ABSTRACT. The Gram-positive, endospore-forming bacterium Pasteuria penetrans is an obligate

Transcription

1 ABSTRACT WATERMAN, JENORA TURNER. Functional genomics of Pasteuria penetrans, an obligate hyperparasite of root-knot nematodes, Meloidogyne spp. (Under the direction of Charles H. Opperman and David McK. Bird.) The Gram-positive, endospore-forming bacterium Pasteuria penetrans is an obligate parasite of root-knot nematodes, Meloidogyne spp., which themselves are parasites of plants. Pasteuria penetrans has a demonstrated ability to control root-knot nematodes, thus making it an ideal biological alternative to chemical nematicides. Currently, the genome of P. penetrans is being sequenced. Comparative genomic analyses between a partial P. penetrans genome and complete genomes of five closely related Bacillus spp. using BLAST homology searches revealed genome colinearity and microsynteny. Conservation of essential genes for basic developmental, metabolic and physiological processes was observed. The presence of putative competence pathway gene members implies that similar regulatory mechanisms may govern these processes in Pasteuria. Transposable elements may have been active during the evolution of these bacterial genomes, a conclusion supported by the presence of genes encoding transposon-like proteins within the genomes and chromosomal inversions. Therefore, it is plausible that P. penetrans is regulated by biochemical processes similar to those controlling its close relatives, the Bacillus spp. This information will provide insight into understanding mechanisms of host recognition, germination and virulence. Codon- and protein-level phylogenetic analyses have been done on 46 single or concatenated sporulation genes from six Bacillus members, including P. penetrans, using maximum likelihood and Bayesian approaches. Concatenated and single gene trees consistently positioned P. penetrans near nonparasitic Bacillus species. The nonsynonymous

2 and synonymous rate ratios were surveyed to infer proteins and sites within proteins under diversifying selection. Phylogenies indicate that P. penetrans diverged prior to its Bacillus relatives and that it is more closely related to nonparasitic members of this group. Overall, the inferred phylogenies of sporulation proteins showed a tendency toward purifying selection, resulting in conservation of amino acid residues. However, certain membrane proteins yielded alternative phylogenies compared to the concatenated set, which may indicate a role in host-parasite interactions. Mass production of endospores remains a challenge to the implementation of P. penetrans as a root-knot nematode biocontrol agent. In vitro culturing investigations with metal titrations have been performed which augmented growth and sporulation of P. penetrans over previous reports. These findings suggest a potential for metalloregulation of growth and sporulation in P. penetrans. The Spo0F protein is a sporulation response regulator that is central for integrating stress signals necessary to trigger the differentiation of vegetative cells into environmentallyresistant, dormant spores. Structural and functional analyses revealed similarities and differences between P. penetrans Spo0F, the well-characterized B. subtilis Spo0F, and Spo0F proteins from other closely related Bacillus spp. All Spo0F proteins assayed have a conserved negatively-charged active site and display a similar three-dimensional conformation as observed by NMR and inferred by in silico modeling methods. P. penetrans Spo0F is surprisingly more hydrophobic and possesses a significantly higher instability index than Bacillus counterparts. The inherent difference of P. penetrans Spo0F, possibly due in part to the ecological niche the bacterium inhabits, affects its stability during in vitro manipulations, but has no apparent compromises to in vivo functionality.

3 FUNCTIONAL GENOMICS OF PASTEURIA PENETRANS, AN OBLIGATE HYPERPARASITE OF ROOT-KNOT NEMATODES, MELOIDOGYNE SPP. By JENORA TURNER WATERMAN A dissertation submitted to the Graduate Faculty of North Carolina State University in partial fulfillment of the requirements for the Degree of Doctor of Philosophy FUNCTIONAL GENOMICS Raleigh 2006 APPROVED BY: Charles H. Opperman Committee Co-Chair David McK. Bird Committee Co-Chair John Cavanagh Committee Member Jeffrey L. Thorne Committee Member

4 DEDICATION For Daddy. ii

5 BIOGRAPHY Jenora Turner Waterman, a 1997 magna cum laude recipient of a Bachelor of Science degree in Biology from Bennett College, was born in Los Angeles, California in She received the Master of Science degree North Carolina A & T State University in May Her thesis was entitled, Bovine Neutrophils Express the Cyclooxygenase-2 Gene When Stimulated with Bacterial Lipopolysaccharide. Her training in immunology and interest genomics led Jenora to pursue the Doctor of Philosophy degree at North Carolina State University, focusing on Functional Genomics. Here, she completed three laboratory rotations and joined the laboratory of Charles H. Opperman. Her work focused on Pasteuria penetrans, an obligate bacterial parasite of root-knot nematodes, Meloidogyne spp. Jenora has made break-throughs with in vitro culturing of P. penetrans and assisted with the P. penetrans genomic sequencing project. As P. penetrans genomic sequence became available, Jenora was able to do comparative genomic analysis with genomes of closely related Bacillus species, perform phylogenetic analysis of sporulation-specific genes and structural and functional analysis of Spo0F, a key response regulator in endospore-forming bacteria. The results of these projects are found in the following pages. iii

