STARCH UTILIZATION I N PAENIBACILLUS SP. STRAIN JDR 2: DEPOLYMERIZATION CAT ALYZED BY CELL ASSOCIATED M ULTI MODULAR AMYLASE By LEI PAN A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2014
Â© 2014 Lei Pan
3 ACKNOWLEDGMENTS I would like to express my deepest gratitude to my committee chair, Dr. James F . Preston. He has been a tremendous mentor for me. I would like to thank him for his time, patience, and understanding and encouraging my research. I would also like to express my appreciation to my committee members Dr. Julie A . Maupin Furlow , Dr. Tony Romeo, Dr. Keelnatham T . Shanmugam for their valuable instructions and insightful comments. This thesis would not have been possible without my great committee. My sincere gratitude also g oes to all current and former lab members. Thanks to Mr. John Rice for putting up with me and training me to be able to walk in a lab. Thanks to Dr. Munsu Rhee, I wish one day I can get molecular and genetic works done with eyes cl osed like him. Thanks to Dr. Virginia Chow for her useful advice for my experiments. Thanks to Lusha, Roy and Victoria. Their technique assistance has been very helpful. Special thanks to Miss Neha Sawhney and Dr. Franz St . John, Casey Crooks for the tran scriptomic studies. pin Furlow technical assistance. Finally, I would like to thank my family and my fellow friends for trusting me, supporting me and encouraging me. Words cannot express my gratefulness.
4 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 3 LIST OF TABLES ................................ ................................ ................................ ............ 3 LIST OF FIGURES ................................ ................................ ................................ .......... 7 ABSTRACT ................................ ................................ ................................ ..................... 8 CHAPTER 1 INTRODUCTION ................................ ................................ ................................ .... 10 Sources and Classification of Amylases ................................ ................................ . 10 Amylase Family ................................ ................................ ................................ ... 11 Starch Utilization in Bacillus subtilis and Paenibacillus sp. Strain JDR 2 ................ 14 2 STARCH UTILIZATION I N PAENIBACILLUS SP. STRAIN JDR 2: DEPOLYMERIZATION CAT ALYZED BY CELL ASSOC IATED MULTI MODULAR AMYLASE ................................ ................................ ......................... 16 Materials and Methods ................................ ................................ ............................ 18 Chemic als and Reagents ................................ ................................ ................. 18 Maintenance of Cultures and Preparation of Inocula ................................ ........ 18 Growth Studies ................................ ................................ ................................ . 19 Total Carbohydrate and Protein Assays ................................ ........................... 20 Modular and 3D Structure Prediction and . 20 Induction of Activity Associated with Cell Surface ................................ ............ 21 Preparation of Fractions from Paenibacillus sp. Strain JDR 2 Cultures ........... 21 Activity Assays ................................ ................................ ................................ . 22 Thin Layer Chromatography ................................ ................................ ............. 23 SDS PAGE Analysis ................................ ................................ ........................ 23 Gel Spot LC MS/MS ................................ ................................ ......................... 24 Starch Uti lization Regulon Identification ................................ ........................... 24 Results ................................ ................................ ................................ .................... 25 Growth and Utilization Analysis of Paenibacillus sp. Strain JDR 2 ................... 25 Modular Architecture and 3D Structure of Amy13A 2 ................................ ......... 26 Phylogenetic Analysis of Amy13A 2 ................................ ................................ ... 27 Activity of Multi modular Amy13A 2 Associated with Paenibacillus sp. strain JDR 2 Cell Surface ................................ ................................ ....................... 28 Distribution of Amylase Activity in Paenibacillus sp. Strain JDR 2 Starch Culture Fractions ................................ ................................ ........................... 29 Analysis of Products from Cell Wall Suspension Digest of Starch .................... 30 SDS PAGE Analysis of Paenibacillus sp. Strain JDR 2 Culture Fractions ....... 31 Protein Identification by Gel Spot LC MS/MS ................................ ................... 31
5 Identification of the Starch Utilization Regulon with RNA seq Evidence .......... 32 Discussion ................................ ................................ ................................ .............. 34 3 CONCLUSION ................................ ................................ ................................ ........ 50 LIST OF REFERE NCES ................................ ................................ ............................... 53 BIOGRAPHICAL SKETCH ................................ ................................ ............................ 60
6 LIST OF TABLES Table page 2 1 Sources and characteristics of sequences used for phylogenetic analysis ........ 38 2 2 Distribution of amylase activity in Paenibacillus sp. strain JDR 2 starch culture fractions . ................................ ................................ ................................ . 39 2 3 Protein identifications based on LC MS/MS. ................................ ...................... 40 2 4 Regulation of candidate genes involved in starch utilization ............................... 41
7 LIST OF FIGURES Figure page 2 1 Growth of and substrate utilization analysis of Paenibacillus sp. strain JDR 2. .. 42 2 2 TLC anal ysis of released products from Paenibacillus sp. strain J DR 2 starch culture supernatant ................................ ................................ ............................. 43 2 3 Modular architecture map of Amy13A 2 . ................................ .............................. 43 2 4 3D structure model of Amy13A 2 ................................ ................................ ......... 44 2 5 Phylogenetic analysis of a set of GH 13 amylase family CD sequences . ........ 45 2 6 Induction of amylase activity and glucanase activity associated with cell surface and in the medium. ................................ ................................ ................ 46 2 7 TLC analysis of pro ducts from cell wall fraction digest of starch . ........................ 47 2 8 SDS PAGE analysis of isolated fractions from Paenibacillus s p. JDR 2 starch induced culture . ................................ ................................ ....................... 48 2 9 SDS PAGE analysis of isolated fractions from Paenibacillus sp. JDR 2 yeast extract or starch culture . ................................ ................................ ..................... 49 2 10 Genomic organization of starch utilization regulon in Paenibacillus sp. strain JDR 2 . ................................ ................................ ................................ ................ 49
8 Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science STARCH UTILIZATION IN PAENIBACILLUS SP. STRAIN JDR 2: DEPOLYMERIZATION CAT ALYZED BY CELL ASSOCIATED M ULTI MODULAR AMYLASE By Lei Pan December 2014 Chair: James F Preston III Major: Microbiology and Cell Science Paenibacillus sp. strain JDR 2 ( Pjdr2 ) is a bacterium derived from decaying wood that secretes enzymes for processing lignocellulose to biofuels and chemicals. Its ability to efficiently digest xylans and metabolize the products of extracellular digestion depends upon secretion of a cell asso ciated GH 10 family endoxylanase containing surface layer homology domains (SLH) that mediate cell association. Genes encoding other glycoside hydrolases identified in the Pjdr2 genome share a common theme for utilization of soluble glucans, including star ch, as well as xylans. One of these encodes a 235 KDa GH 13 family amylase (Amy13A 2 ) composed of carbohydrate binding modules family 48 (CBM 48) and family X25 (CBM like) followed by a GH 13 catalytic domain and three SLH domains on the C terminus. Growth on starch induces the formation of this amylase and resulting in the rapid and complete utilization of starch. Distribution of significant amylase activity in a cell wall fraction and product analysis indicate Amy13A 2 is anchored to the cell surface to depolymerize starch and generates maltose and maltodextrins as major products. A cluster of genes encoding a maltodextrin specific ABC transporter and two amylases was identified by
9 bioinformatic studies and verified by transcript omic studies. This gene cluster is co regulated with and complements amy13A 2 , consisting a starch utilization regulon. This depolymerization and assimilation system provides further insight into utilization of plant polysaccharides catalyzed by extracellul ar glycoside hydrolases anchored via SLH domains. These systems are expected to contribute to the development of biotatalysts for efficient conversion of lignocellulosic biomass to targeted products.
