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Characterization of Arsenic Resistant Bacterial Communities in the Rhizosphere of an Arsenic Hyperaccumulator Pteris vit...

Permanent Link: http://ufdc.ufl.edu/UFE0024083/00001

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Title: Characterization of Arsenic Resistant Bacterial Communities in the Rhizosphere of an Arsenic Hyperaccumulator Pteris vittata L.
Physical Description: 1 online resource (98 p.)
Language: english
Creator: Huang, Anhui
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: Soil and Water Science -- Dissertations, Academic -- UF
Genre: Soil and Water Science thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

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Abstract: 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 CHARACTERIZATION OF ARSENIC RESISTANT BACTERIAL COMMUNITIES IN THE RHIZOSPHERE OF AN ARSENIC HYPERACCUMULATOR Pteris vittata L. By Anhui Huang May 2009 Chair: Lena Q. Ma Cochair: Max Teplitski Major: Soil and Water Science The arsenic (As) hyperaccumulator fern Pteris vittata L. produces large amounts of root exudates, which are hypothesized to solubilize arsenic and maintain a unique rhizosphere microbial community. A group of rhizosphere arsenic resistant bacteria were isolated and identified from two arsenic-contaminated sites where P. vittata growed. Twelve aerobic or facultative anaerobic bacterial isolates (Naxibacter sp. AH4, Mesorhizobium sp. AH5, Methylobacterium sp. AH6, Enterobacter sp. AH10, Pseudomonas sp. AH21, AH34, AH43, AH45, Bacillus sp. AH22, Acinetobacter sp. AH23 and Caryophanon sp. AH28) were resistant to 400 mM arsenic, the highest level of arsenic resistance reported to date. Two levels of arsenic detoxifications were proposed and studied. General resistance mechanisms were investigated by studying microbial growth characteristics under osmotic /oxidative stresses induced by sodium arsenate, sodium chloride, polyethylene glycol 6000 (PEG6000), or hydrogen peroxide. Arsenic specific resistant mechanisms were determined by identifying the two functional arsC or arrA genes based on PCR and Southern Hybridization method. Similar hydrogen peroxide inhibitions with broad-host pathogen Salmonella typhimurium were observed, and bacteria grew better under osmotic stress generated by arsenic than sodium chloride or PEG, suggesting the existence of cross-stress tolerances in the isolates. While no arsC in bacterial isolates was detected to be similar with PAO1 arsC, arrA homologous sequences were cloned from some of the strains, indicating variations of both detoxification mechanisms and functional genes in different bacterial genera.
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 Anhui Huang.
Thesis: Thesis (M.S.)--University of Florida, 2009.
Local: Adviser: Ma, Lena Q.
Local: Co-adviser: Teplitski, Max.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2010-05-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0024083:00001

Permanent Link: http://ufdc.ufl.edu/UFE0024083/00001

Material Information

Title: Characterization of Arsenic Resistant Bacterial Communities in the Rhizosphere of an Arsenic Hyperaccumulator Pteris vittata L.
Physical Description: 1 online resource (98 p.)
Language: english
Creator: Huang, Anhui
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: Soil and Water Science -- Dissertations, Academic -- UF
Genre: Soil and Water Science thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: 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 CHARACTERIZATION OF ARSENIC RESISTANT BACTERIAL COMMUNITIES IN THE RHIZOSPHERE OF AN ARSENIC HYPERACCUMULATOR Pteris vittata L. By Anhui Huang May 2009 Chair: Lena Q. Ma Cochair: Max Teplitski Major: Soil and Water Science The arsenic (As) hyperaccumulator fern Pteris vittata L. produces large amounts of root exudates, which are hypothesized to solubilize arsenic and maintain a unique rhizosphere microbial community. A group of rhizosphere arsenic resistant bacteria were isolated and identified from two arsenic-contaminated sites where P. vittata growed. Twelve aerobic or facultative anaerobic bacterial isolates (Naxibacter sp. AH4, Mesorhizobium sp. AH5, Methylobacterium sp. AH6, Enterobacter sp. AH10, Pseudomonas sp. AH21, AH34, AH43, AH45, Bacillus sp. AH22, Acinetobacter sp. AH23 and Caryophanon sp. AH28) were resistant to 400 mM arsenic, the highest level of arsenic resistance reported to date. Two levels of arsenic detoxifications were proposed and studied. General resistance mechanisms were investigated by studying microbial growth characteristics under osmotic /oxidative stresses induced by sodium arsenate, sodium chloride, polyethylene glycol 6000 (PEG6000), or hydrogen peroxide. Arsenic specific resistant mechanisms were determined by identifying the two functional arsC or arrA genes based on PCR and Southern Hybridization method. Similar hydrogen peroxide inhibitions with broad-host pathogen Salmonella typhimurium were observed, and bacteria grew better under osmotic stress generated by arsenic than sodium chloride or PEG, suggesting the existence of cross-stress tolerances in the isolates. While no arsC in bacterial isolates was detected to be similar with PAO1 arsC, arrA homologous sequences were cloned from some of the strains, indicating variations of both detoxification mechanisms and functional genes in different bacterial genera.
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 Anhui Huang.
Thesis: Thesis (M.S.)--University of Florida, 2009.
Local: Adviser: Ma, Lena Q.
Local: Co-adviser: Teplitski, Max.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2010-05-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0024083:00001


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CHARACTERIZATION OF ARSENIC RESIST ANT BACTERIAL COMMUNITIES IN THE RHIZOSPHERE OF AN ARSENIC HYPERACCUMULATOR Pteris vittata L. By ANHUI HUANG A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2009 1

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2009 Anhui Huang 2

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To my undergraduate university Nankai 3

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ACKNOWLEDGMENTS It has been a great experience for me to work with world-class scie ntists duri ng the past two years. The task at hand was not easy a nd their broad knowledge and patience contributed greatly. I thank all my advisors, Dr. Lena Ma, Dr Max Teplitski, Dr. Bala Rathinasabapathi, and Dr. Arthur Berg. I appreciate help from Dr. Mengsheng Gao, Dr. Uttam Saha, and Shiny Mathews. Without them, none of this could have been accomplished. I wish to acknowledge the debt I owe to my parents, Shengzeng Huang and Liang Du; and my brother, Shi Huang, who supported my every step. 4

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TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4 LIST OF TABLES................................................................................................................. ..........7 LIST OF FIGURES.........................................................................................................................8 ABSTRACT.....................................................................................................................................9 CHAPTER 1 INTRODUCTION................................................................................................................. .11 1.1 Environmental Sources of Arsenic..................................................................................11 1.1.1 Arsenic in the Environment..................................................................................11 1.1.2 Biogeochemistry of Arsenic.................................................................................12 1.2 Arsenic in Plants......................................................................................................... .....13 1.2.1 Plant Arsenic Metabolisms...................................................................................13 1.2.2 Arsenic Resistance Mechanisms in Plants...........................................................15 1.2. 3 Arsenic Hyperaccumulator Pteris Vittata L.........................................................16 1.3 Microbial Arsenic Resistance..........................................................................................17 1.3.1 Arsenic Resistant Bacteria....................................................................................17 1. 3.2 General Arsenic Resistant Mechanisms...............................................................18 1.3.3 Specific Arsenic Resistant Mechanisms...............................................................22 1.4 Arsenic Hyperaccumulator Pteris vitttata L. as a Unique Model...................................25 1.3.1 Arsenic-resistant Microbial Communities.............................................................26 1.3.2 Phosphorus Solubilizing Bacteria..........................................................................27 1.3.3 Arsenic-resistant Bacteria......................................................................................28 1.5 Objectives........................................................................................................................30 2 MATERIALS AND METHODS...........................................................................................31 2.1 Soil Sampling and Arsenic Concentration Analysis......................................................31 2.2 Bacterial Isolation and Enumer ation by Total Heterotrophic Counting........................31 2.3 Bacterial Identification................................................................................................ ..32 2.4 Bacterial Growth Characteriza tion under Arsenic and Osmotic Stress.........................33 2.5 Oxidative Stress Test.....................................................................................................33 2.6 Arsenic Transformation by Arsenic Resistant Bacteria.................................................34 2.7 Arsenate Reductase Assay.............................................................................................35 2.8 Arsenic Detoxifying Genes Detetermination.................................................................35 2.9 Arsenic Detoxifying Plasmid Determination.................................................................36 2.10 Antibiotics Test..............................................................................................................37 2.11 Mesorhizobium sp. AH5 Genomic DNA Library Construction....................................37 5

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3 RESULTS AND DISCUSSIONS...........................................................................................38 3.1 Arsenic Concentrations in Soil and Plant Samples..........................................................38 3.2 Arsenic Resistant Bacteria fr om Arsenic Contaminated Soils........................................38 3.3 Phosphorus Solubilizing Bacteria from Arsenic Contaminated Soils.............................40 3.4 Characterization and Identification of Arsenic Resistant Bacteria..................................43 3.5 General Stress Tolerance Characteri stics of Arsenic Resistant Bacteria.........................47 3.5.1 Growth Characteristic s under Arsenic Stress........................................................47 3.5.2 Arsenic Resistant Bacteria l Growth under Osmotic Stress...................................51 3.5.3 Oxidative Stress Assay of Arsenic Resistant Bacteria..........................................55 3.6 Arsenate Transformation by Arsenic Resistant Bacteria.................................................61 3.6.1 In vitro Arsenic Transformation............................................................................61 3.6.2 Reductase Enzyme Activity Assay and Functional Gene Searching....................64 APPENDIX A TWELVE MOST ARSENIC RESISTANT ISOLATES16S RIBOSOMAL RNA GENE SEQUENCES.........................................................................................................................74 B REFERENCES FOR 16S RIBOSO MAL RNA GENE SEQUENCES.................................78 C ARSENATE REDUCTAS E GENE SEQUENCES...............................................................86 D ARSB GENE SEQUENCES...................................................................................................88 LIST OF REFERENCES...............................................................................................................89 BIOGRAPHICAL SKETCH.........................................................................................................98 6

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LIST OF TABLES Table page 3-1 Percentage of arsenate re sistant colonies recovered in TYEG medium isolated from different soils and under different arsenic concentrations.................................................42 3-2 Percentage of bacteria survived in NBRIP phosphate rock plates under different arsenic concentrations........................................................................................................4 2 3-3 The twelve most arsenic-re sistant bacteria identified from two arsenic contaminated soils.......................................................................................................................... ..........44 3-4 Antibiotic resistance test on the arsenic resistant bacteria isolated from arsenic contaminated soils............................................................................................................. .46 7

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LIST OF FIGURES Figure page 3-1 Arsenic concentrations in soil and Pteris vittata ....................................................................40 3-2 Naxibacter sp. AH1 and Naxibacter sp. AH4 on agarose plates............................................43 3-3 Phylogenetic tree of the 12 most arsenic resistant bacteria....................................................46 3-4 Growth characteristics of eight arsenic-resistant bacter ia in TYEG medium containing 400 mM sodium arsenate or 2.4 M sodium chloride.........................................................49 3-5 Arsenate Resistant Index (ARI) based on ba cterial growth at 400 mM sodium arsenate......51 3-6 Bacterial growth under osmotic stress (-1.5MPa) generated by 400 mM of NaCl, 176 mM of sodium arsenate and 26% of PEG6000..................................................................53 3-7 Oxidation inhibition area around a gla ss disc containing 25 l of 50 mM N,N'Dimethyl-4,4'-bipyridinium dichloride (paraquat)............................................................58 3-8 Growth of S. typhimurium wild type and mutants in M9 minimal medium...........................59 3-9 Oxidation inhibition zone around a glass disc containing 25 l of 3% hydrogen peroxide..............................................................................................................................61 3-10 Arsenic transformation by arsenic-resistant bacteria AH4, AH5 and AH21 during 32 h of growth in TYEG medium spiked w ith 375 g of arsenate or arsenite..........................62 3-11 Growth of arsenic-resistant bacter ia AH4, AH5 and AH21 after 32 h in TYEG medium spiked with 375 g of arsenate or arsenite..........................................................64 3-12 Arsenate reductase assay......................................................................................................68 3-13 The arsC gene Southern Hybridization................................................................................71 3-14 The arrA gene cloning..........................................................................................................72 8

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Abstract of Thesis Presen ted to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science CHARACTERIZATION OF ARSENIC RESIST ANT BACTERIAL COMMUNITIES IN THE RHIZOSPHERE OF AN ARSENIC HYPERACCUMULATOR Pteris vittata L. By Anhui Huang May 2009 Chair: Lena Q. Ma Cochair: Max Teplitski Major: Soil and Water Science The arsenic (As) hyperaccumulator fern Pteris vittata L. produces large amounts of root exudates, which are hypothesized to solubili ze arsenic and maintain a unique rhizosphere microbial community. A group of rhizosphere arsenic resistant bacteria were isolated and identified from two arsenic-contaminated sites where P. vittata growed. Twelve aerobic or facultative anaerobic bacterial isolates ( Naxibacter sp AH4, Mesorhizobium sp. AH5, Methylobacterium sp AH6, Enterobacter sp AH10, Pseudomonas sp. AH21, AH34, AH43, AH45, Bacillus sp. AH22, Acinetobacter sp. AH23 and Caryophanon sp. AH28) were resistant to 400 mM arsenic, the highest le vel of arsenic resistance reported to date. Two levels of arsenic detoxifications were proposed a nd studied. General resistance m echanisms were investigated by studying microbial growth characteristics under os motic /oxidative stresses induced by sodium arsenate, sodium chloride, polyethylene glyc ol 6000 (PEG6000), or hydr ogen peroxide. Arsenic specific resistant mechanisms were dete rmined by identifying the two functional arsC or arrA genes based on PCR and Southern Hybridizati on method. Similar hydrogen peroxide inhibitions with broad-host pathogen Salmonella typhimurium were observed, and bacteria grew better 9

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10 under osmotic stress generated by arsenic than sodi um chloride or PEG, suggesting the existence of cross-stress to lerances in the isolates. While no arsC in bacterial isolates was detected to be similar with PAO1 arsC arrA homologous sequences were cloned from some of the strains, indicating variations of both detoxification mechanisms and functional genes in different bacterial genera.

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CHAPTER 1 INTRODUCTION 1.1 Environmental Sources of Arsenic 1.1.1 Arsenic in the Environment Carcinogenic, mutagenic and teratogenic (Pla nt et al., 2003) arsenic (As) is a major constituent in more than 245 minerals and is ubiquous in the environment (Mandal and Suzuki, 2002). It is responsible for bladder, kidney, liver, lung, and skin cance rs and is listed as a Class A human carcinogen by the USEPA (Chen et al., 2002). Both acute and chronic poisoning to humans has raised great concerns especially in heavily contaminated areas such as Bangladesh and West Bengal, India. The serious health problems were described as the greatest mass poisoning in human history by Worl d Health Organization (Vaughan, 2006). The average concentration of arsenic in terre strial environments is around 1.5 to 3 mg/kg. Arsenic in the environmental comes from natural and anthropogenic sources. Arsenic is present in reducing marine sediment, iron deposits, sedime ntary iron ores and manganese nodules and is commonly associated with iron hydroxides and sulfides. Among the 245 minerals, approximately 60% are arsenates, 20% sulfides and sulfo-salts and the rema ining 20% includes arsenides, arsenites, oxides, silicates and el emental arsenic (Ritchie, 1980). The levels of soil arsenic range from 0.1 to 40 mg/kg in various countries. Anthropogenic sources generally exceed natural sources by 3 to 1in the environment. Arsenic can substitute for Si, Al or Fe in silicates minera ls, therefore, contaminated soils usually have arsenic-rich parent materials (Fitz and Wenzel, 2002).The utilization of natural resources by humans releases arsenic into the air, water and soil. Arsenic may accumula te in soil through use of arsenical pesticides, application of fertilizers, dusts fr om burning of fossil fuels, and di sposal of industrial and animal wastes. It has been estimated that there ar e 41% of the superfund sites in the USA are contaminated with arsenic (EPA, 1997), 1.4 milli on contaminated sites within the European 11

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Community impacted by arsenic (ETCS, 1998), a nd more than 10,000 arsenic contaminated sites reported in Australia (Smith et al., 2002). These anthropogenic sources will adversely affect plants, animals and microorganisms. The main arsenic producers were USA, Russia, France, Mexico, Germany, Peru, Namibia, Sweden, and China, and these countries accounted for about 90% of the world production (Mandal and Suzuki, 2002). In the past, about 80 % of arsenic consumption was for agriculture uses such as insecticides and pesticides. The inorganic arsenical s, primarily, sodium arsenite, were widely used since 1890 as weed killers, particularly as non-sele ctive soil sterilants (Vaughan, 2006). Two thousand and five hundred tons of H3AsO4 were used as desiccants on 1,222,000 acres (about 495,000 ha) of U.S. cotton in 1964 (Fordyce et al., 1995). Fluorchromearsenic-phenol (FCAP), chromated copper arse nate (CCA) and ammonical copper arsenate (ACA) were used in 99% of th e arsenical wood preservatives (P erker, 1981). Several arsenic compounds are currently used for feed additives, such as H3AsO4, 3-nitro-4-hydroxy phenylarsonic acid, 4-nitrophenylarsonic acid etc (Mandal and Suzuki, 2002). 1.1.2 Biogeochemistry of Arsenic Changes in arsenic speciation occurs both ab iotically and biotically, the latter was catalyzed by organisms. Arsenite oxidation can be catalyzed by iron oxides, manganese oxides and organic compounds when the oxidation potential is high e nough and usually at low pH (< 3), though it is slow. Most arsenite is oxidized microbiologically as a detoxification mechanisms or as elector donor, which are known as heterotrophic arsenite oxidizers (HAOs) or chemolithoautotrophic arsenite oxidizers (CAOs) (Oremland and Stolz, 2003). HAOs incorporate a periplasmic enzyme to catalyze the oxidation reaction, which converts arsenite encountered on the cells outer membrane. This presumably makes it less likely to enter the cell. On the other hand, CAOs use 12

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arsenite as elector donor, reducing either oxygen or nitrate to obtai n the energy generated in this process to fix CO2. While arsenate reduction at low pH occurres abiotically and is coupl ed with sulfide (HSor H2S) oxidation, its reduction in neutral environmen ts are mostly cataly zed by microorganisms, for either energy production or arsenic detoxification. Referred to as dissimilatory arsenate-reduci ng prokaryotes (DARPs) (Plant et al., 2003), bacteria incorporating arsena te reduction in respiration usually growing in anaerobic environments and use arsenate as elector acceptor. Therefore, they are found to be able to grow in both oxic and anoxic conditions, where hydrogen as well as a variety of organic carbon sources including acetate, formate, pyruvate, butyrate, citrate, succinate, fumarate, malate, and glucose can be their electr on donors (Dowdle et al., 1996). While the respiratory arsenate reductases re mains to be fully elucidated, the second arsenate reduction-detoxifi cation system, known as an ars operon, is found in many microorganisms and well understood both functio nally and structurally. Since the arsenate/ arsenite oxidation/reduction potential is +135mV (Niggemyer et al., 2001), this type of arsenate reduction needs glutaredoxin, thiore doxin or ferredoxin as cofactor to reduce reaction potential. Although the two resistant mechan isms function differently, the ability to respire arsenate does not preclude the presence of ars operon system. Recently, Shewanella sp. ANA-3 was found to have both respiratory and detoxify ing arsenate reductase s (Fitz et al., 2003). 1.2 Arsenic in Plants 1.2.1 Plant Arsenic Metabolisms Most plants do not take up much arsenic, with average concen trations in plants being <3.6 mg/kg (Kabata-Pendias and Pendias, 2000). This is not only because arsenic concentration is low 13

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and it is highly toxic to organisms, but also, like phosphorus, majority of arsenic in soils is insoluble and thus has low availabil ity to plants (Tu et al., 2004). Most studies on arsenic in plants concentrate on the transformation of arsenical pesticides in crops such as rice, tomato, apple or carrot. All plants growing on both arsenic-contaminated and uncontaminated sites have more than one arse nic species in their tissu es. A range of arsenic compounds are found in plant tissues for example, inorganic arseni te and arsenate, methylated arsenic species, arsenobetaine a nd arseno-sugars. Plant species, wh ich are not resistant to arsenic suffer considerable stress upon exposure, with sy mptoms ranging from inhi bition of root growth to death (Meharg and Hartley-Whitaker, 2002). Inorganic arsenic species are generally highl y toxic to plants. Mechanisms of arsenic uptake by plant roots are not clea rly understood. It may occur eith er through uptake by phosphate transporters in mycorrhizal fungus (Sharples et al., 2000) or directly uptake by plant roots (Abedin et al., 2002). Arsenate acts as a phosphate analogue and is transported across the plasma membrane via phosphate transport sy stems. It competes with phosphate in all biomolecules. Arsenate can be reduced to ar senite non-enzymatically by glutaredoxin or enzymatically by specific arsenate reductase. Arsenite is taken up by the aquaglyceroporin. Following the reduction of arsenate to arsenite in plants, arsenic may be potentially further metabolized to methylated species. Organic arsenic such as MMA, DMA, tetramethylarsonium ions (TETRA), trimethylarsonium oxide (TMAO), as well as arsenobetaine and arseno-sugars are found in plants; however, methylated arsenic species are present as a minor fraction of the arsenic burden in plants. It has not been proven whether these compounds are actually metabolized by the plant or simply taken up in those forms from soil solution. Organic arsenic species are generally considered to be less toxic than inorganic species to organisms; however, it 14

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is suggested that they were more toxic than inor ganic arsenic species in te rrestrial plants (Meharg and Hartley-Whitaker, 2002). Exposure to inorganic arsenic species result s in the generation of reactive oxygen species (ROS), which probably occurs th rough the conversion of arsenate to arsenite and leads to synthesis of enzymatic antioxidants such as supe roxide dismutase, catalase and glutathione-Stransferase, and nonenzymatic antioxidants like glutathione and ascorb ate (Dat et al., 2000). Moreover, methylation is also thoug ht to be redox driven and such reactions could give rise to ROS. 1.2.2 Arsenic Resistance Mechanisms in Plants As an analogue of the m acronutrient phosphorus, arsenic is somewhat unusual comparing with transition metals and metalloids. Plants growing on arsenate contaminated soils will assimilate high levels of arsenate unless they have altered phosphate transport mechanisms (Sharples et al., 2000). In spite of that, arsenate resistance has been identi fied in a number of plant species growing on arseni c contaminated soils including Andropogon scoparius, Agrostis castellana, A. delicatula, A. capillaris, Deschampsia cespitosa, and Plantago lanceolata (Meharg and Hartley-Whitaker, 2002). In those plants, resist ance is generally achieved via suppression of the high affinity phosphate uptake system. It is thought that this suppression reduces arsenate influx to a level at which th e plant can detoxify by constitutive mechanisms (Meharg and Macnair, 1992). Thus, arsenate sensitivity is intimately linked to phosphate nutrition, with increased phosphate status leading to reduced arsenate uptake (Meharg et al., 1994). Indeed, most arsenate resist ant plants always suppress the high affinity uptake system and are insensitive to plant phosphorous status (Meharg and Macnair, 1992). However, arsenic resistant plants can still accumu late considerable levels of arsenic in their tissues. Therefore it is assumed that arsenic resistant plants either compartmentalize and/or 15

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transform arsenic to less phytot oxic arsenic species to withsta nd high cellular arsenic burdens, such as complexing with phytochelatins (PCs) (Meharg et al., 1994). PCs are synthesized from reduced glutathione (GSH) by the transpeptidation of -glutamylcysteinyl dipeptides, through the action of the constitutive enzyme PC synthase (Vatamaniuk et al., 2000). Synthesis of PCs is induced by a range of cations such as Ag+, Cd2+, Cu2+, Hg2+ and Pb2+ and the oxyanions arsenate and selenate (Grill, 1987). Arsenite in many arse nic-resistant plant tissues is complexed with phytochelatins (PCs). For example, X-ray absorption spectroscopy (XAS) of Brassica juncea has determined that arsenic, when present as arsenite, is coordinated with three sulphur groups (Pickering et al., 2000). Meharg and Hartley-Whitaker (2002) s howed that PCs are induced upon exposure to inorganic arsenic in cel l cultures, root cultures, and en zyme preparations of different plants In addition to glutathione's role as the precursor of PCs, it is also an antioxidant. Synthesis of PCs can therefore result in glutathione deplet ion, reducing the amount of antioxidant available for quenching ROS (Hartley-Whitaker et al., 2001). Microorganisms associated with plants are al so involved in heavy me tal resistance. Free living and symbiotic bacteria and arbuscular mycorrh izal fungi play important roles in increasing nutrient storages, amplifying plan t resistance to drought, enhanci ng plant salinity tolerance and cold hardiness, and strengthening plant resist ance to heavy metals (Gyaneshwar et al., 2002; Lucy et al., 2004). 1.2. 3 Arsenic hyperaccumulator Pteris Vittata L. Chinese brake fern ( Pteris Vittata L.) is the first reported arsenic hyperaccumulator. It tolerates soil arsenic concentration up to 1500 mg /kg and rapidly accumulate up to 2.3% in its aboveground biomass from both uncontaminated and contaminated soil (Ma et al., 2001). Most 16

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of the accumulated arsenic is conc entrated in the epidermal layers of the fronds (Lombi et al., 2002) and is probably stored in the vacuoles (Pickering et al., 2006). In addition to the remarkable ability of P. vittata to tolerate high internal arsenic, its extraction of low levels of arsenate from soil is extraordinary, consideri ng that arsenate mobility in soil is limited. On one hand, arsenic detoxification and accumulation mechanisms by P. vittata have been investigated, but it is st ill not fully elucidated. P. vittata can hyperaccumulate arsenite and MMA, suggesting that alteration of phosphate transp orter may not be involved in hyperaccumulation (Meharg and Hartley-Whitaker, 2002). On the ot her hand, explanation from evolutionary aspect has been proposed. Hyperaccumulation may serv e as a means of avoiding competition or a defense strategy against herbivor es and pathogens to gain ecol ogical advantage, since most plants are sensitive to arsenic. Hyperaccumulator turns out to be toxic for herbivores such as grass hoppers (Rathinasabapathi et al., 2007). Arsenic hyperaccumulation by P. vittata may be a strategy for attaining metal resi stance by accumulation and sequestra tion, or may be a result of inadvertent uptake of arsenate, which enha nces its ability in phosphate acquisition (Rathinasabapathi, 2006). 1.3 Microbial Arsenic Resistance 1.3.1 Arsenic Resistant Bacteria Bacteria living under environmental stresses ha ve evolved different systems to withstand the growth restriction, where a number of gene s are activated or repressed to adapt cell physiology or metabolism to the environment. Those genes include both global regulators and some specific functional genes. Th e global regulators regulate a la rge number of genes and result in a very different transcriptional profile, wh ile the specific functional genes encode specific enzymes responsible for counter acting environmental factors. 17