6 ACKNOWLDEGMENTS I thank God for affording me this great opportunity. I appreciate my husband, family and friends for their unfailing support and encouragement. Special thanks to Mrs. Ware and Project Jordan for their support over the years. I thank Tom E. Hewlett and the scientists at Pasteuria Bioscience for hosting me as an intern during the summer of Many thanks to Stella Chang for technical assistance and helpful discussions over the years. Thanks to Drs. Ravisha Weerashinghe and Elizabeth H. Scholl for assistance in preparing DIC image figures and bioinformatics support, respectively. Special thanks to Dr. Keith G. Davies for discussions and collaborations. I am grateful Richele Thompson and the Cavanagh Lab for their help with NMR studies. I would like to express my sincere gratitude to my major professors, Drs. Charles H. Opperman and David McK. Bird, for guiding me through my academic and research endeavors at NC State University. I also thank my advisory committee members Drs. Jeffrey L. Thorne and John Cavanagh for their advice and discussions during my tenure at NC State University. Finally, I thank the National Science Foundation, North Carolina Agricultural Research Service, Rothamsted Research, Ltd. and Syngenta for funding. iv

7 TABLE OF CONTENTS LIST OF TABLES LIST OF FIGURES Page viii ix 1. REVIEW 1 Nematode Biology and Root-Knot Nematodes, Meloidogyne spp 2 Nematodes as Model Organisms 3 Agricultural Impact of Root-Knot Nematodes 4 Taxonomy of Pasteuria 10 Pasteuria penetrans: A Promising Biological Control Agent 11 Comparative Genomic Analysis of Pasteuria penetrans 14 In vitro Culturing of Pasteuria penetrans 15 Molecular Aspects of Sporulation 16 NMR and Protein Functional Studies 19 Conclusions 21 Literature Cited A METHOD FOR ISOLATION OF PASTEURIA PENETRANS ENDOSPORES FOR BIOASSAY AND GENOMIC STUDIES 40 Abstract 42 Introduction 43 Materials and Methods 43 Results and Discussion 47 Literature Cited ADVANCES IN CULTURING METHODS FOR THE ENDOPARASITIC BACTERIUM PASTEURIA PENETRANS 52 Abstract 53 Introduction 54 Materials and Methods 57 In vivo cultures of Pasteuria penetrans 57 Establishing in vitro cultures 57 Varying metal concentrations in Pasteuria growth and sporulation medium 59 In vivo and in vitro P. penetrans Endospore Attachment 59 Results 60 In vitro Cultures 60 Endospore Attachment Bioassay 60 Varied Composition of P. penetrans Growth and Sporulation Medium 60 Effect of Copper on Pasteuria penetrans Growth and Sporulation 62 v

8 Discussion 63 Acknowledgements 64 Literature Cited COMPARATIVE GENOMIC ANALYSIS REVEALS MICROSYNTENY BETWEEN PASTEURIA PENETRANS AND CLOSELY RELATED BACILLUS SPECIES 73 Abstract 74 Introduction 75 Methods 76 Results and Discussion 79 General Features 82 Competence and Sporulation 82 Virulence 84 Transposable Elements 85 Acknowledgements 86 Literature Cited MAXIMUM LIKELIHOOD AND BAYESIAN ANALYSIS OF PASTEURIA PENETRANS SPORULATION GENES 100 Abstract 101 Introduction 103 Methods 107 Results 110 Discussion 113 Acknowledgements 115 Literature Cited STRUCTURAL AND FUNCTIONAL ANALYSIS OF THE PASTEURIA PENETRANS SPORULATION RESPONSE REGULATOR SPO0F 138 Abstract 139 Introduction 140 Materials and Methods 148 Sporulation Phylogeny 148 Pasteuria penetrans Spo0F Homology Modeling and Parameter Estimation 149 Construction of pet 43.1a P. penetrans Spo0F Expression Vector 149 Expression and Purification of P. penetrans Spo0F pet43.1a 150 Removal of the Thrombin Cleavable NusA-tag 151 Alternative Expression and Purification of P. penetrans Spo0F pet43.1a 152 Alternative Removal of the Thrombin Cleavable NusA-tag 152 NMR 15 N HSQC Experiment 154 Results 155 Spo0F Sequence Homology 155 Inference of the Spo0F Protein Phylogeny 156 vi