10 CHAPTER 1 INTRODUCTION Sources and Classification of A mylases Amylases are glycoside hydrolases that break down starch molecules to release smaller sugars , including malto dextrin, maltotriose, maltose and glucose , by hydrolyzing glycosidic bonds. As one of the first enzymes produced industrially (Pandey et al ., 2000) , amylases are one of the most widely used industrial enzymes ac counting for 25% of the global enzyme market (Rajagopalan et al . , 2008) . Applications of amylases have replaced chemical processing in food, detergent, paper and textile industries (Pandey et al . , 2000) due to advantages of amylase catalyzed reactions such as specificity of the reactions, stability of products, low energy investment and no requirement for neutralization steps (Sivaramakrishnan et a l . , 2006) . In addition , the diversified substra te specificity, action pattern and optimal reaction condition of amylases allow adaptation of enzymes with desired properties, contributing to promising application in pharmaceutical and fine chemicals industries. With the expanded spectrum of amylase appl ication into clinical, medicinal and analytical chemistry fields, studies of amylases are gaining sub stantial attention and interest . Amylases have been found in most microorganisms, plants and animals. Three major classes of amylases amylases have been reported . Amylases (endo 1, 4 D glucan glucohydrolase, EC 188.8.131.52) are enzymes that act random ly at locations along the starch chain, breaking down the polysaccharides, and release maltose, maltotriose from amylose, or glucose, maltose and ma ltodextrins from amylopectin. The amylases are widely found in bacteria and fungi. Amylase producing strains from Bacillus sp., including Bacillus amyloliquefaciens and Bacillus
11 licheniformis have been commercial ly applied amylases serve as major digestive enzymes. P resence of amylase in plants has also been reported. Amylase ( 1, 4 glucan maltohydrolase, EC 184.108.40.206) cleave s at the 1, 4 glycosidic bond of non reducing ends of amylose , amylopectin and also glycogen molecules , releasing one maltose ( anomeric form) at a time . Because amylase is unable to digest 1, 6 glycosidic linkages in amylopectin, it results in incomplete degradation of the polysaccharide chain , yielding 50 60 % maltose and a limit dextrin. This form of enzyme is usually from plant origin . Only a few studies have reported amylase from microbes . Amylase (exo 1, 4 D glucan glucanohydrolase, EC 220.127.116.11) hydrolyzes 1, 6 glycosidic linkages as well as 1, 4 glycosidic linkages at the non reducing ends of amylose and amylopectin, releasing a single glucose unit every time. A mylase can be found in a number of sources, such as plants, animals and microorganisms. Even though these three amylases have closely related functions and substrate specificit y, they are structurally different and evolutionarily distant ( , 1994) . The main amylase from amylase and amylase besides their different attacking site and products is the mechanism be hind the hydrolase activity. A mylase uses a retaining mechanism while the other two use an inverting mechanism ( , 1997) . Amylase F amily With their ability to depolymerize starch, enzymes from the amylase family have received the most attention for applications . Starch is one of the most abundant polysaccharide s on the earth . 1, 4 linked glucose polymers, and amylopectin, which is composed of 1, 4 linked glucose 1, 6 linkages. Based on four ty pes of reactions for
12 synthesis or hydrolysis of glucosidic bonds of starch, conventional classification of amylases and related enzymes are divided into four types: 1, 4 1, 6 glucosidic linkages; pullulanase (EC 18.104.22.168) or isoamylase (EC 22.214.171.124); (iii) 1, 4 glucosidic linkages; cyclodextrin glucanotransferase (CGTase, EC 126.96.36.199); and (iv) 1, 6 glucosidic linka ges; branching enzyme ( EC 2.4. 1.18). E xceptional examples of amylases and related enzymes continue to be reported in addition to the main reaction. Some amylase s have been reported to catalyze hydrolysis of 1, 6 glucosidic linkages (Hehre et al . , 1971 ) . P ullulanases from thermophiles have been reported to hydrolyze not only 1, 6 1, 4 glucosidic linkages ( Kuriki et al . , 1988a, Kuriki et al . , 1988b ) , and neopullulanase catalyzes all four types of reactions ( Imanaka and Kuriki , 1989 ). Thus t he boundaries between the four types of enzymes that have been classified specifically 1, 6 1, 4 glucosidic linkages become less clear. Further studies comparing structures revealed four h ighly co nserved regions through out all four types of amylases and related enzymes despite low overall sequence similarity (Nakajima et al . , 1986; , 2002 ) . The four conserved regions contain three amino acids, Asp168, Glu200 and Asp261 ( B. s ubtilis amylase numbering), determined either by X ray crystallography or site directed mutagenesis (Holm L . , 1990; Vihinen et al . , 1990; Klein et al . , 1992 ; Mathupla et al . , 1993; Podkovyrov et al . , 1993; Takata et al . , 1994 ; Penninga et al . , 1995 ) , that were suggested to play important roles in catalysis. The common feature of the structures of
13 all these four types of enzymes is a TIM barrel ( , 1997 ) , a conserved protein strands t hat alter nate along the peptide backbone ( Banner et al., 1975 ). Besides the sequence similarity, a common catalytic mechanism has been identified for all four types of enzymes ( Takata et al . 1992 ) . Based on the sequence similarity and common catalytic am ylase family was re defined ( Kuriki and Imanaka , 1999 ) as enzymes fit four requirements: i) cleavage of glucosidic linkages; ii) retaining mechanism that results in release of anomeric mono or oligosaccharides or formation of gluc osidic li nkages by transglycosylations; iii) four highly conserved regions in their primary structure that contain all catalyti c and substrate binding sites; iv) Glu (p roton donor) and Asp (n ucleophile) amino acid residues serve as catalytic sites in a TIM barrel catalytic domain. amylase family by the new definition have been greatly expanded. Takata and coworkers (1992) demonstrated that amylase, pullulanase, isoamylase, CGTase, the branching enzymes, glucosid ase, oligo 1, 6 glucosidase, amylopullulanase, and neopullulanase belong to the amylase family. Subsequently, Svensson (1994) added cyclodextrinase, dextran glucosidase, amylomaltase, and glycogen debranching enzyme to the family. On the basis of simi larities in amino acid sequences, Henrissat (1991) proposed a classification of glycoside hydrolases into more than 45 families. This classification is useful for indicating the intr insic structural features of enzyme s , which act on several distinct substr ates. The classification system has been updated with the availability of an increasing number of primary sequences ( Henri ssat and Bairoch , 1996; Henrissat and Davies , 1997 ) and has been improved by
14 taking into account the catalytic mechanisms ( Davies and Henrissat , 1995 ) . Accor ding a amylase form their own families , GH 14 and 15 families, respectively, while most members of the amylase family are grouped into the GH 13 family. Starch U tilization in Bacillus subt ilis and Paenibacillus sp. S train JDR 2 The G ram positive soil bacterium B. subtilis can utilize starch as a carbon source. Prior to transport through the cell membrane, the polysaccharide must be depolymerized by the extracellular amylase AmyE ( Ishikura et al ., 1977 ) into oligosaccharides as maltose and maltodextrins . The released maltose and maltodextrins are assimilated and serve as carbon source in B. subtilis . Previous s tudies ( Thompson et al . , 1998 ; Reizer et al . , 1999 ) suggested that maltose uptake occurs via a phosphoenolpyruvate dependent phosphotransferase system (PTS) (Postma et al . , 1993) . Evidence for this uptake is b ased on identification of coding sequence for a NAD(H) dependent phospho 1, 4 glucosidase (MalA) (Thompson et al . , 1998) located in an operon together with a maltose specific enzyme ( IICB Mal ) (Yamamoto et al . , 2001) . In addition, a gene cluster containing a potential maltodextrin specific A TP b inding c assette (ABC) including transporters ( Kamionka an d Dahl , 2001 ) and enzymes for further processing phosphorylated maltose and maltodextrins were identified ( SchÃ¶nert et al . , 1998; SchÃ¶nert et al ., 1999; Cho et al ., 2000 ) . SchÃ¶nert and colleagues ( 2006) proposed the current model of maltose and maltodextrin utilization in B . subtilis : maltose is taken up by the PTS and becomes phosphorylated. The maltodextrin specific ABC transporter composed of maltodextrin binding protein ( MdxE ) and membrane spanning components ( MdxF and MdxG ) takes up maltodextrins without phosphorylation . Cytoplasmic maltose P is hydrolyzed by MalA, resulting in glucose and
15 glucose 6 P. As m altodextrins enter the cell, they are degraded by the concerted action of cytoplasmic maltogenic amylase or neopullulanase ( YvdF ) , maltose phosphorylase (YvdK) , and glucosidase (Mal L ) for subsequent processing through glycolysis. Starch utilization in Paenibacillus sp. strain JDR 2 would be expected t o share similarity with that of B . subtilis consid ering the overall similar genetic background shared by Pjdr2 and Bacillus species. However, the amylase responsible for depolymerization of starch in Pjdr2 is a multi modular enzyme th at is phylogeneticall y distant from well studied AmyE of B . subtilis. In addition, although potential ABC transporter s were identified by homology search of maltodextrin specific ABC transporter of B . subtilis , Pjdr2 not only lacks orthologs of maltose specific enzyme IICB Mal from B . subtilis , but also does not have genes encoding enzymes responsible for processing phosphorylated maltose. T he objective of this study is to determine the role of the multi amyl ase in the extracellular depolymerization process and transport system of oligosaccharides for the utilization of starch by Pjdr2 . Experimental a pproaches included physiological studies to determine the efficiency of starch utilization, bioinformatic studi es to make functional predictions and phylogenic relationships, distribution of amylase activities in cell and extracellular fractions and characterization of the cell associated amylase with respect to product formation. The results presented in this stud y provide an insight into the molecular basis of starch utilization and the role that extracellular glycoside hydrolases anchored via SLH domains play in the efficient utilization of plant polysaccharides in Pjdr2 and other bacteria.
