quality of life for people with type 1 diabetes

DISTRIBUTION AND CONSERVATION OF REDUCED P METABOLISM OPERONS IN BACTERIA

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A Thesis Presented to the Faculty of California State University, Chico

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In Partial Fulfillment of the Requirements for the Degree Master’s of Science in Biological Sciences

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by Betsey Renfro Fall 2012

ACKNOWLEDGEMENTS

The completion of this thesis concludes a decade long quest for a Master of Science degree in Biology. I would never have attained this degree without encouragement and support of numerous faculty. I would like to than Dr. Ailsie McEnteggart and Dr. Jeff Bell, past and present Chairs of the Biology Department, for allowing me the release time required from the media lab to complete the course work for this degree. Than you Dr. Kris Blee, Dr. Larry Hanne, and Dr. Patricia Edelmann, for your help with experimental design, troubleshooting, data analysis and for your encouraging words. Special thanks to Dr. Jeff Bell, who was so generous with his time and helped me with this project in more ways than I can enumerate. The biggest thanks however, must go to my advisor, co-worker and dear friend Dr. Andrea White. I have learned so much under your guidance. You were the perfect advisor for me, and I appreciate your support more than you will ever know. Finally, I would like to thank my family, especially my husband James and my children, Zach, Lauren and Michael. You guys always believed in me, and I love you so much. A huge thanks you to my parents, Craig and Susie Hawes, for all of your help with the kids and your willingness to act as a taxi service while I was busy pursuing my dream. This work was funded by the Office of Research and Sponsored Programs at California State University, Chico.

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TABLE OF CONTENTS

PAGE Acknowledgements ......................................................................................

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List of Tables ................................................................................................

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List of Figures ...............................................................................................

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Nomenclature ...............................................................................................

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Abstract ........................................................................................................

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CHAPTER I. Introduction .........................................................................................

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Questions ........................................................................................

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II. Methods ..............................................................................................

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Pseudomonas stutzeri WM88 Chromosomal Library Construction............................................................................... Selection of Hpt+ and Pt+ Library Clones ....................................... Restriction Analysis of Cosmid Clones............................................ Database Mining for Hypophosphite, Phosphite and Phosphonate Oxidation Operons............................................... III. Results ................................................................................................ Reduced P Oxidation Pathways in Pseudomonas putida ......................................................................................... Distribution and Conservation of Hypophosphite Oxidation Pathways ................................................................... Distribution of Phosphite Oxidation Pathways................................. Conservation of Phosphite Oxidation Pathways.............................. Distribution of C-P Lyase Operons.................................................. Conservation of C-P Lyase Operons...............................................

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26 27 28 28 31

31 36 44 47 52 55

CHAPTER

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IV. Discussion...........................................................................................

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Overview .........................................................................................

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References ...................................................................................................

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Appendix A. Reduced Oxidizing Bacteria Identified in this Study........................

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LIST OF TABLES

TABLE 1. 2.

3. 4. 5.

PAGE Commercial Products Containing Phosphate that are Marketed as Fungicides and Fertilizers ...................................

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Substrate Ranges Cosmid Clones in Glucose Mops minimal media +100 ug/ml Carbenicillin + 0.5mM Phosphorus source after 36 Hours of growth...............................................

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Primers and expected product size for amplified htxA and ptxD products ..............................................................................

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Function of Pseudomonas stutzeri WM88 Phn and Htx Orthologues of the C-P Lyase Operons...................................

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Reduced P Oxidation Genes in bacteria with htx Encoded C-P Lyase Operons

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LIST OF FIGURES

FIGURE

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1. Diagram Depicting the Traditional P Cycle.............................................

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2. Chemical Structure and Oxidation State of Common Organic and Inorganic Phosphorus Compounds ..................................................

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3. Pathways for the Microbial Metabolism of Phosphonates......................

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4. Model for the Degradation of Methylphosphonate Via the C-P Lyase Pathway .................................................................................

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5. Biochemical Pathway for the Oxidation of Hypophosphite and Phosphite .........................................................................................

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6. Arrangement of Genes Involved In Catalysis of Hypophosphite, Phosphite and Phosphonates in Diverse Bacterial Species ............

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7. Pseudomonas Putida AW2 Phosphite Oxidation Pathway ....................

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8. Growth of wild type P. putida AW2, E. coli Epi T100 +pBR20 and wild type E ........................................................................................

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9. Phylogenetic Tree of the HtxA Hypophosphite-2-Oxoglutarate Dioxygenase Catalytic Protein..........................................................

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10. Gene Organization and Protein Similarity of Phosphite Oxidation Pathways Indicate Recent Horizontal Gene Transfer. ......................

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11. Comparison of the htx and phn Encoded C-P Lyase Pathways in Pseudomonas stutzeri WM88 with the htx Encoded C-P Lyase Pathway in Pseudomonas aeruginosa PADK-CF510 .......................

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12. Structure of Two Potential Hypophosphite Oxidation Pathways in Bradyrhizobium BTAi1 and Their Similarity to Hypophophite Oxidation Clusters in Xanthobacter flavis and Alcaligenes faecalis .............................................................................................

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13. Bacteria with Phosphite Oxidation Enzymes are Found in Chemically and Physically Diverse Environments ............................