9 Effects of Copper on Growth and Sporulation of B. subtilis and P. penetrans 156 P. penetrans Spo0F General Properties and Secondary Structure Prediction 157 Structural Analysis of P. penetrans Spo0F Protein 160 Discussion 161 Acknowledgements 165 Literature Cited CONCLUSIONS 185 Introduction 186 Comparative Genomics 186 In vitro Culturing 188 Sporulation Phylogenetics 189 Spo0F Structural Studies 190 Future Directions 191 Mass Production of P. penetrans Endospores 191 Characterization of P. penetrans Virulence Factors 192 Literature Cited APPENDICIES 199 Table A.1.1. Pasteuria species and their nematode hosts 200 Table A.4.1. Bacillus species used in comparative genomic analyses 201 Table A.4.2. Pasteuria penetrans homologs to Bacillus subtilis essential genes 202 Table A.5.1. Outgroups use for sporulation phylogeny reconstruction 209 Supplemental Methods A.6.1: Construction of protein experession vectors for Pasteuria penetrans Spo0F 212 Control File A.4.1: Control file developed to conduct Tera-BLASTX comparisons 220 Control File A.5.1: CodeML control file developed to analyze GeneDoc-edited amino acid multiple sequence alignments 221 Control File A.5.2: CodeML control file developed for Likelihood Ratio Tests 223 Control File A.5.3: CodeML control file developed for Bootstrapped Data 224 Control File A.5.4 : CodeML control file developed to generate Codon Trees 226 Script A.4.1: Perl script developed to parse Tera-BLAST output for a user-defined set of top matches 228 Script A.5.1: Perl script developed to extract phylogenetic rees from batch SeqBoot data containing multiple genes 233 Script A.5.2: Perl script developed to extract phylogenetic trees from SeqBoot data 236 vii

10 LIST OF TABLES 3.1 Average attachments of P. penetrans endospores attached to 20 M. arenaria J Summary of varying Pasteuria penetrans growth conditions Summary of the effect of copper on Pasteuria penetrans growth and spore formation Pasteuria penetrans Sigma Factors Description of sporulation protein used in present study Maximum likelihood estimates of parameters for data set analyzed in this article Log-likelihood values for M7 (H 0 ) vs. M8 (H A ) LRT for Sporulation Genes and positively selected sites Percent identity and similarity between Pasteuria penetrans and Bacillus species Spo0F proteins and e-values as reported by BLASTP Summary of the effect of copper on vegetative cell growth and spore formation in Bacillus subtilis and Pasteuria penetrans ProtParam parameter predictions for Spo0F proteins 175 viii

11 LIST OF FIGURES 2.1 Nomarski differential interference contrast microscopy of the Pasteuria penetrans endospore filtrate Nomarski differential interference contrast microscopy on 14-day-old in vitro cultures of Pasteuria penetrans MegaBLAST comparison of Bacillus subtilis and Pasteuria penetrans genomes Comparison of the genomes of Pasteuria penetrans and Bacillus species Microsynteny between Bacillus subtilis, Pasteuria penetrans and closely related Bacillus spp Genome synteny between Bacillus subtilis and Pasteuria penetrans Maximum likelihood analysis of the 46 concatenated proteins for six bacterial species Bayesian analysis for the 46 concatenated sporulation proteins from six bacterial species SpoVAE single gene trees for seven bacterial species Maximum likelihood analysis of DacB SpoIIAA single gene cladogram inferred by Maximum likelihood analysis Small acid soluble spore protein B single gene tree Bayesian Analysis of SpoIIIAB codons Diagram of proteins involved in the phosphorelay required for initiating the sporulation signal transduction pathway in B. subtilis Spo0F amino acid sequence comparison Bayesian analysis of the response regulator Spo0F Maximum likelihood phylogram of Spo0F PSIPred secondary structure prediction for Pasteuria penetrans Spo0F protein Hydropathy comparison for B. subtilis and P. penetrans 181 ix

12 6.7 SDS-PAGE analysis of Pasteuria penetrans protein Three-dimensional structure conservation in Spo0F proteins Structural homology between B. subtilis and P. penetrans Spo0F Proteins 184 x

13 CHAPTER 1: Review 1

14 NEMATODE BIOLOGY AND ROOT-KNOT NEMATODES, MELOIDOGYNE SPP. Nematodes are roundworms belonging to the phylum Nematoda. Nematodes are the most abundant group in the animal kingdom when considering the combined number of described and estimated unknown species (Blaxter, 1998; Bongers and Bongers, 1998). Currently, there are approximately 20,000 known and an estimated 500,000 (Hammond, 1992) to one million (May, 1988) unknown species. These estimates are substantiated by repeated samplings of single marine and terrestrial habitats (Lawton et al., 1998; Boucher and Lambshead, 1995). Members of this phylum are found worldwide and display extreme diversity. The majority (>50%) are marine forms, but the phylum also includes species that are free-living, including the well known biological model Caenorhabditis elegans, and others which are parasites of plants (e.g. cyst and root-knot nematodes), animals (e.g. filarial worms) and even other nematodes. In essence, their ability to occupy every ecological niche on earth (Blaxter and Bird, 1997) makes them extremely successful animals. Root-knot nematodes (RKN), Meloidogyne spp., are sedentary, obligate plant endoparasites that generally reproduce parthenogenetically, however, some species do reproduce by cross-fertilization (Triantaphyllou, 1985). The life cycle of Meloidogyne spp. is marked by morphologically distinct life stages, including an egg, four juvenile stages and an adult. In free-living roundworms, the eggs hatch into larvae, which eventually grow into adults. In parasitic roundworms, the life cycle is often much more complicated and will be discussed appropriately with emphasis on Meloidogyne spp. and in relation to their impact on agriculture. 2