16 CHAPTER 2 STARCH UTILIZATION I N PAENIBACILLUS SP. STRAIN JDR 2: DEPOLYMERIZATION CAT ALYZED BY CELL ASSOC IATED MULTI MODULAR AMYLASE Paenibacillus sp. strain JDR 2 ( Pjdr2 ) was isolated from decaying sweet gum wood . Its ability to not only rapidly but also complete ly utilize methylglucuronoxylan (MeGXn), the major component of hemicellulose , was characterized (St . John et al ., 2006) and revealed a process of plant polysaccharide utilization initiated by depolymerization catalyzed by a cell associated multi modular glycoside hydrolase followed by immediate assimilation of depolymerization products . Previous studies have shown the xylan utilization regulon of Pjdr2 includes a gene encoding a cell associated multi modular GH 10 family endoxylanase (Xyn 10 A 1 ) that cataly zes production of xylobiose, xylotriose and methylglucuronotriose ( MeGX 3 ) and an aldouronate utilization gene cluster ( Chow et al ., 2007) with genes encoding signal transduction regulatory proteins, ABC transporter proteins, a GH glucuronosidase (Agu 67 A), a GH10 endoxylanase (Xyn 10 A 2 xylosidase (Xyn 43B ) . Expression of this gene cluster and xyn 10 A 1 is regulated at a highly coordinated level in terms of induction by MeGXn and repression by glucose. This regulon confer s the ability for efficient assimilation and intracellular catabolism of products generated by Xyn 10 A 1 on the cell surface. These findings together with the observation of minimal accumulation of extracellular products during exponential growth phase of Pj dr2 in MeGXn and a 2.8 fold greater utilization rate of MeGXn than that of MeGX 3 indicate products of depolymerization catalyzed by Xyn 10 A 1 are assimilated as they are formed at the cell surface (Nong et al ., 2009) . Th is provides a dynamic coupling process that c ould be advantageous to the bacterium in its ecological niche and also advantageous
17 for development of biocatalysts for the rapid and efficient processing of hemicellulosic biomass to targeted products. The sequenced genome (Chow, 20 12) of Pjdr2 allowed identification of a collection of genes encoding glycoside hydrolases catalyzing polysaccharide depolymerization with similar molecular architecture to the Xyn10A 1 , including a GH glucanase, a GH 43 arabinofuranosidase and a GH 13 amylase. Physiological studies of Pjdr2 provide evidence for aggressive utilization of polysaccharides that co mprise hemicelluloses and water soluble glucans such as barley glucan, arabinan and starch , suggesting a common model in Pjdr2 which confer s a significant metabolic potential in utilizing various polysaccharides initiated by cell associated glycoside hydrolases. The common molecular architecture of these glycoside hydrolases , consisting of carbohydrate binding modules ( CBM ) , a catalytic domain an d SLH domains, may represent a paradigm for the efficient extracellular depolymerization of plant derived polysaccharides catalyzed b y cell associated enzymes . To further define this model in Pjdr2 , characterization of these enzymes and transcriptional responses to growth conditions affecting different polysaccharide utilization systems are both necessary and important. In this study, we have investigated properties of secreted modular amylase and the utilization of starch by Pjdr2 . amylase Amy13A 2 that is a candidate to catalyze the depolymerization of starch at cell surface. SLH domains on the C terminal end of the enzyme suggest Amy13A 2 depolymerizes starch a t the cell surface (Matuschek et al ., 1994; Egelseer 1995 ; Mesnage et al ., 2000; May et al ., 2006) and generates mainly maltose and maltodextrins with a small amount of glucose at the cell surface . In contrast to the starch utilization system in B . subtilis ,
18 which transports maltose via PTS , transportation of both maltose and maltodextrins in Pjdr2 may be effected by an ABC transporter. A gene cluster that is co regulated with Amy13A 2 encodes this ABC transporter, together with two additional GH 13 amylases . W e propose that w ith induction of starch, the multi modular enzyme gene and the gene cluster will be expressed. Amy13A 2 is secreted and anchor ed to the cell surface to depolymerize polysaccharide starch. Once the oligosaccharides are r elease d , they may be transported by ABC transporter proteins , followed by intracellular processing of the assimilated oligosaccharides . Materials and Methods Chemicals and Reagents Oat spelt xylan, potato starch, maltose, maltotriose, maltotetraose , DNAse I were obtained from Sigma Aldrich Co. ; LLC. Barley glucan was obtained from Megazyme International Ireland . Components of Zucker Hankin minerals, sodium phosphate buffer, imidazole buffer and chemicals used in phenol sulfuric acid carbohydrates as say , cell lysis and thin layer chromatography were either purchased from Sigma Aldrich Co. LLC . or Thermo Fisher Scientific Inc. BCA assay kit was from Pierce Biotechnology, Inc. Maintenance of C ulture s and Preparation of I nocul a A p rotocol adapted from St . John and coworkers (2006) was used for maintenance of Pjdr2 cultures and preparation of inocula. Growth of Pjdr2 was performed at 30Â°C. Unless stated otherwise, cultures in tubes were set on a tube rack fixed to a New Brunswick G 2 gyratory shaker platfo rm with an approximately 45Â°C angle sh aking at 200 rpm rotation speed . C ultures i n baffle flasks were incubated at 30Â°C with 180 rpm rotation speed. The optical density (OD) readings of cultures were
19 measured with a Beckman DU500 series spectropho tometer a t 600 nm using 1 ml cuvettes and a path of 1.00 cm. Each time a culture was prepared for study, a sample from the cryostored stock culture was transferred and streaked on 0.5% (w/v) oat spelt (OS) xylan Zucker Hankin minerals (ZH) (Zucker and Hankin , 1970) agar medium supplemented with 0.5% yeast extract (YE). After 48 to 72 h of growth, a single colony with an expected size and surrounded by a clearing zone was picked and suspended in 1% YE ZH medium and inoculated into 3 mL of fr esh 1% YE ZH medium. After overnight incubation, an aliquot was taken. Cells were harvested and washed with ZH minerals by centrifugation using a Fisher Scientific accuSpin Micro 17 cen trifuge at 17,000 x g for 5 min and suspended in an aliquot of 3 mL of 1% YE ZH medium and inoculate back. Cultures in exponential growth phase (OD 600 approximately 0.8) were used as inocula for subsequent studies. Growth S tudies All substrates were added to a defined concentration based upon total carbohydrate before inocula tion . Cultures were maintained in 16 by 100 mm test tubes containing 4 mL of 0.2% starch or maltose substrate in ZH minerals supplemented with 0.1% YE. Inocula prepared as described above were harvested by centrifugation using a Fisher Scientific accuSpin Micro 17 centrifuge at 17,000 x g for 5 min to remove supernatant. The pellet was suspended in 4 mL of medium with substrates and inoculated to give a starting OD 600 at around 0.06 . Incubation was performed as described above. A duplicate set of cultures was monitored . Growth of cultures was evaluated by measuring OD 600 as described above. Samples from cultures were harvested by centrifugation as above. All supernatant s and pellets w ere stored at 20Â°C
20 for analysis of total carbohydrates , individual carbohydrates by T LC analysis and protein concentration . Total Carbohydrate and Protein A ssays T otal carbohydrate concentration related to substrate preparations and utilization was determ ined by a phenol sulfuric acid assay ( Dubois , 1956 ) . For biomass determination, cell pellets collected from cultures were suspended in 200 ÂµL of 1 N NaOH and incubated in a water bath at 85Â°C for 10 min. Samples were cooled to room temperature, neutralized with an equal volume of 1 N HCl, and analyzed for total protein concentration by using the bicinchonin ic acid (BCA) protein assay kit with bovine serum albumin (Pierce Chemical Co . ) as the standard. Modular and 3D Structure P rediction and Sequence of Pjdr2_5200 ( Amy13A 2 ) was retrieved from the Pjdr2 genome sequence . Modular architecture of Amy13A 2 was determined with Conserved Domain Database at NCBI (Marchler Bauer et al ., 2005) . The 3D structure of Amy13A 2 without signal peptide was predicted and modeled with RaptorX ( KÃ¤llberg , 2012 ) . The catalytic domain (CD) sequence was extracted and used for phylogenetic analysis. Thirty three amyla ses and amylopullulanases from G ram positive bacteria were selected from p rotein B last ( Altschul , 1990 ) results and the CAZy database (Henrissat, 1991; Davies and Henrissat , 1995 ; Henri ssat and Bairoch , 1996; Henrissat and Davies , 1997 ) . Fifteen of these sequences were trimmed to contain only the highly conserved catalytic domain s and presented in this study. All proteins selected for sequence comparisons have been either experimentally characterized or crystallized . These sequences were aligned using MUSCLE ( Edgar , 2004) and a phylogenetic tree wa s constructed using MEGA 5.5 (Tamura et al ., 2011) . Activity triads
