVMII GGQVKQTTDAVI G AA A VN----GlmR Psuedomonas aeruginosa QA I GRAA AARI REHA-RIII D S G S TTAAMIPE L GHKP---GLV V M TNSLNVVN A L RELEHEPVLLMT GGTWDPHSE S FQ G QV A EQ----190 200 210 220 230 240 250 260 270 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| HVO_1501 Haloferax volcanii -----------------FMERTN FD LLF L G TNALD V E S G L TTPNEDE ARMK ELM VEK A AKVVLV AD----LSKL GRRSFVQFAS L --EEI GlpR Escherichia coli -----------------FISQFRL D FGILG IS G I D SDGSLLEFDYH E VRTK RAIIENSRHVMLVV D ----HSKF GRNAMVNMGSI--SMV DeoR Escherichia coli -----------------TLNNFCP D IAF YSAA G VHVSKG A T CFNLEE LPVK HWAMSM A QKHVLVV D ----HSKF G KVRPARMGD L --KRF A gaR Escherichia coli -----------------SLQNYH FD MLF L G V D AID L E R G VS T HNEDE ARLNRR M CEV A ERIIVVT D ----SSKFN R SSLHKIIDT--QRI SrlR Escherichia coli -----------------AFEHFT FD KLF M G T DG I D LNA G V TTFNEVYT-VSKA M CNA A REVILM AD----SSKF GRKSPNVVCS L --ESV UlaR Escherichia coli -----------------ENSLYAGHWM F TSGK G LTA E G-LYKTDMLTAMAEQK M LSVVGKLVVLV D ----SSKI G ERAGMLFSRA--DQI SgcR Escherichia coli -----------------MINAFSVERA F ISCD ALSL E T G I T NATMFE VGVK TRIIQRSREVILM AD----HSKFDAVEPHAVAT L --SCI DeoR Bacillus subtilis RMVEKDRHIERIIEMGKQANIAL F TVGTVRDEALLFRL G YFNEEEKALLK K QAVGDICSRFFDAKGNICSSAIND R TIGVELQD L RLKER SugR Corynebacterium glutamicum -----------------MISQLRA D IAF VEVD AIQSDTSLC T FFPETIPI K QAM IKN A AFTVAVLSPRSPQDQELQLLKHPFST L --ADF LacR Lactococcus lactis -----------------QLSAYQ F NSAF V G AN G F D N E H G AM T PDHEE AAIK GLAVKQSQNAFIL AD----SSKL G QMSFVKFANS--EEV FruR Leuconostoc citreum -----------------QINHMV F NVSF V G A DG LSV E F G L TTPDLEE AAIK KAVVERSQVSYVL AD----NSKI G TAAFAKAVD L --ERV GlmR Psuedomonas aeruginosa -----------------VLRSYD FD QLF I G A DG I D L E R G T TTFNELLG-LSRV M AEV A REVIVM A E----AD K V GRRIPNQELPW--SSI ....|... HVO_1501 Haloferax volcanii D LFITD-GlpR Escherichia coli D AVYTD -DeoR Escherichia coli D IVVS D -A gaR Escherichia coli D MII V D -SrlR Escherichia coli D KLITD-UlaR Escherichia coli D MLIT G-SgcR Escherichia coli KTI I S D -DeoR Bacillus subtilis SILVAG-SugR Corynebacterium glutamicum DALV TDDH LacR Lactococcus lactis ELITEN-FruR Leuconostoc citreum VLVTSS-GlmR Psuedomonas aeruginosa HTL ITD-

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200 B Figure 61. Continued glpR pfkB fruC fruA ptsH ptsIGroup I : Haloferax volcanii, Haloterrigena turkmenica, Moorella thermoacetica Group III : Haloarcula marismortui Halomicrobium mukohataei Group II : Thermoanaerobacter tengcongensis Symbiobacterium thermophilum Bacillus cereus, Lactobacillus sakei Enterococcus faecalis glpR pfkB fruC fruA glpR pfkB fbpA glpR pfkB fruC fruA ptsH ptsI glpR pfkB fruC fruA ptsH ptsIGroup I : Haloferax volcanii, Haloterrigena turkmenica, Moorella thermoacetica Group III : Haloarcula marismortui Halomicrobium mukohataei Group II : Thermoanaerobacter tengcongensis Symbiobacterium thermophilum Bacillus cereus, Lactobacillus sakei Enterococcus faecalis glpR pfkB fruC fruA glpR pfkB fruC fruA glpR pfkB fbpA glpR pfkB fbpA