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FIGURE

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14. Schematic of Common Arrangements of the ptx Operon and Surrounding Genes in Bacteria..................................................

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15. Phylogenetic Tree of the NAD-Dependent Phosphite Dehydrogenase Enzyme PtxD .........................................................

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16. Maximum Likelihood Tree of Catalytic C-P Lyase Protein PhnJ and HtxH with 100 Bootstrap Replicates ..........................................

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17. The distribution of the organisms into two separate clades indicates separate evolution of the Phn and Htx proteins.................

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18.

Maximum Likelihood Tree of Catalytic Proteins PhnM and HtxL with 100 Bootstrap Replicates ..........................................................

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60

NOMENCLATURE

P

Phosphorus or any compound containing phosphorus

Pi

Inorganic phosphate. Inorganic reduced phosphorus compound (+5 oxidation state)

Hpt

Hypophosphite. Inorganic reduced phosphorus compound (+1 oxidation state)

Pt

Phosphite Inorganic reduced phosphorus compound (+3 oxidation state)

AePn

Aminoethylphosphonate. Organic reduced phosphorus compound (+3 oxidation state)

PH 3

Phosphine gas. (-3 oxidation state)

htxA

Gene encoding 2-oxoglutarate-dependent hypophosphite dioxygenase, which is the enzyme that catalyzes the oxidation of hypophosphite to phosphite.

ptxD

Gene encoding a NAD-dependent phosphite dehydrogenase, which is the enzyme that catalyzes the oxidation of phosphite to phosphate.

HGT

Horizontal gene transfer.PET—photosynthetic electron transport

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ABSTRACT

DISTRIBUTION AND CONSERVATION OF REDUCED P METABOLISM OPERONS IN BACTERIA by Betsey Renfro Master of Science in Biological Sciences California State University, Chico Fall 2012

P has long been considered a biologically inert yet essential element to all living organisms. However, our understanding of how P compounds are converted and made available for growth in the environment is greatly lacking. This deficit in our knowledge of P metabolism in an environmental context is highlighted by recent studies demonstrating that common soil bacteria are capable of oxidizing and reducing P compounds, thus altering P bioavailability in the environment. Understanding the interactions between these reduced P compounds and microbial populations is crucial to our understanding of P nutrient availability and management in the environment. These deficits in our knowledge led to our desire to identify novel reduce P oxidation pathways. Towards this end, DNA sequencing and analysis of the phosphite oxidation pathway in Pseudomonas putida AW2 were completed. The similarity of this x

pathway to previously characterized ptx operons suggested recent horizontal gene transfer. The industrial use of hypophosphite and phosphite is likely leading to increased concentrations of these compounds in the environment. Database mining was used to look for further evidence of horizontal gene transfer of these operons, which would suggest that bacteria are adapting to these environmental changes. Sixty-four organisms were identified that harbor genes allowing the oxidation of hypophosphite, phosphite or both compounds. Recent horizontal gene transfer was evident in both of these pathways. HtxA was 100 percent conserved in four of the five bacteria identified as having HtxA. Seven examples of recent cross genus horizontal gene transfer of PtxD were identified. ptxD was found in association with heavy metal detoxification genes in several organisms, suggesting that it may play a role in the detoxification of phosphite in the environment. Finally, the divergent evolution of two distinct lineages C-P lyase operon, designated phn and htx were demonstrated. These findings indicate that reduced P compounds have been, and are currently important sources of P in the environment, and that diverse bacterial species play an essential role in the bioavailability of P.

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CHAPTER I INTRODUCTION P has long been considered a biologically inert yet essential element to all living organisms. However, our understanding of how P compounds are converted and made available for growth in the environment is greatly lacking. This deficit in our knowledge of P metabolism in an environmental context is highlighted by recent studies demonstrating that common soil bacteria are capable of oxidizing and reducing P compounds, thus altering P bioavailability in the environment. Yet we know very little about which bacteria can do this, how common they are, how they do it and how important these conversions are in changing P flux. As a result, our ability to effectively restore and manage ecosystems, to develop effective and efficient agricultural practices, which rely heavily on P supplementation, and to take advantage of bacterial mediated P cycling, is not possible. Furthermore, more recent industrial and agricultural use of reduced P compounds has undoubtedly resulted in their increased concentrations in the environment. These human activities have resulted in profound impacts on the environment, yet we understand very little of how this has altered P flux and bioavailability in diverse ecosystems. This is in part due to our very limited understanding of how reduced P compounds are altered by environmental microorganisms, and how native populations of microorganisms 1

2 are altered due to the presence of these compounds. It is clear that bacteria have evolved mechanisms for adapting to environmental changes, one of which is the ability to transfer genetic traits to other bacteria via horizontal gene transfer. Evidence for recent horizontal gene transfer of reduced P oxidation genes among bacteria strongly suggests that the P profile in the environment may indeed be changing, likely as a result of human activity. Understanding the interactions between these reduced P compounds and microbial populations is crucial to our understanding of P nutrient availability and management in the environment. In this project the distribution of reduced P compound oxidation pathways among bacteria is explored, as is the spread of these pathways among diverse microbes due to increased concentrations of environmental reduced P compounds. Phosphorus is an essential nutrient required for a diverse set of cellular processes. It is a structural component of nucleic acids and cell membranes; it is part of the primary molecules utilized in energy transfer within cells, such as ATP and GTP; and it is used to regulate the activation state of numerous enzymes via phosphorylation. The primary form of P utilized by cells is inorganic phosphate, Pi (+5 oxidation state), and the biological cycling of P is believed to be done by the inter-conversion of phosphate to phosphate esters and anhydrides (+5 oxidation state), during which no change in oxidation state occurs (Fig. 1). While these reactions are critical to all organisms, Pi is often a limiting nutrient in the environment, and it has long been accepted that P does not undergo biogeochemical redox cycling like other essential nutrients such as C, N and S (2).