21 of these sequences were identified either from Uniprot database (Uniprot , 2008) o r using PDBsum ( Laskowski , 2001 ) . Induction of Activity Associated with Cell S urface Inocula prepared as described above were used to inoculate 4 mL of 0.5% starch or barley glucan in ZH medium supplemented with 0.2% YE. Cultures were incubated as described before . Three ml of mid l og phase cultures (OD 600 approximately 1.0) were harvested by centrifugation using a Sorvall RC 3 general purpose centrifuge at 2 , 200 x g for 30 min at 4 o C. The supernatant was concentrated using Amicon Ultra 15 10K MCO centrifugal Filter Unit and sample v olume was adjusted to 750 ÂµL with 50 mM sodium phosphate buffer , pH 6.8 . The pellet obtained from centrifugation was suspended in 3 mL of 50 mM sodium phosphate , pH 6.8, and harvested again by centrifugation as described above. T he wash ing step was repeated except using 3 mL of 50 mM sodium phosphate , pH 6.8, with 0.5M NaCl. The pellet was suspended in 750 ÂµL of 50 mM sodium phosphate buffer, pH 6.8 . The concentrated supernatant and washed cell suspensions were kept on ice until use . Preparation of F ractions from Paenibacillus sp. S train JDR 2 C ulture s Inocula (0.1 mL) prepared as described above were added to 50 mL of 0.5% of starch in Zucker Hankin minerals medium supplemented with 0.2% yeast extract in 125 mL baffle flasks and incubated as described above . Cells were harvested by centrifugation using a Sorval RC 5B super speed centrifuge with SS 34 rotor at 7 , 650 x g for 20 min at 4 o C. The medium supernatant was concentrated using an Amicon Ultra 15 10K MCO centrifugal Filter Unit by centrif uge using Sorval RC 3 general purpose centrifuge at 2 , 200 x g at 4Â°C and used as medium supernatant concentrate (MSC) . The cell pellets were suspended with cold 50 mM sodium phosphate, pH 6.8, and
22 harvested by centrifugation as described above. The washing step was repeated with cold 50 mM sodium phosphate , pH 6.8 , with 0.5M NaCl. The cell pellet was then suspended in 50 mM sodium phosphate , pH 6.8, and lysed by 2 passes through a French pressure cell at 16000 psi. DNA se I ( Sigma Aldrich Co. LLC. ) was added to 1U /mL and i ncubated for 20 min at room temperature. The lysate was centrifug ed using a Beckman L8 70M centrifuge with 70 Ti rotor at 165 , 000 x g for 20 min at 4Â°C . The supernatant was collected and concentrated as described above and designated as cell free extraction (CFE) . The pellet was washed with and suspended in 50 mM sodium phosphate and designated as cell wall suspension (CWS) . All fractions were stored on ice for subsequent studies. Activity A ssays The glucanase activity assay me thod was adapted from Kenealy and Jeffries (2003) . Reactions were performed in 13 by 100 mm test tubes. Reactions were started by adding 250 ÂµL of prepared supernatant s , cell suspensions or culture fractions to 1 mL of 0.5% starch or barley glucan in 50 m M imidazole , pH 6.8 . Control reactions were set up in the same way except reaction substrates did not contain polysaccharides and the addition of culture fractions were replaced by 50 mM sodium phosphate buffer, pH 6.8 . Reactions were incubated in a 37 o C water bath. For every 20 min a 50 ÂµL sample was taken from each re action and diluted with 450 ÂµL of deionized water and added to 500 ÂµL of BCA working reagent. The working mixtures were then centrifuged at 12,000 x g for 5 min and a triplicate of 200 ÂµL of the supernatants were transferred to 96 well plates. The plates were incubated in oven at 80 o C for 45 min to develop a purple color that correlates with reducing sugar concentration. Maltose at concentrations ranging from 0 to 200 Âµg /mL was used as a sta ndard. Plates were
23 cooled to room temperature and the accumulation of reducing sugar in each reaction was evaluated by monitoring absorbance at 560 nm over time . Thin Layer C hromatography TLC analysis (Bounias , 1980) was performed using 20 by 20 cm, 0.25 m m thickness, Silica Gel 60 plates (EM Laboratories, Inc.). Glucose, maltose, maltotriose and maltotetraose containing 10 nmoles of glucose carbohydrate equivalents were spotted on same lane as standards. Plates were developed twice with chloroform acetic a cid water (6:7:1, vol/vol) using 3 hrs for each run. The plates were air dried overnight and sprayed with 6.5 mM N (1 naphthyl) ethylenediamine dihydrochloride in methanol containing 3% (vol/vol) sulfuric acid. The stained plates were baked in an oven at 90Â°C for 10 min for visualization of carbohydrates. The interpretation of products is approximate due to effects of the components of the medium and reaction buffer on mobility. For analysis of substrates and products in culture supernatants, 50 ÂµL of supernatant samples from starch culture during growth studies were spotted on a plate. For analysis of products from ce ll wall suspension digest of starch, digestion reactions were set up as described in activity assays using cell wall suspension as Amy13A 2 crude extract and starch as substrate. Samples from 0 min, 30 min, 60 min and 120 min were used to perform TLC analys is . SDS PAGE A nalysis Samples from MSC, CL, CFE and CWS were normalized to 20 Âµg of protein with a few exceptions and reducing SDS PAGE was performed using Bio rad Mini with 25 mM Tris, 192 mM glycine, 0.1% SDS, pH 8.3 running buffer and Laemmli loading buffer (Laemmli , 1970 ) . Gels were developed at 100 V for 15 min and then 200 V for 15 min. Gels were incubated in C oomassie blue
24 stain with gentle rocking for 15 min and rinsed twice in deionized water followed by overnig ht incubation in destaining buffer on a rocker. Gel S pot LC MS/MS Protein bands at 240 kDa (STH A), 80 kDa (STH B), 50 kDa (STH C) on CWS fraction from cells grown on starch and at 160 kDa (BGL A) and 30 kDa (BGL B) on CWS fraction from cells induced by barley glucan were excised from SDS PAGE gels and rinsed with filtered deionized water. Samples were sent to Interdisciplinary Cente r for Biotechnology Research ( ICBR ) at UF for Gel Spot LC MS/MS to identify the protein bands ( Blouzard et al ., 2010 ) . Starch Utilization R egulon I dentification Candidate genes encoding glycoside hydrolases that have been shown to participate in starch utilization have been identified from genome sequence of Pjdr2 using IMG database (Markowitz et al ., 2012) . Potential oligosaccharides transporter systems for were identified in two ways: i ) maltose and maltodextrins transporter genes characterized and reported in B . subtilis studies were used to blast ( Altschul , 1990) against the Pjdr2 genome sequence to iden tify putative orthologs . ii ) protein protein interaction s of candidate glycoside hydrolases were predicted using STRING database (Szklarczyk et al ., 2011) to map genes encoding function related proteins. The results from the two methods were combined and r efined to give a preliminary candidates list of starch utilization system in Pjdr2 . RNA samples of Pjdr2 in individual polysaccharides, oligosaccharides and YE control cultures were isolated from mid log phase, purified and sent to the Joint Genome Instit ute , Walnut Creek, CA, for RNA sequencing. RNA sequence data were processed using DNAStar and normalized by determining the ratio of linear RPKM
25 (Reads Per Kilobase per Million reads sequenced) obtained for RNA derived from Pjdr2 cultures grown on 0.5% starch in 0.5% yeast extract compared to 0.5% yeast extract alone (Sawhney et al . , 2014 submitted). Results Grow th and Utilization A nalysis of Paenibacillus sp. Strain JDR 2 Pjdr2 had comparable growth rates with the oligosaccharide maltose and polysaccharide starch as substrates (Figure 2 1A). The cells exhibited a rapid growth phase between 6 and 14 h both on starch and maltose as determined by measuring the OD 600 of the cultures . The rapid decline follow ing the rapid growth phase was cell lysis associated with sporulation. A similar pattern was obtained for growth based on total cell protein assays (Figure 2 1B). Substrate consumption mirrored the growth curves for each substrate (Fig ure . 2 1A), indicating that growth was quantitat ively correlated with the consumption of each substrate. Both substrates were utilized by Pjdr2 with no significant difference in consumption rates and both substrates were depleted in less than 14 h. TLC analysis of medium supernatant samples (Figure 2 2) showed that starch was depolymerized to generate maltose, maltodextrins and a small amount of glucose , which were consumed within 18 h. The results from preliminary growth and utilization analysis reveal that Pjdr2 has an efficient utilization system of s tarch that allows the strain to depolymerize starch to consume the oligosaccharides formed. Transient accumulation of maltodextrins was observed suggesting that the depolymerization of starch and assimilation of the products may not be coupled processes as in the case of utilization of methylglucuronoxylans (Nong et al. , 2009) .