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Arsenic detoxification is one of those stress tolerance mechanisms in bacteria. Although its concentration in crust is about 1 mg/kg, arsenic is widely distributed in natural environments and commonly associated with mineral ores like Cu, A u, Ag, Pb, and Sn, either as part of the mineral structure or as sorbed species (Smedley and Kinniburgh, 2002). It is nontoxic in the insoluble forms before they are chemically /biol ogically mobilized, which would produce high concentration of inorganic arsenite and/or arsenate, especially in some acid mine drainage. On the other hand, biological metabolism converts inorganic arsenic in to monomethylated, dimethylated, and trimethylated organic arsenic species, which are more toxic than inorganic arsenic without further sequestration, though me thylation and sequestration together is considered as one of the arsenic detoxification st rategies (Stolz et al., 2006; Turpeinen et al., 1999). Therefore, bacteria play an important role in arsenic biogeoc hemistry, involving in biological reduction/oxidation, me thylation/demethylation, precipitation/dissolution, and sorption/desorption. In consequence, different detoxifications strategi es are developed to withstand the growth restriction when they expose to arsenic. It is hypothesized that similar to bact erial detoxification principles under all environmental stresses; arsenic resistance systems can be categ orized into two classe s, general and specific systems. While the general systems alleviate ar senic induced cell toxicities such as oxidative burst or osmotic stress damage, the specific sy stems involve in arsenic transformation and sequestration. The two systems together accomplis h arsenic detoxification at different cellular metabolic levels. 1. 3.2 General Arsenic Resistant Mechanisms The first class of resistant mechanisms comprises more general stress related to gene regulations, which prepare bacteria to survive under different environmental stresses. These systems are generally turned on under hyper-osmotic cond ition or low nutrien t environment, and 18

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cross protect from other stresses such as oxidative burst, he avy metal stress and sodium hypochlorite (Pichereau et al., 2000). In fact, multiple types of tolerance occur frequently in bacteria living under osmotic stress or starvation resulting from globa l reprogramming of gene expre ssions. Those results appeared in early literature. For exampl e, Kjelleberg (1993) showed, upon stress, a rapid change in gene expression pattern in non-differentiating bacteria by a two-dimensional el ectrophoresis. Hartke (1998) showed that, induced under complete starvation condition in tap water, Enterococcus faecalis cells become more to lerating to heat, acid and sodium hypochlorite stresses, and was significantly more resistant to UV245 irradiation. Although the func tional genes and physiological pathways were of fundamental interests to under stand the general stress resistant mechanisms, it seemed impossible at that time to study at the tr anscriptional or post-tran scriptional level because of technique and information limitations. Howeve r, as more new technologies being developed, scientists are now able to look into gene up or down regulation in more specific details. These techniques include microarray, quantitative real-time PCR or Northern blot in analyzing transcriptional profiles and more recent discovery, RNA in posttranscriptional regulation. For example, a recent study of gene profile changes responding to variation of pH in Shigella flexneri by whole-genome microarrays differentiated th e expression of 307 genes, including global regulators such as the sigma factors and specific pH dependent and energy metabolic genes that increase acid production and energy generation (Cheng et al., 2007). Northern blot comparison of Listeria monocytogenes mRNA between growth in neutral and alkali environments revealed that expression of about 60 B regulated genes was significantly increased, and bacterial strain was more resistant to subsequent alkaline, osmotic or ethanol stress (Giotis et al., 2008). Those 19

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researches discern that global regulons confer cross-protection against multiple stresses such as oxidative burst, osmotic stress, as well as heavy metal toxicities. Bacterial growth unde r oxidative burst. An example of global regu lators in bacteria is oxyR system, which is a regulon induced typically by hydrogen peroxide and tightly controls hydrogen peroxide level by increasing scave nging activities and limiting hydrogen peroxide generation in the respiratory chain. The conn ection between oxidative stress and arsenic detoxification is supported not only by evoluti onal view, but also by experimental results. On one hand, the earlier age of life was in anaerobic environment; therefore, all fundamental enzymes were integrated into meta bolisms in the absence of selective oxidative pressure, including As detoxifica tion system. But after photosystem II appeared, microorganisms acquired oxygen tolerance and de veloped mechanisms to defend themselves against both As induced oxidative stress and superoxide (O2 ) which are inadvertently generated by-products of aerobic metabolisms (Imlay, 2008). OxyR is one of the functional systems prevailing among bacteria. Although it has been known for a long ti me, its regulation profile isnt clear until microarray technique was employed in recent studie s. For example, Zheng et al. (2001) showed that the regulon was activated by a submicro molar of hydrogen peroxide and up or down regulated a large amount of genes, incl uding 30 genes with >10-fold induction On the other hand, arsenic detoxification is also found to be evolved in the early ag e of life, which is verified by phylogenetic analysis of arsenic resistant genes in both archaea and bacteria (Gihring et al., 2003; Jackson and Dugas, 2003). These studies showed arsenic detoxification results from either variation in ars operon genes or phylogenetical separation of bacterial, archaeal, and eukaryotic in the sa me gene in the operon. Therefore, at early age, metabolisms of 20

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microorganisms living in environments with geol ogical source of arsenic are under influence of both arsenic and superoxide, which lead to overlap in bacterial metabolisms. Arsenic has been shown to mediate its toxicity through induced ge neration of reactive oxygen species. Therefore, it is not a surprise to see the co rrelation between their arsenic resistance and antihydrogen peroxide ability (Gihring et al., 2003; Liu et al., 2001). Moreover, previous study showed that oxyR mutation increased sensitivity to both arsenate and arsenite in a plant pathogenic bacterium, Xanthomonas campestris pv. phaseoli ( Xp ) (Sukchawalit et al., 2005). It is reasonable to assume that, while specific arsenic detoxification systems directly eliminate toxic arsenic, other oxidative respons e systems such as OxyR scavenge downstream oxidative burst toxicity induced by arsenic, and thus reduce toxicities at different levels. Growth under osmotic stress. Many studies on heavy metal resi stance and osmotic stress in bacteria suggested that, bacteria are capable of many chemical transformations of heavy metals, including oxidation, reduction, methylation, demethylation, co mplexation, and precipitation. However, these transformations are sometime s byproducts of normal metabolism (Silver and Misra, 1984), therefore making the effort to s earch for functional genes became difficult. From this angle, cell structural or physiological sp ecialties are also important in studying general resistant mechanisms. For example, detoxification via immobilization can re sult from sorption to biomass or exopolymers, sequestration, crystalliz ation or precipitation as organic and inorganic compounds intracellularly. Reduction/ oxidation of different species may result in immobilization, e.g., MnII to MnIV, CrVI to CrI II, and arsenite to arsenate. Those transformation are not only affected by bacterial functional enzymes, but also the reduct ion/oxidation potential in the environment, which are greatly influenced by inhabited microbial communities (Gadd, 2004). 21

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Deposition of metals onto bact erial cell wall/membrane carboxylic, amino, thiol, hydroxyl, and hydro-carboxylic functional groups are well-known phenomenon. Although there is no direct evidence, a previous study showed arsenate may interfere with the regulation of cell wall biosynthesis, resulting from arsenic binding onto cell walls (Manda l et al., 2008). Moreover, data analysis revealed similar functional heavy metal-binding peptides phytochelatins (PCs) in prokaryotes, such as cyanobacteria nostoc (Anabaena) sp. PCC 7120, Prochlorococcus marinus str. MIT9313 (BX572098), and Anabaena variabilis ATCC29413 (Hirata et al., 2005). Like heavy metal hyperaccumulation plants, PCs in bact eria would confer dram atically heavy metal tolerance. 1.3.3 Specific Arsenic Resistant Mechanisms The second class of arsenic deto xification is via speci fic resistant system, which is induced by sublethal doses of arsenic, and permits surv ival against a challenging arsenic dose. Most arsenite is oxidized microbiologically, either by HAOs or CAOs (Oremland and Stolz, 2003). In terms of arsenate-reduction based mechanisms, so far two mechanisms have been reported, the DARPs and ars operon system. DARPs usually grow in anaerobic environments using arsenate as elector acceptor to incorporate arsenate reduction in respirati on. This happens when redox potential is below +135mV (arsenate/arsenite), allo wing the reaction happen easily with the help of glutaredoxin, thioredoxin or ferredoxin (Plant et al., 2003). However, no obligate DARPs have been found, because all the strains examined can use other electron acceptors for growth, such as sulphate, phosphate, nitrate, MnIV and FeIII (Dowdle et al., 1996). For example, while nitrate was found to be the preferred electron accep tor and inhibited arsenate reduc tion by Dowdle et al. (1996), a laboratory cultured strain Desulfomicrobium sp. Ben-RB was found to reduce arsenate and sulfate at the same time using lactate as electr on donor, where same sulfate concentration did not 22

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inhibit arsenate reduction (Dowdle et al., 1996 ; Macy et al., 2000). Ce rtain DARP species are more sensitive to arsenic than others, for example, haloalkaliphile Bacillus selenitireducens grows well at 10 mM arsenate, while Sulfurospirillum species is only able to grow at 5 mM. This is possibly because the product arsenite is charge d at high pH and cannot exit the cell in alkaline condition where the latter one habited (Ahmann et al., 1994; Switzer Blum et al., 1998), and may be also due to the strong gene ral resistant cell metabolisms. Structural and functional study of arsenate reductase was first carried out in Chrysiogenes arsenatis (Gram negative) and Bacillus selenitireducens (Gram positive), both of which are encoded by typical arr operon. The protein is a heterodimer consisting of 87 kD (arrA) and 29 kD (arrB) subunits, both contain an iron sulfur cluster, placin g it in the dimethylsulfoxide (DMSO) reductase family of mononuclear moly bdenum enzyme (Krafft and Macy, 1998). While the respiratory arsenate reductase remain s to be fully elucidated, the second arsenate reduction-detoxificati on system, known as ars operon, is found in many microorganisms and well understood both functionally and structurall y. Most heterotrophic bacteria have the chromosome ars operon with one set of genes of multiple set of genes. The operon in Escherichia coli SG20136 has both plasmid and chromosoma l loci. The plasmid R733 consists of arsA, arsB, arsC, arsD, and arsR whereas the chromosome has only arsB, arsC, and arsR (Rosen et al., 1988). Arsenate is bond by a cysteine residue near the N-terminal of ArsC, and is reduced with electrons donated by GSH (Rosen, 2002) Although arsenite is more toxic than the oxidized form arsenate, it can be excret ed by an arsenite-specific transporter, ArsB whose function depends on an ATPase arsA in E. coli. On the other hand, the ars operon in plasmid pI258 of Staphylococcus aureus consists of only arsB, arsC, and arsD and the electron donor is reduced thioredoxin (Liu et al., 2004; Newman et al., 1997). Although the ATPase gene arsA is 23

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absent in the operon, arsenite is expelled from the cell via an AT Pase independent ArsB. ArsR and arsD are arsenite res ponsive repressors of the ars operon. In contrast to the DARPs, this system doesnt gain energy in the reduction r eaction and occurs under oxic and anoxic condition (Macur et al., 2001b). Though typical ars operon is comprised of the three-gene structure arsRBC previous study showed a variation in ars operon in different bacteria l genera. For example, while E. coli SG20136 has both plasmid chromosome loci, two complete ars operons ( ars1 and ars2 ) encoding arsRBC, and two orphan genes ( arsB3 and arsC4) are found in Corynebacterium glutamicum genome (Mateos et al., 2006). Ochrobactrum tritici SCII24T also has two ars operons (named as ars1 and ars2) encoding arsenic and antimony resistant genes, ars1 contains five genes encoding arsR, arsD, arsA, CBS-domain-containing protein and arsB, and ars2 encodes two arsR, two arsC, one ACR3 and one ar sH-like protein (Branco et al., 2008). Another study of a large linear plasmid pHZ227 in Streptomyces sp. found two novel genes, arsO and arsT which were coactivated and cotranscribed with arsR, arsB and arsC (Wang et al., 2006). ArsO is a flavin-binding monooxygenase, a nd arsT is a thioredoxin reductase. The sequence, structure and functional analys is showed difference of functional genes among the two arsenate reductases in DARPs or ars operon. In addition, tw o unrelated clades of arsC sequences are found among those ars operon reported. Though their biochemical function is the same, there is no evolutionary relationship (Mukhopadhyay et al., 2002). The representatives of the two enzymes are E. coli SG20136 plasmid R773 arsC (Rosen, 2002) and S. aureus plasmid pI258 arsC (Mukhopadhyay et al., 2002) Both ArsC arsenate reductases are small monomeric protein of about 135 amino acid residues containing three essential cysteine residues, which are involved in a cascade sequence of enzyme activ ity. They use different active 24

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cysteine sites and different co factors, glutaredoxin and thiore doxin respectively, to reduce the reduction potential. The enzyme arsC catalyzing arsenate reduction is located in the cytoplasm and is therefore only able to reduce arsenate that already entered the cell. This reduction-detoxification mechanism has been found in aerobic bacteria isol ated from arsenic-contaminated soils and mine tailings (Jones et al., 2000; Macur et al., 2001a), which indi cates the importance of this mechanism in the biogeochemical cycling of arsenic in nature (Inskeep et al., 2002). 1.4 Arsenic Hyperaccumulator Pteris vitttata L. as a Unique Model Concentration of soluble phosphorus in soil is usually very low, normally at levels of 1 mg/kg or less (Belton et al., 1985). Arsenic concentrations are even lower in uncontaminated soils. However, the ar senic hyperaccumulator P. vittata is found to retrieve more bioavailable arsenic from recalcitrant pool (F ayiga et al., 2007). Understanding the interactions between roots, rhizosphere microbial communities, as well as rh izosphere dynamics of arsenic and phosphorus are important for improving phosphorus nutrition a nd reducing arsenic uptake in food and feed crops, and increase arsenic uptake in the hyperaccumulation plant. The rhizosphere of P. vittata serves as a unique model for such investigations because of the unusual arsenic resistance and hyperaccumulation traits of the plant. The fact that the soil where arsenic hyperaccumulation is first found in P. vittata has been contaminated for about 60 years (Komar, 1999), coupled with the ability of P. vittata to solubilize arsenic and/or phosphorus in the rhizosphere, provides a great o pportunity to discover microbes that may have developed new and/or unique tolerance mechanis ms in arsenic resistance in the rhizosphere. Besides, it is reasonable that such high ability in accumulating arsenic from soil may partially result from the high ability of rhizosphere bacteria associated with the plant in mobilizing and tolerating arsenic. 25

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Rhizosphere microbial communities are contri buting to the biodegradation of old fronds fallen back to soil. While live fronds accumulate about 4000 mg/kg of arsenic, fronds undergoing the process of senescence have only 200-300 mg/kg of arsenic. Th e exploitation of the functional microbial communities involved in the process w ill provide potentially environmental friendly method for identifying arsenic-resistant bacteria and the associated mechanisms. Fitz et al (2002) investigated the changes in the rhizosphere characteristics of P vittata relevant to its use in phytoextraction. They reported that arsenic was mainly acquired from less available pools. Although no informa tion is available on the fate of As in the rhizosphere, it is known that certain environmental microorganisms ha ve adapted to a variety of habitats using arsenic oxyanions for energy generation, either by oxidizing arsenite or by respiring arsenate (Lasat, 2002; Raghu and MacRae, 1966), thus ca talyzing the arsenic biogeo-cycle in the environment. Soil microorganisms have an important role in mobilizing nutrients in the soil from recalcitrant sources. This is of great importance for phytoextracti on of toxic metals because the heavy metal ions are made more available for ro ot uptake by a more activ e rhizosphere microbial community (Schuster et al., 2003). 1.3.1 Arsenic-resistant mi crobial communities The rhizosphere is the area where the roots are interacting with the neighboring plant species for space, water, and mineral nutrients, and with soil-borne microorganisms, including bacteria and fungi feeding on an abundant sour ce of organic material (Ryan et al., 2001). Rhizosphere microbial communities are usually in contact with the root surface, or rhizoplane, providing available P, N and mine ral nutrients to host plants. While exposed to more bioavailable ar senic, rhizosphere microorganisms in P. vittata have to tolerate higher arsenic concen tration. Microbial detoxification based on arsenate reduction has been well studied in E. coli and has also been documented for Staphylococcus, Bacillus, 26

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Acidithiobacillus, Pseudomonas, Shewanella and a large group of bact eria (Mateos et al., 2006). Scientists have successfully isolated high arse nic resistant bacteria and fungi from arsenic contaminated environment. For example, A nderson and Cook (2004) isolated a number of arsenic resistant chemoheterotrophic bacteria from two arsenic contaminated soils in New Zealand, which tolerated up to 7,500 mg/kg arsenate. Rathinasabapathi et al. (2006) isolated bacteria from the phyllosphere of P. vittata which exhibited resistance to arsenate, arsenite, and antimony in the culture medium. Cnovas et al. (2003) isolated a filamentous fungus ( Aspergillus sp. P37) from arsenic contaminated river in Spain, which is able to grow at 15,000 mg/kg arsenic. Among those documented arsenic resistant bacteria, Corynebacterium glutamicum is the most prominent, which tolerated up to 30,000 mg/kg of arsenate (Mateos et al., 2006). Most of those studies have focused on the sp ecific arsenic resistant mechanisms, i.e., functional genes in detoxification mechanisms through transformation of arsenic species and sequestration in either vacuoles or outer membrane; However, little information is available at global metabolic levels such as the osmotic stre ss when arsenic is stored/accumulated in outer membrane, the oxidative stress generated duri ng arsenic exposure, and the impacts of those stresses on cellular growth. 1.3.2 Phosphorus solubilizing bacteria There are considerable popul ations of phosphate-solubilizi ng microorganisms (PSMs) in soil, especially in the rhizosphere. These incl ude both aerobic and anae robic strains, with a prevalence of aerobic strains in submerged soils. PSMs render insoluble phosphate into soluble form via acidification, chelation and exchange re actions. This process no t only reduces the cost of manufacturing fertiliz ers in industry but also mobilizes the fertilizers added to soil (Rodriguez and Reynaldo, 1999). Therefore, many researchers have tried to increase the plant-available 27

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phosphate fraction using PSMs such as Pseudomonas (Suh et al., 1995), Bacillus (Raj et al., 1981), Enterobacter (Laheurte and Berthelin, 1988), Agrobacterium and Aspergillus (Varsha and Patel, 2000; Son et al., 2006). Bacterial genera in this capacity include Pseudomonas, Bacillus, Rhizobium, Burkholderia, Achromobacter, Agrobacterium, Microccocus, Aereobacter, Flavobacterium a nd Erwinia. Species from the genera Pseudomonas, Bacillus and Rhizobium are among the most efficient phosphate solubilizers. The presence of these ba cteria is essential in providing available phosphate to plants (Hilda et al ., 1999). A previous study has s hown more root exudates in P. vittata than non-hyperaccumulator Nephrolepis exaltata consisting mainly of phytic and oxalic acid (Tu et al., 2004). P. vittata takes up more phosphorus when exposed to arsenic, probably resulting from arsenic-induced phosphorus defi ciency (Tu and Ma, 2003). As a nutrient, phytic acid sustains the organic phosphor us solubilizing micr obial community. It is hypothesized that both organic and inorgani c PSMs are more active in the rhizosphere of P. vittata and therefore, increasing phosphorus and/or arsenic availability and possibly allevi ating arsenic to xicity to the plant. However, there is no study conducted to understand the roles of those bacteria in the rhizosphere of P. vittata 1.3.3 Arsenic resistant bacteria Many engineering technologies have been developed for remediation of arsenic contaminated soil and water. Remediating cont aminated water to achieve US Environmental Protection Agencys current sta ndard of 0.01 mg /L arsenic can be achieved using technologies such as precipitation/coprecipitation, membra ne filtration, adsorption, ion exchange and permeable reactive barriers. However, these technologies are expensive and time-consuming, and therefore, are not widely applicable. Phytoe xtraction, the use of green plants to clean up contaminated soil, has attrac ted attention as an environm entally friendly and low-input 28

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remediation technique (Fitz and Wenzel, 2002). Th is technology makes use of hyperaccumulator plants that extract pollutants from the soil and accumulate them in the harvestable above-ground biomass. P. vittata offers a promising resource for phyt oremediation, however, the technology is still in the development and more research is needed. The rate of metal removal is the key to the success of phytoremediation, which depends upon both the plant biomass harvested and meta l concentration in harvested biomass. Interactions between arsenic and phosphorus influence their availabi lity in soils, and thus plant growth and uptake of arsenic and phosphorus. Quantitative analysis of kinetic parameters showed that phosphate inhibited arsenate influx in a directly competitive manner; consistent with the hypothesis that arsenate enters plant roots via phosphate transporters (Wang et al., 2002). However, addition of phosphate substantially increased plant biomass by alleviating arsenate phytotoxicity at high arsenate levels in soils (400 mg/kg) (Rodriguez and Fraga, 1999). Cao et al. (2003) reported that phosphate fertilizer increased soil arsenic availability to P. vittata grown in arsenic contaminated soils. These results s how both arsenic and phosphorus are key factors impacting plant biomass and metal concentration in phytoremediation. It is well known that a consider able number of bacterial spec ies, mostly those associated with the rhizosphere, are able to exert a beneficial effect upon plant growth, by either providing plant nutrients or growth promoters. They ar e called plant growth promoting rhizobacteria (PGPR), however, usually their numbers are not high enough to compete with other bacteria commonly established in the rhizosphere (Rodriguez and Frag a, 1999). Therefore, their impact on plant growth is limited. Inocul ation of plants by a target microorganism at a much higher concentration than that normally found in soil to outcompete with other non-PGPR is necessary to take advantage of the property of plant growth enhancement. By inoculating the fern by target 29

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30 phosphorus and arsenic solubili zing microorganisms to outco mpete with non-contributing communities, the efficiency of phytoremediation by P. vittata could be enhanced. Several studies have been done to analy ze the potential of micr oorganisms on arsenic accumulation by P. vittata ; however, so far the efforts are mostly on abruscular mycorrhizal (AM) fungi. For example, AM fungi increase arsenic uptake by P. vittata when grown on arseniccontaminated soils. This is attributed to enhan ced plant P uptake and better plant growth. Both AM fungi isolated from arsenic-contaminated soils and those commonly found in soils such as Glomus mosseae and Gigaspora margarita functioned similarly. In addition, Liu et al.(2005) reported reduced plant arsenic uptake by P. vittata whereas others showed that AM fungi reduce P. vittata growth (Leung et al., 2006; Trotta et al ., 2006). However, among the references cited here, there is no study focuses on the plant growth promoting bacteria. 1.5 Objectives The overall hypothesis for this st udy is that bacteria in arse nic contaminated sites have evolved strong arsenic detoxification abilities, including those are benefi cial for the arsenic hyperaccumulator. The objectives are therefore to isolate phosphorus solubilizing and arsenicresistant bacteria from arsenic-contaminated soils and understand the mechanisms of their arsenic resistance. Efforts were made to isolate PGPR associated with P. vittata specifically, phosphorus and arsenic solubilizing bacteria, which can poten tially enhance phytoremediation through improving metal removal rate and increasing harvested biom ass. We identified and purified a group of the most As-resistant bacteria, which tolerated up to 30,000mg/kg of arsenic in liquid culture, the higher level reported to date. Experiment result s suggested that arseni c resistance in those bacteria resulted from their efficiency in ar senic transformation and sequestration and their ability in scavenging oxidative burst and count eracting with different osmotic stresses.