Incorporated into Plant tissue

Animals

Rock weathering Fungal and bacterial decomposition (Phosphate and Phosphate-esters, P oxidation = +5)

Inorganic phosphates in soil and solution

Precipitated phosphates FIG. 1. Diagram depicting the traditional P cycle. In the traditional P cycle, the role of bacteria is limited to decomposition of organic matter, while the inter-conversion of P is between inorganic P and P esters, resulting in no change in oxidation state. 3

4 Despite this belief, abundant evidence exists which supports the existence of microbial redox cycling of P, including both the production and oxidation of reduced P compounds (compounds in which P is in a lower oxidation state than that found in Pi) by microbes and eukaryotes. Studies have shown that numerous reduced P compounds are found in the environment. For example, a wide variety of invertebrates synthesize natural organic reduced P compounds in the form of phosphonates (+3) and phosphinates (+1). These compounds are characterized by a direct carbon-phosphorus (C-P) bond that is resistant to chemical and enzymatic hydrolysis, in contrast to the more common C-O-P bond of phosphate esters (67, 75) Aminoethylphosphonate (AEPn) and phosphonoalanine are examples of biologically important phosphonates that serve as structural molecules in certain invertebrates. Both AEPn and phosphoalanine can be found as side groups in glycoproteins and polysaccharides, or as the head group of phosphonolipids in cell membranes (17). In some invertebrates, phosphonates can account for approximately 50% of the total cellular P (55, 56, 70). The marine diazotroph, Tetrahymena, may have up to 30% of its membrane lipids in the form of phosphonolipis, and it has recently been demonstrated that phosphonates comprise approximately 10% of the total P in the marine cyanobacterium Trichodesmium erythareum IMS101 (9, 28). Furthermore, phosphonates have been shown to represent up to 25% of the dissolved organic phosphorus in all investigated marine environments and are preferentially removed from sinking

5 particles relative to phosphate esters, suggesting that phosphonates are a preferred form of P for some organisms (5, 31). Many other naturally occurring phosphonates and phosphinates are produced by common soil microbes (48). A wide range of compounds, the majority of which possess antibiotic properties, are produced by Actinobacteria. For example, members of the genus Streptomyces produce a plethora of phosphonate and phosphinate compounds including phosphonothrixin tripeptide, phosphonthrixin, and fosfomycin, all of which have clinical and commercial applications. In addition to these natural sources, numerous synthetic organic reduced P compounds are manufactured and introduced into the environment each year. Two examples are the herbicides glyphosate and Round Up (active ingredient is glyphosate) which are used extensively both agriculturally to improve crop yields and by home and garden and garden consumers. In 2007 it was estimated that between 95 to125 million pounds of the herbicide Round Up were applied. Currently, there are over 750 products for sale in the United States that contain glyphosate (69). Inorganic P compounds, including phosphine (-3), hypophosphite (+1) and phosphite (+3), whose structures are depicted in Fig. 2, also occur naturally. Phosphine is a toxic gas that has been detected in the atmosphere and in nearly every anaerobic environment, including marine sediments, anaerobic sewage digestion tanks and even in the head space of anaerobic bacterial

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FIG. 2. Chemical structure and oxidation state of common organic and inorganic phosphorus compounds. Source: Reprinted by permission from Andrea White, Department of Biological Sciences. Assistant Professor at California State University, Chico, California.

7 cultures (10, 13, 25, 50, 51, 60). In sewage treatment plants, significant P loss between ingoing and outgoing waste was demonstrated to be up to 45%. It was suggested that this loss might be due to the production of volatile phosphine gas (7). Phosphine production has also been demonstrated in the gastrointestinal tract of mammals, where it is thought to play a role in the development of stomach cancers (13). Additional studies have shown the reduction of phosphate in wetland soils, common soils and during metal corrosion under anaerobic conditions (23, 68, 73). Together, these reports suggest that P reduction occurs in nature, and it is suspected that microorganisms play a major role, albeit an unclear one, in these processes. Phosphine is important commercially as it is used extensively as a chemical fumigant in grain storage vessels. The treatment of these vessels involves filling them with phosphine gas, then venting it directly into the atmosphere. This process likely results in large quantities of phosphine being introduced into areas of the environment where it would not normally occur. Phosphine is spontaneously oxidized to phosphate when exposed to oxygen, presumably through hypophosphite and phosphite intermediates, although this has not been definitively demonstrated. However, two studies support that this is indeed occurring. The first study found Hpt and Pt were products of phosphine oxidation (59) and the second demonstrated that 40 days after phosphine treatment 70% of the phosphine had oxidized to Pi, while the remaining 30% is presumably present in the environment as Hpt and/or Pt (18). As a result, these