26 Modular Architecture and 3D S tructure of Amy13A 2 Search of the Pjdr2 genome sequence for starch degrading enzyme s led to the identification of amy13A 2 , a large modular amylase gene of 6,483 nucleotides ( encoding 2160 amino acids). Conserved domain identification reveals Amy13A 2 is a multi modular protein like other glycosyl hydrolases identified and characterized in Pjdr2 (Figure 2 3 ). The protein contains a triplicate of SLH modules at the C terminal end, suggesting a secreted Amy13A 2 that may be integrated into surface protein layer and display ed on the surface of cell wall. Other domains within the N terminal region include duplicates of a CBM 48 family domain and a X25/CBM X family domain , in a n CMB 48 X25/CBM X X25/CBM X CBM 48 order. CBM 48 family domains are found in enzymes with a GH 13 family catalytic domain . CBM X family domains, or X25 domains, were first identified in Bacillus acidopullulyticus pullulanase (Turken burg et al ., 2009) . Both CBM X and 48 domains are suggested to target mixed alpha 1, 4/alpha 1, 6 linked D glucan polysaccharides. A single GH 13 catalytic domain and two fibronectin type III (FN3) domains were also identified . The GH 13 family catalytic domain has an activity triad composed of three amino acids: an Asp as a nucleophile, a Glu as a proton donor and an Asp as transition state stabilizer. Unlike the inverting mechanism, which usually utilize s two amino acid resid ues that act as acid and base , respectively , to cleave the glycoside bond in one step, Amy13A 2 is a retaining hydrolase that operate s in a two step process. The nucleophile Asp attack s the glycoside bond , resulting in the formation of a glycosyl enzym e int ermediate with acidic assistance prov ided by the acidic proton donor Glu, followed by the now deproton at ed acidic carboxylate which acts as a base and assists a water molecule to hydrolyze the glycosyl enzyme intermediate, releasing the hydrolyzed product. The fibronectin type III domain is usually found in animal
27 extracellular and intracellular proteins but also appear s in bacterial extracellular glycosyl hydrolases (Little et al ., 1994) . It s function in bacterial glycosyl hydrolases has been suggested to be promoting hydrolysis by modifying the surface of substrate ( Kataeva et al ., 2002 ) . The predicted 3D structure of Amy13A 2 is modeled base d on templates of each domain obtained by aligning domain sequences with known PDB entries (Berman et al ., 2000) . The templates were rendered using RaptorX to construct the final 3D structure. The 3D structure demonstrates that the CBMs reach out and fold back to GH 13 CD to form an active pocket while SLHs are exposed for anchoring. Phylogenetic A nalysis of Amy13A 2 The Amy13A 2 sequence together with fifteen selected GH 13 family enzymes (Table 2 1 ) were trimmed to only contain the catalytic domain and used for multiple alignment analysis (Figure 2 5A ). The alignment results are consistent with previous reports of a few highly conserved sequence regions and four completely invariant amino acids of members of amylase family ( Nakajima et al . , 1986; , 2002 ) . All the sequences show four well conserved sequence regions (the regions I, II, III, IV). T he four invariant amino acids Arg166 plus the activity triad Asp168, Glu200 and Asp261 ( B . subtilis strain amylase numbering ) are conserved throughout the selected sequences. There are three other c onserved regions (the regions V , VI and VII) that have been show n to conserve in amylases ( Svensson , 1988 ; MacGregor and Svensson, 1989; Jespersen et al ., 1991; Jespersen et al ., 1993 ). However, region V seems to be variable in the alignment , which is inconsistent with the previous reports . R egion VI and VII are well conserved throughout all sequences (alignment not shown), thus only provid ing limited information. Of particular interest is conserved sequence region II. This region contains one invariant Arg locate d at a position two amino acids before the nucleophile
28 Asp and has been shown to contain signature amino acids for different amylase family members (Macgregor et al ., 2001) . The alignment result of region II shows the sequence of last four amino acids of this region fall into 3 different groups (VANE, VENE a nd AVKH). Although there are small variations (substitution s with amino acid of similar physiochemical properties ), generally sequences that have been characterized with specificity on alpha 1, 4 and alpha 1, 6 linkage fall into the first 2 groups and these sequences have been correlated with specificity on alpha 1, 4 linkage belong to the third group. This indicates that Amy13A 2 , unlike well characterized B . subtilis strain GH 13 amylase, may hydrolyze both alpha 1, 4 and alpha 1, 6 linkages. The dist ance between nucleophile (Asp in region II) and proton donor (Glu in region III) of these sequences (Table 2 1 ) varies significantly. Three sequences have high similarity with Amy13A 2 share a 39 amino acids distance while others, including several modular sequences from Thermoanaerobacterium species, share a distance of approximately 29 amino acids (~28 to 32 amino acids). The phylogenetic tree derived from sequence alignments of Amy13A 2 and orthologs has identified three highly similar sequences that form clade s for modular sequences from Thermoanaerobacterium and Bacillus species, while single domain GH 13 enzymes from Bacillus species form s another clade . The multiple alignment and phylogenetic tree results suggest the starch utilization system of Pjdr2 d iffers significantly from that of B . subtilis . Activity of M ulti modular Amy13A 2 Associated with Paenibacillus sp. strain JDR 2 Cell S urface The SLH domains in the multi modular glycoside hydrolase structures indicate these enzymes could be integrated into the surface protein layer and depolymerize polysaccharides at the cell surface. To test this, Pjdr2 was grown in starch or barley
29 glucan to induce amylase or glucanase activities associated wi th the cell surface. The mid log phase cultures were harvested and the cells were washed with buffers twice and directly evaluated for hydrolase activity. The culture supernatants were also examined for secreted glycoside hydrolases. Activity was determine d by measuring accumulation of reducing sugar termini in reacti on mix ture using BCA copper method (Figure 2 6). The washed cells from starch culture demonstrated significant amylase activity (Figure 2 6A) indicating that the Amy13A 2 is anchored to and fu nctions on the cell surface. Activity was also detected in the medium supe rnatant, al though it was only 26% of that found in whole cell preparation. These results suggest that another amylase is secreted into medium that cooperat es with Amy13A 2 to accomplish depolymerization of starch. In barley glucan culture, the cell associated glucanase activity was also detected, although it is not the major hydrolase activity compared with activity detected in the medium supernatant (Figure 2 6B). The e xpression of the amylase s or glucanase s is regulated by the presence of corresponding polysaccharides. The amylase activity was only detected when the cells and medium supernatants were from a starch culture while only the cells and medium supernatan ts from barley glucan culture demonstrate glucanase activity (Figure 2 6 ). When Pjdr2 was cultured in minimum medium supplemented with only yeast extract, neither o f these two glycoside hydrolase activities was detected (data not shown). Distribution of Amylase A ctivity in Paenibacillus sp. Strain JDR 2 S tarch Culture F ractions Amylase activity in fractions from Pjdr2 starch culture was examined to determine activity distribution in the cell wall fraction, cytoplasmic fraction and medium
30 supernatant. Based upon amino acid sequence and predicted structure, Amy13A 2 is presumed to be anchored to the cell surface . Search for glycoside hydrolase homologs in Pjdr2 genome revealed there is another putative extracellular amylase and a third putative amylas e that is intracellular . T here fore, if the other two amylase genes are expressed, activity should be detected in both medium supernatant, on the cell wall and in the cytoplasm ic fraction . Pjdr2 starch culture was used to isolate different fractions. These fractions could serve as crude extracts of Amy13A 2 and the two other putative amylases to evaluate distribution of activity. Major amylase activity was detected in cell wall fraction. The ce ll wall fraction showed activity with 0. 13 U nit per 10 8 cells , slightly lower than the activity detected from whole cell suspension (0. 17 U nit per 10 8 cells ). The specific activity of cell wall suspension was almost 2.5 fold higher than that of whole cell suspension since the fraction isolation process partially purified the Amy13A 2 that anchored to cell wall. The cell free extract had 0.0 8 U nit per 10 8 cells activity indicat ing the putative intracellular amylase may play a role in processing depolymerization products inside the cells when Pjdr2 is cultured on starch. The medium supernatant also showed amylase activity, albeit a rather low level , 0.01 U nit per 10 8 cells. The cell associated activity is 13 times that found in the medium (Table 2 2). Analysis of Products from Cell Wall Suspension Digest of S tarch TLC analysis of products from the cell wall suspension digest of starch ( Figure 2 7 ) showed that starch was depolymerized to generate maltodextrins and a small amount of glucose . The digestion profile is consistent with profile of depolymerization products accumulated during exponential phase of Pjdr2 starch culture, suggesting that Amy13A 2 as the major enzyme account ing s for the generation of these product s.