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CHAPTER 2 MATERIALS AND METHODS 2.1 Soil Sampling and Arsenic Concentration Analysis A total of 12 soil samples (bulk and rhizosphe re) and 3 plants were collected from two arsenic contaminated sites where P. vittata grew naturally in south Florida in April of 2007. The first site (CCA site) was contaminated from chromated copper arsenate, which was previously used for pressure treat lumber fr om 1951 to 1962 (Komar et al., 1998). The second site (RES site) was a residentia l site in central Florida, wher e CCA treated woods were used for stairs and decks. Rhizosphere soil was define d as the soil attached to the roots, which was removed from the roots by shaking gently. The bulk soil was collected from site without plants influence. Soil samp les were kept at 4C. For arsenic concentration analysis, soil and plant samples were air dried (22C), mixed thoroughly and digested by nitric acid / hydrogen peroxide (USEPA Method 3051) in a heating block (Environmental Express, Ventura, CA). Arsenic concentrations in the solutions were analyzed by graphite furnace atomic absorption spectroscopy (GFAAS, Perkin Elmer SIMMA 6000, Perkin-Elmer Corp., Norwalk, CT). Analysis was carried out in triplicates. 2.2 Bacterial Isolation and Enumeration by Total Heterotrophic Counting To select arsenic-resistant bacteria, thr ee soil samples from CCA site and two soil samples from RES site, with different arseni c concentrations were used. The CCA soils included two bulk soils (92.8 a nd 167 mg/kg As) and one rhizosphere soil (80.0 mg/kg As) and the RES soils included one bulk soil ( 12.9 mg/kg As) and one rhizosphere soil (28.2 mg/kg As). Soil samples (0.3 g) were suspended in 10 mL of sterilized water and vortexed rigorously. Soil suspensions (2, 20, and 200 l) were plated onto two agarose media: modified TYEG (1/10 strength) or NBRIP agarose medium. The TYEG medium contained 1g/L tryptone, 0.3g/L yeast extr act, 0.5/L glucose and 1% agarose. The NBRIP agarose 31

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media contained 5g glucose, 5g MgCl2H2O, 0.25g MgSO4H2O, 0.2g KCl, 0.1g (NH4)2SO4; 10mg FeSO4, 14mg nitric acid, and 1% agarose, which was spiked with inorganic or organic phosphorus sources (5g of phosphate rock: Ca10(OH)2(PO4)6(F, Cl); or sodium phytic acid: Na12C6H6O24P6). After 2 days, bacteria produc ed most single colonies among 3 inoculum volumes on the media were inoculat ed to the respective media agarose plates containing different levels of arsenic (10, 50 100, 200, 300 and 400 mM arsenic as sodium arsenate). Number of survived bacterial col onies was counted after two days of growth. Since arsenic availability in agar media (s emi solid) may be limited, bacteria grew on 400 mM arsenic agar plate were inoculated into 400 mM arseni c TYEG liquid medium to test bacterial arsenic tolerance in liquid culture. All bacterial inc ubations were conducted at room temperature (22C). 2.3 Bacterial Identification The twelve most arsenic resistant bact eria were identified by 16s rRNA gene sequencing method. Bacterium genomic DNA were extracted with phenol, then phenol/chloroform/isoamyl alcohol (pH 8.0 25: 25:1), and chloroform/isoamyl (24:1). DNA was precipitated in 3M sodium acetate (pH 5.2) and ice cold ethanol, pelleted by centrifugation, ethanol washed 3 times, and then resuspended in water. 16s rRNA genes were PCR amplified by primers 8F 5-AGA GTT TGA TCC TGG CTC AG-3 and 1489R 5-TAC CTT GTT ACG ACT TCA-3, PCR condition included: initial de naturation for 7 min at 95 C, 30 cycles of 95 C for 1 min, 51C for 1min, 72C for 1min, and a fina l extension step at 72C for 10 min, with 0.2 M final primer conc entration (Bruneel et al., 2006). PCR products were cloned by TOPO TA Cloning Kit (In vitrogen Inc.) and sequenced by ICBR Sequencing Lab at University of Florida. 16s rDNA phylogenetic tree was constructed by using the BLAST (Altschul et al., 1990), clusta lX (Jeanmougin et al., 1998), and Treeview program (Page, 1996). 32

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2.4 Bacterial Growth Characterization under Arsenic and Osmotic Stress Bacterial isolates, which tolerated to 400mM sodium arsenate in liquid culture, belonged to 8 different genera a ccording to 16s rRNA sequences ( Naxibacter sp. AH4 Mesorhizobium sp. AH5 Methylobacterium sp. AH6 Enterobacter sp. AH10, Pseudomonas sp. AH21, Bacillus sp. AH22, Acinetobacter sp. AH23 and Caryophanon sp. AH28) were chosen for the growth characteristics study. Their arsenic resistance mechanisms were examined by measuring their growth characteris tics under arsenic or osmotic stresses in two experiments. The first experiment compared b acterial growth characte ristics under same ionic strength using sodium arsenate or sodium chloride. The second experiment was conducted under -1.5M Pa of osmotic stress using 3 different sources: sodium arsenate, sodium chloride and polyethylene glycol 6000 (PEG 6000). Both experiments were conducted with three replicates. In the first experiment, 2.4 M of NaCl or 400 mM of sodium arsenate were used to compare bacterial growth (Naydenov et al., 2006) with control bacter ia growing in TYEG medium with no osmotic stress. Bacteria were grown at room temp erature (22C) with constant agitation (150 rpm). B acterial growths were measured at 0, 6, 20 and 26 hours by a spectrometer (SHIMADZA BioSpec-Mini) at 600 nm (OD600) (1 cm light-path length) after inoculation. The second experiment was done similarly ex cept using 176 mM of sodium arsenate (calculated by MINTEQA2, (Allison et al., 1999)), 400 mM of NaCl or 26% of PEG (Sosa et al., 2005), which produced -1.5M Pa of osmotic stress. Bacterial growths were monitored over 42 hours. The extra time in the second expe riment allowed comparing stationary phase growth and Cltoxicity in the isolates. 2.5 Oxidative Stress Test Preliminary experiment was done to identify Salmonella typhimurium mutants. Strains of S. typhimurium SF1005 (unmarked rpoS mutation), S. typhimurium cc1000 ropS:: tet and S. 33

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typhimurium GS014 oxyR ::Tn10 were tested using hydrogen pe roxide (Aaron Industries Inc., SC, USA) and N,N'-Dimethyl-4,4'-bipyridinium dichloride (paraquat) (Sigma) either by measuring inhibition area on Petri dishes, or by measuring bacterial growth in 96-well cell culture plates. Measuring inhibition area on Petri dishes. Aliquots of 50 l of overnight-grown bacterial culture were plated in TYEG agar media and bacteria were allowed to grow for 4 hours. A 6 mm sterile paper disc was placed on each plate, to which 25 l of 3% hydrogen peroxide or 50 mmol/L of para quat were applied. Plates were incubated at 22C, and the inhibition zone of bacterial growth was measured after 24 and 48 hours. The experiments were carried out in triplicates. Measuring bacterial growth in 96-well cell culture plates. Bacterial isolates were inoculated to R2A medium (proteose peptone 0.5g/L, casamino acids 0.5 g/L, yeast extract 0.5 g/L, glucose 0.5 g/L, so luble starch 0.5 g/L, K2HPO4 0.3 g/L, MgSO4H2O 0.05 g/L, sodium pyruvate 0.3 g/L and 1.5% of agarose, fina l pH7.2 at 25 C) to st arve the cells. Cells were scratched from the surface of the plat e using cotton swab, then suspended in M9 minimal medium (12.8g Na2HPO4H2O, 3g KH2PO4, 0.5g NaCl, 1.0g NH4Cl, 2mM MgSO4, 100M CaCl2) with 0.02% of glucose as carbon source and 0.03% of tetrazolium violet (TV) (Sigma) as a bacterial respiratory indicator. TV was stocked in etha nol stock solution. 300l of final volume with 50mM of paraquat was used to test in 96 well plates. Initial cell concentration was OD590=0.02. Bacterial growth was determ ined by measuring absorption of suspension at 590 nm after 24 hours (Tracy et al., 2002). S. typhimurium LT2 oxyR::Tn10 and 14028 wild type were used as controls af ter checking the growth of all the mutations. 2.6 Arsenic Transformation by Arsenic Resistant Bacteria Arsenic transformation by three arse nic-resistant bacterial isolates ( Naxibacter sp. AH4, Mesorhizobium sp. AH5, Pseudomonas sp. AH21) and a non-arsenic resistant bacterium Sinorhizobium meliloti MG32 were analyzed. The experi ment included a control without 34

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bacterium, and 3 replicates for each treatme nt. Bacterial medium contained either 1mM arsenite or arsenate and started at same cell density (OD600 = 0.1). Cell cultures were grown at room temperature with 150 rpm constant sh aking. Shake cultures were sampled after 4, 8, 16 and 32 hours. Total arsenic was analyzed by a graphite furnace atomic absorption spectrophotometer (240Z, Vari an, Walnut Creek, CA). As speciation was done by arsenic speciation cartridge (Metal Soft Center, Highland Pa rk, NJ) (Meng et al., 2001). 2.7 Arsenate Reductase Assay Cell culture of three arsenic-resistant bacteria Naxibacter sp. AH4, Mesorhizobium sp. AH5, Pseudomonas sp. AH21and control bacteria Escherichia coli DH5 were grown in TYEG and LB media overnight, re spectively, before inoculated into 50 mL of TYEG or LB media. Protein expression was induced by 0.1 mM sodium arsenate after the lag phase (about 3 hours), then shaken for another 4 hours befo re harvesting the cells by centrifugation. Cell pellets were resuspended in reacti on buffer (10 mM Tris, pH 7.5, 1 mM Na2EDTA, 1 mM MgCl2, 1 mM DTT) (Anderson and Cook, 2004) w ith 0.1 mM of proteinase inhibitor phenylmethylsulfonyl fluoride (PMSF, SIGMA); Total proteins were extracted by glass beads and centrifugation in 4 C, and the concentration was determined by Bio-Rad Protein Assay (Bio-Rad Laboratories, USA). Arsenate reductase assay solu tion contained 150 l protein extracts, 1 ml reaction buffer, 10 M sodium arsenate, 0.5 mM NADPH, 1 mM GSH, 2U yeast glutathione reductase and 0.02M E. coli Glutaredoxin 2. Enzyme activity was determined by measuring the absorption at 340 nm (Gladysheva et al., 1994). 2. 8 Arsenic Detoxifying Gene Determination The details for degenerated or regular primers for arsR arsB and arsC gene amplifications were described below: arsR forward: 5ATGMTYAMCCCTCCCCARGTCTTYAAAT-3 and arsR reverse: 5TYAACAACAASCGGCKGCGCGCW-3. PCR cond ition included: initial denaturation for 7 min, 40 cycles of 94C for 45s, 58C for 30s, 72C for 1min, and a final extension step at 35

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72C for 10 min, with 0.2 M final primer concentration (Mandal et al., 2008); arsB forward: 5ATGGCAACCGAAAGGTTTAG-3, arsB reverse: 5GTTGGCATGTTGTTCATAAT3. PCR condition was: 94C fo r 7 min and 29 cycles at 94C for 30 s, 55C for 30 s, 72C for 1 min, and a final extension at 72C for 10 min (Anderson and Cook, 2004), with 0.2M of primers; arsC forward: 5GCATTCTTTCCGAAGCCATGTTCAA3, arsC reverse: 5AGCTCACGCTTGAGCTGGTCGCGAT3, which were designed based on Pseudomonas aeruginosae PAO1 arsC gene. A Pseudomonas aeruginosae PAO1 strain was used as a positive control for both arsB and arsC, and the PCR products from this organism were confirmed by sequencing and then used as probes for Southern hybridization. PCR condition was: 94C for 7 min and 29 cycles at 94C for 1min, 56.8C for 1mins, 72C for 1 min, and a final extension at 72C for 10 min, with 0.2M of primers. Southern Blot followed the method of Russell (2001) and user manual of DIG DNA labeling & Detecting Kit (Roche Diagnostics, USA. REF: 11093657910 Cat: 1093657). Primers for arrA gene cloning were ArrA forward: 5AAGGTGTATGGAATA AAGCGTTT gtbgghgaytt3 and ArrA reverse: 5CCTGTGATTTCAGGTGCCcayt yvggngt-3) (lower case indi cated the degenerated parts). Shewanella sp. ANA3 was used as arrA positive control. PCR reactions included incubation at 95C for 10 minutes, followed by 40 cycles of 95C for 15 seconds, 50C for 40 seconds, and 72C for one minute. The final primer concentration was 0.5 M (Malasarn et al., 2004) 2. 9 Arsenic Detoxifying Plasmid Determination Arsenic resistant isolates were divided in to 10 groups and each group containing 5 mL of mixed cell culture in TYEG medium was used for plasmid extraction. Plasmid extraction performed by the guideline of QIAprep Spin Miniprep Kit (QIAGEN Science, Maryland, USA). Plasmids were transformed into chemical competent E. coli DH5 and plated on LB media containing 50 mM sodium arsenate to test their arsenic tolerance. Plasmid in As-r E. 36

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coli DH5 was further isolated and transformed again into E. coli DH5 to confirm plasmid encoded arsenic resistant functional genes. Isolates in groups with As-r plasmid were further tested one by one to identify where the plasmid was from. 2.10 Antibiotics Test All identified isolates were tested with th eir kanamycin resistance at 50g/ml in TYEG with 1% of agarose plates. Negatives results furt her tested their resistance with carbenicillin, nalidix acid, ampicillin and neomycin at concentration of 50 g/ml. 2.11 Mesorhizobium sp. AH5 Genomic DNA Library Construction Bacterial isolate Mesorhizobium sp. AH5 was chosen to construct a DNA library to search for functional genes contributing to ar senic resistance. An am ount of 1.5g bacterial DNA was extracted and partial digested w ith EcoRI or BamHI at 37C for one hour. Digestion was stopped by loading on ice for 5 minutes. DNA fragments at about 10 thousand base pairs were purified by Gel Band Purificatio n Kit (GE Healthcare, USA) and ligated with CopyControl Vector (CopyControlTM BAC Cloning Kit, EPICENTR Wisconsin). The vector was then transformed into competent E. coli DH5 and selected with LB plates added with 40mg/ml of x-gal, 0.4mmol/L of Isopropyl -D-1-thiogalactopyranoside (IPTG) and 12.5g/mlof chloramphenicol. Positive colonies were inoculated into plates containing 50mM of sodium arsenate. GenBank Accession Numbers of the 16S rRNA gene sequences of the isolates: AH4: FJ621305; AH5: FJ621306; AH6: FJ621307; AH10: FJ621308; AH21: FJ621309; AH22: FJ621310; AH23: FJ621311; AH25: FJ621311; AH28: FJ621313; AH34: FJ621314; AH43: FJ621315; AH45: FJ621316. 37

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CHAPTER 3 RESULTS AND DISCUSSIONS 3.1 Arsenic Concentrations in Soil and Plant Samples Total arsenic concentrations in soil and Pteris vittata samples from two contaminated sites (CCA and RES) are showed in Figure 3-1. The data show that soils were heterogeneous with varying arsenic concentrations in the contaminated sites, with rhizosphere soils generally containing higher arsenic concentration than those in bulk soils. Arsenic concentrations in the fronds of P. vittata were substantially higher than those in the roots. Translocation factor (TF), which is defined as the ra tio of metal concentrations in the fronds to those in the roots, was 31.7 fo r plants growing in CCA soil, and 14.7-21.9 for plants growing in RES soil. Bioaccumulation factor (BF), which is defined as the ratio of metal concentration in plant biomass to that in the soil, ranged from 21.5 of the CCA plant, to 171, 110 of the two RES plants. While TF has been used to determine the effectiveness of plants in translocating metals from the roots to the shoots, BF has been used to determine the effectiveness of plants in removing metals from soils. 3.2 Arsenic Resistant Bacteria from Contaminated Soils Culturable bacteria account for only less than 1% of natural bacterial communities (Amann et al., 1995). Techniques exploring phospholipid fatty acid (PLFA) or nucleic acids such in terminal restriction fragment length polymorphism (TFLP), denaturing gradient gel electrophoresis (DGGE) are more robust in unveiling microbial community structures. However, arsenic resistance bacteria are special case. It is known that the formation of microbial biofilm protects bacterial community from toxic agents. Although there are no direct data showing formation of biofilm help bacteria resist higher arsenic concentrations, it has been showed that antimicrobial agents such as chlorine and many antibiotics failed to penetrate the biofilm (Mah and O'Toole, 2001). Therefore, it is reasonable to assume that fo rmation of microbial biofilm may also protect 38

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bacterial community from arse nic toxicity. Due to the hete rogeneous property of soil, microbial communities in contaminated sites are not necessarily having higher ability in arsenic detoxification. Therefore, obtaining pure bacterial cultur e is still the first choice in selecting environmental bacteria possessing the ability in arsenic detoxification. In this experiment, arseni c-resistant bacteria were isolated from two arseniccontaminated soils (CCA and RES), which were plated on two media TYEG and NBRIP containing up to 400 mM arsenate. In both sites, P. vittata rhizosphere soils sustained substantially higher number of arse nic resistant bacteria, compared to bulk soils with similar arsenic concentrations (Table 3-1, 3-2). For example, in CCA site, 42.5% of the tested colonies were able to grow in TYEG containing 400mM of arsenate, while the numbers in the two bulk soils from the same site were 16.9% and 3.4%, respectively, all starting with about 80 single colonies (Table 3-1). In RES site, the percentages of survivals in 400 mM TYEG plates were 13.3% versus 3.1% fo r rhizosphere soil and bulk so il respectively. Totally fiftythree isolates were obtained from plates cont aining 400mM of arsena te, and designated as AH1 to AH53. Among them, AH1 to AH4 were isolated by NBRIP and the rest were by TYEG medium. Previous study revealed that majority of arsenic that P. vittata took up were solubilized from recalcitrant fractions in soil (51-71% we re from Ca-bound arsenic) (Fayiga et al., 2007). Therefore, rhizosphere bacteria may be exposed to higher bioavailable arsenic concentration than those from bulk soils. Under this selec tive pressure for decades, microbial community developed sophisticate arsenic detoxificat ion mechanisms to overcome the growth restrictions. In Table 3-1 and 32, arsenic resistant bacteria presented as percentage of the bacterial colony number tested, which mi nimizes the effects of nutrition and soil heterogeneity. The fact that higher percentage of arsenic resistant bacteria were from rhizosphere soils indicated that, during pl ant arsenic accumulation, higher bioavailable 39

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arsenic mobilized from soil indu ced higher ability of arsenic toleration in the associated microbial community. 0 50 100 150 200 SB1SB2SB3SR1SR2CB1CB2CB3CB4CB5CB6CRSoil SampleAs(mg/kg)a 0 500 1000 1500 2000 2500 3000 3500 4000 C_rootC_frondS_root_1S_frond_1S_root_2S_frond_2Plant SampleAs(mg/kg)bFigure 3-1. Arsenic concentrations in soil (a) and Pteris vittata (b) samples from two arsenic contaminated sites. S: RES site; C: CCA site; B: bulk soil; R: rhizosphere soil. Two plants from RES site and one plant from CCA site were analyzed. Soil_53 is standard soil sample with 151.0 mg/kg of ar senic. Points are means and standard errors of 3 replicates. 3.3 Phosphorus Solubilizing Bacteria from Arsenic Contaminated Soils A group of phosphorus solubilizing bacteria was isolated. Thos e bacterial isolates were capable of mobilizing phosphorus from either orga nic or inorganic sources. They were also 40

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arsenic tolerant (Table 3-2), though not as tolerant as those cultured in TYEG media. Those bacteria can used to increase P availabili ty in agricultural soils as well as arsenic contaminated soils to improve plants P nutrition. Phytic acid has been identified as one of the major compounds of P. vittata root exudates (Tu et al., 2004). In addition, phosphorus ha s been identified to play a major role in arsenic detoxification by P. vittata Only several bacterial colonies, which were identified as Naxibacter sp. AH1 and AH4, were able to grow on NBRIP with phytic acid. They were isolated from CCA 3R and RES 5R, showing the existe nce of organic phosphorus solubilizing bacterial communities in rhizosphere soil (Figure 3-2). Two of them with unique yellow and red pigments were identified as Naxibacter species by 16s rDNA sequence method (Sequences are available in Appendix A). Figure 3-2 shows the two bacteria on N BRIP plates, with their 16s rRNA gene sequences sharing 98% of similarity. Bact erium AH1 produced red pigment (A) and was more sensitive to arsenic, which cant surv ive in 400 mM of sodium arsenate (data not shown). Naxibacter is a recently described bacterial genus by Xu et al.(2005) and at present the genus comprises only one species, Naxibacter alkalitolerans (Kampfer et al., 2008). However, 16s rRNA gene of bacteria AH1 and AH4 shared <97% of similarity with the published Naxibacter species, which is a border line to define a species by 16s rRNA. It is possible that AH1 and AH4 are new bacteria that have not been fully described. However, no bacteria were recovered on N BRIP media without additional glucose as C source, showing that no chemoheterotrophic bacterium utilizing phytic acid as carbon source was isolated from the soils, although bacteria were able to use phytic acid as P source. Compared to the large number of arseni c-resistant bacteria recovered by TYEG medium with arsenic con centrations up to 400 mM in agar pl ates, those bacteria able to use organic or inorganic phosphorus from the immobilized sources ( phytic acid or rock phosphate) 41

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in NBRIP medium were much less in quantity an d much more sensitive to arsenic. Table 3-2 shows percentage of arsenic resistant bacteria am ong those isolates that were able to use rock phosphate as phosphorus source. This may imply th at bacterial ability to solubilize P was not related to its ability to tolerate arsenic or th e bacteria couldnt obtain sufficient amount of P to help detoxify arsenic since P availability in phosphate rock was much lower than that provided in TYEG growth media. However, one of phosphorus solubilizing bacteria AH4 was identified as one of the 12 most arsenic resistant bacteria (Table 3-3). Table 3-1. Percentage of arse nate resistant colonies recove red in TYEG medium isolated from different soils and under di fferent arsenic concentrations*. Arsenic concentrations in TYEG medium (mM) Soil used Soil As mg/kg 10 50 100 200 300 400 CCA 1B* 92.5 62.3 46.8 42.9 40.3 36.4 16.9 CCA 2B 167 40.9 37.5 36.4 21.6 15.9 3.40 CCA 3R 80.0 68.1 66.0 57.4 46.8 42.5 42.5 RES 4B 12.9 15.9 9.50 4.80 3.10 3.10 3.10 RES 5R 28.0 78.7 76.0 74.7 72.0 69.3 13.3 Table 3-2. Percentage of bacteria survived in NBRIP + phosphate rock plates under different arsenic concentrations Arsenic concentrations in NBRIP medium (mM) Soil used Soil As mg kg-1 10 50 100 200 300 400 CCA 1B* 92.5 87.2 38.5 25.6 15.4 5.1 0 CCA 2B 167 98.9 73.3 14.4 4.40 0 0 CCA 3R 80.0 54.3 F* 20.0 20.0 0 0 RES 4B 12.9 0 F 0 0 0 0 RES 5R 28.0 53.7 F 24.1 0 0 0 B = bulk soil; R= rhizosphere soil; CCA site = ch romated copper arsenate contaminated site, and RES = residential contaminated site; *F: No data, media precipitation. 42

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Figure 3-2. Naxibacter sp. AH1 (A) and Naxibacter sp. AH4 (B) on agarose plates. 3.4 Characterization and Identificati on of Arsenic Resistant Bacteria Rhizosphere soil is typically aerated and nut rient rich. This experiment attempted to isolate chemoheterotrophic bacteria from rhizosphere, which were able to tolerate high concentration of arsenic. However, the method di dnt preclude that the is olates would also be facultative anaerobics. Totally, 53 isolates that can grow on plates containing 400mM arsenate were obtained from two CCA contaminat ed sites. These strains were designated as AH1 to AH53. Because TYEG is nutrient-rich medium and NBRIP is defined medium, bacteria recovered by NBRIP were all able to gr ow in TYEG. Therefore, for arsenic resistant mechanism study, TYEG was used for all experiments. Considering that arsenic availability in agaros e plates is lower than that in liquid culture, 53 isolates were then screened in TYEG li quid culture containing 400 mM sodium arsenate. Those were able to grow were further separa ted and identified base d on 16s rRNA analyses (Table 3-3). Based on the BLAST results of th e 16s rRNA gene, the 12 mo st arsenic resistant soil isolates were identified as Naxibacter sp. AH4, Mesorhizobium sp. AH5, Methylobacterium sp. AH6, Enterobacter sp. AH10, Pseudomonas sp. AH21, Bacillus sp. AH22, Acinetobacter sp. AH23, Pseudomonas sp. AH25, Bacillus sp. AH28, Pseudomonas sp. AH34, Pseudomonas sp AH43, and Pseudomonas sp. AH45. 43

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A phylogenetic tree of the isol ates and references from GenBank were shown in Figure 3-3. The references in Figure 3-3 were all fr om culturable isolates and well identified. GenBank access numbers and DNA sequences are available in Appendix B. In this study, total hetero trophic arsenic resistant bact erial counts from arsenic contaminated sites showed a higher quantity and quality of arsenic toleration in rhizosphere soil compared to bulk soils (Table 3-1, 3-2). The 8 genera of the 12 most arsenic resistant bacteria belong to the two phyla: Proteobacter ia and Firmicutes (Figure 3-3). Consistent results are found in literature, which show that majority of the 16S rRNA gene sequences of cultured prokaryotes are from Proteobacteria ( ca. 50%), Actinobacteria, Firmicutes (ca. 15%), and Bacteroidetes within GenBank (Riesenfeld et al., 2004). Therefore, th e isolates in this study represent the cultivable aerobic heterotrophic, whic h are arsenic resistant. Table 3-3. The 12 most arsenic-resistant bacter ia identified from two arsenic contaminated soils ID CCA/RES* site R/B* Naxibacter sp AH4 RES R Mesorhizobium sp. AH5 CCA B Methylobacterium sp. AH6 CCA B Enterobacter sp. AH10 CCA B Pseudomonas sp. AH21 CCA R Bacillus sp. AH22 CCA R Acinetobacter sp. AH23 CCA R Pseudomonas sp. AH25 CCA R Bacillus sp. AH28 CCA R Pseudomonas sp. AH34 CCA R Pseudomonas sp. AH43 RES R Pseudomonas sp. AH45 RES R *CCA: chromated copper arsenate contaminat ed site; RES: residential contaminated site; R: rhizosphere soil; B: bulk soil. 44

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45 Up to date, Corynebacterium glutamicum was the only known microorganism, which tolerated up to 400 mM sodium ar senate (Mateos et al., 2006). C. glutamicum is in the phylum of Actinobacteria, G+, aerobic or facultatively anaerobic. In fact C. glutamicum is one of the biotechnologically most important bacterial species in us e today. The group of arsenic resistant bacteria isolated in this study are either able to solubilize phosphorus or arsenic, or are potentially beneficial bacteria for arsenic phytoremediation by P. vittata In addition, they also had the potentials for biol eaching to remove arsenic or phosphorus from soils. Tn5 transposon mutation library has been used to search for bacteria functional genes. However, in using Tn5 transposon, bacteria need to be sensitive to kanamycin. Most arsenic resistant bacteria should have an tibiotic resistance, so their an tibiotics resistant profiles were examined. The results in Table 3-4 showed that, except for Mesorhizobium sp. AH5, Pseudomonas sp. AH36, and Pseudomonas sp. AH47 (identified by 16s rRNA gene sequences, sequence are listed in Appendix A), all arsenic resistant bacteria identified in this study carried kanamycin resistant genes. The resi stant profiles of the 3 bacteria were shown in Table 3-4. E. coli DH5 is an experimental control. Th e results showed broad antibiotics resistance in arsenic resistant bacteria. Tn5 transposon mutation library should be carried out in future study based on the results on bacterial genomic DNA library in E. coli DH5 Indeed, the two methods represent the concepts of forward genetics and reverse genetics. Because different arsenate reductase genes require different cofactors or other host differences, there is no guarantee that functional genes can be found by forward genetics of genomic DNA library in E. coli DH5 Therefore, the reverse genetics of a Tn5 tran sposon mutation library may be another approach.