8 inorganic reduced P compounds are present in areas where phosphine is being produced or used commercially. Additional sources of environmental hypophosphite arise from its use in metal plating applications, and the black market usage of hypophosphite as a methamphetamine precursor. Phosphite is more abundant in the environment than Hpt due to broader commercial usage. Phosphite is used extensively as an alternative P source in fertilizers and as an anti-fungal agent (Table 1) (36). It is also the only accepted treatment for Sudden Oak Death, which is caused by the fungal pathogen, Phytophthora ramorum, and has decimated oak groves throughout Northern California. The treatment of this disease involves spraying phosphite directly onto the trunks of shrubs and trees until the point of runoff, resulting in significant amounts of phosphite being added to the environment (30). While there is indirect evidence of reduced P compounds in the environment, the actual concentrations in complex environments such as soil have not been measured. However, ion chromatography has been used to detect hypophosphite and phosphite in simulated geothermal waters (39), and more recently, phosphite has been detected in naturally occurring geothermal pools at a concentration of ~0.06 μM using suppressed-ion chromatography coupled with tandem conductivity and electrospray mass spectrometry (53). Given that reduced P compounds occur naturally in the environment and are introduced through human activities, it is not surprising that many bacteria possess the ability to metabolize these compounds. Microbial

TABLE 1. Commercial products containing phosphate that are marketed as fungicides and fertilizers Product Aliette Nutri-Phite Ele-Max ProPhyt Nutrol Phostrol Agrifos Foli-r-fos 400 Fosphite Lexx-a-phos Trafos line Phytos'K Phosfik line Fosfisan, Vigorsan Geros-K Kalium Plus Frutogard Foliaphos

Company Bayer Cropscience Biagro Western Sales Helena Chemical Luxembourg-pamol Lidochem NuFarm America Liquid Fert Pty (Agrichem) UiM Agrochemicals Jh Biotech Foliar Nutrients Inc Tradecorp Valagro Biolchim Agrofill L-Gobbi Lebosol Spiess Urania Plantin

Active Ingredient Fosetyl-Al (Aluminum phosphite) Phosphites & Organic acids Phosphorus acid Monopotassium Phosphite Potassium phosphite Phosphorus acid Monopotassium Phosphite Monopotassium Phosphite Monopotassium Phosphite Monopotassium Phosphite Potassium phosphite Potassium phosphite Phosphorus acid Potassium phosphite Potassium phosphite Potassium phosphite Potassium phosphite Potassium phosphite

Marketed as Fungicide Fertilizer Fertilizer Fungicide Fungicide and Fertilizer Pesticide Fungicide Fungicide Fungicide Fungicide Fertilizer & defense stimulator Biostiumulant Fertilizer Fertilizer & defense stimulator Fertilizer Fertilizer Fertilizer Fertilizer

Source: Courtesy of Leymonie, Jean-Pierre. 2007. Phosphites and phoshates: When distributors and growers alike get get confused!! UK Representative Office. The New Ag International Sarl. September edition.

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10 catabolism of reduced organic P compounds for use as a P and/or C source has been the most extensively studied and appears to be quite common among environmental bacteria. Four well-characterized pathways for phosphophonate catabolism have been described: A) C-P lyase, B) phosphonopyruvate hydrolase, C) phosphonoacetate hydrolase, and D) phosphonoacetaldehyde hydrolase (phosphonatase) (Fig. 3). The C-P lyase pathway encodes proteins that cleave the direct C-P bond, yielding Pi and a corresponding hydrocarbon. In E. coli, the C-P lyase pathway is encoded by a fourteen-gene operon designated phnCDEFGHIJKLMNOP, and exhibits broad substrate specificity, allowing oxidation of compounds such as phenylphosphonate, methylphosphonate and aminoethyl phosphonate. In E. coli, it also allows oxidation of phosphite. Although the enzymatic mechanism of the C-P lyase pathway has not been fully characterized, (26) in vitro reproduction of activity and identification of reaction intermediates of some of the enzymatic subunits in E. coli have been reported (27). It is believed that phnCDE encode ABC type transporters, phnF encodes a repressor and phnNOP encode proteins with regulatory or accessory function (20, 21, 46, 72) and the role of phnK is unknown (27). The catalytic subunits are encoded by phnGHIJKLM. It has recently been demonstrated that PhnJ catalyzes the core reaction, the S-adenosyl-L-methionine-dependent radical cleavage of 5-phosphoribosyl-1-phosphate yielding 5-phosphoribosyl-1,2-cyclic phosphate and the corresponding alkane (Fig. 4) (20, 26, 27). Several

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FIG. 3. Pathways for the microbial metabolism of phosphonates: A) C-P lyase pathways (3), B) 2-AEP catabolism by 2-AEP transaminase (4) and phosphonoacetaldehyde hydrolase (5), C) Phosphonoacetate catabolism by phosphonoacetate hydrolase (6), D) 2-AEP catabolism by 2-AEP transaminase (4), phosphonoacetaldehyde dehydrogenase (7) and phosphonoacetate hydrolase (6). Source: Adapted from Villarreal-Chiu J. F., J. P. Quinn, and J. W. McGrath. 2012. The genes and enzymes of phosphonate metabolism by bacteria, and their distribution in the marine environment. Front. Microbiol. 3:19.