31 SDS PAGE A nalysis of Paenibacillus sp. S train JDR 2 Culture F ractions SDS PAGE analysis of protein s in each isolated fraction from Paenibacillus s p. JDR 2 starch induced culture identified a prote in band approximately 240 kDa on the cell wall suspensi on (Figure 2 8) . The size of this protein band is consistent w ith the estimated size of multi modular amy lase Amy13A 2 ( estimated M r 235 kDa). Also, the most significant band at approximately 80 kDa position is most likely composed of S layer proteins (encoded by Pjdr2_5781) . Another major band approximately 50 kDa matches the size of the extracellular solute binding protein (47 kDa) (encoded by Pjdr2_0771) from an ABC transporter . This transporter cluster shows significant homology with maltodextrin specific ABC transporters in B . subtilis ( SchÃ¶nert et al ., 2006 ) . When culture d in minim al medium supplemented with YE, the 240 kDa band was not observed, and the 80 kDa band and 50 kDa band s were significantly reduced (Figure 2 9) . The difference of protein profile s in cell wall fractions from starch and yeast extract cultures indicate that the three proteins could be associated with the cell wal l and involved in starch depolymerization and assimilation. Similarly, the CWS fraction from cultures grown on barley glucan showed the presence of a protein band wit h an estimated size of 160 kDa and this band was not present when Pjdr2 was cultured in mi nimum medium supplemented only with YE (data not shown). Protein Identification by Gel S pot LC MS/MS The identification of proteins were evaluated by total unique peptide count and sequence coverage percentage recovered from LC MS/MS results (Table 2 3) . The protein band at 240 kDa ( STH A ) has 36% sequence coverage and 67 peptide sequences matching the multi modular amylase Amy13A 2 . It also has 16% sequence coverage and 11 unique peptide sequences matching S layer protein . The protein band
32 at 80 kDa sho ws 65% sequence coverage and 51 unique peptide sequences that match the S layer protein encoded by locus Pjdr2_5781 in Paenibacillus sp. JDR 2 genome sequence. The NEAr transporter appeared in this same band with 37% sequence coverage and 26 unique peptide matches. The STH C sample with protein band at 50 kDa has shown several possible identifications. All of them are related to ABC transporters including the extracellular solute binding protein encoded by Pjdr2_0771 that was up regulated on starch . An exce ption is one annotated as a RNA binding metal dependent phosphohydrolase. This is the likely due to the fact that there were proteins of similar size with in that band that was excised and analyzed . Of these only the product of Pjdr2_0771 appears to be up r egulated from the transcriptome data. The protein bands on CWS from barley glucan induced cells were also examined. A protein band at 160 kDa, designated BGL A, shows only 15% sequence coverage, but has 11 uniq ue peptide counts to support it s identity as a glucanase encoded by gene at locus Pjdr2_0951. The 30 kDa protein sample did not return any useful information regarding its identity. Overall, the protein identification results support the hypot hesis that multi modular hydrolases are induced in the pr esence of appropr iate polysaccharide and are displayed at the surface of cells . Oligosaccharides released from multi modular hydrolase s catalyzed depolymerization process may be assimilated through ABC transporters into cell for further metabolism. Identification of the Starch U tilization R egulon with RNA seq E vidence In addition to the multi modular amylase gene amy13A 2 , f our genes encoding 1, 4 glucans have been identified from Pjdr2 genome sequence . These include Pjdr2_0774 encoding a GH 13 amylase with signal
33 peptide, Pjdr2_0783 encoding a GH 13 amylase, Pjdr2_1045 encoding a GH 13 amylase and Pjdr2_5276 encoding a GH 31 glucosidase. From protein protein interaction prediction s and a search f or homolog ues of maltose and maltodextrin transporter genes of B . subtilis in Pjdr2 genome, Pjdr2_0771, Pjdr2_0772, Pjdr2_0773 and Pjdr2_0961, Pjdr2_0962, Pjdr2_0963 are identified as two gene clusters encoding ortholog s of maltodextrin ABC transporters of B. subtilis . However, unlike maltose assimilation that proceeds via a PTS system in B. subtilis , no maltose specific PTS proteins were found. Pjdr2 genome lack s genes encoding enzymes for intracellular processing of phosphorylated maltose. This suggests that transportation of maltose and maltodextrins in Pjdr2 are dependent on ABC transporters. The two identified ABC transporters and GH 13 hydrolases may complement the cell bound multi modular amylase Amy13A 2 to provide an efficient starch utilization s ystem. RNA sequencing data verified this hypothesis (Table. 2 4) . The expression level of amy13A 2 was significantly up regulated in starch and maltose culture while down regulated in barley glucan culture . Transcript of genes Pjdr2_1045 ( GH 13 amylase ) , Pjdr2_5276 ( GH 31 glucosidase ) , Pjdr2_ 0961, Pjdr2_0962 and Pjdr2_0963 (ABC transporter) remain ed at similar levels or was down regulated in starch, maltose and barley glucan cultures, suggesting that the se genes do not play a functional role in sta rch utilization. The expression of Pjdr2_0774 and Pjdr2_0783 (designated as amy13A 1 and amy13A 3 respectively) encoding GH 13 amylases were significantly up regulated in starch and maltose cultures compared to barley glucan and YE control culture s . T he po tential ABC transporter genes Pjdr2_0771, Pjdr2_0772 and Pjdr2_0773 (designated mdb , mdp 1 and mdp 2 ) were co regulated with the glycoside hydrolases and
34 were highly up regulated in starch and maltose cultures. It is interesting that expression level s of the three amylases and the ABC transporter was also slightly up regulated in sweet gum xylan culture. In fact, this is a common observation for genes involve d in other polysaccharide systems, which are also up regulated in cu ltures containing sweetgum MeGXn (Sawhney et al., 2014 submitted) . Given the information obtained from gene identification and RNA seq, we propose a regulon conferring the ability to efficiently utilize starch in Pjdr2 ( Figure 2 10). With the co expression of the genes, Amy13A 2 is secret ed and anchor ed to the cell surface, cooperating with secreted Amy13A 1 to depolymerize starch polymers. Once the maltodextrins are release d , they may be transported by ABC transporters composed of Mdb, Mdp 1 , Mdp 2 proteins. The intracellular Amy13A 3 may the n process the oligosacc harides for further metabolism. Discussion Based upon growth and substrate utilization analysis, Pjdr2 efficie ntly utilize s the polysaccharide starch . G rowth rate on starch was comparable to that of disaccharide maltose (Figure 2 1). However, detection of transient accumulation of oligosaccharides in medium (Figure2 2) during the exponential growth phase indicates extracellular depolymerization of starch and assimilation of generated products was not a strongly c oupled process as in the case of utilization of methylglucuronoxylans. Phylogenetic analysis identified Amy13A 2 as a typical GH 13 amylase. The enzyme sequence has all four highly conserved domains and four invariable amino acid residues for am ylase family members (Figure 2 5) . S ignature amino acids grouped Amy13A 2 with three other multi amylase family
35 members that amylase family members from Thermoanaerobacterium that hydrolyze both a group of modular sequences from Thermoanaerobacterium species and Bacillus species , share a distance around 29 amino acids The SLH domains in the multi modular Amy13A 2 structure indicate that this enzyme may be anchored to the cell surface. (Figure 2 3, Figure 2 4) . To test this, we adapted a BCA copper method based assay to examine amylase activity. A w hole cell suspension was prepared with washing steps to remove possible residual activity from amylase activity. The results support that the amylase activity is associated with cell surface. Furthermore, cell wall f raction s were isolated and used to examine activity. Similar levels of amylase activity in the whole cell suspension and cell wall fraction indicate that most of the amylase activity is associated with cell wall (Figure 2 6, Table 2 2) . Analysis of cell wall suspension digestion products of starch showed that starch was depolymerized by cell wall fraction and released maltose and maltodextrins as major products. To further determine if the activity detected in cell wall suspension was due to Amy13A 2 , the isolated cell wall fraction was subject ed to SDS PAGE analysis to determine the protein profile.