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Table 3-4. Antibiotic resistance test on the arsenic resistant bacteria isolated from arsenic contaminated soils Antibiotic Mesorhizobiu m sp. AH5* Pseudomonas sp. AH36 Pseudomonas sp. AH47 E. coli DH5 Carbenicillin + + + Nalidix acid + + + Ampicillin + + + Neomycin + + + Kanamycin Figure 3-3. Phylogenetic tree of the 12 most arsenic resist ant bacteria based on 16s rRNA sequences. F. cheniae 16s rRNA gene obtained from GenBank was used as an outgroup (Accession no. EF407880). Numbers represent per centages of 1000 bootstraps and are shown only for bootstrap value<80%. The scale bar re presents 1 nucleotide substitution per 100 nucleotides of 16s rRNA sequence. 46

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3.5 General Stress Tolerance Characteri stics of Arsenic Resistant Bacteria 3.5.1 Growth Characteristics under Arsenic Stress The growth characteristics of eight most arse nic-resistant bacteria were compared in the presence of 0.4 M sodium arsenate or 2.4 M NaCl (with similar ionic strengths) to determine if bacterias arsenic resistance was related to salt tolerance. All eight strains had a longer lag phase in 0.4 M sodium arsenate comparing to the growth curv e in TYEG medium, and the exponential growth started after 6 hours (Figure 3-4). Except for Naxibacter sp. AH4, which had a higher OD600 in TYEG medium with 2.4 M NaCl than 0.4 M sodium arsenate, all the other strains were not able to grow at such hi gh NaCl concentration. This result indicted that they were not tolerant to such high salt co ncentration. In comparis on, after 25 hours, there was little difference in growth betw een arsenic and TYEG treatment. The fact that the growth curve as measured by OD600 after 26 hours under arsenate stress in strain AH5, AH10 and AH28 were almost the same as the TYEG control indicated limited toxicity effect of arsenate on the is olates. This was confirmed in the follow up experiment, where bacteria were grown in 1.5MPa osmotic stress. After 25 hours, there was little difference in growth between As and TY EG treatment (Fig. 3-6). This result also showed a higher OD600 in sodium arsenate than NaCl or PEG (Figure 3-6). Bacteria also grew better in NaCl than under same osmotic stress of PEG. Arsenic resistant index (ARI), which was defined as the ratio of the growth rate in medium with 400 mM arsenic to that in the control medium, was developed to normalize bacterial arsenic detoxification (F igure 3-5). The closer ARI is to 1, the smaller toxicity of arsenic is to the soil bacter ia. The ARI index showed that, Mesorhizobium sp. AH5, Enterobacter sp. AH10 and Bacillus sp. AH28 were the most efficient in arsenic detoxification ( = 0.01) and their growths were only less that 30% slower in 400 mM of sodium arsenate than TYEG without arsenic. The ability of the soil isolates in arsenic detoxification was also show n in the osmotic stress test. Though the control strain P. 47

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48 fluorescens CHAO was able to grow at -1.5M Pa of osmotic stress, it didnt grow under arsenic stress (Figure 3-6). Those bacteria were shown to be efficient in arsenate reduction (Fig. 3-10). Under the model of arsenate reduction detoxification unde r aerobic condition, bacteria take up arsenate through phosphate transporter, and reduce it to arsenite by specific enzy me with the help of glutaredoxin, thioredoxin or fe rredoxin, and arsenite is then extruded out by membrane pumper (Rosen, 2002). The efficiency of phosphate transporter is positively correlated with cellular growth; after normalized with cellu lar growth, ARI indicates the comprehensive effects of bacterias ability in reducing and ex truding arsenic in this model. The fact that bacterial isolates Mesorhizobium sp. AH5, Enterobacter sp. AH10 and Bacillus sp. AH28 had the highest score of ARI indicates their hi ghest ability in arsenic detoxification. In this study, we proposed, for the first tim e, the model of multiple levels of arsenic tolerance. In other words, while specific functional enzymes take up, reduce and extrude arsenic, global gene regulations are turned on to counteract ar senic-induced growth stresses, such as oxidative burst or osmotic stress. From the angle of overall cellular metabolisms, ARI also indicates bacterias ability in overcoming cellular toxicity. The closer ARI is to 1, the higher its ability in s cavenging oxidative burst and osmotic stress that the arsenic resistant bacteria have. For example, the ARI of Methylobacterium sp. AH6 is 0.29, which means overall cellular metabolism was reduced by 71% from arsenic toxicity. The ARI of Bacillus sp. AH28 was 0.75, suggesting that 25% of ce llular metabolism was spent on arsenic detoxification.

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-1 -0.8 -0.6 -0.4 -0.2 0 0.2 0.4 0510152025lg(OD600)A -1.2 -1 -0.8 -0.6 -0.4 -0.2 0 0.2 0.4 0510152025lg(OD600)B -1.2 -1 -0.8 -0.6 -0.4 -0.2 0 0.2 0510152025lg(OD600)C -1.2 -1 -0.8 -0.6 -0.4 -0.2 0 0510152025lg(OD600) TYEG NaCl As D Figure 3-4. Growth characteris tics of eight arsenic-resistan t bacteria in TYEG medium containing 400 mM sodium arsenate or 2.4 M sodium chloride. The legend diamond denotes TYEG medium without additional salts, rectangle denotes TYEG with 2.4 M of NaCl, and cross de notes TYEG medium with 400 mM of sodium arsenate. A: Naxibacter sp AH4; B: Mesorhizobium sp. AH5; C: Methylobacterium sp AH6; D: Enterobacter sp AH10; E: Pseudomonas sp. AH21; F: Bacillus sp. AH22; G: Acinetobacter sp. AH23; H: Caryophanon sp. AH28. Points are means and standa rd errors of 3 replicates. 49

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-1.2 -1 -0.8 -0.6 -0.4 -0.2 0 0.2 0.4 0510152025lg(OD600)E -1.4 -1.2 -1 -0.8 -0.6 -0.4 -0.2 0 0.2 0510152025lg(OD600)F -1.4 -1.2 -1 -0.8 -0.6 -0.4 -0.2 0 0.2 0510152025lg(OD600)G -1.4 -1.2 -1 -0.8 -0.6 -0.4 -0.2 0 0.2 0510152025 Hourslg(OD600) TYEG NaCl As HFigure 3-4. Continued. 50

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0 0.2 0.4 0.6 0.8 1 AH4AH5AH6AH10AH21AH22AH23AH28StrainArsenic Resistance Indexab abab c c d dFigure 3-5. Arsenate Resistant Index (ARI) based on bacteria l growth at 400 mM sodium arsenate ( = 0.01). ARI is defined as the ratio of the growth rate in medium with 400 mM arsenate to that in the control medium. Columns are means and standard errors of 3 replicates. 3.5.2 Arsenic Resistant Bacterial Growth under Osmotic Stress None of the isolates were able to grow in NaCl under similar ionic strength of 400 mM of sodium arsenate except AH4 (Figure 3-4). In stead of using lethal dose of NaCl, the second growth experiment used -1.5M Pa of osmotic st ress that the eight arse nic-resistant bacteria can tolerate to, which was equal to 400 mM of NaCl, 26% of polyethylene glycol 6000 (PEG6000) and 176 mM of sodium arsenate (All ison et al., 1999; Sosa et al., 2005). Growth results showed that the isolates grew better in NaCl than PEG at the same osmotic stress level (Figure 3-6). All bacteria except P. fluorescens CHAO grew in the presen ce of sodium arsenate, exhibiting their tolerance to hi gh arsenic concentrati on (Figure 3-6). All the strains except P. fluorescens CHAO were tolerant to -1.5MPa or higher osmotic stress, which was proved by growth in the presence of NaCl and PEG. With the same amount of inoculation, the initial absorption in medium with PEG was smaller than TYEG, NaCl or arsenate in all strains. This suggested that the impermeable solute reduced cell absorption or led to cell break down. 51

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However, bacteria AH10 and AH22 grew better in PEG than NaCl at the end of the experiment. Compared to TYEG, NaCl and PEG, there was a lack phase for arsenic resistant bacteria to grow in the presence of arsenic, indicated that some of the detoxification related genes were not housekeeping genes. Exponentia l growth started 6 hours after inoculation except for bacteria AH22 and AH23, which was because that, while others were inoculated from fresh cell culture, the two strains were inoc ulated from cultures that had been kept in 4 C. After 25 hours, there was little differen ce in growth between NaCl, PEG and TYEG treatment. After 35 hours, bacteria in the medium with arsenic a ll reached to stationary phase. After 45 hours, all bacteria grew better with arsenic than with NaCl or PEG except AH23. NaCl and PEG represent two type of osmotic stress: salt and water stresses. Both water and salt stress inhibited cellular growth to some extent and they have a common effect, lowering the osmotic potential of the cellular environment. While water stress is caused by using impermeable solutes (such as PEG 6000), salt stress is caused by various ions, mainly Na+ and Cl-, which can be transported into and out of cells. Thus, salt stress has additional specific effect of ions present in the environment. The fact that arsenic resistant bacteria grow better in NaCl indicates the existence of cr oss protection between arsenic and other ion generated osmotic stress. This suggests bacter ias ability in tolerating arsenic may also because of maintenance a homeostasis of intracellular ionic strength. However, this result doesnt mean that the ability of arsenic-resistant bacteria in detoxifying arsenic is because of resistant to osmotic stress, nor expression of As-induced genes alleviated ionic osmotic stress. Indee d, cellular growth bene fited from both arsenic stress and salt stress (Figure 3-6). As laboratory strain, P. fluorescens CHAO tolerated to 1.5M Pa of osmotic stress, but was not able to grow in the presence of arsenic. 52

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-3 -2.5 -2 -1.5 -1 -0.5 0 0.5 0510152025303540lg(OD600)A -2.5 -2 -1.5 -1 -0.5 0 0.5 0510152025303540lg(OD600)B -2.5 -2 -1.5 -1 -0.5 0 0.5 0510152025303540lg(OD600)C -3 -2.5 -2 -1.5 -1 -0.5 0 0.5 0510152025303540 Hourlg(OD600) TYEG NaCl PEG As DFigure 3-6. Bacterial growth under osmotic stress (-1.5MPa) generated by 400 mM of NaCl, 176 mM of sodium arsenate and 26% of PEG6000. The legend diamond denotes TYEG medium without additional osmotic source, rectangle denotes TYEG with NaCl, triangle denotes TYEG with PEG, and cross denotes TYEG medium with sodium arsenate. Pseudomonas fluorescens CHAO is a laboratory control strain. A: Pseudomonas fluorescens CHAO; B: Naxibacter sp AH4; C: Mesorhizobium sp. AH5; D: Methylobacterium sp AH6; E: Enterobacter sp. AH10; F: Pseudomonas sp. AH21; G: Bacillus sp. AH22; H: Acinetobacter sp. AH23; I: Caryophanon sp. AH28. Points are means and standa rd errors of 3 replicates. 53

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54 -3.5 -3 -2.5 -2 -1.5 -1 -0.5 0 0510152025303540lg(OD600)E -3 -2.5 -2 -1.5 -1 -0.5 0 0510152025303540lg(OD600)F -3 -2.5 -2 -1.5 -1 -0.5 0 0.5 0510152025303540lg(OD600)G -3 -2.5 -2 -1.5 -1 -0.5 0 0.5 0510152025303540lg(OD600)H -3 -2.5 -2 -1.5 -1 -0.5 0 0.5 0510152025303540 Hourslg(OD600) TYEG NaCl PEG As IFigure 3-6. Continued.

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3.5.3 Oxidative Stress Assay of Arsenic Resistant Bacteria N,N'-Dimethyl-4,4'-bipyridinium dichloride (p araquat), one of the most widely used quaternary ammonium herbicides in the world, is extremely poi sonous to organisms. It is easily reduced to a radical ion, which genera tes superoxide radical that reacts with unsaturated membrane lipids. Therefore, it is quick-acting, non-select ive, and kills green plant tissue on contac t conditions (Goel and Aggarwal, 2007). It has been shown that inhibition of Photorhabdus luminescens growth on Petri dishes by 10 l of 50 mM paraquat, and reduction of P. luminescens mRNA relative abundance in 50 min by 1 mM of paraquat in cell culture (Cha labaev et al., 2007). Figure 3-7 shows the result of paraquat growth inhibition of Salmonella typhimurium wild type and mutants. The mutants used are S. typhimurium SF1005, unmarked rpoS mutation, S. typhimurium cc1000 rpoS::tet (Tetracycline resistant gene) and S. typhimurium GS014 oxyR ::Tn10 (Tn10 transposon). However, the mutants are not confirmed. RpoS is a sigma factor, which is thought to be expressed only in sta tionary phase or cell starvation. However, it is also suggested that rpoS is induced by high cell density and that cells growth at these high densities seem to have undergone the general stress response, as judged by the production of trehalose (an osm oprotectant) and catalase (Mah and O'Toole, 2001). OxyR regulon is induced typically by hydrogen peroxide and tightly controls hydrogen peroxide level by increasing scavengi ng activities and limiting hydrogen peroxide generation in the respiratory chain (Gonzalez-Flecha and Demple, 2000). The result in Figure 3-7 shows the inhibition zone around a glass disc containing 25 l of 50 mM paraquat. The inhibition diameter reveals that S. typhimurium GS014 was more sensitive than wild type and S. typhimurium SF1005, suggesting reverse mutation in S. typhimurium SF1005 or synergistic reac tion between paraquat and rpoS unmarked mutation. To confirm the result, bacterial growth under paraquat stress in 96 well culture plates was investigated. 55

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Figure 3-8 shows the growth of S. typhimurium wild type and mutants in M9 minimal medium with 0.02% of glucose and 0.1, 0.3, 0.6, 0.9, 1.2, 1.5 or 2 mM of paraquat in 96-well cell culture plates at 37C. The 96 well plates can be easily contaminated because of strong evaporation at 37C, which explains the grow th in 0.1 mM in Figure 3-8 A and 2 mM in Figure 3-8 B. Figure 3-8 B, C and D showed th e increase of growth inhibition as paraquat concentration increased; however, paraquat had a smaller inhibition compared to relevant mutants. Among the mutants, S. typhimurium GS014 was more sensitive to paraquat than S. typhimurium cc1000 and Salmonella typhimurium SF1005. Both results confirmed reverse mutation in S. typhimurium SF1005 and S. typhimurium cc1000. The fact that better growth in S. typhimurium mutants than the wild type was observed may result from synergistic reaction of paraquat and the rpoS mutations. Expressions of stress-related regulon regulated genes are quite energy consuming; however, in the mutants, low paraquat concentration may not be able to affect normal cellular proliferation. Structural alteration in reverse mutation may lead to insens itivity to paraquat induced oxidative stress. Besides, as a broad host pathogen, S. typhimurium had stronger tolerance to oxidative burst than P. luminescens; however, confirmation of synergis tic effects is beyond this study. Therefore, S. typhimurium wild type and S. typhimurium GS014 were chosen as control to study oxidative stress growth of arsenic resistan t bacteria. Because of the possible synergistic effect of paraquat and oxyR mutants, the following experiment was carried out with hydrogen peroxide. Compared to paraquat, hydrogen peroxi de exerts internal oxidative stress, which is generated in cellular re spiration under specific conditions, allowing this experiment to bypass the side effects of external reagents. Arsenate at concentrations of 176 mM or 400 mM exerte d great oxidative stress for bacteria, the 8 isolates belonging to 8 different genera were te sted for anti-oxidative stress by measuring hydrogen peroxide inhibition zone with controls of S. typhimurium wild type and a 56

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mutant having a Tn10 transposon inserted in oxyR gene. The result showed that, arsenic resistant bacteria were as se nsitive to hydrogen peroxide as S. typhimurium wild type, except AH4, which was as sensitive as the oxyR mutant (Figure 3-9) (p -value <0.001). Bacterium AH4 was the only one that was able to grow in the presence of 2.4M NaCl (Figure 3-4). Induced oxidative burst is a plant defense mechanism wh en encountering bacterial invasion (Bolwell and Wojtaszek, 1997). Meanwhile in animals, phagocytic cells generate superoxide and other reactive oxygen species, wh ich are involved in antibacterial activity (Hassett and Cohen, 1989). Usually pathogens are able to surv ive through those host defense systems. As a broad host of pathogen, S. typhimurium possess antioxidant defenses that allow its survival in inflammatory foci in either animal or plant hosts. The defenses include antioxidant enzymes such as superoxide di smutase and catalase, DNA repair systems, scavenging substrates, and competition with phagocytes for molecular oxygen. These defenses are regulated by global regulon such as OxyR in a coordina ted fashion (Hassett and Cohen, 1989). The fact that the arsenic resi stant bacteria were as sens itive to hydrogen peroxide as S. typhimurium wild type indicates the st rong ability of the bacteria in scavenging oxidative burst. This result suggests that the ability of ar senic-resistant bacteria in detoxifying arsenic was both because that expression of arsenic-indu ced genes alleviates ce llular oxidative stress and the ability of the bacterial isolates in scavenging oxidative burst. The comprehensive effects were indicated by ARI in Figure 3-5. Naxibacter sp. AH4 was as sensitive to hydrogen peroxide as S. typhimurium mutant. With such high sensitivity to hydrogen peroxide, AH4 needed to either sequestrate/immobilize all arsenic before it indu ced oxidative burst inside cells or prevent arsenic uptake, which is nearly impossible. Th is is because the chemical property of arsenic is similar to phosphorus, and arsenate is ta ken up nonspecifically by phosphate transporters 57

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(Rosen, 2002). Therefore, lack of downstream arsenic detoxification ability in AH4 suggested a different arsenic detoxification mechanism in the isolate. In addition, AH4 was the only isolate that tolerated 2.4 M of NaCl (Figure 34), but not 26% of PEG (Figure 3-6). Further study is needed to unveil the specific genes that confer the unique det oxification ability to this strain. 34 36 38 40 42 44 46 48 SF1005cc1000GS014WT (mm) 24 Hours 48 Hours Figure 3-7. Oxidation inhibiti on area around a glass disc cont aining 25 l of 50 mM N,N'Dimethyl-4,4'-bipyridinium dichloride (paraquat) ( = 0.05); SF1005: Salmonella typhimurium SF1005 unmarked rpoS mutant; cc1000: S. typhimurium cc1000 rpoS:: tet ; GS014: S. typhimurium GS014 oxyR :: Tn10; WT: S. typhimurium 14028 wild type. Y axis is diameter of th e inhibition area on Petri dishes. Columns are means and standard errors of 3 replicates. 58

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mMLogOD590 A LogOD590 B LogOD590 C Figure 3-8. Growth of S. typhimurium wild type and mutants in M9 minimal medium with 0.02% of glucose and 0.1, 0.3, 0.6, 0.9, 1.2, 1.5 or 2mM of N,N'-Dimethyl-4,4'bipyridinium dichloride (paraquat) in 96-well cell culture plates at 37 C; A: Salmonella typhimurium 14028 wild type; B: S. typhimurium GS014 oxyR :: Tn10; C: S. typhimurium cc1000 rpoS:: te; D: S. typhimurium SF1005 unmarked rpoS mutant. Columns are means and standard errors of 3 replicates. 59

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-3 -2.5 -2 -1.5 -1 -0.5 0 0.5 1 0.10.30.60.91.21.52 mMLogOD590 1day 2days 3days Figure 3-8. Continued. 60

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0 10 20 30 40 50 AH4AH5AH6AH10AH21AH22AH23AH28GS014WT (mm) 24h 48hA A BB C CD DE EE E Figure 3-9. Oxidation inhibiti on zone around a glass disc containing 25 l of 3% hydrogen peroxide ( = 0.05). AH4-AH28 are soil arseni c resistant isolates; GS014: Salmonella typhimurium LT2 oxyR :: Tn10; WT: S. typhimurium 14028 wild type. Y axis is diameter of the inhibition ar ea on Petri dishes. Columns are means and standard errors of 3 replicates. 3.6 Arsenate Transformation by Arsenic Resistant Bacteria 3.6.1 In vitro Arsenic Transformation It has been reported that majority of bact eria carry functional enzymes to transform arsenic, either reduce arsenate to arsenite or oxidize arsenite to arsena te. The arsenic resistant bacteria isolated from arsenic contaminated soil s were tested for their ability in transforming arsenic during 32 hours of grow th in TYEG medium, which was spiked with 375 g of arsenate or arsenite. Figure 3-10-1 shows the speciation of ar senic in TYEG medium without bacterial inoculation, which confirms that both arsenate and arsenite were stable in TYEG medium with agitation. Figure 310-5 shows the speciation of arsenic in medium with a laboratory strain Sinorhizobium sp. MG32, Figure 3-10-2, 3, 4 show the high efficiency of the soils isolates Naxibacter sp. AH4, Mesorhizobium sp. AH5, Pseudomonas sp. AH21in reducing arsenate to arsenite. Al l arsenate was reduced to arsenite after 32 hours, with the half life of arsenate in presence of As resi stant bacteria being 22 hours for AH4, 18 hours for AH5 and 12 hours for AH21 in this study. 61

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Figure 3-11 shows the final OD at 600 nm afte r 32 hours of growth in the presence of AsV or AsIII. MG32 growth was inhibited by 1 mM of arsenite, though it was resistant to 1 mM of arsenate. In comparis on, arsenic-resistant bacteria Naxibacter sp. AH4, Mesorhizobium sp. AH5, Pseudomonas sp. AH21 tolerated both arsenate and arsenite. While there was neither oxidation nor reduction happe ned in the medium with MG32, the arsenic resistant bacteria reduced arsena te to arsenite efficiently (Fig ure 3-10). There was no arsenate detected after 32 hours of growth in the medium started with 1 mM of sodium arsenate (Fig. 3-10). 1a-100 0 100 200 300 400 410162228As (g) 1b-100 0 100 200 300 400 410162228HourAs (g) AsV AsIII Figure 3-10. Arsenic transformation by arse nic-resistant bacteria AH4, AH5 and AH21 during 32 h of growth in TYEG medium sp iked with 375 g of arsenate (AsV) or arsenite (AsIII). A laboratory strain Rhizobium sp. MG32 served as control. 1: no bacterium; 2: Naxibacter sp. AH4; 3: Mesorhizobium sp. AH5; 4: Pseudomonas sp. AH21; 5: Sinorhizobium sp. MG32. a: started with AsV; b: started with AsIII. Points are means and standard errors of 3 replicates. 62

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2a-100 0 100 200 300 400 410162228As (g) 2b-100 0 100 200 300 400 410162228As (g) 3b-100 0 100 200 300 400 410162228As (g) 3a-100 0 100 200 300 400410162228As (g) -100 0 100 200 300 400 410162228As (g) a 4b-100 0 100 200 300 400 410162228As (g) 5a-100 0 100 200 300 400 410162228As (g) -100 0 100 200 300 400 410162228 HoursAs (g) AsV AsIII b Figure 3-10 Continued. 63

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A AH4AH5AH21MG32OD600 B AH4AH5AH21MG32OD600 Figure 3-11. Growth of arse nic-resistant bacteria AH4, AH 5 and AH21 after 32 h in TYEG medium spiked with 375 g of arsenate (A) or arsenite (B). A laboratory strain Rhizobium sp. MG32 served as control. Points are means and standard errors of 3 replicates. 3.6.2 Reductase Enzyme Activity Ass ay and Functional Gene Searching While the arsenic transformation experiment showed efficient reduction by arsenic resistant bacteria isolated from arsenic contam inated soils, this experiment tested enzymatic arsenate reduction, specifically the glutaredoxin (grx, specifically, E. coli grx2) dependent arsenate reductase activity. During arsenic reduction, glutaredoxin provi des electron to arsena te, and the oxidized form of glutaredoxin is then reduced by GSH. Finally oxidized GSSG is reduced again by yeast glutathione reductase, wh ich uses NADPH as electron donor and results in the decrease 64

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of absorption. Figure 3-12 A and B are backgr ound control, which showed a very small arsenate reduction by E. coli glutaredoxin, confirming that gl utaredoxin is able to reduce arsenate slowly (Shi et al ., 1999). Figure 3-12 C, D, E are enzymatic reduction by total protein from E. coli DH5 Pseudomonas sp. AH25 and Pseudomonas sp. AH45. The total protein concentrations were 13.5, 94.5 and 203 g, respectively. Chemical transformation results and enzymatic assay together prove the existence of a specific gene system to transform and detoxify arsenic by arsenic resistant bacteria. Although the isolation method used in this study recovered aerobic heterotrophi c bacteria from the rhizosphere of the arsenic hyperaccumulator P. vittata, the isolates could be also facultative anaerobics; therefore, both arsenic specific resi stant mechanisms were studied. Several pairs of primers had been tried to clone ars operon genes or arrA gene. Those primers were either reported in previous studies or designed based on ars operon sequence of P. aeruginosae PAO1. All positive controls were sequenced to confirm if the primers were working. There was no positive PCR product amplified by the primers based on arsR arsB and arsC genes in Pseudomonas aeruginosae PAO1 in tested arsenic re sistant bacteria. Southern hybridization with arsC probe from PAO1 also showed a negative result (Figure 3-13). Figure 3-13 A shows DNA in agarose gel and Figure 3-13 B shows hybridization results in nylon membrane. The lanes in the upper half are arsC PCR products from the genomic DNA templates of Naxibacter sp. AH4, Mesorhizobium sp. AH5, Methylobacterium sp. Enterobacter sp. AH10 Pseudomonas sp. AH21, Bacillus sp. AH22, Acinetobacter sp. AH23, Pseudomonas sp. AH25, Caryophanon sp. AH28, Pseudomonas sp. AH33, Pseudomonas sp. AH34, Pseudomonas sp. AH45, and P. aeruginosae PAO1, respectively, with primers based on PAO1 arsC gene. The probe was confirmed by sequencing. 65