FIG. 4. Model for the degradation of methylphosphonate via the C-P lyase pathway. Proteins PhnI, PhnG, PhnH, PhnL, PhnM and PhnJ are required. PhnJ catalyzes the core reaction, the S-adenosyl-L methioninedependent radical cleavage of 5-phosphoribosyl-1-phosphate yielding 5-phosphoribosyl-1,2-cyclic phosphate and methane. Source: Reprinted by permission from Macmillan Publishers Ltd: [NATURE], Kamat S. S., H. J. Williams, and F. M. Raushel. Intermediates in the transformation of phosphonates to phosphate by bacteria. Nature 480:570–573. 2011. doi:10.1038/nature10622

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13 variations of this operon with different substrate ranges have been described in numerous environmental bacteria (22, 77). All C-P lyase pathways described to date, including those of E. coli, Pseudomonas stutzeri, and T. erythreaum, are regulated as part of the Pho regulon in response to phosphorus starvation and are therefore known as phosphate starvation inducible (psi) genes (45, 57, 71, 76). In E. coli, psi genes are regulated by the two component regulatory system PhoBR. PhoR is a sensor histidine kinase and PhoB is a response regulator that becomes activated under Pi limiting conditions. When inorganic phosphate levels are high, phosphate is transported into bacterial cells via low affinity inorganic phosphate transporters. Once environmental phosphate becomes limiting, phosphate specific transporters, which consist of a high affinity periplasmic phosphate binding protein, two transmembrane transporter subunits and an ATPase, collectively known as PST-SCAB, are activated. Upon activation of the transporters, PhoU, a putative repressor protein dissociates from the PST-SCAB transporter complex. This signal induces autophosphorylation of PhoR using ATP. PhoR then transfers its phosphoryl group to PhoB, activating it. PhoB-P binds the promoter regions of multiple genes, resulting in a global change in gene expression. P starvation induces the synthesis of proteins involved in Pi scavenging and utilization of alternative P sources up to 1500 fold, indicating the importance of these processes to bacterial cell survival.

14 Two other C-P degrading enzymes, phosphonopyruvate hydrolase and phosphonoacetate hydrolase are activated by the presence of their substrates, rather than by limiting phosphate, which suggests that the physiological role of these enzymes is to provide carbon, not phosphate. Phosphonopyruvate hydrolase cleaves the direct C-P bond of this compound to yield Pi and pyruvate ((66, 69) while phophonoacetate hydrolase (41, 70), yields Pi and acetate. The regulation of phosphonoacetaldehyde hydrolase is organism specific: in some cases, it is activated by the presence of its substrate, and in others it is regulated via the Pho regulon. The end products of the phosphonoacetaldehyde hydrolase reaction are acetaldehyde and Pi , although this reaction occurs in two steps (8, 33, 70). First, phosphonoacetaldehyde hydrolase, encoded by phnX, catalyzes the transamination of aminoethylphosphonate using pyruvate as an amino acceptor yielding 2phosphonoacetaldehyde and alanine. Next, the phnW gene product, phosphonatase, hydrolyzes 2-phosphonoacetaldehyde to acetaldehyde and Pi. Recently, phnA, which encodes phosphonoacetate hydrolase, has been found in an operon with phnW, which encodes a 2-AEP:pyruvate aminotransferase, and a gene encoding a novel NAD-dependent phosphonoacetaldehyde dehydrogenase, designated PhnY. Together these enzymes degrade phosphonates to phosphonoacetate (3, 29). Thus, this pathway comprises a biogenic route for phosphonoacetate production (6, 70).

15 In addition to the four well-characterized pathways described above, several novel pathways allowing C-P bond cleavage have been recently described: two proteins, a novel 2-oxoglutarate dioxygenase designated PhnY* (it is a different protein then the PhnY described above) and a phosphonohydrolase designated PhnZ, isolated from a genomic fragment of the marine cyanobacteria Proclorococcus marinus were reported to confer AEPn utilization to E. coli via heterologous expression (37, 38), a Sinorhizobium haukuii isolate that can utilize the phosphonate antibiotic fosfomycin as a sole C and P source (40, 42, 43), Mendez, et al. (44), and Ford, et al. (11) report Pi independent phenylphosphonate degradation in Campylobacter and Helicobacter sp., while Gomez-Garcia, et al. (15) have reported previously uncharacterized Pn catabolism by Synechooccus sp. The existence of several distinct pathways that allow phosphonate utilization by bacteria, indicates the importance of phosphonate compounds to bacteria. The recent discovery of several novel pathways suggests that there may be additional pathways that are yet to be discovered, and that our knowledge of reduced P metabolism remains incomplete. Several pathways allowing utilization of inorganic reduced P compounds such as Hpt and Pt, have also been described. A hypophosphite oxidase that requires NAD and a respiratory chain component for activity was identified in Bacillus caldolyticus (16), and another Bacillus strain that is able to grow anaerobically using Hpt as a sole P source (12) has been described.