36 Comparison of protein profile s of cell wall fraction s from culture s grown on starch and yeast extract with those grown on yeast extract alone showed formation of a 240 kDa protein band in cell wall suspension from starch culture (Figure 2 8, Figure 2 9) . The size of the band was consistent with the theoretical M r of Amy13A 2 . The identification of this band was confirmed to be Amy13A 2 by gel spot LC MS/MS (Table 2 3) . In the absence of genetic manipulation methods with Pjdr2 , effects of gene knockout s on phenotypes c ould not be performed to provide direct evidence for the role of Amy13A 2 . H owever, the presence of 13 times greater amylase activity the cell wall fraction compared to the medium (Table 2 2) indicates that Amy13A 2 , with its SLH domains, is the enzyme responsible for the extracellular depolymerization of starch in Pjdr2 . Through bioinformatic studies , candidate assimilation system genes for oligosaccharides were identified in Pjdr2 (Figure 2 10). A mylase activity was detected in the medium supernatant, cell wall fraction and cytoplasm ic fraction only when Pjdr2 was cultured in starch or maltose. When Pjdr2 was cultured in yeast extract alone , SDS PAGE analysis demonstrated that protein bands in the cell wall fraction from starch culture s identified by gel spot LC MS/MS as Amy13A 2 , s urface layer protein and extracellular binding component of a putative ABC transporter either could not be observed or were significantly reduced (Figure 2 9) . These results suggested that a co regulated system is involved in depolymerization of starch and assimilation of generated products. With the support of transcriptome data (Table 2 4), we propose a maltodextrin ABC transporter composed of Mdb, Mdp 1 and Mdp 2 proteins, an extracellular amylase Amy13A 1 and an intracellular amylase Amy13A 3 are complementing Amy13A 2 to form a starch utilization regulon. This regulon is induced by starch or maltose and not by
37 grown on other polysaccharides. The starch utilization system in Pjdr2 may transport both maltose and maltodextrins through the proposed ABC transporter s since genes encoding maltose specific enzyme (IICB Mal ) or maltose P processing enzyme were not found in Pjdr2 . Al though starch utilization in Pjdr2 may not be a highly coupled process of extracellular depolymerization and product assimilation as for me thylglucuronoxylans , t he results obtained in this study prov ide insight into efficient starch utilization with multi modular amylase s and the role that extracellular glycoside hydrolases anchored via SLH domains play in the utilization of plant polysaccharides in Pjdr2 and other bacteria.
38 Table 2 1 . Sources and characteristics of sequences a ,b,c used for phylogenetic analysis Sequence No. Source Organism Swiss Prot/ GenPept Accession N o. Sequence Nucleophile proton donor distance d Nucleophile Proton donor 1 Paenibacillus sp. JDR 2 ACT03811.1 GWRL D V IILG E EW 39 2 Bacillus sp. XAL601 BAA05832.1 GWRL D V LILG E IW 39 3 Geobacillus stearothermophilus AAG44799.1 GWRL D V LILG E IW 39 4 Geobacillus stearothermophilus ABR26448.1 GWRL D V ALLG E IW 39 5 Thermoanaerobacter pseudethanolicus P38939 GWRL D V PMIA E LW 29 6 Thermoanaerobacterium saccharolyticum P36905 GWRL D V PMIA E NW 29 7 Thermoanaerobacterium thermosulfurigenes P38536 GWRL D V PMIA E NW 29 8 Thermoanaerobacterium thermosulfurigenes AAB00841.1 GWRL D V PMIA E NW 29 9 Bacillus acidopullulyticus P32818 GWRL D V YILG E IW 29 10 Bacillus circulans P08137 GIRV D A FTFG E WF 28 11 Bacillus subtilis P00691 GFRF D A FQYG E IL 32 12 Geobacillus stearothermophilus P06279 GFRL D A FTVG E YW 30 13 Bacillus amyloliquefaciens P00692 GFRI D A FTVA E YW 30 14 Bacillus licheniformis P06278 GFRL D A FTVA E YW 30 15 Bacillus sp. KSM K38 Q93I48 GYRL D A FVVG E YW 30 16 Bacillus sp. O82839 GFRI D A FAVA E FW 30 a: Sequences have been trimmed to contain only the highly conserved catalytic domain b: All sequences have been either experimentally characterized or crystallized except the Amy13A 2 sequence from this study c: All sequences used for comparison were selected from either high score pBlast hits or directly from CAZy database GH 13 family. d: Nucleophile and proton donor is in bold. Distance in number of amino acids.
39 Table 2 2 Distribution of amylase activity in Paenibacillus sp. strain JDR 2 starch culture fractions a . a: Isolated fractions used in reaction were normalized to from 1 mL of culture. b : Total activity is calculated for 50 mL of starch culture with OD 600 approx . 1.0 . c : One unit is defined as amount of protein release d 1 Âµmole of maltose equivalents in 1 min. *: Does not apply
40 Table 2 3. Protein identifications based on LC MS/MS . *: The threshold used for protein identification is 5 unique peptide or 25% sequence coverage.
41 Table 2 4 . Regulation a of candidate genes involve d in starch utilization Locus tag No. Product Name S/YE b S/ YE p val M/YE b M/YE p val B/YE b B/YE p val SG/YE b SG/YE p val c Pjdr2_ 0771 Substrate binding protein Mdb 86.37 < 0.0 1 24.13 < 0.0 1 0.06 < 0.0 1 13.18 < 0.0 1 Pjdr2_0772 Permease Mdp 1 94.27 < 0.0 1 34.73 < 0.0 1 0.21 < 0.0 1 14.23 < 0.0 1 Pjdr2_0773 Permease Mdp 2 134.78 < 0.0 1 58.25 < 0.0 1 0.13 < 0.0 1 22.02 < 0.0 1 Pjdr2_0774 GH 13 amylase (extracellular) Amy13A 1 114.11 < 0.0 1 69.68 < 0.0 1 0.20 < 0.0 1 28.70 < 0.0 1 Pjdr2_0783 GH 13 amylase (intracellular) Amy13A 3 114.11 < 0.0 1 69.68 < 0.0 1 0.20 0.0 1 69.07 < 0.0 1 Pjdr2_5200 GH 13 amylase (Cell bound) Amy13A 2 56.77 < 0.0 1 4.20 < 0.0 1 0.21 < 0.0 1 4.10 < 0.0 1 a: RNA sequence data was processed using DNAStar and normalized by determining the ratio of linear RPKM obtained for RNA derived from Pjdr2 cultures grown on 0.5% starch in 0.5% yeast extract compared to 0.5% yeast extract in minimum medium alone. b: S, starch ; M, maltose; B, barley glucan; YE, extract; SG, sweetgum MeGXn. c: p valu e.