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The lanes in the lower half are EcoR I partial digested genomic DNA from Naxibacter sp. AH4, Mesorhizobium sp. AH5, Methylobacterium sp. Enterobacter sp. AH10 Pseudomonas sp. AH21, Bacillus sp. AH22, Acinetobacter sp. AH23, Pseudomonas sp. AH25, Caryophanon sp. AH28, Pseudomonas sp. AH33, Pseudomonas sp. AH34, Pseudomonas sp. AH45, PCR2.1 with arsC insertion (uncut), and Pseudomonas aeruginosae PAO1. While there was strong signal in the positive controls, there was no hybridization between genomic DNAs and arsC from P. aeruginosae PAO1, which may because of low sensitivity of DIG DNA labe ling method and low copy of arsC from the arsenic resistant bacteria, or because there was no similar arsC as P. aeruginosae PAO1 in arsenic resistant bacteria. If so, the strong hybridization signals between P CR products resulted from the primer sequence hybridizations. Phylogeneti c analysis of bacterial and archaeal arsC gene sequences has shown that arsC phylogeny is complex and is likel y the result of a number of evolutionary mechanisms, in which inconsistencies between arsC and 16S rRNA are apparent for some taxa, and other isolated taxa possess arsC genes that would not be expected based on known evolutionary relations hips (Jackson and Dugas, 2003). Therefore, the experiment result suggest s novel functional arsenate re ductase genes or very low similarity between different strains, resulting in the difficulties to lo cate the target gene. ArrA gene amplification was positive based on the arsenate respiration marker designed by D. Malasarn (Malasarn et al., 2004). The PCR result is shown in Figure 3-14. Positive control was confirmed by sequencing. Ho wever, except partial sequences of arrA gene were found in some of the isolates such as Pseudomonas sp. AH25 and Pseudomonas sp. AH45, all other clones showed novel sequences, whic h had never been reported and there was no similar sequences available in GenBank. Blast result of Pseudomonas sp. AH25, which has the highest similarity among those sequenced so il isolates PCR products is shown in Figure 66

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3-14 b. Not only there was low sim ilarity between soil isolates arrA genes and Shewanella sp. arrA but also, upon the reported arrA genes, there was only a fragment of 103 nucleotides similarity between Shewanella sp. arrA and Bacillus selenitireducens strain MLS10 arrA gene (Genbank accession no. AY283639). The cloning results is the conserve region of the 2.5k base pairs arrA gene according to D. Malasarn (Malasarn et al., 2004). However, to further confirm the existence of the arr operon, it is required to eith er test bacterial enzymatic arsenate reduction in strict anaerobic condition or empl oying better cloning strategy. Though arsenic resistant plasmid determination was negative, there is no guarantee that the plasmid functional genes will confer arsenic resistance to E. coli DH5 which may cause false negative results. This can be caused by the difference between plasmid hosts, such as copy number restriction and gene transcripti on regulation. In additi on, different arsenate reductase genes require different co factors. Arsenate reductase in Pseudomonas species may not favor glutaredoxin in E. coli DH5 Moreover, some of the plasmid encoding ars operon genes are huge and wont be able to be rec overed by the plasmid extr action method used here. In this case, those plasmids are considered as genomic DNA while we were searching for functional genes. A genomic DNA library was al so tried to search for functional genes. However, due to the fact that different arsenate reductase genes require different cofactors and other host differences, there is no functional gene found so far. 67

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Figure 3-12. Arsenate reductase assay of arsenic resistant ba cteria isolated from arsenic contaminated soils by measuring NADPH ab sorption at 340 nm. This method is to analysis the enzymatic activity of the glutaredoxin (grx) dependent arsenate reductase activity. Reaction solution in 2mL cuvette contains 150 l bacterial protein extracts, 1 ml reaction buffer, 10 M sodium arsenate, 0.5 mM NADPH, 1 mM GSH, 2U yeast glutat hione reductase and 0.02M E. coli Glutaredoxin 2. A and B are background control, which show s a very small arsenate reduction by E. coli glutaredoxin. C, D, E are enzymatic reduction by tota l protein from E. coli DH5 Pseudomonas sp. AH25 and Pseudomonas sp. AH45. The total protein concentrations were 13.5, 94.5 and 203 g, respectively. 68

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Figure 3-12. Continued. 69

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70 Figure 3-12. Continued.

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Figure 3-13. arsC gene Southern Hybridization. A s hows DNA in agarose gel and B shows hybridization results in nylon membrane. The lanes in the upper half are arsC PCR products from the genomic DNA templates of Naxibacter sp. AH4, Mesorhizobium sp. AH5, Methylobacterium sp. AH6, Enterobacter sp. AH10, Pseudomonas sp. AH21, Bacillus sp. AH22, Acinetobacter sp. AH23, Pseudomonas sp. AH25, Bacillus sp. AH28, Pseudomonas sp. AH33, Pseudomonas sp. AH34, Pseudomonas sp. AH45, and Pseudomonas aeruginosae PAO1, respectively. The lanes in the lower half are EcoRI partial digested genomic DNA from Naxibacter sp. AH4, Mesorhizobium sp. AH5, Methylobacterium sp. AH6, Enterobacter sp. AH10, Pseudomonas sp. AH21, Bacillus sp. AH22, Acinetobacter sp. AH23, Pseudomonas sp. AH25, Bacillus sp. AH28, Pseudomonas sp. AH33, Pseudomonas sp. AH34, Pseudomonas sp. AH45, PCR2.1 with arsC insertion (uncut), and Pseudomonas aeruginosae PAO1. 71

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Figure 3-14. arrA gene cloning. A: PCR pr oducts in agarose gel. Lanes are PCR product from genomic DNA templates of Naxibacter sp. AH4, Mesorhizobium sp. AH5, Methylobacterium sp. AH6, Enterobacter sp. AH10, Pseudomonas sp. AH21, Bacillus sp. AH22, Acinetobacter sp. AH23, Pseudomonas sp. AH25, Bacillus sp. AH28, Pseudomonas sp. AH34, Pseudomonas sp. AH43, Pseudomonas sp. AH45, and Shewanella sp. ANA-3, respectively. Shewanella sp. ANA-3 arrA PCR product was confirmed by sequencing. B: BLAST result of Pseudomonas sp. AH25 arrA gene. Left side sequences are identical with arrA forward primer. No similarity between right side sequences and arrA primers. Similar BLAST results in AH34 and AH45 (data not show, sequences availabl e in Appendix C), which suggested novel arrA genes. Sequence in B: 1. HRD9 arsenate respiratory reductase (arrA ) gene. GenBank accession No. AY707754.1 a: Identitie s = 33/33 (100%), Gaps = 0/33 (0%); b: Identities = 35/38 (92%), Gaps = 1/38 (2%). 2. GL-ARR A1 arsenate respiratory reductase-like (arrA ) gene. GenBank access No.EF014944.1. Identities = 32/33 (96%), Gaps = 0/33 (0%). 3. HRR23 arsenate respiratory reductase ( arrA ) gene. GenBank access No.AY707770.1. a: Identities = 32/33 (96%), Gaps = 0/33 (0%); b: Identities = 32/33 (96%), Gaps = 0/33 (0%). 4. HRR20 arsenate respiratory reductase ( arrA ) gene. GenBank accession No. AY707768.1, Identities = 28/30 (93%), Gaps = 0/30 (0%). 5. Homo sapiens cDNA, GenBank access No AK307664.1, Identities = 25/27 (92%), Gaps = 0/27 (0%). This study isolated a group of bacteria from arsenic contaminated soils where P. vittata grew. This included phosphorus and arsenic solu bilizing bacteria, which can potentially enhance phytoremediation through improving metal removal ra te and increasing harvested biomass. In addition, we have identified and purified the most arsenic resistant bacteria, which were able to tolerate up to 30,000mg/kg of arsenic in liquid culture, the highest level reported to date. 72

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73 Most of studies on arsenic resistant bacteria have focused on specific arsenic resistant mechanisms, i.e., functional genes in detoxification mechanisms th rough transformation and sequestration of arsenic species in either vacuoles (if the organisms have) or outer membrane; However, little information is available at global metabolic levels such as the osmotic stress between membranes when arsenic are stored/accu mulated, oxidative stress generated during the exposure, and the impacts of those stresses in cellular growth. In this study, for the first time, multiple types of arsenic resistance were proposed. Experiment results support that hi gh arsenic resistant ab ility of arsenic resistant bacteria was due to not only their efficiency in transformation and sequestration arsenic, bu t also their ability in scavenging oxidative burst and c ounteracting with different osmo tic stresses. In other words, bacteria growth benefited from both arsenic indu ced genes that alleviat e arsenic toxicity and from general high level of cell metabolisms in counteracting cellular grow th stresses. Arsenic resistant index (ARI) was developed to qualify the two level of arseni c detoxification. In the index, ARI indicated the comprehensive effects of bacterias ability in reducing and extruding arsenic besides bacterias ability in overcoming toxicity at cellular level.

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APPENDIX A TWELVE MOST ARSENIC RESISTANT ISOLATES16S RIBOSOMAL RNA GENE SEQUENCES Bacteria are denoted by > follows with strain names. >AH1 ggcatgctttacacatgcaagtcgaacggcagcacgggcttcggcctggtggcgagtggcgaac gggtgagtaatacatcggaacgtacccagaa gtgggggataacgtagcgaaagttacgctaat accgcatacgttctacggaagaaagtgggggat cgcaagacctcatgcttttggagcggccgatg tctgattagctagttggtggggtaaaggctcaccaaggcgacgat cagtagctggtctgagaggacgaccagccacactggaactgagacacggtc cagactcctacgggaggcagcagtggggaattttgg acaatgggcgcgagcct gatccagcaatgccgcg tgagtgaagaagg ccttcgggttgt aaagctcttttgtcagggaagaaacggctgaggctaatatcctcggct aatgacggtacctgaagaataagcaccggctaactacgtgccagcagcc gcggtaatacgtagggtgcaagcgttaatcggaattactgggcgtaaagcgtgcgcaggcggttttgtaag tctgatgtgaaatccccgggctcaacc tgggaattgcattggagactgcaaggctggagtctggcaga >AH4 ttagataccctggtagtccacgccctaaacgatgtctactagttgtcgggtttt aattaacttggtaacgcagctaacgcgtgaagtagaccgcctgggg agtacggtcgcaagattaaaactcaaaggaattgacggggacccgcacaagcggtggatgatgtggattaa ttcgatgcaacgcgaaaaaccttacc tacccttgacatgtcaggaatcctcgagagatcgaggagtgcccgaaaggg agcctgaacacaggtgctgcatggctgtcgtcagctcgtgtcgtga gatgttgggttaagtcccgcaacgagcgcaacccttg tcattagttgctacgcaagagcactctaat gagactgccggtgacaaaccggaggaaggt ggggatgacgtcaagtcctcatggcccttatgggtagggcttcacacgtcatacaatggtacatacagagggccgccaacccgcgagggggagcta atcccagaaagtgtatcgtagtccggatcgcagtctgcaactcgactgcgtga agttggaatcgctagtaatcgcg gatcagcatgccgcggtgaata cgttcccgggtcttgtacacaccgcccgtcacaccatgggagcgggttttaccagaagtaggtagcttaacc >AH5 ttagataccctgggtagtccacgctgtaaacgatggaagctagccgtcggc aagtttacttgtcggtggcgcagct aacgcattaagcttcccgcctgg ggagtacggtcgcaagattaaaactcaaaggaattgacgggggcccgcacaa gcggtggagcatgtggtttaattc gaagcaacgcgcagaacctt accagcccttgacatcccggtcgcggcctagagaga tttaggccttcagttcggctggacc ggtgacaggtgctgcatgg ctgtcgtcagctcgtgtc gtgagatgttgggttaagtcccgcaacgagcg caaccctcgcccttagttgccatcattcagttgggcact ctaaggggactgccggtgataagccga gaggaaggtggggatgacgtcaagtcctcatggcccttacgggctgggctacacacgtgctacaat ggtggtgacagtgggc agcgagaccgcg aggtcgagctaatctccaaaagccatctcagttcggattgcactctgcaactcg agtgcatgaagttggaatcgctag taatcgcggatcagcatgccg cggtgaatacgttcccgggccttgtacacaccgcccgt cacaccatgggagttggttttacccgaaggcgctgtgctaacc >AH6 ttagataccctggtagtccacgccgtaaacgatg aatgccagctgttggggtgcttgcacctcagta gcgcagctaacgctttaagcattccgcctggg gagtacggtcgcaagattaaaactcaaaggaa ttgacgggggcccgcacaa gcggtggagcatgtggtttaattcgaagcaacgcgcagaacctta ccatcccttgacatggcatgttacccggagagattcggggtccacttcggtgg cgtgcacacaggtgctgcatggctgtcgtcagctcgtgtcgtgag atgttgggttaagtcccgcaacgagcgcaacccacgt ccttagttgccatcatttagttgggcactctag ggagactgccggtgataagccgcgagga aggtgtggatgacgtcaagtcctcatggcccttacgggatgggct acacacgtgctacaatggcggtgacagtgggacgcgaaggagcgatctgg agcaaatccccaaaaaccgtctcagttcagattgcactctgc aactcgagtgcatgaaggcggaatcgct agtaatcgtggatcagcatgccacggtg aatacgttcccgggccttgtacacaccgcccgtcacaccatg ggagttggtcttacccgacggcgctgcgccaacc >AH10 ttagataccctggtagtccacgccgtaaacgatgt cgatttggaggttgtgcccttgaggcgtggc ttccggagctaacgcgttaaatcgaccgcctgg ggagtacggccgcaaggttaaaact caaatgaattgacgggggcccg cacaagcggtggagcatgtggtttaattcggtgc aacgcgaagaacctt acctggtcttgacatccacagaactttccagagatggattggtgccttcgggaact gtgagacaggtgctgcatggctgtcgtcagctcgtgttgtgaa atgttgggttaagtcccgcaacgagcgcaaccc ttatcctttgttgccagcggttaggccggg aactcaaaggagactgccagtgataaactggagg agggtggggatgacgtcaagtcatcatggcccttacgaccagggct acacacgtgctacaatggcgcatacaaagagaagcgacctcgcgagagc aagcggacctcataaagtgcgtcgtagtccgga ttggagtctgcaactcgactccatgaagtcggaatcgctagtaatcgtagatcagaatgctacgg tgaatacgttcccgggccttgtacacac cgcccgtcacaccatgggagtggg ttgcaaaagaagtaggtagcttaacc >AH20 ggcggcgtgcctaatacatgcaagtcgagcg aatggattaagagcttgctcttatgaagtta gcggcggacgggtgagtaacacgtgggtaacctgc ccataagactgggataactccgggaaaccggggctaataccggataacattttgaactgcatggttcga aattgaaaggcggcttcggctgtcatttat ggatggacccgcgtcgcattagctagttggtgaggtaacggct caccaaggcaacgatgcgtagccgacctgagagggtgatcggccacactggg actgagacacggcccagactcctacgggaggcag cagtagggaatcttccgcaatggacgaaagt ctgacggagcaacgccgcgtgagtgatga aggctttcgggtcgtaaaactctgttgttaggaaagaacaagtgctag ttgaataagctggcaccttgacgg tacctaaccagaaagccacggctaact acgtgccagcagccgcggtaatacgtaggtggcaagcgttatccgg aattattgggcgtaaagcgcgcgcaggtggtttcttaagtctgatgtgaaa gcccacggctcaaccgtggagggtcattggaaactggg 74

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>AH21 ttagataccctggtagtccacgccctaaacgatgt caactggttgttgggaggg tttctttctcagtaacgtagctaacg cgtgaagttgaccgcctggg gagtacggccgcaa ggttgaaactcaaaggaattgacgggg acccgcacaagcggtggatgatgtgg tttaatccgatgcaacgcgaaaaacctta cctacccttgacatgtctggaatcctgaagagatttgggagtgctcgaaag agagccagaacacaggtgctgcatggccgtcgtcagctcgtgtcgtg agatgttgggttaagtcccgcaacgagcgcaaccc ttgtcattagttgctacgaaagggcactctaat gagactgccggtgacaaaccggaggaagg tggggatgacgtcaggtcatcatggcccttatgggtagggctacacacgtcatacaatggccgggacagagggctgccaacccgcgagggggag ctaatcccagaaacccggtcgtagtccggatcgtagtct gcaactcgactgcgtggagtcggaatcgct agtaatcgcggatcagcttgccgcggtg aatacgttcccgggtcttatacacaccgcccgtcacaccatgggag cgggttctgccagaagtagttagcctaacc >AH22 ttagataccctggtagtccacgccgtaaacgatgagtgctaagtgttaga gggtttccgccctttagtgctgaagttaacgcattaagcactccgcctgg ggagtacggccgcaaggctgaaactcaaaggaattgacgggggcccgcacaagcggtggagcatgtggtttaa ttcgaagcaacgcgaagaacc ttaccaggtcttgacatcctctgaaaaccctagagatagggcttctccttc gggagcagagtgacaggtggtgcatgg ttgtcgtcagctcgtgtcgtg ggatgttgggttaagtcccgcaacgagcgcaaccc ttgatcttagttgccatcatt aagttgggcactctaaggtgactgccggtgacaaaccggagg aaggtggggatgacgtcaaatcatcatgccccttatgacctgggctacacac gtgctacaatggacggtacaaagagctgcaagaccgcgaggtgg agctaatctcataaaaccgttctcagttcgg attgtaggctgcaactcgcctacatgaagctggaat cgctagtaatcgcggatcagcatgccgcggtg aatacgttcccgggccttgtacacaccgcccgtcacaccacga gagtttgtaacacccgaagtcggtggggtaacc >AH23 ttagataccctggtagttccatgccgtaaacgatgtctactagccgttgggg cctttgaggctttagtggcgcagctaacgcgataagtagaccgcctg gggagtacggtcgcaagactaaaactcaaatgaattgacgggggcccg cacaagcggtggagcatgtggtttaattcgatgcaacgcgaagaacct tacctggtcttgacatagtaagaactttccagagatggattggtgccttcgggaacttacatacaggtgctgcatggctgtcgtcagctcgtgtcgtgag atgttgggttaagtcccgcaacgagcgcaacccttttccttatttgccatcg ggttaagccgggaactttaaggatactgccagtgacaaactggagga aggcggggacgacgtcaagtcatcatggcccttacgaccagggct acacacgtgctacaatggtcggtacaaaggg ttgctacctcgcgagaggat gctaatctcaaaaagccgatcgtagtccggattggagtctgcaactcgactccatgaagtcggaatcgct agtaatcgcggatcaaaatgccgcggtg aatacgttcccgggccttgtacacaccgcccgtcacaccatg ggagtttgttgcaccagaagtaggtagtctaacc >AH25 ttagataccctggtagtccacggccgtaaacgat gtcaactagccgttggaatccttgagattttagtggcgcagctaacgcattaagttgaccgcctgg ggagtacggccgcaaggttaaaact caaatgaattgacgggggcccg cacaagcggtggag catgtggtttaattcgaag caacgcgaagaacctt accaggccttgacatgcagagaactttccagagatgg attggtgccttcgggaactctgacacaggtgc tgcatggctgtcgtcagctcgtgtcgtga gatgttgggttaagtcccgtaacgagcgcaaccc ttgtccttagttaccagcacgtcatggtgggcact ctaaggagactgccggtgacaaaccgga ggaaggtggggatgacgtcaagtcatcat ggcccttacggcctgggctacacacgtgctacaat ggtcggtacagagggttgccaagccgcgagg tggagctaatctcacaaaaccgatcgtagtccggatcgcagtctgcaactcgactgcgtgaagtcggaatcgctag taatcgcaaatcagaatgttgc ggtgaatacgttcccgggccttgtacacaccgcccgtcacaccat gggagtgggttgcaccagaagtagctagtctaacc >AH28 ttagataccctggtagtccacgccgtaaacgatgagtgctaagtgttg gggggtttccgcccctcagtgctgcagctaacgcattaagcactccgcctg gggagtacggtcgcaagactgaaactcaaaggaattgacgggggcccgcacaagcggtggagcat gtggtttaattcgaagcaacgcgaagaac cttaccaggtcttgacatcccggtgaccactatggagacatagtttccccttc gggggcaacggtgacaggtggtg catggttgtcgtcagctcgtgtc gtgagatgttgggttaagtcccgcaacgagcg caacccttattcttagttgccat cattcagttgggcactctaaggagactgccggtgataaaccgga ggaaggtggggatgacgtcaaatcatcatgccccttatgacctgg gctacacacgtgctacaatggacggtacaaacggttgccaacccgcgaggg ggagctaatccgataaaaccgttctcagttcggattgtaggctgcaactcgcctaca tgaagccggaatcgctagtaat cgcggatcagcatgccgcg gtgaatacgttcccgggccttg tacacaccgcccgtcacaccacgagagtttgtaacacccgaagtcggtgaggtaacc >AH30 ggcggcgtgcttaacacatgcaagtcgaacgatgatctccagcttgctggggggattagtggcgaacgggtg agtaacacgtgagtaacctgccctt gactctgggataagcctgggaaactgggtctaataccggatatgacca ttccacgcatgtggtggtggtggaaa gcttttgcggttttggatggactcg cggcctatcagcttgttggtggggtaat ggcctaccaaggcgacgacgggt agccggcctgagagggtgaccg gccacactgggactgagacac ggcccagactcctacgggaggcagcagtgggg aatattgcacaatgggcggaagcctgatgcagcgacgccgcgtgagggatgacggccttcg ggttgtaaacctctttcagtagggaagaagcgt aagtgacggtacctgcagaagaagcgccgg ctaactacgtgccagcagccgcggtaatacgta gggcgcaagcgttatccggaattattgggcgtaaagagctcgtaggcgg tttgtcgcgtctgctgtgaaagaccggggctcaactccggttctgcagt gggtacgggcagactagagtgcagtaggggagactggaattc >AH34 ttagataccctggtagtccacgccgtaaacgatgtcaactagccgtttgg aatccttgagattttagtggcgcagctaacgcattaagttgaccgcctgg ggagtacggccgcaaggttaaaact caaatgaattgacgggggcccg cacaagcggtggag catgtggtttaattcgaag caacgcgaagaacctt accaggccttgacatgcagagaactttccagagatgg attggtgccttcgggaactctgacacaggtgc tgcatggctgtcgtcagctcgtgtcgtga gatgttgggttaagtcccgtaacgagcgcaaccc ttgtccttagttaccagcacgtcatggtgggcact ctaaggagactgccggtgacaaaccgga ggaaggtggggatgacgtcaagtcatcat ggcccttacggcctgggctacacacgtgctacaat ggtcggtacagagggttgccaagccgcgagg 75

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tggagctaatctcacaaaaccgatcgtagtccggatcgcagtctgcaactcgactgcgtgaagtcggaatcgctag taatcgcaaatcagaatgttgc ggtgaatacgttcccgggccttgtacacaccgcccgtcacaccat gggagtgggttgcgccagaagtagctagtctaacc >AH37 ggcggcaggcctaacacatgcaagtcggacggtag cacagagagcttgctctcgggtgacgagtg gcggacgggtgagtaatgtctggggatctg cccgatagagggggataaccactggaaacggtggc taataccgcataacgtcgcaagaccaaagaggg ggaccttcgggcctctcactatcggat gaacccagatgggattagctagtaggcggggtaatggcccacctaggcgacgatccctagctggtctgagaggatgaccagccacactggaactg agacacggtccagactcctacgggaggcagcagtggggaatattgcacaatgggcgcaagcctga tgcagccatgccgcgtgtatgaagaaggc cttcgggttgtaaagtactttcagcggggaggaaggcgatgcggtt aataaccgcgtcgattgacgttacccgcagaagaagcaccggctaactccg tgccagcagccgcggtaatacggagggtgcaagcgttaatcggaattact gggcgtaaagcgcacgcaggcggtc tgttaagtcagatgtgaaatc cccgggcttaacctgggaactgcatttgaaactggcaggcttgagtc >AH43 ttagataccctggtagtccacgccctaaacgatgt caactggttgttgggaggg tttcttctcagtaacgtagctaacgcgtgaagttgaccgcctggg gagtacggccgcaa ggttgaaactcaaaggaattgacgggg acccgcacaagcggtggatgatgtgg tttaattcgatgcaacgcgaaaaacctta cctacccttgacatgtctggaatcctgaagagatttgggagtgctcgaaag agagccagaacacaggtgctgcatggccgtcgtcagctcgtgtcgtg agatgttgggttaagtcccgcaacgagcgcaaccc ttgtcattagttgctacgaaagggcactctaat gagactgccggtgacaaaccggaggaagg tggggatgacgtcaggtcatcatggcccttatgggtagggctacacacgtcatacaatggccgggacagagggctgccaacccgcgagggggag ctaatcccagaaacccggtcgtagtccggatcgtagtct gcaactcgactgcgtgaagtcggaatcgct agtaatcgcggatcagcttgccgcggtg aatacgttcccgggtcttgtacacaccgcccgtcacaccatgggagcgggttctg ccagaagtagttagcctaacc >AH45 ttagataccctggtagtccacgccgtaaacgat gtcaactagccgttggaatccttgagatttta gtggcgcagctaacgcattaagttgaccgcctggg gagtacggccgcaaggttaaaactcaaatgaa ttgacgggggcccgcacaagcggt ggagcatgtggtttaattc gaagcaacgcgaagaacctta ccaggccttgacatgcagagaactttccagagatggattggtgccttcggg aactctgacacaggtgctgcatggctgtcgtcagctcgtgtcgtgag atgttgggttaagtcccgtaacg agcgcaacccttgtccttagttaccagcacgtcatggt gggcactctaaggagactgccggtgacaaaccggag gaaggtggggatgacgtcaagtcatcatgg cccttacggcctgggctacacacgtgctacaatggtcggtacagagggttgccaagccgcgaggt ggagctaatctcacaaaaccgatcgtagtccggatcgcagtctgcaactcgact gcgtgaagtcggaatcgctagtaatcgcaaatcagaatgttgcg gtgaatacgttcccgggccttgtacacaccgcccgtcacaccatgggagtgggttgcaccagaagtagctagtctaacc >AH47 ggcggcaggcctaacacatgcaag tcgagcggatgacgggagcttgctccttgattcagcg gcggacgggtgagt aatgcctaggaatctgcctg gtagtgggggacaacgtttcgaaaggaacgctaataccgcatacgtcctacgggag aaagcaggggaccttcgggccttgcgctatcagatgagcc taggtcggattagctagtaggtgaggtaat ggctcacctaggcgacgatccgt aactggtctgagaggatgat cagtcacactggaactgagacacg gtccagactcctacgggaggcagcagtggggaat attggacaatgggcgaaagcctgatccagccat gccgcgtgtgtgaagaaggtcttcggatt gtaaagcactttaagttgggaggaagggttgt acgctaataccgtgcaattttgacg ttaccgacagaataagcaccggctaactctgtgccagcagc cgcggtaatacagagggtgcaagcgttaatcgg aattactgggcgtaaagcgcgcgtaggtgg ttcgttaagttggatgtgaaagccccgggctcaa cctgggaactgcatccaaaactggcgagctagagtatgg >AH49 aggcctaacacatgcaagtcggacggtagcacagagag cttgctcttgggtgacgagtggcggacg ggtgagtaatgtctggggatctgcccgata gagggggataaccactggaaacggtggctaat accgcataacgtcgcaagaccaaagaggg ggaccttcgggcctctcactatcggatgaaccca gatgggattagctagtaggcggggtaatgg cccacctaggcgacgatccctagctggtctga gaggatgaccagccacactggaactgagacacg gtccagactcctacgggaggcagcagtggggaata ttgcacaatgggcgcaagcctgatgcagccatg ccgcgtgtatgaagaaggccttcgggtt gtaaagtactttcagcggggaggaaggcgatg cggttaataaccgcgtcgattgacgttacccg cagaagaagcaccggctaactccgtgccagca gccgcggtaatacggagggtgcaagcgttaatcggaattactgggcgtaaagcgcacgcaggc ggtctgttaagtcag atgtgaaatccccgggct taacctgggaactgcatttgaaactggcaggcttgagtcttgtag >AH51 ggcaggcctaacacatgcaagtcgagcggatgaagagagcttgctctctga ttcagcggcggacgggtgagtaat gcctaggaatctgcctggtag tgggggacaacgtttcgaaaggaacgc taataccgcatacgtcctacgggagaaa gcaggggaccttcgggccttg cgctatcagatgagcctagg tcggattagctagttggtgaggtaatggctcaccaaggcgacgatccgtaactggtctgagaggatgatca gtcacactggaactgagacacggtcc agactcctacgggaggcagcagtggggaatatt ggacaatgggcgaaagcctgatccagccatgccg cgtgtgtgaagaaggtcttcggattgtaa agcactttaagttgggaggaagggttgtaga ttaatactctgcaattttgacgttaccgacagaataagcaccggctaactctgtgccagcagccgcgg taatacagagggtgcaagcgttaatcggaa ttactgggcgtaaagcgcgcgtaggtggtttgttaagttggatgtgaaagccccgggctcaacctgg gaactgcattcaaaactgacaagctagag >AH52 ggcaggcctaacacatgcaagtcgagcggatgaagagagcttgctctctga ttcagcggcggacgggtgagtaat gcctaggaatctgcctggtag tgggggacaacgtctcgaaagggacgctaataccg catacgtcctacgggagaaagcaggggaccttc gggccttgcgctatcagatgagcctag 76