16 However, the mechanism allowing these activities was not determined. The first fully characterized enzyme that confers utilization of Hpt as a sole P source is a hypophosphite-2-oxoglutarate dioxygenase, encoded by htxA. This enzyme catalyzes the oxidation of hypophosphite to phosphite concomitant with the decarboxylation of 2-oxoglutarate yielding succinate and CO 2 , in a ferrous ion and oxygen dependent manner as described in Fig. 5. HtxA was first identified in Pseudomonas stutzeri WM88 and was shown to be a member of the Pho regulon (47, 76, 77). A putative hypophosphite dehydrogenase, encoded by htxXY, which is homologous to soluble NAD-dependent formate dehydrogenases was identified in Xanthobacter flavus WM2814, but attempts to fully characterize the enzymes were unsuccessful (79). The two known mechanisms for oxidizing Pt to Pi are carried out by the enzymes bacterial alkaline phosphatase (BAP) encoded by phoA and a NAD dependent phosphite oxidoreductase encoded by ptxD. Although BAP has only been shown to oxidize Pt in E.coli, (80) PtxD mediated Pt oxidation has been demonstrated in a variety of organisms including P. stutzeri, A. faecalis, X. flavus, Desulftignum phosphitoxidans and Prochlorococcus marinus 9301 (38, 61, 75, 79, 80). In Pseudomonas stutzeri, hypophosphite is oxidized to phosphate through a phosphite intermediate and the genes encoding these functions are found in two discreet loci designated htx (for hypophosphite oxidation), and ptx, (for phosphite oxidation). The htx locus is comprised of genes htxA-N, which form

FIG. 5. Biochemical pathway for the oxidation of hypophosphite and phosphite by the enzymes hypophosphite 2-oxoglutarate dioxygenase (HtxA) and the NAD-dependent phosphite oxidoreductase (PtxD) in Pseudomonas stutzeri WM88. Source: Reprinted by permission from Andrea White, Department of Biological Sciences. Assistant Professor at California State University, Chico, California.

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18 a single transcriptional unit (47, 77). As described previously, htxA encodes a hypophosphite 2-oxoglutarate dioxygenase that catalyzes the oxidation of hypophosphite to phosphite. The genes htxB-E encode an ABC transporter that is believed to function as a transporter of both hypophosphite and phosphonates. The genes htxF-L encode catalytic proteins for the metabolism of phosphonates via the C-P lyase pathway, and htxMN encode proteins with unknown function (47, 77). Therefore, in this organism, the htx operon encodes pathways for the oxidation of both hypophosphite to phosphite (catalyzed by HtxA) and phosphonates to phosphate (catalyzed by C-P lyase, encoded by htxF-L). Pseudomonas stutzeri also has a second complete chromosomal copy of a C-P lyase operon similar to the phn operon in E. coli that confers the ability to oxidize phosphonates, but not hypophosphite (77). The phosphite oxidation pathway in Pseudomonas stutzeri consists of a five-gene operon designated ptxABCDE (47, 77). The genes ptxABC encode an ABC-type phosphite transporter, ptxD encodes a phosphite dehydrogenase, and ptxE encodes a putative LysR type regulatory protein (47, 77). Since the discovery and characterization of Hpt oxidation in P. stutzeri WM88, the genetic pathways for Hpt and Pt oxidation in several other bacterial species with this ability have been identified, and these arrangements are depicted in Fig. 6. Alcaligenes faecalis possesses one operon that encodes genes similar to those of P. stutzeri, designated htxABCDptxDE, which allows for the transport and oxidation of hypophosphite to phosphate (80). In Xanthobacter

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FIG. 6. Arrangement of genes involved in catalysis of hypophosphite, phosphite and phosphonates in diverse bacterial species. Source: Reprinted by permission from Andrea White, Department of Biological Sciences. Assistant Professor at California State University, Chico, California. flavus, the hypophosphite and phosphite oxidation genes are separated on the chromosome and are divergently transcribed (79). The phosphite oxidation pathway in Proclorococcus marinus MIT9301 consists of the genes ptxABCD but the putative transcriptional regulator ptxE is absent (38). Finally, in the strictly anaerobic marine organism Desulfotignum phosphitoxidans, phosphite oxidation

20 genes ptxED, which are orthologs to those previously described in P. stutzeri WM88, are found in conjunction with genes designated ptdFCDHI. The ptd locus encodes genes believed to be involved in anaerobic respiration using phosphite as an electron donor and sulfate as a terminal electron acceptor (63). The fact that in this organism Pt plays a role in energy metabolism as well as P acquisition implies that phosphite is abundant in its natural environment and likely composes a major fraction of P available in anaerobic marine environments. Until recently, the abundance of bacteria capable of utilizing reduced P compounds in natural environments had not been investigated. Stone and White (65) used Most Probable Number analysis to quantify the number of reduced phosphorus oxidizing bacteria in twelve common aquatic and terrestrial environments. Each site was sampled and bacteria were cultured with Pi, Hpt, Pt or AEpn as a sole P source. This study demonstrated that reduced P oxidizing bacteria are common and easily isolated from natural environments. Bacteria capable of utilizing Pi and AEpn as a sole P source were equally abundant (1x106 per gram of sample) while the concentrations of Hpt and Pt oxidizers were lower (1x105 per gram of sample), but still significant (65). Previously it was observed that the hypophosphite oxidation genes htxABCD of A. faecalis WM2072 and P. stutzeri WM88 share 99.5% identity, with only 22 nucleotide changes in a 4.2 kbp region (80). This high nucleotide identity strongly suggests horizontal gene transfer of these genes. Horizontal gene transfer (HGT), which is the exchange of genes between bacterial species, as