42 A) B) Figure 2 1. Growth of and substrate utilization analysis of Paenibacillus sp. strain JDR 2 . Substrate was 0.2% potato starch or maltose in ZH minerals supplemented with 0.1% YE. A). Growth determined by OD 600 for cultures grown on starch ( filled diamond), maltose ( fil led squares ) and substrate utilization determined by total carbohydrates assay, starch (open triangle) or maltose (open circle). B). Amount of total protein of cells grown on starch (filled diamond) or maltose (filled square). 0 150 300 450 600 750 0 10 20 30 40 Cell protein Âµ g/mL) Time (h) 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 0 20 40 60 80 100 120 0 10 20 30 40 OD 6 00 Substrate remaining (%) Time (h)
43 Figure 2 2 . TLC analysis of released products from Paenibacillus sp. strain JDR 2 starch culture supernatant. Samples were taken every several hours during the growth studies. Same volume of supernatant samples were concentrated and spotted. G, glucose; M2, maltose; M3, maltotriose; M4, maltotetraose. Figure 2 3. Modular architect ure map of Amy13A 2 . SigP, signal peptide; CBM 48, carbohydrate binding module 48 family; GH 13 CD, glycoside hydrolase 13 family catalytic domain; X25/CBM X, uncharacterized module 25/unc haracterized carbohydrate biding module; FN3, fibronectin type III domain; SLH, surface layer homology module .
44 Figure 2 4. 3D structure model of Amy13A 2 . Model was predicted by se condary and tertiary structures, solvent acces sibility and disordered regions and visualized by Chimera (Pettersen et al ., 2004)
45 A) B) Figure 2 5 . Phylogenetic analysis of a set of GH 13 amylase family CD sequences (Table 2 1 ). A) Multiple alignment of conserved regions of analyzed sequences. Activity triad is marked in red, NCP, nucleophile; PD, proton donor; TS S , transit ion state stabilizer. B) Neighbor joining/bootstrap p hylogenetic tree analysis of GH13 family amylase s . AA, amino acid .
46 A ) B ) Figure 2 6. Induction of amylase activity and glucanase activity associated with cell surface and in the medium. Activity was measured by determining accumulation of reducing termini in reaction mixture using BCA copper method. Cells and medium supernatants used for reaction were normalized to the same level that from 1 mL culture with OD 600 approximately 1.0 . Filled symbols, starch substrates ; Open symbols: barley glucan subs t rates . A) Squares: treated cells from starch culture ; Circle s : concentrated supernatant from starch culture ; B) Triangle s : treated cells from starch culture ; Diamond s : concentrated s upernatant from barley glucan culture . -0.1 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 0 10 20 30 40 50 60 70 A 560 Time (min) -0.1 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 0 10 20 30 40 50 60 70 A 560 Time (min)
47 Figure 2 7. TLC a nalysis of products from CWS digest of starch . G, glucose ; M2, maltose; M3, mal totriose; M4, maltotetrarose . Same volume of samples from 0, 30, 60 and 120 min of digestion reaction were spotted .
48 Figure.2 8 . SDS PAGE analysis of isolated fractions from Paenibacillus sp. JDR 2 starch induced culture. Lane designation: MSC, medium supernatant concentrate; Wash I and Wash II, supernatant concentrates from 2 stpe wash; CFE, cell free extraction; CWS, cell wall suspension. Protein amounts loaded were normalized to 20 Âµ g e xcept wash II is 3 Âµg . Bands designated are based on size and may include more than one protein. Identified gene products are based upon sequences determined by LC MS/MS (Table 2 3 ) .
49 Figure 2 9 . SDS PAGE analysis of isolated fractions from Paenibacillus sp. JDR 2 yeast extract or starch culture. Lane designation: MSC, medium supernatant concentrate; CL, cell lysates; CFE, cell free extractions; CWS, cell wall suspensions. Protein amounts loaded were normalized to 20 Âµg except the two MSCs are 5 Âµg . Bands designated are based on size and may include more than one protein. Identified gene products are based upon sequences determined by LC MS/ MS (Table 2 3 ) . Figure 2 10 . Genomic organization of starch utilization regulon in Paenibacillus sp. strain JDR 2 .
50 CHAPTER 3 CONCLUSION P aenibacillus sp. strain JDR 2 efficiently utilizes starch at a comparable growth rate with the disaccharide maltose. TLC analysis of products of starch depolymerization in medium supernatant samples showed maltose, maltodextrins and small amount s of glucose. The accumulation of maltodextrins, albeit transient, indicates the depolymerization of starch and assimilation of the products was not a coupled process as in the case of util ization of methylglucuronoxylans (Nong et al . , 2009) . Search of the Pjdr2 genome sequence for starch degrading enzyme s led to the identification of amy13A 2 , a large modular amylase gene of 6,483 nucleotides ( encoding 2160 amino acids). Amy13A 2 is a multi modular protein like other glycosyl hydrolases identified and characterized in Pjdr2 (Figure 2 3 ). The protein contains a triplicate of SLH modules at the C terminal end. Other domains within the N terminal region include duplicates of CBM 48 family domain and X25/CBM X family domain . Both CBMs are suggested to target mixed alpha 1, 4/alpha 1, 6 linked D glucan polysaccharides. Following these domains is a GH 13 CD and two fibronectin type III domains. The predicted 3D structure indicates that the CB Ms may reach out and fold back to GH 13 CD to form an active pocket while SLHs are exposed for anchoring. These studies collectively support a process in which the Amy13A 2 is secreted and anchored to the cell surface to depolymerize starch polymers. Phylogenetic analysis of Amy13A 2 placed this enzyme a s a member of the amylase family. However, the signature amino acids in conserved sequence region II and the 39 amino acid distance between nucleophile Asp and proton donor Glu grouped Amy13A 2 with sev eral multi modular GH 13 amylases hydrolyze both alp ha 1, 4 and
51 alpha 1, 6 linkages that are distant from B. subtilis amylase. Th is suggest s starch utilization system of Pjdr2 differs significantly from that of B. subtilis . To determine whether Amy13A 2 cou ld depolymerize starch at the cell surface , activity assays were performed with washed whole cells and isolated cell wall fractions. The assay results indicate that the major Pjdr2 starch culture . The level of amylase in the cell wall fraction is 13 times that found in the medium, indicating Amy13A 2 is the major enzyme responsible for efficient depolymerizati on. The observation of activity in medium supernatant and cytoplasm ic fraction s and bioinformatic searches provide preliminary information on other participants of starch utilization system in expression and the formation of cell associated amylase is induced by starch. conferring ability to efficiently utilize starch. With the co expression of the se genes, Amy13A 2 is secreted and anchor ed to the cell surface, cooperating with Amy13A 1 in the medium to depolymerize
52 starch polymers. ABC transp orters composed of Mdb, Mdp 1 and Mdp 2 proteins may transport the generated maltodextrins . The intracellular Amy13A 3 may then process the oligosaccharides for further metabolism. This system does not involve PTS transportation of maltose as in B. subtilis . In this study, the role of the cell associated multi amylase in the extracellular depolymerization process and assimilation of oligosaccharides was determined. The presented work further defined metabolic potential of
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60 BIOGRAPHICAL SKETCH Lei Pan was born and raised in Hengyang, Hunan, China. He received a Bachelor of Science degree from Henan Normal University and the College of Life Sciences with a major in Biotechnology. After graduation, he entered graduate school at the University of F completion of his Master of Science degree, he will continue studies and research at the Department of Microbiology and Cell Science at the University of Florida to pursue Doctor of Philosophy deg ree.