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201 C Figure 6 1. Continued HVO 1501 Haloferax volcanii HborDRAFT 0052 Halogeometricum borinquense Htur 2761 Haloterrigena turkmenica rrnAC0341 Haloarcula marismortui Hmuk 2660 Halomicrobium mukohataei STH793 Symbiobacterium thermophilum TTE2588 Thermoanaerobacter tengcongensis Hm1 2674 Heliobacterium modesticaldum CBC 0871 Clostridium botulinum AgaR Escherichia coli SrlR Escherichia coli GlmR Pseudomonas aeruginosa SugR Corynebacterium glutamicum cgR 1764 Corynebacterium glutamicum DeoT Escherichia coli FruR Leuconostoc citreum LacR Lactococcus lactis FruR Lactococcus lactis GlpR Escherichia coli SFV_3430 Shigella flexneri c4824 Escherichia coli LacR Staphylococcus aureus LacR Streptococcus mutans IolR Lactobacillus casei IolR Bacillus subtilis b1770 Escherichia coli SFV 2884 Shigella flexneri HI0615 Haemophilus influenzae DeoR Escherichia coli SgcR Escherichia coli HI1009 Haemophilus influenzae UlaR Escherichia coli DeoR Bacillus subtilis SorC Klebsiella pneumoniae SorC Lactobacillus casei Sty0448 Salmonella enterica F box protein Arabidopsis thaliana AraC Rhizobium leguminosarum 99 99 99 99 99 99 99 97 99 99 99 99 99 99 61 94 81 73 99 63 98 92 99 54 88 96 80 83 77 99 95 0.1 Haloarchaea Firmicutes Firmicutes Proteobacteria & Actinomycetes Proteobacteria Firmicutes Proteobacteria Eukaryotes HVO 1501 Haloferax volcanii HborDRAFT 0052 Halogeometricum borinquense Htur 2761 Haloterrigena turkmenica rrnAC0341 Haloarcula marismortui Hmuk 2660 Halomicrobium mukohataei STH793 Symbiobacterium thermophilum TTE2588 Thermoanaerobacter tengcongensis Hm1 2674 Heliobacterium modesticaldum CBC 0871 Clostridium botulinum AgaR Escherichia coli SrlR Escherichia coli GlmR Pseudomonas aeruginosa SugR Corynebacterium glutamicum cgR 1764 Corynebacterium glutamicum DeoT Escherichia coli FruR Leuconostoc citreum LacR Lactococcus lactis FruR Lactococcus lactis GlpR Escherichia coli SFV_3430 Shigella flexneri c4824 Escherichia coli LacR Staphylococcus aureus LacR Streptococcus mutans IolR Lactobacillus casei IolR Bacillus subtilis b1770 Escherichia coli SFV 2884 Shigella flexneri HI0615 Haemophilus influenzae DeoR Escherichia coli SgcR Escherichia coli HI1009 Haemophilus influenzae UlaR Escherichia coli DeoR Bacillus subtilis SorC Klebsiella pneumoniae SorC Lactobacillus casei Sty0448 Salmonella enterica F box protein Arabidopsis thaliana AraC Rhizobium leguminosarum 99 99 99 99 99 99 99 97 99 99 99 99 99 99 61 94 81 73 99 63 98 92 99 54 88 96 80 83 77 99 95 0.1HVO 1501 Haloferax volcanii HborDRAFT 0052 Halogeometricum borinquense Htur 2761 Haloterrigena turkmenica rrnAC0341 Haloarcula marismortui Hmuk 2660 Halomicrobium mukohataei STH793 Symbiobacterium thermophilum TTE2588 Thermoanaerobacter tengcongensis Hm1 2674 Heliobacterium modesticaldum CBC 0871 Clostridium botulinum AgaR Escherichia coli SrlR Escherichia coli GlmR Pseudomonas aeruginosa SugR Corynebacterium glutamicum cgR 1764 Corynebacterium glutamicum DeoT Escherichia coli FruR Leuconostoc citreum LacR Lactococcus lactis FruR Lactococcus lactis GlpR Escherichia coli SFV_3430 Shigella flexneri c4824 Escherichia coli LacR Staphylococcus aureus LacR Streptococcus mutans IolR Lactobacillus casei IolR Bacillus subtilis b1770 Escherichia coli SFV 2884 Shigella flexneri HI0615 Haemophilus influenzae DeoR Escherichia coli SgcR Escherichia coli HI1009 Haemophilus influenzae UlaR Escherichia coli DeoR Bacillus subtilis SorC Klebsiella pneumoniae SorC Lactobacillus casei Sty0448 Salmonella enterica F box protein Arabidopsis thaliana AraC Rhizobium leguminosarum 99 99 99 99 99 99 99 97 99 99 99 99 99 99 61 94 81 73 99 63 98 92 99 54 88 96 80 83 77 99 95 0.1 Haloarchaea Firmicutes Firmicutes Proteobacteria & Actinomycetes Proteobacteria Firmicutes Proteobacteria Eukaryotes

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202 A B Figure 6-2. Genomic organization and transcript analysis of pfkB encoding phosphofructokinase (PFK) and glpR encoding GlpR, a putative transcriptional repressor of the DeoR/GlpR family in H. volcanii A) Schematic representation of glpR and pfkB on the H. volcanii genome and location of annealing sites (represented as vertical lines) for (q)RT-PCR primers. B) qRTPCR reveals pfkB and glpR are co-transcribed fr om a common promoter. Total RNA from parent H26 was ex tracted and reverse transcribed to generate cDNA, which was used as a template for PCR (lane 1). RNA which had not undergone reverse transcription was used as a negative control template for PCR (lane 2). Genomic DNA was used as a positive control template for PCR (lane 3). 100 bp Qu ick Load DNA markers and molecular sizes are indicated on left. C) Absolute quantification of transcripts specific for pfkB and glpR reveal that both genes are fr uctoseand glucose-inducible. Transcript levels were derived from qR T-PCR. D) Relative quantification of transcript levels specific for pfkB in glpR mutant KS8, KS8 with plasmid vector control (pJAM202c) and KS8 with glpR in trans (pJAM2682) compared to parent H26. PFK transcript is increased in the absence of fructose (in Gly) in KS8 compared to H26. Transcript le vels were derived by qRT-PCR. Calculations are based on the N-fold induc tion of transcript levels in KS8 (and subsequent control and complimentary strains) compared to parent H26. Results were normalized with the internal control, ribL Experiments were performed in triplicate, and the means SD were calculated. qRTPCR primers pfkB 2000 bpqRTPCR primers glpR fruB ptsI ptsH1 fruA fruC pfkB glpR leuB RT-PCR primers glpR pfkB qRTPCR primers pfkB 2000 bp 2000 bpqRTPCR primers glpR fruB ptsI ptsH1 fruA fruC pfkB glpR leuB RT-PCR primers glpR pfkB 517 400 300bp200 100 1 2 3 1000 517 400 300bp200 100 1 2 3 1000