21 opposed to the vertical inheritance of genetic material, is a primary process driving bacterial evolution. HGT results in the rapid transfer of genes, allowing bacteria to exploit new ecological niches. Three distinct processes are necessary for successful horizontal gene transfer. DNA must first be transferred into the cytoplasm of the recipient bacterium through either conjugation, mediated by a plasmid, transduction by a bacteriophage or transformation, which involves uptake of exogenous DNA from the environment. Once the DNA is in the cytoplasm, it must be either be integrated into the host chromosome or be on a replicative plasmid in order to be maintained. Integration into the host genome can occur through homologous recombination, but is more likely mediated by transposases, integrases, or site-specific recombinases. Finally, following integration into the genome, there must be a selective pressure for maintaining the newly acquired genes (35). Numerous studies have shown that mutational bias leads to a reduction in genome size that increases fitness and that within bacterial species the size of the genome is limited (4, 32, 78). Therefore, it follows that genes acquired through horizontal gene transfer will be quickly lost, if they do not confer an advantage to the host. An example of genes encoding a metabolic pathway that has been spread via horizontal gene transfer are the genes of the C-P lyase pathway.

22 Previous studies have demonstrated that the C-P lyase operon has a long evolutionary history encompassing both vertical and horizontal gene transfer (22, 72). The dissemination and maintenance of this operon among bacterial species strongly suggests that the ability to metabolize phosphonates confers a selective advantage to bacteria in the natural environment. Evidence of recent horizontal gene transfer of other reduced P oxidation operons would suggest that the importance of these compounds as a source of P to bacteria is increasing, possibly in response to increased amounts of these compounds in the environment as a result of human activities. The diversity of pathways so far characterized in only a handful of organisms, coupled with high concentrations of bacteria capable of utilizing reduced P in natural environments, suggests that oxidation of reduced P compounds is not a unique ability limited to relatively few organisms. Additionally, the abundance of reduced P compounds that occur naturally and the introduction of these compounds to the environment via human activities indicate that reduced P compounds may be an important source of P in the environment. Furthermore, the regulation of reduced P oxidation pathways via phoBR in response to Pi starvation, indicates that possessing these pathways would confer a competitive advantage to environmental bacteria when Pi is limiting. Therefore, understanding the mechanisms of bacterial mediated redox cycling of P in the environment may elucidate novel mechanism of P flux, which could have profound agricultural and ecological implications. This is particularly important for

23 Hpt and Pt utilization, which only a handful of bacteria have been shown to have the ability to utilize. In order to fully understand the mechanisms of environmental P flux, it is crucial to identify the mechanisms by which P is transformed and moved through the environment by microorganisms. This requires a thorough understanding of the roles that both reduced P compounds and the bacteria that oxidize them play in the environment. To this end, it is essential to elucidate the distribution, conservation and genetic transfer of reduced oxidation pathways among bacteria and throughout diverse environments. To determine all possible P conversion pathways, the P oxidation pathways of more organisms need to be studied. Although the evolution of phosphonate degradation operons and their distribution in the environment have been investigated, there have been no studies that have addressed the evolution of inorganic reduced P metabolism operons or their prevalence in diverse environments. These deficits led to the questions addressed in this study. Questions This study will address the following questions: 1. What are the hypophosphite and phosphite oxidation pathways utilized by the bacterium Pseudomonas putida? A bacterial isolate from the local sewage treatment plant identified as Pseudomonas putida was chosen for study due to its robust growth on both Hpt and Pt. Sequenced P. putida strains found in public databases did not have any

24 of the previously characterized genes for Hpt and Pt oxidation, suggesting that this organism might possess a unique pathway. Identification and characterization of this pathway would further add to our understanding of bacterial metabolism of reduced P compounds. 2. What is the distribution and conservation of reduced P oxidation pathways in environmental metagenomic samples and sequenced organisms? The abundance of Hpt and Pt oxidizers, as represented in the public sequence databases, has not been investigated. Mining sequence databases for reduced P oxidation genes could provide a wealth of information regarding the distribution and diversity of these pathways among organisms and across environments, which could not be obtained with culture-based approaches. Determining the distribution and conservation of reduced P metabolism genes in bacteria and across diverse environments may provide significant insights into the importance of these processes in bacterial metabolism and therefore to higher organisms, and of the prevalence of reduced P compounds in the environment. 3. Is there evidence of recent horizontal gene transfer of reduced P oxidation pathways? Evidence of horizontal gene transfer and subsequent gene maintenance provides strong support for the importance of particular genes to the organisms in which they are found. Horizontal gene transfer of the htx and ptx pathways has been suggested by the similarities in gene arrangement and

25 identity among the three bacterial species that have been characterized. Mining the sequence databases for additional, highly similar pathways may provide crucial insights into the evolution and transfer of these genes among bacteria, thus providing a greater understanding of their evolutionary history and current importance in the environment. Evidence of horizontal gene transfer would also demonstrate bacterial adaptation to environmental changes caused by the recent addition of reduced P compounds in the environment.