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77 gtcggattagctagttggtgaggtaatggctcaccaaggcgacgatccgtaactggtctgagagg atgatcagtcacactggaactgagacacggtc cagactcctacgggaggcagcagtggggaatatt ggacaatgggcgaaagcctgatccagccatgccg cgtgtgtgaagaaggtcttcggattgta aagcactttaagttgggaggaagggcagtaaatt aatactttgctgttttgacgttaccgacagaataagc accggctaactctgtgccagcagccgcg gtaatacagagggtgcaagcgttaatcggaatt actgggcgtaaagcgcgcgtaggtggtttgttaagttggatgtgaaatccccgggctcaacctgg gaactgcattcaaaactgacaagctaga >AH53 aggcctaacacatgcaagtcgagcggatgacaggagcttgctcctgaattcag cggcggacgggtgagtaatgcct aggaatctgcctggtagtgg gggacaacgtttcgaaaggaacgctaataccgcatacgtcctacgggagaaa gcaggggaccttcgggccttgcgctatcagatgagcctaggtcg gattagctagttggtgaggtaatggctcaccaaggcgacgatccgtaactggtctgagaggatgatcagtcacactggaactgagacacggtccaga ctcctacgggaggcagcagtggggaatattgg acaatgggcgaaagcctgatccagccatgccgcg tgtgtgaagaaggtcttcggattgtaaagc actttaagttgggaggaagggttgtagattaatactctgcgattttgacgttaccgacagaataag caccggctaactctgtgccagcagccgcggtaa tacagagggtgcaagcgttaatcggaatt actgggcgtaaagcgcgcgtaggtggtttgttaag ttggatgtgaaagccccgggctcaacctgggaa ctgcattcaaaactgacaagctagagta

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APPENDIX B REFERENCES FOR 16S RIBOSOMAL RNA GENE SEQUENCES References 16s rDNA sequences in Figure 3-2 and corresponding GenBank accession numbers. Bacteria are denoted by > follows w ith GenBank accession number, genus name, family name and strain name if it ha s one. Only informatic nucleotides using in constructing phylogenetic tree showed. > EF407880 Flavobacterium cheniae 961 aacagttttt tcttcggaca atttacaagg tgctgcatgg ttgtcgtcag ctcgtgccgt 1021 gaggtgtcag gttaagtcct ataacgagcg caacccctgt cgttagttgc cagcgagtca 1081 tgtcgggaac tctaacgaga ctgccagtgt aaactgtgag gaaggtgggg atgacgtcaa 1141 atcatcacgg cccttacgtc ctgggccaca cacgtgctac aatggtaggt acagagagca 1201 gccactgcgc gagcaggagc g aatctacaa aacctatctc agttcggatc ggagtctgca 1261 actcgactcc gtgaagctgg aatcg ctagt aatcggatat cagccatgat ccggtgaata 1321 cgttcccggg ccttgtacac accgccc gtc aagccatgga agctgggggt gcctgaagtc >EF535812 Mesorhizobium ciceri strain GA-2 ttagataccc 721 tggtagtcca cgccgtaaac tatgag agct agccgtcggc aagtttactt gtcggtggcg 781 cagctaacgc attaagctct ccgcctgggg agtacggtcg caagattaaa actcaaagga 841 attgacgggg gcccgcacaa gc ggtggagc atgtggttta attcgaagca acgcgcagaa 901 ccttaccagc ccttgacatc ccggtcgcgg tttccagaga tggatacctt cagttcggct 961 ggaccggtga caggtgctgc atggc tgtcg tcagctcgtg tcgtgagatg ttgggttaag 1021 tcccgcaacg agcgcaaccc tcg cccttag ttgccagcat taagttgggc actctaaggg 1081 gactgccggt gataagccga ga ggaaggtg gggatgacgt caagtcctca tggcccttac 1141 gggctgggct acacacgtgc tacaatgg tg gtgacagtgg gcagcgagac cgcgaggtcg 1201 agctaatctc caaaaaccat ctcagttc gg attgcactct gcaactcgag tgcatgaagt 1261 tggaatcgct agtaatcgcg ga tcagcatg ccgcggtgaa tacgttcccg ggccttgtac 1321 acaccgcccg tcacaccatg gg agttggtt ttacccgaag gcgctgtgct aacc >EU410950 Mesorhizobium amorphae CCNWGS0015-1 ttag ataccctggt agtccacgcc gtaaacgatg 721 gaagctagcc gttggcaagt ttac ttgtcg gtggcgcagc taacgcatta agcttcccgc 781 ctggggagta cggtcgcaag att aaaactc aaaggaattg acg ggggccc gcacaagcgg 841 tggagcatgt ggtttaattc gaagcaacgc gcagaacctt accagccctt gacatcccgg 901 tcgcggtttc cagagatgga atccttcagt tcggctggac cggtgacagg tgctgcatgg 961 ctgtcgtcag ctcgtgtcgt gagatgttgg gttaagtccc gcaacgagcg caaccctcgc 1021 ccttagttgc cagcattaag ttggg cactc taaggggact gccggtgata agccgagagg 1081 aaggtgggga tgacgtcaag t cctcatggc ccttacgggc tgggctacac acgtgctaca 1141 atggtggtga cagtgggcag cgagaccgcg aggtcgagct aatctccaaa agccatctca 1201 gttcggattg cactctgcaa ctcgag tgca tgaagttgga atcgctagta atcgcggatc 1261 agcatgccgc ggtgaatac g ttcccgggcc ttgtacacac cgcccgtcac accatgggag 1321 ttggttttac ccgaaggcgc tgtgctaacc >EU707154 Mesorhizobium huakuii CCBAU15514 tt agataccctg 661 gtagtccacg ccgtaaacga tggaag ctag ccgttggcaa gtttacttgt cggtggcgca 721 gctaacgcat taagcttccc gcctggggag tacggtcgca agattaaaac tcaaaggaat 781 tgacgggggc ccgcacaagc ggt ggagcat gtggtttaat tcgaagcaac gcgcagaacc 841 ttaccagccc ttgacatccc ggtcgcggtt tccagagatg gataccttca gttcggctgg 901 accggtgaca ggtgctgcat ggctgtc gtc agctcgtgtc gtgagatgtt gggttaagtc 961 ccgcaacgag cgcaaccctc g cccttagtt gccagcattc agttgggcac tctaagggga 1021 ctgccggtga taagccgaga gg aaggtggg gatgacgtca agtcctcatg gcccttacgg 1081 gctgggctac acacgtgcta caatggt ggt gacagtgggc agcgagaccg cgaggtcgag 1141 ctaatctcca aaagccatct cagttcgg at tgcactctgc aactcgagtg catgaagttg 1201 gaatcgctag taatcgcgga t cagcatgcc gcggtgaata cgttcccggg ccttgtacac 1261 accgcccgtc acaccatggg agttggtttt acccgaaggc gctgtgctaa cc 78

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>EU130445 Mesorhizobium septentrionale CCBAU11244 ttagatac cctggtagtc cacgccgtaa 721 acgatggaag ctagccgttg gcaagtttac ttgtcggtgg cgcagctaac gcattaagct 781 tcccgcctgg ggagtacggt cgcaagatta aaactcaaag gaattgacgg gggcccgcac 841 aagcggtgga gcatgtggtt t aattcgaag caacgcgcag aaccttacca gcccttgaca 901 tcccggtcgc ggtttccaga gatg gattcc ttcagttcgg ctggaccggt gacaggtgct 961 gcatggctgt cgtcagctcg tg tcgtgaga tgttgggtta agtcccgcaa cgagcgcaac 1021 cctcgccctt agttgccagc attaagttgg gcactctaag gggactgccg gtgataagcc 1081 gagaggaagg tggggatgac gt caagtcct catggccctt acgggctggg ctacacacgt 1141 gctacaatgg tggtgacagt g ggcagcgag accgcgaggt cgagctaatc tccaaaagcc 1201 atctcagttc ggattgcact ctgcaact cg agtgcatgaa gttggaatcg ctagtaatcg 1261 cggatcagca tgccgcggtg aatacgttcc cgggccttgt acacaccgcc cgtcacacca 1321 tgggagttgg ttttacccga aggcgctgtg ctaacc >AB302928 Methylobacterium adhaesivum DSM17169 ttagata ccctggtagt 721 ccacgccgta aacgatgaat gctagctgtt ggggtgc ttg caccgcagta gcgcagctaa 781 cgctttaagc attccgcctg gggagtacgg tcgcaagatt aaaactcaaa ggaattgacg 841 ggggcccgca caagcggtgg ag catgtggt ttaattcgaa gcaacgcgca gaaccttacc 901 atcccttgac atgtcgtgcc atccggagag atccggggtt cccttcgggg acgcgaacac 961 aggtgctgca tggctgtcgt cagctcgtgt cgtgagatgt tgggttaagt cccgcaacga 1021 gcgcaaccca cgtccttagt tgccat catt tagttgggca ctctagggag actgccggtg 1081 ataagccgcg aggaaggtgt ggat gacgtc aagtcctcat ggcccttacg ggatgggcta 1141 cacacgtgct acaatggcgg tgaca gtggg acgcgaaacc gcgaggttga gcaaatcccc 1201 aaaaaccgtc tcagttcaga ttgcact ctg caactcgagt gcatgaaggc ggaatcgcta 1261 gtaatcgtgg atcagcatgc cacggtgaat acgttcccgg gccttgtaca caccgcccgt 1321 cacaccatgg gagttggtct tacccgacgg cgctgcgcca acc >AJ250801 Methylobacterium fujisawaense DSM5686 tta 721 gataccctgg tagtccacgc cgtaaacgat gaatgccagc tgttggggtg cttgcaccgc 781 agtagcgcag ctaacgcttt ga gcattccg cctggggagt acggtcgcaa gattaaaact 841 caaaggaatt gacgggggcc cgcacaagcg gtggagcatg tggtttaatt cgaagcaacg 901 cgcagaacct taccatcctt tgacatggcg tgttacccag agagatttgg ggtccacttc 961 ggtggcgcgc acacaggtgc tgcat ggctg tcgtcagctc gtgtcgtgag atgttgggtt 1021 aagtcccgca acgagcgcaa cccacg tcct tagttgccat cattcagttg ggcactctag 1081 ggagactgcc gg tgataagc cgcgaggaag gtgtggatga cgtcaagtcc tcatggccct 1141 tacgggatgg gctacacacg tg ctacaatg gcggtgacag tggg acgcga aagagcgatc 1201 tggagcaaat ccccaaaagc cgtctcag tt cggattgcac tctgcaactc gagtgcatga 1261 aggcggaatc gctagtaatc gtggat cagc atgccacggt gaatacgttc ccgggccttg 1321 tacacaccgc ccgtcacacc atgg gagttg gtcttacccg acggcgctgc gccaacc >AM910531 Methylobacterium radiotolerans F2 ttagata ccctggtagt 721 ccacgccgta aacgatgaat gcca gctgtt ggggtgcttg caccgcagta gcgcagctaa 781 cgctttgagc attccgcctg gggagtacgg tcgcaagatt aaaactcaaa ggaattgacg 841 ggggcccgca caagcggtgg ag catgtggt ttaattcgaa gcaacgcgca gaaccttacc 901 atcctttgac atggcgtgtt acccagag ag atctggggtc cccttcgggg gcgcgcacac 961 aggtgctgca tggctgtcgt cagctcgtgt cgtgagatgt tgggttaagt cccgcaacga 1021 gcgcaaccca cgtccttagt tgccatcatt cagttgggca ctctagggag actgccggtg 1081 ataagccgcg aggaaggtgt ggat gacgtc aagtcctcat ggcccttacg ggatgggcta 1141 cacacgtgct acaatggcgg tgacagtggg aggcgaagga gcgatctgga gcaaatcccc 1201 aaaagccgtc tcagttcgga ttgcact ctg caactcgagt gcatgaaggc ggaatcgcta 1261 gtaatcgtgg atcagcatgc cacggtgaat acgttcccgg gccttgtaca caccgcccgt 1321 cacaccatgg gagttggtct tacccgacgg cgctgcgcca acc >AM910537 Methylobacterium mesophilicum F42 ttaga taccctggta 721 gtccacgccg taaacgatga atgccagctg ttggggtgct tgcacctcag tagcgcagct 79

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781 aacgctttga gcattccgcc tggggagtac ggtcgcaaga ttaaaactca aaggaattga 841 cgggggcccg cacaagcggt ggag catgtg gtttaattcg aagcaacgcg cagaacctta 901 ccatcctttg acatggcgtg ttat ggggag agattcccag tccacttcgg tggcgcgcac 961 acaggtgctg catggctgtc gt cagctcgt gtcgtgagat gttgggttaa gtcccgcaac 1021 gagcgcaacc cacgtcctta gttg ccatca ttcagttggg cactctaggg agactgccgg 1081 tgataagccg cgaggaaggt gt ggatgacg tcaagtcctc atggccctta cgggatgggc 1141 tacacacgtg ctacaatggc ggtg acagtg ggacgcgaag gggcgacctg gagcaaatcc 1201 ccaaaagccg tctcagttcg gattg cactc tgcaactcgg gtgcatgaag gcggaatcgc 1261 tagtaatcgt ggatcagcat gccacggtga atacgttccc gggccttgta cacaccgccc 1321 gtcacaccat gggagttggt cttacccgac ggcgctgcgc caacc >EU730910 Methylobacterium brachiatum 182 tta gataccctgg tagtccacgc 721 cgtaaacgat gaatgccagc tgttggggtg cttgcacctc ag tagcgcag ctaacgcttt 781 gagcattccg cctggggagt acggtcgcaa gattaaaact caaaggaatt gacgggggcc 841 cgcacaagcg gtggagcatg tggtttaatt cgaagcaacg cgcagaacct taccatcctt 901 tgacatggcg tgttatgggg ag agattccc agtcctcttc ggaggcgcgc acacaggtgc 961 tgcatggctg tcgtcagctc gtgt cgtgag atgttgggtt aagtcccgca acgagcgcaa 1021 cccacgtcct tagttgccat cattcag ttg ggcactctag ggagactgcc ggtgataagc 1081 cgcgaggaan ggtgtggatg acgtcaagtc ctcatggccc ttacgggatg ggctacacca 1141 cgtgctacaa tggcgnnngt g acagtggga cgcgaagggg cgacctggag caaatcccca 1201 aaagccgtct cagttcggat tgcactct gc aactcgggtg catgaaggcg gaatcgctag 1261 taatcgtgga tcagcatgcc acggtgaata cgttcccggg ccttgtacac accgcccgtc 1321 acaccatggg agttggtctt acccgacggc gctgcgccaa cc >EU849019 Enterobacter cloacae FR tt 781 agataccctg gtagtccacg ccgtaaacga tgtcgatttg gaggttgtgc ccttgaggcg 841 tggcttccgg agctaacgcg ttaaatcgac cgcctgggga gtacggccgc aaggttaaaa 901 ctcaaatgaa ttgacggggg cccg cacaag cggtggagca tgtggtttaa ttcgatgcaa 961 cgcgaagaac cttacctggt cttgacatcc acagaacttt ccagagatgg attggtgcct 1021 tcgggaactg tgagacaggt gctg catggc tgtcgtcagc tcgtgttgtg aaatgttggg 1081 ttaagtcccg caacgagcgc aaccc ttatc ctttgttgtc agcggtccgg ccgggaactc 1141 aaaggagact gccagtgata aact ggagga aggtggggat gacgtcaagt catcatggcc 1201 cttacgacca gggctacaca cgtgctacaa tggcgcatac aaagagaagc gacctcgcga 1261 gagcaagcgg acctcataaa gtgc gtcgta gtccggattg gagtctgcaa ctcgactcca 1321 tgaagtcgga atcgctagta at cgtagatc agaatgctac ggtgaatacg ttcccgggcc 1381 ttgtacacac cgcccgtcac accat gggag tgggttgcaa aagaagtagg tagcttaacc >EF059830 Enterobacter sakazakii E413 ttaga taccctggta gtccacgccg taaacgatgt cgatttggag 781 gttgtgccct tgaggcgtgg cttccggagc taacgcgtta aatcgaccgc ctggggagta 841 cggccgcaag gttaaaactc aaatg aattg acgggggccc gcacaagcgg tggagcatgt 901 ggtttaattc gatgcaacgc gaagaacctt acctggtctt gacatccaca gaacttnnca 961 gagatgnntt ggtgccttcg ggaact gtga gacaggtgct gcatggctgt cgtcagctcg 1021 tgttgtgaaa tgttgggtta ag tcccgcaa cgagcgcaac cc ttatcctt tgttgccagc 1081 ggtnnggccg ggaactcaaa gg agactgcc agtgataaac tggaggaagg tggggatgac 1141 gtcaagtcat catggccctt acgaccaggg ctacacacgt gctacaatgg cgcatacaaa 1201 gagaagcgac ctcgcgagag caa gcggacc tcataaagtg cgtcgtagtc cggattggag 1261 tctgcaactc gactccatga agtc ggaatc gctagtaatc gtagatcaga atgctacggt 1321 gaatacgttc ccgggccttg tacacaccgc ccgtcacacc atgggagtgg gttgcaaaag 1381 aagtaggtag cttaacc >EU661378 Klebsiella pneumoniae K42 ttagata ccctggtagt ccacgccgta 781 aacgatgtcg atttggaggt tgtg cccttg aggcgtggct tccggagcta acgcgttaaa 841 tcgaccgcct ggggagtacg gccgcaaggt taaaactcaa at gaattgac gggggcccgc 901 acaagcggtg gagcatgtgg ttt aattcga tgcaacgcga agaaccttac ctggtcttga 961 catccacaga actttccaga gatg gattgg tgccttcggg aactgtgaga caggtgctgc 1021 atggctgtcg tcagctcgtg ttg tgaaatg ttgggttaag tccc gcaacg agcgcaaccc 80

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1081 ttatcctttg ttgccagcgg tta ggccggg aactcaaagg agactgccag tgataaactg 1141 gaggaaggtg gggatgacgt caag tcatca tggcccttac gaccagggct acacacgtgc 1201 tacaatggca tatacaaaga gaagcg acct cgcgagagca agcggacctc ataaagtatg 1261 tcgtagtccg gattggagtc tg caactcga ctccatgaag tcggaatcgc tagtaatcgt 1321 agatcagaat gctacggt ga atacgttccc gggccttgta cacaccgccc gtcacaccat 1381 gggagtgggt tgcaaaagaa gtaggtagct taacc >FM179768 Acetobacter pasteurianus AUC25 t tagataccct ggtagtccac gccgtaaacg atgagtgcta agtgttagag 841 ggtttccgcc ctttagtgct g aagttaacg cattaagcac tccgcctggg gagtacggcc 901 gcaaggctga aactcaaagg aattgacggg ggcccgcaca agcggtggag catgtggttt 961 aattcgaagc aacgcgaaga accttaccag gtcttgacat cctctgaaaa ccctagagat 1021 agggcttctc cttcgggagc agagtgacag gtggtgcatg gttgtcgtca gctcgtgtcg 1081 tgagatgttg ggttaagtcc cg caacgagc gcaacccttg atcttagttg ccatcattaa 1141 gttgggcact ctaaggtgac tgccg gtgac aaaccggagg aaggtgggga tgacgtcaaa 1201 tcatcatgcc ccttatgacc tgggctacac acgtgctaca atggacggta caaagagctg 1261 caagaccgcg aggtggagct aatctcataa aaccgttctc agttcggatt gtaggctgca 1321 actcgcctac atgaagctgg aatcg ctagt aatcgcggat cagcatgccg cggtgaatac 1381 gttcccgggc cttgtacaca ccgcccgtca caccacgaga gtttgtaaca cccgaagtcg 1441 gtggggtaac c >EU862564 Bacillus cereus AB31 ttagat accctggtag tccacgccgt 841 aaacgatgag tgctaagtgt ta gagggttt ccgcccttta gtgctgaagt taacgcatta 901 agcactccgc ctggggagta cg gccgcaag gctgaaactc aaaggaattg acgggggccc 961 gcacaagcgg tggagcatgt ggtttaattc gaagcaacgc gaagaacctt accaggtctt 1021 gacatcctct gaaaacccta gagatagggc ttctccttcg ggagcagagt gacaggtggt 1081 gcatggttgt cg tcagctcg tgtcgtgaga tgttgggtta agtcccgcaa cgagcgcaac 1141 ccttgatctt agttgccatc attaag ttgg gcactctaag gtgactgccg gtgacaaacc 1201 ggaggaaggt ggggatgacg tcaaatcatc atgcccctta tgacctgggc tacacacgtg 1261 ctacaatgga cggtacaaag agctgcaaga ccgcgaggtg gagctaatct cataaaaccg 1321 ttctcagttc ggattgtagg ctg caactcg cctacatgaa gctgga atcg ctagtaatcg 1381 cggatcagca tgccgcggtg aatacgttcc cgggccttgt acacaccgcc cgtcacacca 1441 cgagagtttg taacacccga agtcggtggg gt >EU871042 Bacillus cereus JL ttagat accctggtag 781 tccacgccgt aaacgatgag tgct aagtgt tagagggttt ccgcccttta gtgctgaagt 841 taacgcatta agcactccgc ctg gggagta cggccgcaag gctgaaactc aaaggaattg 901 acgggggccc gcacaagcgg tggagcatgt ggtttaattc gaagcaacgc gaagaacctt 961 accaggtctt gacatcctct g aaaacccta gagatagggc ttctccttcg ggagcagagt 1021 gacaggtggt g catggttgt cgtcagctcg tgtcgtga ga tgttgggtta agtcccgcaa 1081 cgagcgcaac ccttgatctt agttgccatc attaagttgg gcactctaag gtgactgccg 1141 gtgacaaacc ggaggaaggt ggggatgacg tcaaatcatc atgcccctta tgacctgggc 1201 tacacacgtg ctacaatgga cggtacaaag agctgcaaga ccgcgaggtg gagctaatct 1261 cataaaaccg ttctcagttc ggattg tagg ctgcaactcg cctacatgaa gctggaatcg 1321 ctagtaatcg cggatcagca tgccgcggtg aatacgttcc cgggccttgt acacaccgcc 1381 cgtcacacca cgagagtttg taacacccga agtcggtggg gtaacc >EU812752 Bacillus thuringiensis H04-1 ttagata ccctggtagt ccacgccgta aacgatgagt 781 gctaagtgtt agagggtttc cg ccctttag tgctgaagtt aacgcattaa gcactccgcc 841 tggggagtac ggccgcaagg ctgaaactca aaggaattg a cgggggcccg cacaagcggt 901 ggagcatgtg gtttaattcg aagcaacgcg aagaacctta ccaggtcttg acatcctctg 961 aaaaccctag agatagggct tct ccttcgg gagcagagtg acaggtggtg catggttgtc 1021 gtcagctcgt gtcgtgagat gttgggttaa gtcccgcaac gagcgcaacc cttgatctta 1081 gttgccatca ttaagttggg cactctaagg tgactg ccgg tgacaaaccg gaggaaggtg 1141 gggatgacgt caaatcatca tg ccccttat gacctgggct acacacgtgc tacaatggac 1201 ggtacaaaga gctgcaagac cg cgaggtgg agctaatctc ataaaaccgt tctcagttcg 1261 gattgtaggc tgcaactcgc ctacatg aag ctggaatcgc tagtaatcgc ggatcagcat 81