15 As aforementioned, nematodes are an ecologically diverse group of metazoans including free-living members, which eat bacteria, algae, fungi and protozoans, and a variety of parasitic species. Parasitic nematodes can be pathogens of other animals including dogs (Dirofilaria immitis; heartworm), insects (Heterorhabditis bacteriophora on bee moths), fish (Anisakis simplex; herring worm), plants (Meloidogyne spp. on peanut and others) and humans (Brugia malayi, causative agent of lymphatic filariasis or elephantiasis) (Kennedy and Harnett, 2001). NEMATODES AS MODEL ORGANISMS The nematode Caenorhabditis elegans was adopted as a model organism in the mid 1960s by Brenner (1974). The fact that C. elegans is a nematode makes it the obvious model system for comparative studies with other nematodes (Blaxter, 1998, 2003). However, studies on this organism can provide insight into understanding processes in more complex eukaryotes such as humans. This simple, yet extensively-studied worm can be used as a biological model for physiological processes such as immunity (Kurz and Ewbank, 2003), organ and tissue development development (Sulston and Horvitz, 1977) and host-parasite interactions (Bird and Opperman, 1998; Abally and Ausubel, 2002). C. elegans has many advantages which make it an ideal model organism including its small size (1mm in length), simple growth conditions, self-fertilization, and rapid growth time (~ 3 days at 20 o C) with an invariant cell lineage, and it was the first multicellular animal to have its genome completely sequenced (C. elegans Sequencing Consortium, 1998). There are a lot of resources available for C. elegans including a publicly available complete genomic sequence with an abundant database and literature resources (e.g. 3

16 WormBase, Stein et al., 2001). Furthermore, several unique molecular biological tools exist for C. elegans that are not available in other higher eukaryote models. The developmental lineage of every somatic cell (959 in the adult hermaphrodite; 1031 in the adult male) has been mapped (Sulston and Horvitz, 1977). In addition, C. elegans is one of the simplest organisms with a nervous system and its network of 302 neurons was completely mapped (White et al., 1986) twenty years ago. More recently, functional studies of the entire C. elegans genome by RNA interference, in which every gene in the worm was systematically knocked out, produced an RNAi library of nearly 17,000 reusable bacterial clones (Kamath et al., 2003). The wealth of resources available for C. elegans renders it a well-qualified model for higher eukaryotes, including human. In recent years, some plant-parasitic nematodes, such as root-knot and cyst nematodes have emerged as genetic and genomic models for other plant-parasitic nametodes (Opperman and Bird, 1998; Tytgat et al., 2000; Mitreva et al., 2005) and genomic tools have been applied to accelerate the acquisition of genomic data (for examples see Hammond and Bianco, 1992, McCarter et al., 2000). In response to an ever-growing need for genomic resources for plant parasitic nematodes, we have begun to sequence the genome of M. hapla, which will be the first plant parasitic nematode to have its genome sequenced. AGRICULTURAL IMPACT OF ROOT-KNOT NEMATODES Parasitic nematodes are responsible for an annual loss of over $100 billion annually (Sasser and Freckman, 1987), impacting both the quantity and quality of marketable yields. The majority of damage is caused to food and fiber crops and ornamentals, by a small number of nematode species, chiefly the sedentary endoparasites Meloidogyne spp. (root- 4

17 knot), Globodera and Heterodera spp. (cyst) and migratory nematodes (such as Pratylenchus spp.) (Nickle, 1991). Development in RKN begins within embryogenesis, followed by one molt within the egg and hatching of the second stage juvenile (J2). The Meloidogyne infection cycle commences when the J2 hatches and migrates through the soil in search of host roots. The J2 penetrates the root in the zone of elongation, just behind the root tip and migrates to the developing vascular system (Gheyson and Fenoll, 2002). Nematodes that develop into males will leave the root as adults in search of females with which to mate, females will remain the rest of their lives. Invasion of J2 into the vasculature evokes local morphological changes within the cells immediately around the head of the nematode causing them to enlarge and become multinucleate cells called giant cells (Gheyson and Fenoll, 2002). For over seven decades, researchers have suggested the formation of the feeding site is induced by the release of an induction signal from the nematode pathogen (Linford, 1937; Bird, 1962, 1967, 1968, 1969; Hussey, 1989; Jammes et al., 2005). Development of the nematode-induced feeding site is accompanied by synchronous rounds of mitotic division, resulting in an increase in the density and area of the cytoplasm without cytokinesis; the presence of numerous, irregular nuclei and thickening of giant cell walls (Bird, 1961; Sijmons et al., 1994; Williamson and Hussey, 1996). Upon establishing a feeding site, generally within 4 days in Meloidogyne spp. (Bird, 1961), J2 begins to feed on giant cells, enlarge and molt thrice more. The final molt results in sexually mature adults: a non-mobile female with a characteristic pear-body shape and vermiform males. As females become progressively swollen with eggs, they may cause lysis of the surrounding root material, allowing access to adult males for egg fertilization. 5