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203 C D Figure 62. Continued 0 5000 10000 15000 20000 25000 30000 35000 1 mRNA Copy Number Fru MM Gly Fru MM Gly MM Glu MM Gly Glu MM pfkB glpR 0 5000 10000 15000 20000 25000 30000 35000 1 mRNA Copy Number Fru MM Gly Fru MM Gly MM Glu MM Gly Glu MM pfkB glpR 0 5000 10000 15000 20000 25000 30000 35000 1 mRNA Copy Number Fru MM Gly Fru MM Gly MM Glu MM Gly Glu MM pfkB glpR 0 5000 10000 15000 20000 25000 30000 35000 1 mRNA Copy Number 0 5000 10000 15000 20000 25000 30000 35000 1 mRNA Copy Number Fru MM Gly Fru MM Gly MM Glu MM Gly Glu MM pfkB glpR pfkB glpR pfkB glpR -1 1 3 5 7 9 11 13 15 1 N-fold Induction of Transcript Fru MM Gly Fru MM Gly MMKS8 ( glpR ) KS8-pJAM202c ( glpR with vector control) KS8-pJAM2682 ( glpR with glpR in trans ) S 3 S -1 1 3 5 7 9 11 13 15 1 N-fold Induction of Transcript Fru MM Gly Fru MM Gly MMKS8 ( glpR ) KS8-pJAM202c ( glpR with vector control) KS8-pJAM2682 ( glpR with glpR in trans ) S 3 S

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204 AB Figure 6-3. PCR and Souther n blot confirmation of H. volcanii glpR mutant strain KS8 (H26 glpR). A) Confirmation of the glpR knockout KS8 by PCR. Primer pairs that annealed outside the genomic regi on cloned in suicide plasmid pJAM2677 were used for confirmation of the glpR gene knockout by PCR. HiLo DNA markers and molecular masses are indicated on left. Genomic DNA from the following strains served as template: Lane 1. Parent strain H26, Lane 2. KS8 (H26 glpR). B) Southern blot confirmation of the glpR knockout in strain KS8 (H26 glpR). Genomic DNA was digested with MluI and HpaI and hybridized with a DIG-labeled probe specific for glpR. The following strains served as the source of genom ic DNA: Lane 1. Parent strain H26, Lane 2. KS8 (H26 glpR). kb 10 8 6 4 3 2 1.5 1 0.5 12 1.4kb 10 8 6 4 3 2 1.5 1 0.5 12 1.4MluIand HpaI with glpR probe kb124 3 2 1.5 MluIand HpaI with glpR probe kb124 3 2 1.5

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205 Figure 64. PFK activity increases when cells are grown on glycerol minimal medium after deletion of glpR Specific activity of PFK was determined from cell lysate of H. volcanii H26 (parent), KS8 ( glpR mutant), KS8 pJAM202c ( glpR mutant with vector control) and KS8pJAM2682 ( glpR mutant with glpR in trans ) cells grown to log phase in Fru, Gly Fru and Gly as indicated. Experiments were perf ormed in triplicate, and the means SD were calculated. 0 100 200 300 400 500 600 700 800 1 Phosphofructokinase Specific Activity (mU 1 ) Fru MM Gly Fru MM Gly MM KS8 ( glpR ) KS8-pJAM202c ( glpR with vector control) KS8-pJAM2682 ( glpR with glpR in trans ) H26 0 100 200 300 400 500 600 700 800 1 Phosphofructokinase Specific Activity (mU 1 ) 0 100 200 300 400 500 600 700 800 1 Phosphofructokinase Specific Activity (mU 1 ) Fru MM Gly Fru MM Gly MM KS8 ( glpR ) KS8-pJAM202c ( glpR with vector control) KS8-pJAM2682 ( glpR with glpR in trans ) H26 KS8 ( glpR ) KS8-pJAM202c ( glpR with vector control) KS8-pJAM2682 ( glpR with glpR in trans ) H26

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206 Figure 65. GlpR is not required for reduction of PFK activity on peptide media in the absence of fructose or glucose. PFK specific activity was determined as previously described for H. volcanii H26 (parent), KS8 ( glpR deficient strain), KS8 pJAM202c ( glpR mutant with vector control) and KS8pJAM2682 ( glpR deficient strain with glpR in trans ) cells grown in YPC, YPC Fru and YPC Glu media. 0 50 100 150 200 250 300 350 400 450 1 PFK Specific Activity (mU-1) YPC YPC GluH26 KS8 ( glpR ) KS8-pJAM202c ( glpR with vector control) KS8-pJAM2682 ( glpR with glpR in trans ) YPC Fru 0 50 100 150 200 250 300 350 400 450 1 PFK Specific Activity (mU-1) 0 50 100 150 200 250 300 350 400 450 1 PFK Specific Activity (mU-1) YPC YPC GluH26 KS8 ( glpR ) KS8-pJAM202c ( glpR with vector control) KS8-pJAM2682 ( glpR with glpR in trans ) H26 KS8 ( glpR ) KS8-pJAM202c ( glpR with vector control) KS8-pJAM2682 ( glpR with glpR in trans ) YPC Fru