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1321 gccgcggtga atacgttccc gggccttgta cacaccgccc gtcacaccac gagagtttgt 1381 aacacccgaa gtcggtgggg taacc >EU841483 Acinetobacter lwoffii 412 ttag ataccctggt agtccatgcc gtaaacgatg tctactagcc 781 gttggggcct ttgaggcttt agtg gcgcag ctaacgcgat aag tagaccg cctggggagt 841 acggtcgcaa gactaaaact caaat gaatt gacgggggcc cgcacaagcg gtggagcatg 901 tggtttaatt cgatgcaacg cg aagaacct tacctggtct tgacatagta agaactttcc 961 agagatggat tggtgccttc g ggaacttac atacaggtgc tgcatggctg tcgtcagctc 1021 gtgtcgtgag atgttgggtt aagtcccgca acgagcgcaa cccttttcct tatttgccag 1081 cgggttaagc cgggaacttt aa ggatactg ccagtgacaa actggaggaa ggcggggacg 1141 acgtcaagtc atcatggccc ttacgaccag ggctacacac gtgctacaat ggtcggtaca 1201 aagggttgct acctcgcgag aggatgctaa tctcaaaaag ccgatcgtag tccggattgg 1261 agtctgcaac tcgactccat gaagtc ggaa tcgctagtaa tcgcggatca gaatgccgcg 1321 gtgaatacgt tcccgggcct tgtacacacc gcccgtcaca ccatgggagt ttgttgcacc 1381 agaagtaggt agtctaacc >AF181576 Pseudomonas cf. monteilii ttagata 781 ccctggtagt ccacgccgta aacg atgtca actagccgtt ggaatccttg agattttagt 841 ggcgcagcta acgcattaag ttg accgcct ggggagtacg gccgcaaggt taaaactcaa 901 atgaattgac gggggcccgc acaagcggtg gagcatgtgg tttaattcga agcaacgcga 961 agaaccttac caggccttga catgcagaga actttccaga gatggattgg tgccttcggg 1021 aactctgaca caggtgctgc atggctgtcg tcagctcgtg tcgtgagatg ttgggttaag 1081 tcccgtaacg agcgcaaccc ttgt ccttag ttaccagcac gttatggtgg gcactctaag 1141 gagactgccg gtgacaaacc ggagga aggt ggggatgacg tcaagtcatc atggccctta 1201 cggcctgggc tacacacgtg ct acaatggt cggtacagag ggttgccaag ccgcgaggtg 1261 gagctaatct cacaaaaccg atcgta gtcc ggatcgcagt ctgcaactcg actgcgtgaa 1321 gtcggaatcg ctagtaatcg caaatcagaa tgttgcggtg aatacgttcc cgggccttgt 1381 acacaccgcc cgtcacacca tgggagtggg ttgcaccaga agtagctagt ctaacc >AJ006086 Bacillus silvestris tt 781 agataccctg gtagtccacg cc gtaaacga tgagtgctaa gt gttggggg gtttccgccc 841 ctcagtgctg cagctaacgc a ttaagcact ccgcctgggg ag tacggtcg caagactgaa 901 actcaaagga attgacgggg gcccgcacaa gcggtggagc atgtggttta attcgaagca 961 acgcgaagaa ccttaccagg tc ttgacatc ccggtgacca ctatggagac atagtttccc 1021 cttcgggggc aacggtgaca ggtggtgcat ggttgtcgtc agctcgtgtc gtgagatgtt 1081 gggttaagtc ccgcaacgag cg caaccctt attcttagtt gccat cattc agttgggcac 1141 tctaaggaga ctgccggtga t aaaccggag gaaggtgggg atgacgtcaa atcatcatgc 1201 cccttatgac ctgggctaca cacg tgctac aatggacggt acaaacggtt gccaacccgc 1261 gagggggagc taatccgata aaaccgttct cagttcggat tgtaggctgc aactcgccta 1321 catgaagccg gaatcgctag taat cgcgga tcagcatgcc gcggtgaata cgttcccggg 1381 ccttgtacac accgcccgtc acaccacg ag agtttgtaac acccgaagtc ggtgaggtaa 1441 cc >AJ491302 Caryophanon latum DSM14151T tta gataccctgg 781 tagtccacgc cgtaaacgat gagt gctaag tgttgggggg ttt ccgcccc tcagtgctgc 841 agctaacgca ttaagcactc cg cctgggga gtacggtcgc aagactgaaa ctcaaaggaa 901 ttgacggggg cccgcacaag cggtggagca tgtggtttaa ttcgaagcaa cgcgaagaac 961 cttaccaggt cttgacatcc cgttgaccac tatggagaca tagttttccc ttcggggaca 1021 acggtgacag gtggtgcatg gttgtc gtca gctcgtgtcg tgagatgttg ggttaagtcc 1081 cgcaacgagc gcaacccttg tccttagttg ccagcattta gttg ggcact ctagggagac 1141 tgccggtgac aaaccggagg aag gtgggga tgacgtcaaa tcatcatgcc ccttatgacc 1201 tgggctacac acgtgctaca atggacggta caaacggttg ccaacccgcg agggggagcc 1261 aatccgataa agccgttctc agttc ggatt gtaggctgca actcgcctac atgaagccgg 1321 aatcgctagt aatcgcggat cagcat gccg cggtgaatac gttcccgggc cttgtacaca 1381 ccgcccgtca caccacgaga gtttgtaaca cccgaagccg gtggggtaac c 82

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>AJ491303 Caryophanon tenue DSM-14152T ttagatac cctggtagtc cacgccgtaa acgatgagtg ctaagtgttg gggggtttcc 841 gcccctcagt gctgcagcta acg cattaag cactccgcct ggggagtacg gtcgcaagac 901 tgaaactcaa aggaattgac gggggcccgc acaagcggtg gagcatgtgg tttaattcga 961 agcaacgcga agaaccttac caggtcttga catcccgctg accgctatgg agacatagct 1021 ttcccttcgg ggacagtggt gacaggtggt gcatggttgt cgtcagctcg tgtcgtgaga 1081 tgttgggtta agtcccgcaa cgagcgcaac ccttgtcctt agttgccatc atttagttgg 1141 gcactctagg gagactgccg gtg acaaacc ggaggaaggt ggggatgacg tcaaatcatc 1201 atgcccctta tgacctgggc tacacacgtg ctacaatgga cgatacaaac ggttgccaac 1261 ccgcgagggg gagccaatcc gat aaagtcg ttctcagttc ggattgtagg ctgcaactcg 1321 cctacatgaa gccggaatcg ctagt aatcg cggatcagca tgccgcggtg aatacgttcc 1381 cgggccttgt acacaccgcc cg tcacacca cgagagtttg taacacccga agccggtggg 1441 gtaacc >EU194334 Pseudomonas plecoglossicida XJUHX-16 t tagataccct ggtagtccac gccgtaaacg 781 atgtcaacta gccgttggaa tccttgagat tttagtggcg cagctaacgc attaagttga 841 ccgcctgggg agtacggccg caaggttaaa actcaaatga a ttgacgggg gcccgcacaa 901 gcggtggagc atgtggttta a ttcgaagca acgcgaagaa ccttaccagg ccttgacatg 961 cagagaactt tccagagatg gattg gtgcc ttcgggaact ctgacacagg tgctgcatgg 1021 ctgtcgtcag ctcgtgtcgt gagatgttgg gttaagtccc gtaacgagcg caacccttgt 1081 ccttagttac cagcacgtta tggtgg gcac tctaaggaga ctgccggtga caaaccggag 1141 gaaggtgggg atgacgtcaa gtcat catgg cccttacggc ctgggctaca cacgtgctac 1201 aatggtcggt acagagggtt gccaagccgc gaggtggagc taatctcaca aaaccgatcg 1261 tagtccggat cgcagtctgc aact cgactg cgtgaagtcg gaatcgctag taatcgcgaa 1321 tcagaatgtc gcggtgaata cgttcccggg ccttgtacac accgcccgtc acaccatggg 1381 agtgggttgc accagaagta gctagtctaa cc >AF368755 Pseudomonas saccharophila ttag ataccctggt 781 agtccacgcc ctaaacgatg t caactggtt gttgggaggg tttcttctca gtaacgtagc 841 taacgcgtga agttgaccgc ct ggggagta cggccgcaag gttgaaactc aaaggaattg 901 acggggaccc gcacaagcgg tg gatgatgt ggtttaattc gatgcaacgc gaaaaacctt 961 acctaccctt gacatgtctg g aatcctgaa gagatttggg agtgctcgaa agagaaccag 1021 gacacaggtg ctgcatggcc gtcgtcag ct cgtgtcgtga gatgttgggt taagtcccgc 1081 aacgagcgca acccttgtca ttag ttgcta cgaaagggca ctctaatgag actgccggtg 1141 acaaaccgga ggaaggtggg gatgac gtca ggtcatcatg gcccttatgg gtagggctac 1201 acacgtcata caatggccgg gacagagggc tgccaacccg cgagggggag ctaatcccag 1261 aaacccggtc gtagtccgga tcgtagtctg caactcgact gcgtgaagtc ggaatcgcta 1321 gtaatcgcgg atcagcttgc cgc ggtgaat acgttcccgg gtcttgtaca caccgcccgt 1381 cacaccatgg gagcgggttc tgccagaagt agttagccta acc >AM501435 Pelomonas aquatica CCUG52575T t 721 tagataccct ggtagtccac gccctaaacg atgtcaactg gttgttggga gggtttcttc 781 tcagtaacgt agctaacgcg tg aagttgac cgcctgggga gtacggccgc aaggttgaaa 841 ctcaaaggaa ttgacgggga cccg cacaag cggtggatga tgtg gtttaa ttcgatgcaa 901 cgcgaaaaac cttacctacc cttgacatgc caggaatcct gaagagattt gggagtgctc 961 gaaagagagc ctggacacag gtgc tgcatg gccgtcgtca gctcgtgtcg tgagatgttg 1021 ggttaagtcc cg caacgagc gcaacccttg tcattag ttg ctacgaaagg gcactctaat 1081 gagactgccg gtgacaaacc ggagga aggt ggggatgacg tcaggtcatc atggccctta 1141 tgggtagggc tacacacgtc atacaatggc cgggacagag ggctgccaac ccgcgagggg 1201 gagctaatcc cagaaacccg gtcgtagt cc ggatcgtagt ctgcaactcg actgcgtgaa 1261 gtcggaatcg ctagtaatcg c ggatcagct tgccgcggtg aatacgttcc cgggtcttgt 1321 acacaccgcc cgtcacacca tgggagcggg ttctgccaga agtagttagc ctaacc >AM501432 Pelomonas saccharophila IAM14368T t 721 tagataccct ggtagtccac gccctaaacg atgtcaactg gttgttggga gggtttcttc 781 tcagtaacgt agctaacgcg tg aagttgac cgcctgggga gtacggccgc aaggttgaaa 83

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841 ctcaaaggaa ttgacgggga cccg cacaag cggtggatga tgtg gtttaa ttcgatgcaa 901 cgcgaaaaac cttacctacc cttgacatgc caggaatcct gaagagattt gggagtgctc 961 gaaagagaac ctggacacag gtgctg catg gccgtcgtca gctcgtgtcg tgagatgttg 1021 ggttaagtcc cg caacgagc gcaacccttg tcattag ttg ctacgaaagg gcactctaat 1081 gagactgccg gtgacaaacc ggagga aggt ggggatgacg tcaggtcatc atggccctta 1141 tgggtagggc tacacacgtc atacaatggc cgggacagag ggctgccaac ccgcgagggg 1201 gagctaatcc cagaaacccg gtcgtagt cc ggatcgtagt ctgcaactcg actgcgtgaa 1261 gtcggaatcg ctagtaatcg c ggatcagct tgccgcggtg aatacgttcc cgggtcttgt 1321 acacaccgcc cgtcacacca tgggagcggg ttctgccaga agtagttagc ctaacc >EU921228 Pseudomonas mosselii BM-F1 ttagatacc ctggtagtcc acgccgtaaa cgatgtcaac tagccgttgg aatccttgag 781 attttagtgg cgcagctaac gcattaagtt gaccgcctgg ggagtacggc cgcaaggtta 841 aaactcaaat gaattgacgg gggcccgcac aagcggtgga g catgtggtt taattcgaag 901 caacgcgaag aaccttacca ggccttgaca tgcagagaac tttccagaga tggattggtg 961 ccttcgggaa ctctgacaca ggtgc tgcat ggctgtcgtc agctcgtgtc gtgagatgtt 1021 gggttaagtc ccgtaacgag cg caaccctt gtccttagtt accagcacgt tatggtgggc 1081 actctaagga gactgccggt gacaa accgg aggaaggtgg ggatgacgtc aagtcatcat 1141 ggcccttacg gcctgggcta cacacgtgct acaatggtcg gtacagaggg ttgccaagcc 1201 gcgaggtgga gctaatctca caaaaccgat cgtagtccgg atcgcagtct gcaactcgac 1261 tgcgtgaagt cggaatcgct agt aatcgcg aatcagaatg tcgcggtgaa tacgttcccg 1321 ggccttgtac acaccgcccg tcacaccat g ggagtgggtt gcaccagaag tagctagtct 1381 aacc >EU239475 Pseudomonas plecoglossicida XJUHX-15 ttaga taccctggta gtccacgccg taaacgatgt 781 caactagccg ttggaatcct tg agatttta gtggcgcagc taacgcatta agttgaccgc 841 ctggggagta cggccgcaag g ttaaaactc aaatgaattg acg ggggccc gcacaagcgg 901 tggagcatgt ggtttaattc gaagcaacgc gaagaacctt accaggcctt gacatgcaga 961 gaactttcca gagatggatt ggtg ccttcg ggaactctga cacaggtgct gcatggctgt 1021 cgtcagctcg tgtcgtgaga tg ttgggtta agtcccgtaa cgagcgcaac ccttgtcctt 1081 agttaccagc acgttatggt g ggcactcta aggagactgc cggtgacaaa ccggaggaag 1141 gtggggatga cgtcaagtca t catggccct tacggcctgg gctacacacg tgctacaatg 1201 gtcggtacag agggttgcca agcc gcgagg tggagctaat ctcacaaaac cgatcgtagt 1261 ccggatcgca gtctgcaact cgactgc gtg aagtcggaat cgctagtaat cgcgaatcag 1321 aatgtcgcgg tgaatacgtt cccgggcctt gtacacaccg cccgtcacac catgggagtg 1381 ggttgcacca gaagtagcta gtctaacc >EU239464 Pseudomonas putida XJUHX-1 ttag ataccctggt agtccacgcc gtaaacgatg 781 tcaactagcc gttggaatcc ttg agatttt agtggcgcag ctaacgcatt aagttgaccg 841 cctggggagt acggccgcaa ggttaaaact caaatgaatt gacgggggcc cgcacaagcg 901 gtggagcatg tggtttaatt cg aagcaacg cgaagaacct taccaggcct tgacatgcag 961 agaactttcc agagatggat t ggtgccttc gggaactctg acacaggtgc tgcatggctg 1021 tcgtcagctc gtgtcgtgag at gttgggtt aagtcccgta acgagcgcaa cccttgtcct 1081 tagttaccag cacgttatgg t gggcactct aaggagactg ccggtgacaa accggaggaa 1141 ggtggggatg acgtcaagtc atcatggccc ttacggcctg ggctacacac gtgctacaat 1201 ggtcggtaca gagggttgcc aag ccgcgag gtggagctaa tctcacaaaa ccgatcgtag 1261 tccggatcgc agtctgcaac tcgactgcgt gaagtcggaa tcgctagtaa tcgcgaatca 1321 gaatgtcgcg gtgaatacgt tcccgggcct tgtacacacc gcccgtcaca ccatgggagt 1381 gggttgcacc agaagtagct agtctacc >EF178450 Pseudomonas entomophila 2P25 tta gataccctgg tagtccacgc cgtaaacgat gtcaactagc 781 cgttggaatc cttgagattt tagt ggcgca gctaacgcat t aagttgacc gcctggggag 841 tacggccgca aggttaaaac t caaatgaat tgacgggggc ccgcacaagc ggtggagcat 901 gtggtttaat tcgaagcaac gcgaagaacc ttaccaggcc ttgacatgca gagaactttc 961 cagagatgga ttggtgcctt cg ggaactct gacacaggtg ctgcatggct gtcgtcagct 1021 cgtgtcgtga gatgttgggt taagtcccgt aacgagcgca acccttgtcc ttagttacca 1081 gcacgttatg gtgggcactc taagga gact gccggtgaca aaccggagga aggtggggat 84

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85 1141 gacgtcaagt catcatggcc ctt acggcct gggctacaca cgtgctacaa tggtcggtac 1201 agagggttgc caagccgcga ggtggagcta atctcacaaa accgatcgta gtccggatcg 1261 cagtctgcaa ctcgactgcg tgaagt cgga atcgctagta atcgcaaatc agaatgttgc 1321 ggtgaatacg ttcccgggcc ttgtacacac cgcccgtcac accatgggag tgggttgcac 1381 cagaagtagc tagtctaacc >EU857417 Pseudomonas taiwanensis BF-S2 ttagata ccctggtagt ccacgccgta aacgatgtca actagccgtt 781 ggaatccttg agattttagt ggcgc agcta acgcattaag ttgaccgcct ggggagtacg 841 gccgcaaggt taaaactcaa atg aattgac gggggcccgc acaagcggtg gagcatgtgg 901 tttaattcga agcaacgcga agaaccttac caggccttga catgcagaga actttccaga 961 gatggattgg tgccttcggg aactctgaca caggtgctgc atgg ctgtcg tcagctcgtg 1021 tcgtgagatg ttgggttaag tcccgtaacg agcgcaaccc ttgtccttag ttaccagcac 1081 gttatggtgg gcactctaag gagactgccg gtgacaaacc ggaggaaggt ggggatgacg 1141 tcaagtcatc atggccctta cg gcctgggc tacacacgtg ctacaatggt cggtacagag 1201 ggttgccaag ccgcgaggtg ga gctaatct cacaaaaccg atcgtagtcc ggatcgcagt 1261 ctgcaactcg actgcgtgaa gtcg gaatcg ctagtaatcg cgaatcagaa tgtcgcggtg 1321 aatacgttcc cgggccttg t acacaccgcc cgtcacacca tgggagtggg ttgcaccaga 1381 agtagctagt ctaacc

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APPENDIX C ARSENATE REDUCTASE GENE SEQUENCES Sequencing results of arsenate reduc tase genes, partical sequences. > Shewanella sp. ANA-3 arrA 1 aaagtaacgc tctatagggc gaattgggcc ctctagagca tgctcgagcg 51 gccgccagtg tgatggatat ct gcagaatt cggcttaagg tgtatggaat 101 aaagcgtttg tgggtgactt tattgagggt aaaaacctgt ttaaagcagg 151 taaaaccgtc agtgtcgaga gctttaaaga aacccatacc tacggtttag 201 tcgaatggtg gaaccaggcc c ttaaagatt acactcctga atgggcacct 251 gaaatcacag gaagccgaat t ccagcacac tggcggccgt tactagtgga 301 tccgagctcg gtaccaagct tggcgtaatc atggtcatag ctgtttcctg 351 tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc 401 ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat 451 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc 501 tgcattaatg aatcggccaa cgcg cgggga gaggcggttt gcgtattggg >AH25 arrA 1 aattgatcga ctagtatagg gc gaattggg ccctctagat gcatgctcga 51 gcggccgcca gtgtgatgga tatctgcaga attcggctta aggtgtatgg 101 aataaagcgt ttgtgggcga tttg ctttcc gaggcagtca tctgtggctt 151 cactactttg tcttcacagg ctgtggtcgc tcactgtcaa cagcaaatgt 201 gtgtcaggac aaaatattaa cccctaaatg ggcacctgaa atcacaggaa 251 gccgaattcc agcacactgg cggccgttac tagtggatcc gagctcggta 301 ccaagcttgg cgtaatcatg gtcat agctg tttcctgtgt gaaattgtta 351 tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag 401 cctggggtgc ctaatgagtg ag ctaactca cattaattgc gttgcgctca 451 ctgcccgctt tccagtcggg aaacct gtcg tgccagctgc attaatgaat >PAO1 arsC 1 cggtgaacga ctctataggg cgaattgggc cctctagatg catgctcgag 51 cggccgccag tgtgatggat atctgcagaa ttcgcccttc tgttcatgtg 101 cacggccaac ggccgctggc tttcatggcg ataggtgata gcatagagcc 151 atcattcgca gcgacgctct gc cgcgaaga ccacgaaaga agcgcgtcat 201 gatgaccgag cacgatgacc cgaccctgga ccgcctgaag caccacttcg 251 cccagcgagt gatcaaccag gcgcgccagg ttctggaggt ctggcaacgc 301 ctgacccgcg cggagtggaa cagcgacggc atggaagaac tggccgacgc 351 caccctgcgc ctgcagcgct acg ccgaacg cttcgagcaa gccgagcatg 401 cccagttggc cgtgcacatg aacagaaggg cgaattccag cacactggcg 451 gccgttacta gtggatccga gc tcggtacc aagcttggcg taatcatggt 501 catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac 551 atacgagccg gaagcataaa gt gtaaagcc tggggtgcct aatgagtgag 601 ctaactcaca ttaattgcgt tgcg ctcact gcccgctttc cagtcgggaa 651 acctgtcgtg ccagctgcat taat gaatcg gccaacgcgc ggggagaggc 701 ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg 751 ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa 801 tacggttatc cacagaatca gggg ataacg caggaaagaa catgtgagca 851 aaaggccagc aaaaggccag gaaccg taaa aaggcccgcg ttgctggcgt 901 ttttccatag gctccgcccc ccctgacgag catcacaaaa tcgacgctca 951 gtcagaggtg gcgaacccga ca ggactata aagataccag gcgttccccc 1001 tggaagcctc cctcgtgcgc tc tctgttcg gacctgccgc ttaccggata 1051 cctgtcccgc cttctcctcg gg aagcggtg gcggcttctc atagctcacg 1101 ctgtaggtat tctcaagttc g gggtagttc ggtcgctcca aagcctgctg 1151 ggtgcgaccc cgttcgagcc cggacgcttg cgccttatat ccggtgta >AH4 arsC 1 cacgtgtatc gactactata gggc gaattg ggccctctag atgcatgctc 51 gagcggccgc cagtgtgatg gatatctgca gaattcgccc ttgcattctt 86

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87 101 tccgaagcca tgttcaagta cacgtcccac gctggcgagg gccacaccat 151 cgctgaaatg gacgatgaaa caaagaagga actgtggcag aagaagctgc 201 ctcgactccc cggccatcgc agcg agctcg acaaagtcaa gatcgagaag 251 gtcaagcccg acaagaagga agcgcttctt cgagtcgagg actacattca 301 caccaaggcc aatcatacga ctttcacgga ggccctcacc atttgcccgt 351 tcgaccttct cgagaacgac gcc ttgaaga ccgaaatggt ccgtggcgga 401 atacagcttt tcggttccct cccgacggcg caagacgccc aagagaagct 451 agacgcgctt gagagcgtct ttcctcagtg gaacgccatc gcgaccagct 501 caagcgtgag ctaagggcga >AH34 arsC 1 acggagtatc gactctatag ggcgaattgg gccctctaga tgcatgctcc 51 gagcggccgc cagtgtgatg gatatctgca gaattcgccc ttgcattctt 101 tccgaagcca tgttcaaggc cgcctacgga gaatctccat acaactacct 151 catgacacgg cgcatcgagc g ggccatggc cctgctccgc gcgggaacca 201 gcgtcaccga tgcctgcatg gaag tcggct gtacctcgtt gggctcgttc 251 agcacgcgtt tcaccgaaat cgtg ggaatc aaccccagcg agtaccgcgc 301 ccgggagcac cacgctgtga a ggccatgcc caactgcatc gcgaccagct >AH45 arsC 1 ctaacgtgaa acgactccta tagggcgaat tgggccctct agatgcatgc 51 tcgagcggcc gccagtgtga tggatatctg cagaattcgc ccttgtattc 101 tttccgaagc catg ttcaag ttcagggcct gcaggggcgg caacccgggg 151 gtgtagcgcc gggcgatcac ttcat cgcgc ggctgttggt cgccatggaa 201 gaacaggcac gcggaaagcc cgaggcaacg cgaacggaat tcgccgatgt 251 agtacgcctt gcctgcttcc aggt gcatgg gcacaagaaa ctgttcccgt 301 gcccggatgg agctgtagcc accgttgtag ccgttccaga agaactggaa 351 gtcatagaac tcgtagtcgc cg ggcttgag gggcatgacg aacacgctgg 401 cctcgccggg cagcgcctgc ttgtcggggc cacgcacctc ttcgatgtcc 451 ttcggggtgt gggccacttc ggcgaacccc caccatgccg ccgcgccatc 501 ctgcgcgcca cgtttgcgca ggaggatgcg ttggttggtg taggcactat 551 cgcgaccagc tcaagcgtga gct