18 However, most RKN species are parthenogenetic, therefore males are not needed for reproduction. In a separate, but related phenomenon, cortical and pericycle cells at the periphery of feeding sites are also induced to enlarge and form disfigured root structures called galls, also referred to as knots (Agrios, 1997). Since giant cells are nematode-induced structures it is reasonable that they exhibit unique gene expression profiles (for reviews see Fenoll et al., 1997; Gheyson and Fenoll, 2002). Recent work by Jammes et al. (2005) reported the differential expression of nearly 3,400 genes between healthy root tissues and galls at different developmental stages. Nematode development is temperature and hostsensitive and the entire life cycle may be completed in as few as 17, but up to 57 days (Agrios, 1997) and optimum growth is observed in soil with temperature range of 20 to 30 C. Upon becoming adults, root-knot nematode females will begin to lay eggs (up to a 1000 or more) which are contained in a proteincious matrix at the posterior end of the body (in Meloidogyne spp.) or they become encased in the dead female body in cyst nematodes (Gheyson and Fenoll, 2002). Under optimal conditions, these eggs hatch and subsequently re-infect the host plant, resulting in multiple generations of nematodes infecting the host simultaneously during a single growing season. Cumulative infestations of these kind account for the devastating losses in crop yields. Prior to wide spread use of chemicals to control plant parasitic nematodes in the 1940s, nematode pests were controlled by physical methods such as flooding or burning the soil surface, sterilization or deinfestation of planting material and soil (Newhall, 1940, 1955; Heald, 1987). The role of physical methods in controlling nematode populations has been discussed (Jones, 1978) and the advantage of these methods is that they can be used 6

19 against all species of nematodes and they do not have hazardous effects associated with chemical methods. While effective on a small scale, they are not very practical for farmers. Other methods of controlling nematode pests include rotating plant hosts with resistant and/or non-host crops. Cultural practice can aid in controlling plant parasitic nematode populations (Johnson, 1982; Trivedi and Barker, 1986) and crop rotations remain an important method for controlling root-knot damage. For example groundnut, maize and sorghum have been reported to reduce incidence of M. incognita when planted prior to tobacco (Muro, 1975). The use of plant varieties that are resistant to RKN is also effective. For example the Mi-1 gene in tomato plants protects them from M. incognita, M. arenaria and M. javanica (Vos et al., 1998). Nematode resistance genes are present in several crop species (Roberts, 1992) and are an important part of many breeding programs including tomato, potato, soybean and cereals (Roberts, 1992; Williamson and Hussey, 1996). Moreover, the structure and chemical composition of nematode eggs make them capable of survival in stressful environments (Bird and Bird, 1991). Therefore, it is plausible that the eggs may persist in the soil for extended periods of time, months or years waiting for a plant host, a fact that potentially complicates crop rotation. Chemical fumigants are the most effective way of controlling RKN and other plantparasitic nematodes (Wright, 1981). In general, fumigants provide excellent control of nematodes in soil. As they volatilize, the gas diffuses through the spaces between soil particles and nematodes living in these spaces are killed. Methyl Bromide (Dowfume) is the most effective gas pesticide in use worldwide and is highly efficient in controlling a variety of soilbourne pests, including nematodes (Katan, 1999). However, many nematicides are no 7

20 longer available mainly due to their toxic effects on the environment and they can be extremely costly (Jatala, 1986; Hague and Gowen, 1987). For example, methyl bromide contributes to the depletion of the ozone layer and an international agreement was made to completely phase out its use by 2005 (Katan, 1999). In fact, the United States Environmental Protection Agency (EPA) implemented a plan which incrementally reduced methyl bromide use until its phaseout took effect on Jan. 1, 2005, except for allowable exemptions (www. epa.gov/ozone/mbr). Many non-fumigant nematicides are soluble in water making them readily mobile in soil. Several researchers have reported on the contamination of ground water with organic chemicals including nematicides such as carbofuran, ethoprop, and aldicarb. Fumigants such as DBCP, EDB, and the 1,2 dichloropropane component of DD (Peoples et al., 1980; Wixted et al., 1987) have also been found. Continuing environmental problems associated with nematicides use have resulted in restriction on their use (Khan and Khan, 1991) and many countries have enacted plans to completely ban the use of many nematicides (Katan, 1999). Over the last several decades, an awareness of the hazardous effects of nematicides on humans and the environment has lead investigators to focus their efforts on integrating biological mechanisms for the control of nematode pests (Krizkova et al., 1976; Mankau, 1972, 1975; Jatala, 1986; Stirling, 1991; Chen et al., 1997a, 1997b; Kerry, 2000). Biological control through host resistance (Roberts, 1995; Williamson, 1998; Vos et al., 1998); organically-nematicidal soils (Akhtar and Malik, 2000) and/or microbial pathogens (Akhtar and Malik, 2000; Kerry, 2000) may be the most promising alternative for controlling RKN and other plant-parasitic nematodes. Several organisms are known to antagonize RKN including bacteria (e.g., Pasteuria penetrans), fungi (e.g., Arthrobotrys oligospora), other 8