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207 A B Figure 66. Genomic organization and transcript analysis of KDGK genes located on the chromosome and megaplasmid pHV4 of H. volcanii. A) Schematic representations of chromosomal kdgK1 (upper) and related pHV4carried kdgK2 (lower) genes and their neighbors and location of annealing sites (represented as vertical lines) for qRT PCR primers. B) Absolute quantification of transcripts specific for kdgK1 and kdgK2 reveals that both genes are glucoseinducible, regardless of glycerol supplementation. Transcript specific for kdgK1 and kdgK2 were upregulated 12fold and 4fold, respectively, in the presence of glucosecontaining media compared to glycerol alone. Transcript levels were derived from qRT PCR as described in Methods and Materials. C) Relative quantification of transcript levels specific for kdgK1 and kdgK2 Chromosomal kdgK1 transcripts are increased in the presence of glycerol regardless of glucose supplementation in a glpR mutant (KS8) compared to parent H26. Transcript levels were derived by qRT PCR. Calculation s are based on the N fold induction of transcription in the designated minimal media for glpR deficient cells compared to parent H26. Results were normalized to the nfold induction of the internal control, ribL Experiments were performed in triplicate, and the means SD were calculated. mutL HVO_0550 kdgK1 nhaC HVO_0547 qRTPCR primers kdgk1 2000 bp lacA HVO_A0327 kdgK2 kdgA mviM qRTPCR primers kdgk2 mutL HVO_0550 kdgK1 nhaC HVO_0547 qRTPCR primers kdgk1 2000 bp lacA HVO_A0327 kdgK2 kdgA mviM qRTPCR primers kdgk2 0 50 100 150 200 250 300 350 1 mRNA Copy Number Glu MM kdgK1 kdgK2 Gly Glu MM Gly MM 0 50 100 150 200 250 300 350 1 mRNA Copy Number Glu MM kdgK1 kdgK2 Gly Glu MM 0 50 100 150 200 250 300 350 1 mRNA Copy Number Glu MM kdgK1 kdgK2 kdgK1 kdgK2 kdgK1 kdgK2 Gly Glu MM Gly MM

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208 C Figure 6 6 Continued 1 1 3 5 7 9 11 13 15 1 N fold Induction of Transcript Glu MM Gly Glu MM Gly MM kdgK1 kdgK2 1 1 3 5 7 9 11 13 15 1 N fold Induction of Transcript 1 1 3 5 7 9 11 13 15 1 N fold Induction of Transcript Glu MM Gly Glu MM Gly MM kdgK1 kdgK2 kdgK1 kdgK2