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APPENDIX D ARSB GENE SEQUENCES >AH23 arsB 1 acagaagatc cgctactata gggc gaattg ggccctctag atgcatgctc 51 gagcggccgc cagtgtgatg gatatctgca gaattcggct tgcggaaata 101 gaggaacagc accacgggca ggacagcgtg gacgtcctcg acggcaacga 151 tggggaaccc gatgatctgg at cttgtccg tacgcgtgaa gtggccgacg 201 cgcaaacggt gcatgccgat ggcacgcccg acgtaatgcc gcttccagtt 251 ttcggtggct tcggacgggt tgccggggtt ttccagccag tcgatcttgc 301 cgccttcggg gtcagcgtgg tggatggttc cgcccggacc ataaagctgg 351 tagcaaacca ccaggtccgt ggtg ccgtca ccagtgatgt caccgaagtc 401 catgccgacg ggttctttga tcttgtccag caccagcttc ttttcccagg 451 tggggttttt gtaccagtag atttcaccga ctttcaggcc gtagccaacg >AH25 arsB accagtatcgactactatagggcgaattgggccctctagatgcatgctcgagcgg ccgccagtgtgatggatatctgcagaat tcggcttgcggaaatagaggaacagc accacgggcaggacagcgtggacgtcct cgacggcaacgatggggaacccgatgatctggatcttgt ccgtacgcgtgaagtggccgacgcgcaaacggtgcatgc caatggcacgcccgacgtaatgccg cttccagttttcggtggcttcggacgggttg ccggggttttccagccagtcgatcttgccg ccttcggggtcagcgtggtggatg gttccgcccggaccataaagctggtagcaaaccaccaggtccgtggtgccgtcacc ggtgatgtcaccgaagtccatgccg acgggttctttgatcttgtccagcaccag cttcttttcccaggtggggtttttgtaccagtagatttcaccgactttcaggccgtagcc aacgaggtccggcttgccgtcgccgtc aatatctgccgcttccagccagtagc cgtcgcgcaagaagtccgtgactgtctcggttccgaa taccggttcggtgacatccagagtgggtgtggtcttggggatcgctggtgaagtcatgagagtacctttcgtgt gagtacgggtgatggtgctgttcctctatttccg caagccgaattccagcacactggcggccgttactagtggatccgagctcggtaccaagcttggcgtaatcatggtcat agctgtttcctgtgtgaaattgttatccg 88

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LIST OF REFERENCES Abedin, M.J., M.S. Cresser, A.A. Meharg, J. Feldmann, and J. Cotter-Howells. 2002. Arsenic Accumulation and Metabolism in Rice (Oryza sativa L.). Environ. Sci. Technol. 36:962968. Ahmann, D., A.L. Roberts, L.R. Krumholz, a nd F.M. Morel. 1994. Microbe grows by reducing arsenic. Nature 371:750. Allison, J.D., D.S. Brown, and K.J. Novo-Gradac. 1999. MINTEQA2, A geochemical assessment data base and test cases for e nvironmental systemsver. 4.0. Report EPA/600/391/-21. Altschul, S.F., W. Gish, W. Miller, E.W. Myer s, and D.J. Lipman. 1990. Basic local alignment search tool. J Mol Biol 215:403-10. Amann, R.I., W. Ludwig, and K.H. Schleifer. 1995. Phylogenetic identi fication and in situ detection of individual microbial cells without cultivation. Microbiol Rev. 59:143-69. Anderson, C.R., and G.M. Cook. 2004. Isolation a nd characterization of arsenate-reducing bacteria from arsenic-contaminated sites in New Zealand. Curr. Microbiol. 48:341-7. Belton, J.C., N.C. Benson, M.L. Hanna, and R.T. Taylor. 1985. Growth inhibitory and cytotoxic effects of three arsenic compounds on cultured Ch inese hamster ovary cells. J. Environ. Sci. Health, Part A ; Vol/Issu e: A20:1:Pages: 37-72. Bolwell, G.P., and P. Wojtaszek. 1997. Mechan isms for the generati on of reactive oxygen species in plant defence a broad perspect ive. Physiol. Mol. Plant Pathol. 51:347-366. Branco, R., A.P. Chung, and P.V. Morais. 2008. Sequencing and expression of two arsenic resistance operons with diffe rent functions in the highl y arsenic-resistant strain Ochrobactrum tritici SCII24T. BMC Microbiol. 8:95. Bruneel, O., R. Duran, C. Casiot, F. Elbaz-P oulichet, and J.C. Personne. 2006. Diversity of microorganisms in Fe-As-rich acid mine dr ainage waters of Carnoules, France. Appl Environ. Microbiol. 72:551-6. Canovas, D., C. Duran, N. Rodriguez, R. Amils, and V. de Lorenzo. 2003. Testing the limits of biological tolerance to arse nic in a fungus isolated fr om the River Tinto. Environ. Microbiol. 5:133-8. Cao, X., L.Q. Ma, and A. Shiralipour. 2003. Effects of compost and phosphate amendments on arsenic mobility in soils and ar senic uptake by the hyperaccumulator, Pteris vittata L. Environ. Pollut. 126:157-67. 89

PAGE 90

Chalabaev, S., E.T. Jean-Fran, C. Abdelkader, N. Sylvie, P. Alain, G. Emma, B.-F.A. Danchin, and F. Biville. 2007. The HcaR regulatory prot ein of Photorhabdus luminescens affects the production of proteins involved in oxidative stress and toxemia. Proteomics 7:4499-4510. Chen, M., L.Q. Ma, and W.G. Harris. 2002. Arseni c Concentrations in Florida Surface Soils: Influence of Soil Type and Properties. Soil. Sci. Soc. Am. J. 66:632-640. Cheng, F., J. Wang, J. Peng, J. Yang, H. Fu, X. Zhang, Y. Xue, W. Li, Y. Chu, and Q. Jin. 2007. Gene expression profiling of the pH response in Shigella flexneri 2a. FEMS Microbiol. Lett. 270:12-20. Dat, J., S. Vandenabeele, E. Vranova, M. Va n Montagu, D. Inze, and F. Van Breusegem. 2000. Dual action of the active oxygen species during plant stress responses. Cell Mol. Life Sci. 57:779-95. Dowdle, P.R., A.M. Laverman, and R.S. Orem land. 1996. Bacterial Dissimilatory Reduction of Arsenic(V) to Arsenic(III) in Anoxic Sediments. Appl. Environ. Microbiol. 62:1664-1669. EPA, U.S. 1997. Recent Developments for In Situ Treatment of Metal Contaminated Soils. EPA542-R-97-004 p. 8. ETCS. 1998. Topic reportContaminated sites. European Topic Centre Soil. European Environment Agency. Fayiga, A.O., L.Q. Ma, and Q. Zhou. 2007. Effects of plant arsenic uptake and heavy metals on arsenic distribution in an arsenic-cont aminated soil. Envi ron. Pollut. 147:737-742. Fitz, W.J., and W.W. Wenzel. 2002. Arsenic transformations in the soil-rhizosphere-plant system: fundamentals and potential application to phytoremedia tion. J. Biotechnol. 99:259-278. Fitz, W.J., W.W. Wenzel, H. Zhang, J. Nurmi, K. Stipek, Z. Fischerova, P. Schweiger, G. Kollensperger, L.Q. Ma, and G. Stingeder. 2003. Rhizosphere characteristics of the arsenic hyperaccumulator Pteris vittata L. and monitoring of phyt oremoval efficiency. Environ. Sci. Technol. 37:5008-5014. Fordyce, F.M., T.M. Williams, A. Paijitp apapon, and P. Charoenchaisei. 1995. British Geological Survey. Keyworth, Nottinghamshire. London. Gadd, G.M. 2004. Microbial influence on metal m obility and application for bioremediation. Geoderma. 122:109-119. Gihring, T.M., P.L. Bond, S.C. Peters, and J. F. Banfield. 2003. Arseni c resistance in the archaeon Ferroplasma acidarmanus ": new insights into the structure and evolution of the ars genes. Extremophiles 7:123-30. 90

PAGE 91

Giotis, E.S., M. Julotok, B.J. Wilkinson, I.S. Blair, and D.A. McDowell. 2008. Role of sigma B factor in the alkaline tolerance response of Listeria monocytogenes 10403S and crossprotection against subsequent ethanol a nd osmotic stress. J. Food Prot. 71:1481-5. Gladysheva, T.B., K.L. Oden, and B.P. Rosen. 1994. Properties of the Arsenate Reductase of Plasmid R773. Biochemistry 33:7288-7293. Goel, A., and P. Aggarwal. 2007. Pesticide poisoning. Natl. Med. J. India. 20:182-91. Gonzalez-Flecha, B., and B. Demple. 2000. Genetic responses to free radicals. Homeostasis and gene control. Ann. NY Acad. Sci. 899:69-87. Grill, E. 1987. Phytochelatins, the heavy metal bi nding peptides of plants: characterization and sequence determination. E xperientia. Suppl. 52:317-22. Gyaneshwar, P., G.N. Kumar, L.J. Parekh, and P.S. Poole. 2002. Role of soil microorganisms in improving P nutrition of plan ts. Plant and Soil 245:83-93. Hartke, A., J.C. Giard, J.M. Laplace, and Y. Auffray. 1998. Survival of Enterococcus faecalis in an oligotrophic microcosm: changes in morphology, development of general stress resistance, and analysis of protein synthesis. Appl. Environ. Microbiol. 64:4238-45. Hartley-Whitaker, J., G. Ainswo rth, R. Vooijs, W.T. Bookum, H. Schat, and A.A. Meharg. 2001. Phytochelatins Are Involved in Di fferential Arsenate Tolerance in Holcus lanatus Plant Physiol. 126:299-306. Hassett, D.J., and M.S. Cohen. 1989. Bacterial adap tation to oxidative stress: implications for pathogenesis and interaction with ph agocytic cells. FASEB J. 3:2574-2582. Hirata, K., N. Tsuji, and K. Miyamoto. 2005. Bi osynthetic regulation of phytochelatins, heavy metal-binding peptides. J. Biosci. Bioeng. 100:593-9. Imlay, J.A. 2008. Cellular Defenses against S uperoxide and Hydroge n Peroxide. Annu. Rev. Biochem. 77:755-776. Inskeep, W.P., T.R. McDermott, and S. Fendorf. 2002. Environmental Chemistry of Arsenic:183. Jackson, C.R., and S.L. Dugas. 2003. Phylogene tic analysis of bacterial and archaeal arsC gene sequences suggests an ancient, common origin for arsenate reducta se. BMC Evol. Biol. 3:18. Jeanmougin, F., J.D. Thompson, M. Gouy, D.G. Higgins, and T.J. Gibson. 1998. Multiple sequence alignment with Clustal X. Trends Biochem. Sci. 23:403-5. 91

PAGE 92

Jones, C.A., H.W. Langner, K. Anderson, T.R. McDermott, and W.P. Inskeep. 2000. Rates of Microbially Mediated Arsenate Reduction and Solubilizati on. Soil. Sci. Soc. Am. J. 64:600-608. Kabata-Pendias, A., and H. Pendias. 2000. Trace elem ents in soils and plants. 3rd ed. CRC Press, Boca Raton, FL. Kampfer, P., E. Falsen, and H.-J. Busse. 2008. Naxibacter varians sp. nov. and Naxibacter haematophilus sp. nov., and emended de scription of the genus Naxibacter. Int. J. Syst. Evol. Microbiol. 58:1680-1684. Kjelleberg, S., N. Albertson, K. Flardh, L. Holm quist, A. Jouper-Jaan, R. Marouga, J. Ostling, B. Svenblad, and D. Weichart. 1993. How do non-diffe rentiating bacteria adapt to starvation? Antonie Van Leeuwenhoek 63:333-41. Komar, K., L.Q. Ma, D. Rockwood, and A. Sye d. 1998. Identification of arsenic tolerant and hyperaccumulating plants from arsenic cont aminated soils in Florida. Agronomy Abstract:343. Krafft, T., and J.M. Macy. 1998. Purification and characterization of the respiratory arsenate reductase of Chrysiogenes arsenatis Eur. J. Biochem. 255:647-53. Lasat, M.M. 2002. Phytoextraction of Toxic Meta ls: A Review of Biological Mechanisms. J. Environ. Qual. 31:109-120. Leung, H.M., Z.H. Ye, and M.H. Wong. 2006. Inte ractions of mycorrhizal fungi with Pteris vittata (As hyperaccumulator) in As-contaminated soils. Environ. Pollut. 139:1-8. Liu, A., E. Garcia-Dominguez, E.D. Rhine, and L.Y. Young. 2004. A nove l arsenate respiring isolate that can utilize aromatic subs trates. FEMS Microbiol. Ecol. 48:323-332. Liu, S.X., M. Athar, I. Lippai, C. Waldre n, and T.K. Hei. 2001. Induction of oxyradicals by arsenic: implication for mechanism of genotoxicity. Proc. Nat.l Acad. Sci. USA 98:1643-8. Liu, Y., Y.G. Zhu, B.D. Chen, P. Christie, and X.L. Li. 2005. Influence of the arbuscular mycorrhizal fungus Glomus mosseae on uptake of arsenate by the As hyperaccumulator fern Pteris vittata L. Mycorrhiza 15:187-92. Lombi, E., F.J. Zhao, M. Fuhrmann, L.Q. Ma, and S.P. McGrath. 2002. Arsenic distribution and speciation in the fronds of the hyperaccumulator Pteris vittata New Phytol. 156:195-203. Lucy, M., E. Reed, and B.R. Glick. 2004. Appli cations of free living plant growth-promoting rhizobacteria. Antonie van Leeuwenhoek 86:1-25. Ma, L.Q., K.M. Komar, C. Tu, W. Zhang, Y. Cai, and E.D. Kennelley. 2001. A fern that hyperaccumulates arsenic. Nature 409:579. 92

PAGE 93

Macur, R.E., J.T. Wheeler, T.R. McDermott, and W.P. Inskeep. 2001a. Microbial populations associated with the reduction and enhanced mobilization of arsenic in mine tailings. Environ. Sci. Technol. 35:3676-82. Macur, R.E., J.T. Wheeler, T.R. McDermott, and W.P. Inskeep. 2001b. Microbial Populations Associated with the Reduction and Enhanced Mobilization of Arseni c in Mine Tailings. Environ. Sci. Technol. 35:3676-3682. Macy, J.M., J.M. Santini, B.V. Pauling, A.H. O'Neill, and L.I. Sly. 2000. Two new arsenate/sulfate-reducing bacteria: mechanis ms of arsenate reduc tion. Arch. Microbiol. 173:49-57. Mah, T.-F.C., and G.A. O'Toole. 2001. Mechanisms of biofilm resistance to antimicrobial agents. Trends Microbiol. 9:34-39. Malasarn, D., C.W. Saltikov, K.M. Campbell, J.M. Santini, J.G. Hering, and D.K. Newman. 2004. arrA is a reliable marker for As (V) respiration. Science 306:455. Mandal, B.K., and K.T. Suzuki. 2002. Arsenic round the world: a review. Talanta 58:201-235. Mandal, S.M., B.R. Pati, A.K. Das, and A.K. Ghosh. 2008. Characterizati on of a symbiotically effective Rhizobium resistant to arsenic: Isolated from the root nodules of Vigna mungo (L.) Hepper grown in an arsenic-contaminated field. J. Gen. Appl. Microbiol. 54:93-9. Mateos, L.M., E. Ordonez, M. Letek, and J.A. Gil. 2006. Corynebacterium glutamicum as a model bacterium for the bioremediation of arsenic. Int. Microbiol. 9:207-15. Meharg, A.A., and M.R. Macnair. 1992. Suppression of the High Affini ty Phosphate Uptake System: A Mechanism of Arsenate Tolerance in Holcus lanatus L. J. Exp. Bot. 43:519-524. Meharg, A.A., and J. Hartley-Whitaker. 2002. Tansley Review No. 133. Arsenic Uptake and Metabolism in Arsenic Resistant and Nonresis tant Plant Species. New Phytol. 154:29-43. Meharg, A.A., J. Bailey, K. Breadmore, and M.R. Macnair. 1994. Biomass allocation, phosphorus nutrition and vesicular-a rbuscular mycorrhizal infection in clones of Yorkshire Fog, Holcus lanatus L. (Poaceae) that differ in their phosphate uptake kinetics and tolerance to arsenate. Plant and Soil 160:11-20. Meng, X., G.P. Korfiatis, C. Jing, and C. Ch ristodoulatos. 2001. Redox Transformations of Arsenic and Iron in Water Treatment Sludge during Aging and TCLP Extraction. Environ. Sci. Technol. 35:3476-3481. Mukhopadhyay, R., B.P. Rosen, L.T. Phung, and S. Silver. 2002. Microbial arsenic: from geocycles to genes and enzymes. FEMS Microbiol. Rev. 26:311-25. 93

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Naydenov, C.L., E.P. Kirazov, L.P. Kirazov, and T.T. Genadiev. 2006. New approach to calculating and predicting the io nic strength generated during carrier ampholyte isoelectric focusing. J Chromatogr. A 1121:129-139. Newman, D.K., E.K. Kennedy, J.D. Coates, D. Ahmann, D.J. Ellis, D.R. Lovley, and F.M. Morel. 1997. Dissimilatory arsenate and sulfate reduction in Desulfotomaculum auripigmentum sp. nov. Arch. Microbiol. 168:380-8. Niggemyer, A., S. Spring, E. Stackebrandt, and R.F. Rosenzweig. 2001. Isolation and Characterization of a Novel As(V)-Reduci ng Bacterium: Implications for Arsenic Mobilization and the Genus Desulfitobacterium Appl. Environ. Microbiol. 67:5568-5580. Oremland, R.S., and J.F. Stolz. 2003. The ecology of arsenic. Science 300:939-44. Page, R.D. 1996. TreeView: an application to di splay phylogenetic trees on personal computers. Comput. Appl. Bi osci. 12:357-8. Perker, C.L. 1981. USEPA Contract No. 68-01-5965. The Mitre Corporation:1. Pichereau, V., A. Hartke, and Y. Auffra y. 2000. Starvation and osmotic stress induced multiresistances: influence of extracellular compounds. Int. J. Food Microbiol. 55:19-25. Pickering, I.J., R.C. Prince, M.J. George, R.D. Smith, G.N. George, and D.E. Salt. 2000. Reduction and Coordination of Arsenic in Indian Mustard. Plant Physiol. 122:1171-1178. Pickering, I.J., L. Gumaelius, H.H. Harris, R.C. Prince, G. Hirsch, J.A. Banks, D.E. Salt, and G.N. George. 2006. Localizing the biochemical transformations of arsenate in a hyperaccumulating fern. Environ. Sci. Technol. 40:5010-5014. Plant, J.A., D.G. Kinniburgh, P.L. Smedley, F.M. Fo rdyce, B.A. Klinck, D.H. Heinrich, and K.T. Karl. 2003. Arsenic and selenium Treatise on Geochemistry:17. Raghu, K., and I.C. MacRae. 1966. Occurrence of P hosphate-dissolving Micro-organisms in the Rhizosphere of Rice Plants and in Submer ged Soils. J. Appl. Microbiol. 29:582-586. Rathinasabapathi, B. 2006. Arsenic Hyperaccu mulating Ferns and their Application to Pnytoremediation of Arsenic Contaminated S ites. floriculture, ornamental and plant biotechnology 3. Rathinasabapathi, B., S.B. Raman, G. Ke rtulis, and L. Ma. 2006. Arsenic-resistant proteobacterium from the phyllosphere of arsenic-hyperaccumulating fern ( Pteris vittata L.) reduces arsenate to arsenite Can. J. Microbiol. 52:695-700. Rathinasabapathi, B., M. Rangasamy, J. Froeba, R.H. Cherry, H.J. McAuslane, J.L. Capinera, M. Srivastava, and L.Q. Ma. 2007. Arsenic hyperaccumulation in the Chinese brake fern 94

PAGE 95

( Pteris vittata L.) deters grasshopper ( Schistocerca americana ) herbivory. New Phytol. 175:363-369. Riesenfeld, C.S., P.D. Schloss, and J. Hande lsman. 2004. Metagenomics: genomic analysis of microbial communities. Annu. Rev. Genet. 38:525-52. Ritchie, A.R. 1980. Handbook of geochemi stry. Earth-Science Reviews 16:59-60. Rodriguez, H., and R. Fraga. 1999. Phosphate solubi lizing bacteria and their role in plant growth promotion. Biotechnol. Adv. 17:319-39. Rosen, B.P. 2002. Biochemistry of arse nic detoxification. FEBS Lett. 529:86-92. Rosen, B.P., U. Weigel, C. Karkaria, and P. Gangola. 1988. Molecular characterization of an anion pump. The arsA gene product is an arsenite(antim onate)-stimulated ATPase. J. Biol. Chem. 263:3067-3070. Russell, S.a. 2001. Molecula r Cloning 3rd Edition 1. Ryan, P., E. Delhaize, and D. Jones. 2001. Function and Mechanism of Organic Anion Exudation from Plant Roots. Annu Rev Plan t Physiol Plant. Mol. Biol. 52:527-560. Schuster, M., C.P. Lostroh, T. Ogi, and E.P. Greenberg. 2003. Identification, Timing, and Signal Specificity of Pseudomonas aeruginosa Quorum-Controlled Ge nes: a Transcriptome Analysis. J. Bacteriol. 185:2066-2079. Sharples, J.M., A.A. Meharg, S.M. Chambers, and J.W.G. Cairney. 2000. Evolution: Symbiotic solution to arsenic contam ination. Nature 404:951-952. Shi, J., A. Vlamis-Gardikas, F. Aslund, A. Holmgren, and B.P. Rosen. 1999. Reactivity of glutaredoxins 1, 2, and 3 from Escherichia coli shows that glutaredoxin 2 is the primary hydrogen donor to ArsC-catalyzed arse nate reduction. J. Biol. Chem. 274:36039-42. Silver, S., and T.K. Misra. 1984. Bacterial transformati ons of and resistances to heavy metals. Basic Life Sci. 28:23-46. Smedley, P.L., and D.G. Kinniburgh. 2002. A review of the source, behaviour and distribution of arsenic in natural waters. Appl. Geochem. 17:517-568. Smith, E., R. Naidu, and A.M. Alston. 2002. Chemistr y of Inorganic Arsenic in Soils: II. Effect of Phosphorus, Sodium, and Calcium on Arse nic Sorption. J. Environ. Qual. 31:557-563. Sosa, L., A. Llanes, H. Reinoso, M. Reginat o, and V. Luna. 2005. Osmotic and specific ion effects on the germination of Prosopis strombulifera Ann. Bot. (Lond.) 96:261-7. 95

PAGE 96

Stolz, J.F., P. Basu, J.M. Santini, and R.S. Oremland. 2006. Arsenic and Selenium in Microbial Metabolism. Annu. Rev. Microbiol. 60:107-130. Sukchawalit, R., B. Prapagdee, N. Charoe nlap, P. Vattanaviboon, and S. Mongkolsuk. 2005. Protection of Xanthomonas against arsenic toxicity i nvolves the peroxide-sensing transcription regulator OxyR. Res. Microbiol. 156:30-4. Switzer Blum, J., A. Burns Bindi, J. Buzzelli, J.F. Stolz, and R.S. Oremland. 1998. Bacillus arsenicoselenatis sp. nov., and Bacillus selenitireducens sp. nov.: two ha loalkaliphiles from Mono Lake, California that respire oxya nions of selenium and arsenic. Arch. Microbiol. 171:19-30. Tracy, B., K. Edwards, and A. Eisenstark. 2002. Carbon and nitrogen substrate utilization by archival Salmonella typhimurium LT2 cells. BMC Evol. Biol. 2:14. Trotta, A., P. Falaschi, L. Cornara, V. Minganti, A. Fusconi, G. Drava, and G. Berta. 2006. Arbuscular mycorrhizae increase the arse nic translocation factor in the As hyperaccumulating fern Pteris vittata L. Chemosphere 65:74-81. Tu, C., and L.Q. Ma. 2003. Effects of arsenate and phosphate on their accumulation by an arsenic-hyperaccumulator Pteris vittata L. Plant and Soil 249:373-382. Tu, S.X., L. Ma, and T. Luongo. 2004. Root exuda tes and arsenic accumulation in arsenic hyperaccumulating Pteris vittata and non-hyperaccumulating Nephrolepis exaltata Plant and Soil 258:9-19. Turpeinen, R., M. Pantsar-Kallio, M. Haggblom, and T. Kairesalo. 1999. Influence of microbes on the mobilization, toxicity and biomethylation of arsenic in soil. Sc i. Total. Environ. 236:173-80. Vatamaniuk, O.K., S. Mari, Y.-P. Lu, and P. A. Rea. 2000. Mechanism of Heavy Metal Ion Activation of Phytochelatin (PC) Synthase. Blocked Thiols are Sufficient for Pc SynthaseCatalyzed Transpeptidation of Glutathione a nd Related Thiol Peptides. J. Biol. Chem. 275:31451-31459. Vaughan, D.J. 2006. Arsenic. Elements 2:71-75. Wang, J., F.-J. Zhao, A.A. Meharg, A. Raab, J. Feldmann, and S.P. McGrath. 2002. Mechanisms of Arsenic Hyperaccumulation in Pteris vittata Uptake Kinetics, Interactions with Phosphate, and Arsenic Speciat ion. Plant Physiol. 130:1552-1561. Wang, L., S. Chen, X. Xiao, X. Huang, D. Y ou, X. Zhou, and Z. Deng. 2006. arsRBOCT arsenic resistance system encoded by linear plasmid pHZ227 in Streptomyces sp. strain FR-008. Appl. Environ. Microbiol. 72:3738-42. 96

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97 Xu, P., W.-J. Li, S.-K. Tang, Y.-Q. Zhang, G.-Z. Chen, H.-H. Chen, L.-H. Xu, and C.-L. Jiang. 2005. Naxibacter alkalitolerans gen. nov., sp. nov., a novel member of the family 'Oxalobacteraceae' isolated from China. Int. J. Syst. Evol. Microbiol. 55:1149-1153. Zheng, M., X. Wang, L.J. Templeton, D.R. Sm ulski, R.A. LaRossa, and G. Storz. 2001. DNA Microarray-Mediated Transcriptional Profiling of the Escherichia coli Response to Hydrogen Peroxide. J. Bacteriol. 183:4562-4570.

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BIOGRAPHICAL SKETCH Mr. Anhui Huang graduated from No. 7 High School in Quanzhou, P.R.China, in 2002, and then entered the College of Life Sciences at Nankai University for his undergraduate study. In 2003, He was admitted to the National Training Base in Life Science and Technology in Nankai University, where he had my specific education and training in DNA recombination technology, microbial engineering and biochemical engineering. He received his bachelorous degree in Biotechnology in 2006 and has been a gr aduate student in Soil and Water Science Department at the University of Florida since th en. He is scheduled to obtain his Master of Science degree in May 2009. 98