21 nematodes (e.g., Labronema vulvapapillatum), insects (e.g., Entomobyroides dissimilis) and mites (e.g., Hypoaspis aculeifer) (Stirling, 1991). Little is known about the life cycle and general biology of most predatory nematodes and most of the information on their dietary habits originates from chance observations (Small, 1987). There have been some extreme reports of insect predation on nematodes in vitro. For example, a collembolan insect, Entomobyroides dissimilis, was reported to consume more than 1000 nematodes in a 24-hour period (Gilmore, 1970). There are also other invertebrates that prey on nematodes. Some interest has focused the protozoan Theratromyxa weberi, a large amoeboid organism with a creeping-trophic form capable of engulfing nematodes (Stirling, 1991). Particular attention has been given to nematode trapping fungi for their ability to indiscriminately trap, digest and consume nematodes, including plant and animal parasitic species (Kerry, 2000; Larsen, 2000). Nematophagous fungi capture nematodes in specialized structure called traps, of which there are four species-dependent categories: adhesive knobs, adhesive nets, constricting rings, and adhesive branches (Stirling, 1991; Ahren and Tunlid, 2003). Despite the apparent ubiquitous distribution of these fungi (Peterson and Katznelson, 1965; Mankau,1972), there have been few reports of plant parasitic nematodes having been trapped by nematode trapping-fungi in nature (Cooke, 1962, 1963). Most fungi that antagonize nematodes are slow colonizers and must first produce traps (simple or complex) prior to trapping nematodes. It has been suggested (Jansson and Nordbring-Hertz, 1980) that trap production depends on the presence of nematodes. However, once fungi have established traps in the soil, they can persist for long periods of time, but old traps tend to become non-functional and ineffective at trapping nematodes (Barron, 1977). Taken 9

22 together, these facts highlight limitations for the effective use of nematode-trapping fungi as biocontrol agents of RKN. Of the nematode antagonists mentioned above one genus of bacterial pathogens, Pasteuria, has several characteristics which make it a potentially useful agent to control RKN. Populations parasitic in Meloidogyne spp. not only prevent nematode reproduction, but also reduce the infectivity of J2 in the soil by spore-encumberment (Stirling, 1991). When juveniles are encumbered with 15 endospore (Davies et al., 1988) root penetration is reduced by 86%. When Pasteuria endospore levels are high enough (Stirling, 1984; Stirling et al., 1990) J2 may be prevented form entering the roots. Furthermore, endospore of P. penetrans display structural traits (Williams et al., 1989) which make it suited for the harshest of environmental extremes. For example, P. penetrans spores can be air-dried (Stirling and Wachtel, 1980), repeatedly frozen and thawed (Bird et al., 1990), boiled for at least 30 minutes at 100 o C (Dutky and Sayre, 1978; Stirling et al., 1986), however, heating spores at this temperature greatly reduces but does not prevent infectivity (Williams et al., 1989). TAXONOMY OF PASTEURIA Pasteuria penetrans is Gram-positive, endoparasite of root-knot nematodes (Mankau, 1975; Imbriani and Mankau, 1977) and the presence of P. penetrans in adult female nematodes without egg masses and in nematode suppressive soils (for examples see Bird and Brisbane, 1988; Fould et al., 2001; Cetintas and Dickson, 2004) has been well documented. There are five known Pasteuria species, four species that are endoparasites of nematodes, including Pasteuria nishizawae (Heterodera and Globodera, cyst nematodes), Pasteuria thornei (Practylenchus, Lesion nematodes), Pasteuria penetrans (Meloidogyne, root-knot 10

23 nematodes), and Pasteuria usage (Belonolaimus, sting nematode). The first described member Pasteuria ramosa, is an endopathogen of Daphnia (water fleas). The genus name Pasteuria given in honor of Louis Pasteur, was first described by Metchnikoff (1888) infecting water fleas (Sayre, 1993; Ebert, et al., 1996). A look into the systematics of Pasteuria penetrans reveals several changes in the classification of this organism. It was first described as the protozoan Duboscqia penetrans by Thorne in Following extensive microscopy studies (Mankau, 1975; Imbriani and Makau, 1977) the parasite was renamed Bacillus penetrans. However, Sayre and Starr (1985) drew attention to the similarities shared by Bacillus penetrans and P. ramosa (Metchnikoff 1888; Sayre et al., 1979). There are still problems with this classification because there are noticeable differences between the Bacillus life cycle and that of Pasteuria, yet that are marked similarities such as the presence of Bacillus-like vegetative rods (Hewlett, T. E. and Davies, K. G., unpublished data datadatadatadataresults). Pasteuria are commonly referred to as actinomycete-like bacteria due to their life cycle similarities with members of the actinomyces (Atibalentja et al., 2000). However, they do not form true mycelia. Moreover, phylogenetic studies in our lab have resolved the placement of P. penetrans in the tree of life (Charles et al., 2005). P. penetrans is indeed a member of the Bacillus-Clostridium clade of bacteria. There still remains considerable confusion about the taxonomy owing to criteria used for distinguishing species. PASTEURIA PENETRANS: A PROMISING BIOLOGICAL CONTROL AGENT Pasteuria penetrans, an obligate hyperparasite of RKN has been shown to effectively control nematode populations (Hewlett et al., 1997; Chen and Dickson, 1998). The life/infection cycle of P. penetrans begins when J2 of Meloidogyne spp. migrating through 11