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209 Figure 6-7. Phylogenetic di stribution of PFK and KDGK in Bacteria and Archaea. Protein sequences were retrieved usi ng the NCBI database (Benson et al., 2010) and subsequently Nand C-termi nally trimmed and aligned using CLUSTAL W (Thompson et al., 1994). Pa irwise comparisons were performed between sequences and mean genetic distance was evaluated using pdistance (gaps were analyzed using pairwise deletion). The best neighborhood-joining tree was then constr ucted using MEGA 4.0. Interior branch test values are indicated at t he internal nodes and were obtained by performing 1,000 replicates. Biochemical ly characterized proteins are indicated by Members belonging to each clus ter are color-coded (green, Group I KDGK; yellow, Group II KDGK; purple, uncharacterized sugar kinases; red, PFK). H. volcanii proteins HVO_1500 (PfkB), HVO_A0328 (KdgK2), HVO_0549 (KdgK1), and HVO_2612 (sugar kinase) are indicated in bold, large font. Accession numbers fo r protein sequences are provided in Chapter 2. HVO 0549 Haloferax volcanii HborDRAFT0980 Halogeometricumborinquense Hlac0463 Halorubrumlacusprofundi Hlac2870 Halorubrumlacusprofundi HVO A0328 Haloferax volcanii Htur3911 Haloterrigenaturkmenica HacjB3 14395 Halalkalicoccusjeotgali rrnAC0545 Haloarcula marismortui Hmuk2509 Halomicrobiummukohataei VNG0158G Halobacterium salinarium HQ1455A Haloquadratum walsbyi Huta2283 Halorhabdusutahensis Htur3215 Haloterrigenaturkmenica Nmag1292 Natrialbamagadii Huta0006 Halorhabdusutahensis rrnAC2551 Haloarcula marismortui Hmuk0377 Halomicrobiummukohataei KdgK Thermotogamaritima LCABL 28640 Lactobacillus casei KdgK Escherichia coli SBO 3525 Shigella boydii KdgK Erwiniachrysanthemi HI0049 Haemophilusinfluenzae KdgK Bacillus subtilis CA CO395 Clostridium acetobutylicum KdgK Thermusthermophilus Cmaq0369 Caldivirgamaquilingensis KdgK Thermoproteustenax KdgK Sulfolobus sulfataricus KdgK Sulfolobus tokodaii Htur0569 Haloterrigenaturkmenica Nmag2964 Natrialbamagadii VNG1851G Halobacterium salinarium Hmuk2764 Halomicrobiummukohataei HborDRAFT2235 Halogeometricumborinquense HVO 2612 Haloferax volcanii NP3184A Natronomonaspharaonis Hlac2117 Halorubrumlacusprofundi Hlac2162 Halorubrumlacusprofundi Huta0501 Halorhabdusutahensis Htur4085 Haloterrigenaturkmenica HborDRAFT0358 Halogeometricumborinquense HacjB3 09545 Halalkalicoccusjeotgali Htur1630 Haloterrigenaturkmenica Nmag3485 Natrialbamagadii rrnAC0342 Haloarcula marismortui Hmuk2661 Halomicrobiummukohataei HVO 1500 Haloferax volcanii Htur2760 Haloterrigenaturkmenica HacjB3 10180 Halalkalicoccusjeotgali Huta0650 Halorhabdusutahensis Huta1103 Halorhabdusutahensis FruB Lactococcus lactis FruK Bacillus licheniformis FruK Spiroplasmacitri PfkB Borreliaburgdorferi FruK Pseudomonas putida HIAG 00717 Haemophilusinfluenzae FruK Escherichia coli FruK Shigella flexneri PfkM Homo sapiens PfkB Musmusculus 99 99 99 99 99 99 99 99 99 99 99 99 99 63 99 68 99 99 99 79 96 99 72 81 99 99 99 54 99 95 99 99 83 66 99 99 97 99 99 99 61 99 77 99 71 94 99 99 99 98 93 99 91 99 36 99 59 0.1 Haloarchaea Firmicutes Crenarchaeota Firmicutes Proteobacteria Haloarchaea Firmicutes Proteobacteria Eukaryotes HVO 0549 Haloferax volcanii HborDRAFT0980 Halogeometricumborinquense Hlac0463 Halorubrumlacusprofundi Hlac2870 Halorubrumlacusprofundi HVO A0328 Haloferax volcanii Htur3911 Haloterrigenaturkmenica HacjB3 14395 Halalkalicoccusjeotgali rrnAC0545 Haloarcula marismortui Hmuk2509 Halomicrobiummukohataei VNG0158G Halobacterium salinarium HQ1455A Haloquadratum walsbyi Huta2283 Halorhabdusutahensis Htur3215 Haloterrigenaturkmenica Nmag1292 Natrialbamagadii Huta0006 Halorhabdusutahensis rrnAC2551 Haloarcula marismortui Hmuk0377 Halomicrobiummukohataei KdgK Thermotogamaritima LCABL 28640 Lactobacillus casei KdgK Escherichia coli SBO 3525 Shigella boydii KdgK Erwiniachrysanthemi HI0049 Haemophilusinfluenzae KdgK Bacillus subtilis CA CO395 Clostridium acetobutylicum KdgK Thermusthermophilus Cmaq0369 Caldivirgamaquilingensis KdgK Thermoproteustenax KdgK Sulfolobus sulfataricus KdgK Sulfolobus tokodaii Htur0569 Haloterrigenaturkmenica Nmag2964 Natrialbamagadii VNG1851G Halobacterium salinarium Hmuk2764 Halomicrobiummukohataei HborDRAFT2235 Halogeometricumborinquense HVO 2612 Haloferax volcanii NP3184A Natronomonaspharaonis Hlac2117 Halorubrumlacusprofundi Hlac2162 Halorubrumlacusprofundi Huta0501 Halorhabdusutahensis Htur4085 Haloterrigenaturkmenica HborDRAFT0358 Halogeometricumborinquense HacjB3 09545 Halalkalicoccusjeotgali Htur1630 Haloterrigenaturkmenica Nmag3485 Natrialbamagadii rrnAC0342 Haloarcula marismortui Hmuk2661 Halomicrobiummukohataei HVO 1500 Haloferax volcanii Htur2760 Haloterrigenaturkmenica HacjB3 10180 Halalkalicoccusjeotgali Huta0650 Halorhabdusutahensis Huta1103 Halorhabdusutahensis FruB Lactococcus lactis FruK Bacillus licheniformis FruK Spiroplasmacitri PfkB Borreliaburgdorferi FruK Pseudomonas putida HIAG 00717 Haemophilusinfluenzae FruK Escherichia coli FruK Shigella flexneri PfkM Homo sapiens PfkB Musmusculus 99 99 99 99 99 99 99 99 99 99 99 99 99 63 99 68 99 99 99 79 96 99 72 81 99 99 99 54 99 95 99 99 83 66 99 99 97 99 99 99 61 99 77 99 71 94 99 99 99 98 93 99 91 99 36 99 59 0.1 Haloarchaea Firmicutes Crenarchaeota Firmicutes Proteobacteria Haloarchaea Firmicutes Proteobacteria Eukaryotes

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210 Figure 68. KDGK activity is increased by deletion of glpR in cells grown on glycerol minimal medium. KDGK specific activities were determined from cell lysate of log phase H. volcanii H26 (parent), KS8 ( glpR mutant), KS8pJAM202c ( glpR mutant with vector control) and KS8pJAM2682 ( glpR mutant with glpR in trans ) grown on Glu Gly Glu and Gly, as indicated. Experiments were performed in triplicate, and the means SD were calculated. 0 10 20 30 40 50 60 1 KDGK Specific Activity (mU 1 ) Glu MM Gly Glu MM Gly MM H26 KS8 ( glpR ) KS8-pJAM202c ( glpR with vector control) KS8-pJAM2682 ( glpR with glpR in trans ) 0 10 20 30 40 50 60 1 KDGK Specific Activity (mU 1 ) Glu MM Gly Glu MM Gly MM 0 10 20 30 40 50 60 1 KDGK Specific Activity (mU 1 ) Glu MM Gly Glu MM Gly MM H26 KS8 ( glpR ) KS8-pJAM202c ( glpR with vector control) KS8-pJAM2682 ( glpR with glpR in trans ) H26 KS8 ( glpR ) KS8-pJAM202c ( glpR with vector control) KS8-pJAM2682 ( glpR with glpR in trans )