CHAPTER II METHODS Pseudomonas stutzeri WM88 Chromosomal Library Construction Pseudomonas putida genomic DNA was isolated using standard DNA isolation and manipulation techniques described in the Stratagene SuperCos 1 Cosmid Vector Kit. The library was constructed using Epicentre pWEB-TNCTM Cosmid Cloning Kit following manufacturer’s recommendations with the following modifications: 1) DNA was precipitated following gel purification and resuspended in 25ul TE, and this concentrated DNA was used in the ligation reaction; 2) the packaging reaction was not diluted in phage buffer prior to mixing with E. coli EPI100-T1R host cells for transduction. The Poisson distribution was used to determine the number of clones needed to be 99% certain that the entire P. putida genome was represented in our library. The Poisson distribution formula is as follows: N = ln(1-P)/ln(1-F) where P is the desired probability, f is the proportion of the genome contained in a single clone, and N is the desired number of cosmid clones. The P. putida genome is 6.18 x 106 bases, and each cosmid clone should have an insert of 3.8 x 104 bases, so 746 clones were required in the primary library to be 99% certain that the entire P. putida genome is represented. 26

27 E. coli EPI100 –T1R clones harboring P. putida insert DNA were selected on LB + 50 ug/ml Carbenicillin (Agilent Technologies, Stratagene Product Division, 2008). Wild Type P. putida, which is naturally resistant to Carbenicillin and wild type E. coli EPI100 –T1R , which is Carbenicillin sensitive, were also plated on LB + 50 ug/ml Carbenicillin as positive and negative controls, respectively. The primary library was pooled in 1X “M” buffer (40mM 3-(Nmorpholino)propanesulfonic (MOPS) based) and vortexed. Aliquots of library suspensions were frozen in 50% glycerol and stored at -80C. One aliquot was left unfrozen and 10 fold dilutions from 10-3 to 10-9 were plated on 0.2% Glucose MOPS minimal media supplemented with 100 ug/ml leucine , 10 ug/ml thiamine, 50 ug/ml Carbenicillin with 1mM Pi to determine the optimal dilution to use for future screening on reduced P compounds. Selection of Hpt+ and Pt+ Library Clones To isolate clones harboring genes involved in reduced P oxidation, 10 fold dilutions of the library were plated on MOPS minimal media with 50 ug/ml Carbenicillin and with 1mM Pi, Hpt, Pt or AEPn as a sole P source, and incubated for 7 days. Colonies of the E. coli host harboring library clones which grew on Hpt, Pt or AEPn were purified, and the substrate range for each was determined by inoculating each clone into MOPS Carbenicillin broth containing Pi, Hpt, Pt or AEPn. Pi free broth was used as a negative control. Growth in reduced P media was scored at 24, 48 and 36 hours after incubation and was compared to the level of growth for each clone grown in Pi broth.

28 Restriction Analysis of Cosmid Clones Cosmid DNA was isolated from each reduced P oxidizing clone and digested with the restriction enzymes BamH1 and EcoRV. The banding pattern attained after digestion allowed the determination of the number of unique clones isolated. Fisherbrand 1KB ExactGene Ladder was used as a marker for determining the size of insert DNA. Cosmid DNA from clone pBR20, which was positive for both hpt and pt oxidation, was isolated and submitted to San Diego State Micro Core Chemical Facility for sequencing. Sequencing was initiated using standard M13 Forward and T7 Reverse Promoter primers. The remainder of the insert was sequenced via internal primers designed from subsequent sequences. Contiguous DNA sequences were assembled using Serial Cloner 2.1, by F.Perez/Serial Basics (54). Genes involved in phosphite oxidation were identified using BlastN algorithm (1). Database Mining for Hypophosphite, Phosphite and Phosphonate Oxidation Operons The following methods were used to identify sequenced organisms harboring genes for the oxidation of hypophosphite, phosphite and phosphonates to phosphate: 1) The nucleotide sequence of previously identified genes, htxA, htxX, htxY, ptxD and phnJ, were used to query the Comprehensive Microbial Resource (http://www.cmr.jcvi.org/) and NCBI (http://www.ncbi.nlm.nih.gov/) (Nucleotide Collection (nr/nt), and whole-genome shotgun reads (wgs)). Metagenomic sequences were identified using the same nucleotide sequences to

29 search the NCBI database (Metagenomic sequences (env_nr)); 2) The protein sequence corresponding to the genes referenced above were used to probe the CMR and NCBI protein databases (Non-redundant protein sequences (nr)). Once similar genes from sequenced organisms were identified, the corresponding genomic region was examined to determine if the catalytic genes were associated with genes normally found in Reduced P operons, such as transporters and regulatory elements. If an entire putative operon was present, the protein sequences corresponding to the operon genes were collected and used to construct operon maps. Protein alignments were initially done using ClustalW (34). Phylogenetic trees were constructed using MEGA5 (66). The MUSCLE algorithm in MEGA5 was used to align the protein sequences. Hyper variable N and C termini were manually trimmed when necessary, so only relatively conserved regions of the proteins were analyzed. Pairwise amino acid identity analysis using a substitution model was used to calculate the p-distance of the alignment and verify its validity, with a p-distance of