24 the soil in search of host roots become encumbered with endpspores (Stirling, 1981). Upon contact, endospore attach to the nematode cuticle and the nematode may become encumbered with as many as 100 spores, although one spore is sufficient for infection. This initial attachment, however, is non-specific and a specific recognition step is necessary for germination to occur. There is evidence to suggest a role of spore surface proteins in this recognition step (Davies and Redden, 1997). Once a nematode host begins feeding on nutrient rich giant cells within the root vasculature, spores germinate. The time it takes for spores to germinate may range from 4-10 days post-root invasion (Sayre and Wergin, 1977). After the hypodermal tissue has been penetranted, the germ tube develops into rhizoids which divide to produce vegetative, sperical microcolonies, consisting of dichotomously branched septate mycelia (Stirling, 1991). These microcolonies may divide into smaller single cell units vegetative rods (unpublished data datadatadatadataresults Davies, K. G. and Hewlett, T.E.). As the host nutrient supply is depleted and development progresses, the bacterial cells undergo sporulation and divide into multi-lobed colonies consisting of two (doublets) to four (tetrads) segments. The tetrads will then break into doublets, all of which undergo sporogenesis and produce endospore housed in sporangia. The sporogenesis portion of Pasteuria life cycle is similar to that in Bacillus spp. (Chen et al., 1997a) and other endospore-forming bacteria (Stirling, 1991). A polar septum in formed to produce two asymmetric daughter cells, the mother cell and the prespore; followed by spore maturation, marked by the formation of a multilayered protein coat, spore elongation, and broadening of parasporal fibers (Sayre and 12

25 Wergin, 1977; Imbriani and Mankau, 1977). The process from spore to spore takes about 45 days, but is more dependent on the temperature and nutrients available from nematode host (Hatz and Dickson, 1992). Growth of Pasteuria within the nematode inhibits female nematodes from producing and laying eggs. In effect completion of the life-cycle of Pasteuria within the nematode effectively controls nematode populations, thus making it an ideal candidate as a biological nematicide. The level of nematode suppression by Pasteuria stems from its ability to proliferate within the ovaries of female nematodes, thereby reducing fecundity and by encumbering J2 and limiting the number of nematodes that actually penetrate the root and infect plant roots (Davies et al., 1988). The life cycle of Pasteuria has evolved with that of its nematode host and as a result Pasteuria spp. have become host-specific pathogens (Hatz and Dickson, 1992). Understanding which genes are interacting between the host and parasite may give clues for gene targets for manipulating Pasteuria host range. Pasteuria spp., especially P. penetrans, have been extensively studied for s ability to control plant parasitic (root-knot) nematodes in the field (Bird & Brisbane, 1988; Dickson et al., 1991; Dickson et al., 1994; Minton & Sayre, 1989; Davies et al., 1990) and laboratory/glasshouse (Brown & Smart, 1985; Channer & Gowen, 1988; Chen et al., 1996; Davies et al., 1988; Oostendorp et al., 1991; Tzortzakakis et al., 1997). Members of the genus Pasteuria have been reported on a variety of nematode hosts ( for review see Chen and Dickson, 1998) and in many different climates and environments throughout the world (Oostendorp et al., 1990; Hewlett et al., 1994; Sayre & Starr, 1988; Stirling, 1988; Chen and Dickson, 1998). These bacteria naturally occur in the soil and are economically and 13

26 environmentally friendly alternatives to chemical methods (Stirling 1991; Katan, 1999; Kerry, 2000). Challenges that impede the use of P. penetrans spores for the control of RKN on a larger scale are the difficulties associated with mass production of spores and limits in host range. However, we believe that obtaining of the P. penetrans genome and comparative genomics with other relevant organisms will provide insight into tackling these hurdles. COMPARATIVE GENOMIC ANALYSIS OF PASTEURIA PENETRANS The fastidious nature of Pasteuria penetrans make it unamenable to traditional culturing techniques, however, such obstacles make this organism an ideal candidate for genomic analysis, hence, we have undertaken the challenge of obtaining the entire DNA sequence of this bacterium (Bird et al., 2003). Genomics, the study of an organism s complete set of genes and genetic material, and Bioinformatics, the use of applied mathematics and computer science to solve biological problems, have provided a platform on which to study P. penetrans at levels once unattainable. A first step in genomic analysis of an organism is obtaining good quality DNA samples; a step that has been extremely challenging in P. penetrans. Once there is at least a four to five-fold depth of coverage to the genome, genomic comparisons can be performed. Comparative genomics is the comparison and analysis of genomes from different organisms in an attempt to gain information on how species have evolved and to better understand the function of coding and noncoding regions of genomes. Genomic comparisons reveal similarities and differences in genes and proteins which may reveal how selection pressures act on lineages. Generally, features which are responsible for similarities in different organisms are conserved over time (purifying 14