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211 Figure 69 GlpR is not required for reduction of KDGK activity on peptide media in the absence of fructose or glucose. KDGK specific activity was determined as previously described for H. volcanii H26 (parent), KS8 ( glpR mutant), KS8pJAM202c ( glpR mutant with vector control) and KS8pJAM2682 ( glpR mutant with glpR in trans ) cells grown in YPC, YPC Fru and YPC Glu media. 0 2 4 6 8 10 12 14 16 18 1 KDGK Specific Activity (mU-1) YPC YPC Fru YPC GluKS8-pJAM2682 ( glpR with glpR in trans ) H26 KS8 ( glpR ) KS8-pJAM202c ( glpR with vector control) 0 2 4 6 8 10 12 14 16 18 1 KDGK Specific Activity (mU-1) YPC YPC Fru YPC Glu 0 2 4 6 8 10 12 14 16 18 1 KDGK Specific Activity (mU-1) 0 2 4 6 8 10 12 14 16 18 1 KDGK Specific Activity (mU-1) YPC YPC Fru YPC GluKS8-pJAM2682 ( glpR with glpR in trans ) H26 KS8 ( glpR ) KS8-pJAM202c ( glpR with vector control) KS8-pJAM2682 ( glpR with glpR in trans ) H26 KS8 ( glpR ) KS8-pJAM202c ( glpR with vector control) H26 KS8 ( glpR ) KS8-pJAM202c ( glpR with vector control)

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212 Figure 610. Deletion of GlpR does not impact glycerol or glucose utilization in H. volcanii The parent strain H26 and GlpR deficient strain KS 8 exhibit similar growth rates, cell yields and carbon utilization patterns when grown in Gly Glu MM. Furthermore, GK ( glpK ) mutant KS4 and cells deficient in both GlpR and GK ( H26 glpK glpR KS10 ) exhibit similar growth rates, cell yields and carbon utilization patterns when grown in Gly Glu MM. Growth at 42C (200 RPM ) was monitored by an increase in OD600, where 1 U was equivalent to approximately 109 CFU per ml for all strains. At various time points, supernatant fractions were withdrawn from all cultures and analyzed by HPLC for glucose or glycerol consumpt ion as previously described (Sherwood et al., 2009) Experiments were performed in triplicate, and the means SD were calculated. Cell growth and carbon utilization levels are indicated. growth glucose glycerol 0 5 10 15 20 25 0 20 40 60 80 Time (h) Carbon Source Concentration (mM) 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 Cell Growth (OD600) H26 0 5 10 15 20 25 0 10 20 30 40 50 60 70 Time (h) Carbon Source Concentration (mM) 0 0.2 0.4 0.6 0.8 1 1.2 1.4 Cell Growth (OD600) H26 glpK 0 5 10 15 20 25 0 10 20 30 40 50 60 70 Time (h) Carbon Source Concentration (mM) 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 Cell Growth (OD600) H26 glpK glpR 0 5 10 15 20 25 0 10 20 30 40 50 60 70 80 Time (h) Carbon Source Concentration (mM) 0 0.2 0.4 0.6 0.8 1 1.2 1.4 Cell Growth (OD600) H26 glpR growth glucose glycerol growth glucose glycerol 0 5 10 15 20 25 0 20 40 60 80 Time (h) Carbon Source Concentration (mM) 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 Cell Growth (OD600) H26 0 5 10 15 20 25 0 20 40 60 80 Time (h) Carbon Source Concentration (mM) 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 Cell Growth (OD600) H26 0 5 10 15 20 25 0 10 20 30 40 50 60 70 Time (h) Carbon Source Concentration (mM) 0 0.2 0.4 0.6 0.8 1 1.2 1.4 Cell Growth (OD600) H26 glpK 0 5 10 15 20 25 0 10 20 30 40 50 60 70 Time (h) Carbon Source Concentration (mM) 0 0.2 0.4 0.6 0.8 1 1.2 1.4 Cell Growth (OD600) H26 glpK 0 5 10 15 20 25 0 10 20 30 40 50 60 70 Time (h) Carbon Source Concentration (mM) 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 Cell Growth (OD600) H26 glpK glpR 0 5 10 15 20 25 0 10 20 30 40 50 60 70 Time (h) Carbon Source Concentration (mM) 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 Cell Growth (OD600) H26 glpK glpR 0 5 10 15 20 25 0 10 20 30 40 50 60 70 80 Time (h) Carbon Source Concentration (mM) 0 0.2 0.4 0.6 0.8 1 1.2 1.4 Cell Growth (OD600) H26 glpR 0 5 10 15 20 25 0 10 20 30 40 50 60 70 80 Time (h) Carbon Source Concentration (mM) 0 0.2 0.4 0.6 0.8 1 1.2 1.4 Cell Growth (OD600) H26 glpR

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213 A Figure 6-11. Glycerol and fruc tose are co-metabolized in H. volcanii The parent strain H26 and glycerol kinase ( glpK) mutant KS4 exhibit similar growth rates, cell yields and carbon utilization patterns when grown on Fru MM and Gly Fru MM with each carbon source supplemented at 20 mM (A) or 5 mM (B). Growth at 42C (200 RPM) was monitored by an increase in OD600, where 1 U was equivalent to approximately 109 CFU per ml for all strains. At various time points, supernatant fractions were withdrawn from both parent H26 and KS4 cultures and analyzed by HPLC for fruc tose or glycerol consumption as previously described (Sherwood et al ., 2009). Experiments were performed in triplicate, and the means SD were calculated. Cell growth and carbon utilization levels are indicated.

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214 B Figure 6-11. Continued

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215 Figure 6 12. Genomic regions upstream of glpR pfkB and kdgK1 include a conserved inverted repeat that may serve in GlpR binding and transcriptional repression Regions upstream of glpR pfkB and kdgK1 were analyzed for potential GlpR binding site(s) such as inverted repeats near putative Shine Dalgarno (SD) sites and promoter elements including the TFB responsive element (BRE) and TATA box (consensus sequence CRNA AT for BRE and TTTAWA for TATA box wher e W is A or T, R is A or G and N is any nucleotide base). Residues matching the consensus BRE/TATA box sequence are boxed and highlighted in grey. SD sites are indicated by a line above the DNA sequence, and the translational start codon is double underlined. An inverted hexameric repeat with a consensus sequence TCSNCN(3 4)SSN GGA (where S is C or G and N is A, C, G, or T ) was found common to the glpR pfkB and kdgK1 promoter regions. Inverted repeats are indicated with arrows displaying directionality. TCGGAATCCAA C A TAAA CAA T T AA CACTACTCCCCGACCTACCTTCCTCGTAACG AGGA A TCGCCACCG ATG AATCAACCGCGGGCGCGAACCTCGTCGGCAACG C C G G AC G TTTATA TCGCGCCTGCG A ACG G CCGTCC ATG Putative GlpR Binding motif : TCSnC SSn GGA glpR C CT TC C T C GTAA CG A GGA AT C kdgK1 TC G TC G G C AAC GC C GGA CGT BRE/TATA BRE/TATA SDglpR kdgK1 TCGGAATCCAA C A TAAA CAA T T AA CACTACTCCCCGACCTACCTTCCTCGTAACG AGGA A TCGCCACCG ATG AATCAACCGCGGGCGCGAACCTCGTCGGCAACG C C G G AC G TTTATA TCGCGCCTGCG A ACG G CCGTCC ATG Putative GlpR Binding motif : TCSnC SSn GGA glpR C CT TC C T C GTAA CG A GGA AT C kdgK1 TC G TC G G C AAC GC C GGA CGT BRE/TATA BRE/TATA SDglpR kdgK1

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216 A B Figure 6-13. H. volcanii GlpR purifies as a tetramer by gel filtration. A) Standards of thyroglobulin, -amylase, alcohol dehydrogenase, BSA, carbonic anhydrase, and cytochrome c were used determine t he molecular weight of GlpR. The regression obtained upon plotting Kav (x-axis) and log(mo lecular weight) (yaxis) was linear (R2 = 0.9904). B) After StrepT actin chromatography, GlpR was purified to homogeneity by Superde x-200 chromatography using a low salt buffer (100 mM Tris pH 8.0 with 150 mM NaCl). GlpR eluted at a single peak consistent with a tetramer based on the molecular weight standards and the predicted molecular weight of GlpR. 0 0.5 1 1.5 2 2.5 3 00.10.20.30.40.50.60.7 Kavlog(Molecular Weight)Thyroglobulin -amylase Alcohol dehydrogenase BSA Cytochrome anhydrase Cytochrome c GlpR R2= 0.9904 0 0.5 1 1.5 2 2.5 3 00.10.20.30.40.50.60.7 Kavlog(Molecular Weight)Thyroglobulin -amylase Alcohol dehydrogenase BSA Cytochrome anhydrase Cytochrome c GlpR Thyroglobulin -amylase Alcohol dehydrogenase BSA Cytochrome anhydrase Cytochrome c GlpR R2= 0.9904 0 0.002 0.004 0.006 0.008 0.01 0.012 0.014 0.016 0.018 0.02 05101520253035 Elution Volume (ml)Absorbance at 280 nm

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217 Figure 6 14. H. volcanii GlpR purifies to homogeneity after tandem StrepT actin and gel filtration chromatograp hy GlpR was Cterminally StrepII tagged and purified by StrepT actin chromatography (Lane 1, 0.75 g ) followed by gel filtration chromatography (Lane 2, 2.3 g ). Purified fractions Precision Plus ProteinTM Kaleidoscope molecular mass marker ( Mr Std diluted at a ratio of 1:20 according to the S uppliers recommendations ) were boiled for 10 min in 20 l of SDS PAGE buffer and were separated by 12% SDS PAGE. Gels were stained with Coomassie Blue Under denaturing conditions, the GlpR monomer was es timated to be approximately 30 kDa based on gel migration, consistent with the predicted size based on primary amino acid sequence analysis. 230 150/100 80 60 50 40 30 25 20kDa1 2 MrStd 230 150/100 80 60 50 40 30 25 20kDa1 2 MrStd

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218 CHAPTER 7 SUMMARY AND CONCLUSIONS Summary of Findings The goals of this study ar e to advance our understanding of the molecular mechanisms surrounding carbon metabolism and its regulation in haloarchaea using H. volcanii as a model organism. A combination of bioinformatic and molecular techniques were employed to identify and characterize the enzymes responsible for glycerol catabolism and the mechanisms controlling their regulation. A DeoR/ GlpRtype transcriptional repressor of glucose and fructose metabolism was additionally identified and characterized. This study demonstrated that H. volcanii glycerol metabolism proceeds primarily through an ATP dependent GK and a chromosomally encoded NAD+dependent G3PDH. Neither a mutant deficient in glpK or gpdA1 was able to grow in minimal medi um containing glycerol as the sole carbon source, although both knockout mutations could be complimented by providing the appropriate gene in trans Interestingly, a gpdA1 muta t ion could also be complimented by expressing gpdA2 in trans under the control of the constitutive rRNA P2 promoter from H. cutirubrum suggesting that GpdA2 also encodes a functional G3PDH The glpK and gpdA1 transcripts as well as the specific enzymatic activity of their gene products were strongly induced by glycerol and not repressed by glucose. Rather, glycerol was preferentially metabolized over glucose w hen parent cells were grown in minimal media containing equimolar concentrations of glucose and g lycerol. Although glycerol was preferentially used over glucose, glycerol and fructose were coutilized in both parent and mutant strains. B oth glpK and gpdA1B1C1 were transcriptionally linked in an operon which

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219 also contains a putative glycerol facilitator, glpX and a homolog of the bacterial PTS protein Hpr, ptsH2 This operon was further shown to be under the control of a glycerol inducible gpdA1 promoter. This study has also provided evidence that a DeoR/ GlpRtype transcriptional repressor regulates sugar metabolic enzymes in H. volcanii. GlpR repress ed both pfkB encoding PFK and kdgK1 encoding chromosomal KDGK in the presence of glycerol. Repression of pfk B and kdgK1 was relieved when fructose or glucose was provided, respectively. The GlpR interaction site was limited to a 188bp glpR pfkB promoter region and an 89bp kdgK1 promoter region through transcriptional promoter reporter fusion assays. A putative GlpR binding site was predicted within both interaction sites which consist ed of an inverted hexameric repeat, TCSnCn(3 4)SSnGGA (where S is G or C and n is any nucleotide) which either overlaps or is downstream of putative promoter elements. GlpR has be en purified to homogeneity as a tetramer whose monomeric subunits are approximately 30 kDa under both high and low salt from rich, peptidecontaining media. Future Directions Future investigation of H. volcanii glycerol metabolism will focus on its regulation and biotechnological application. Northern blot analysis will be employed to verify the regulation of the primary glycerol metabolic operon by the proposed promoter As previously mentioned, glycerol rich waste streams have been an attractive carbon source for bioconversion to more valuable chemicals. H. volcanii readily grows aerobically on biodiesel waste as a sole carbon source (Figure 71), however, anaerobic fermentation of glycerol has not been characterized. H. volcanii can grow anaerobically using external electron acceptors such as DMSO, trimethylamineN oxide, and nitrate

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220 (Oren and Trper, 1990; van Ooyen and Soppa, 2007) Thus, anaerobic glycerol metabolic pathways in H. volcanii will also be investigated, focusing on the bioindustrial applications. Although the EI and EIIBFru homologs in H. volcanii are needed for fructose metabolism, a more detailed understanding of thei r roles along with additional PTS homologs such as Hpr will be crucial to understanding the evolution and biological function of the PTS in archaea. Future investigation of the GlpR repressor will focus on additional transcriptional targets using microarray analysis of parent and glpR mutant strains grown in the presence of glycerol. The mode of GlpR repression will also be characterized, specifically focusing on the promoter binding si te using florescence anisotropy, electrophoretic mobility shift assays, and DNaseI footprinting assays. In addition, the effector molecule will be demonstrated using calorimetry

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221 Figure 71 H. volcanii grows aerobically on biodiesel waste as a sole carbon source. Minimal media was prepared according to the Halohandbook (DyallSmith, 2008) and as detailed in Chapter 2, with biodiesel waste serving as a the sole carbon sourc e. Glycerol content of the biodiesel waste was analyzed by HPLC and suppl emented in the media to a final concentration of 20 mM. Growth at 42C (200 RPM ) was monitored by an increase in OD600, where 1 U was equivalent to approximately 109 CFU per ml. Experiments were performed in triplicate, and the means SD were calculated. 0.01 0.1 1 10 0 15 30 45 60 Time (h) Cell Growth ( x 109 CFU-1) 0.01 0.1 1 10 0 15 30 45 60 Time (h) Cell Growth ( x 109 CFU-1)

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257 BIOGRAPHICAL SKETCH Katherine Sherwood Rawls was born to Stephen and Frances Sherwood at the Indian River Memorial Hospital in Vero Beach, Florida. From August of 1998 to June of 2002, Katherine attended Vero Beach High School during which time she competed at local, state and international levels of science fair competition in the category of Microbiology After graduating as Salutatorian of her high school class in June of 2002, she received scholarships from the Florida Bright Futures Foundation, the Gator Boosters Foundation, the First Baptist Church of Vero Beach, Florida and the Daughters of the American Revolution to pursue a Bachelors of Science degree in Microbiology and Cell Science at the Univ ersity of Florida. During her undergraduate career at the University of Florida, she performed research in Dr. Julie MaupinFurlows laboratory under the guidance of Gosia Gil Ramadas where she examined the genomic organization of proteasomal genes in H. v olcanii, resulting in her first publication In 2004, Katherine received the University of Florida Presidential Scholar award and was inducted into the Golden Key International Honor Society. I n 2005, she received the University of Florida Institute of Food and Agricultural Sciences (IF AS) Research Internship Award, allowing her to perform research in Dr. Julie MaupinFurlows lab, where she focus ed on the overproduction of H. volcanii TrmA in E. coli She graduated Cum Laude from the University of Florida in May of 2006, after which she received an Alumni Fellowship Award to continue her graduate studies in Dr. Julie MaupinFurlows laboratory. As a graduate student, Katherine has attended numerous conferences including the American Society of Microbiology (ASM) Florida Branch and National Annual Meetings, the Florida Genetics Institute Research Symposium and the Gordon Research Conference. She has additionally served as the graduate conference

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258 organizer for the first, annual Department of Microbiology and Cell Science Undergraduate Research Symposium and as an organizer for the Department of Microbiology and Cell Science Fall 2007 and Spring 2008 seminar series She has received various graduate honors including the Grand Speaker Award (at the 2007 and 2008 Florida Branch ASM Meetings), the first place poster award (at the 2010 University of Florida Departments of Microbiology and Cell Science and Molecular Genetics and Microbiology Joint Graduate Research Symposium), a nomination for the 2007 University of Florida Jack L. Fry Excellence in Teaching award, the Richard and Mary Finkelstein Student Travel Grant (at the 2008 National ASM Meeting), the 2008 IFAS Travel Grant Scholarship, and the 20092010 Doris Lowe and Earl and Verna Lowe Graduate Scholarship. T he work presented in this dissertation has resulted in the publication of two, peer reviewed primary author research articles with a third research article and a review in preparation. On October 24, 2009, Katherine married Colin Douglas Rawls, who recently received a Masters degree in Economics from the University of Florida. Upon completion of her degree, Katherine plans to pursue a government or academic career in molecular biology.