27 selection). Those features which are responsible for differences among species tend to be divergent, indicative of diversifying selection. Chapter 4 of this thesis deals with genomic comparisons of P. penetrans with closely related Bacillus species. Comparing genomes can reveal genes under positive, Darwinian selection. Genes generally found to be under diversifying selection fall into three categories; genes involved in host-pathogen interactions, (i.e. genes for host defense and immunity against pathogens or pathogen genes involved in evading host defense, proteins or pheromones involved in reproduction and those which acquire new functions following duplication events (Yang, 2005). Chapter 5 of this thesis reports efforts to predict sporulation genes potentially under diversifying selection using Bayesian approaches. Knowledge of proteins under diversifying selection may give clues to help unravel the genetic differences that distinguish one species from another, namely the parasitic nature of P. penetrans and closely related parasites, B. anthracis and B. thuringiensis. IN VITRO CULTURING OF PASTEURIA PENETRANS Pasteuria penetrans is an obligate parasite of root knot nematodes. Due to its fastidious nature, in vitro culturing of this organism has been challenging. Recent breakthroughs with in vitro culturing strategies by a company, Pasteuria Bioscience LLC. (Alachua, FL) appear promising for obtaining vast amounts of endospore. This group was able to successfully grow P. penetrans using a co-culture technique with Enterobacter cloacae (Gerber and White, 2001), which is a rhizobacterium whose interactions have been shown to be beneficial for plant growth, P. penetrans reproduction and RKN biocontrol (Duponnois et al., 1999). However, a major challenge to the mass production of endospore is the tendency and apparent preference of P. penetrans to grow as a biofilm making it 15

28 especially difficult to grow liquid cultures (T. E. Hewlett, unpublished data). Our group has expanded on methods described by Gerber and White (2001) which increased growth and sporulation several orders of magnitude. Details of these studies are described in Chapter 3 of this dissertation. MOLECULAR ASPECTS OF SPORULATION Many bacteria undergo complex developmental cycles which are reminiscent of higher organisms. One example of this is endospore formation in Gram-positive bacteria. Sporulation in Bacillus subtilis is the best understood cellular developmental system in bacteria (Stragier and Losick, 1996; Snyder and Champness, 2003; Iber et al., 2006). An enormous repertoire of genetic and developmental information available for studying sporulation exists for Bacillus subtilis, which makes it an ideal model for studying development in P. penetrans and other Gram-positive bacteria. Sporulation is a wellorchestrated process involving intercellular and intracellular communication (Piggot and Losick, 2002; Eichenberger et al., 2004), the cooperation of several pathways to integrate environmental signals (Grossman, 1995; Fabret et al., 1999; Hoch and Varughese, 2001, Perego and Hoch, 2002) and compartmentalization of gene expression (Hilbert and Piggot, 2004; Piggot and Hilbert, 2004; Yudkin and Clarkson, 2005) in an effort to transform vegetative cells into dormant spores (Burbulys et al., 1991; Errington, 1993, 2003). Over the past decade, it has become increasingly clear that the response of bacteria to their environment is governed by two-component signal transduction pathways (Stock et al., 1989; Stock et al., 1990), of which the phosphorelay of sporulation is a complex version (Stephenson and Hoch, 2002; Stephenson and Lewis, 2005). 16

29 The sporulation phosphorelay is a major regulatory pathway which results in the activation of the master regulator of sporulation initiation, Spo0A (Fujita and Losick, 2005). Recent experiments have showed that more than 500 genes are under direct and indirect regulation by Spo0A (Fawcett et al., 2000; Molle et al., 2003). Entry into sporulation is marked by the development of an axial filament which is characterized by the longitudinal arrangement of two chromosomes produced from DNA replication. Followed by the development of an extreme polar septum which asymmetrically divides the cell into a mother cell (equivalent to sporangium, in which the spore matures), engulment of the prespore by the mother cell, endospore maturation and mother cell lysis, liberating the free endospore (Stragier and Losick, 1996; Piggot and Losick, 2002; Piggott and Hilbert, 2004). Sporulation proceeds in essentially the same manner in Gram-positive bacteria, consisting of distinct morphological stages that convert a cell into a two chambered sporangium in which a spore is produced (Stragier and Losick, 1996; Piggot and Losick, 2002). A complex two component signal transduction system involving coordinated activities of several kinases, response regulators, phosphatases and a phophotransferase to transition from cells from vegetative growth to sporulation (Perego and Hoch, 2002) in a six to seven stage process. The decision to sporulate actually begins at the gene level and is governed by the sigma factor protein components which give promoter specificity to RNA polymerase attached to RNA polymerase (RNAP) (Kroos et al., 1999; Snyder and Champness, 2003). The B. subtilis genome encodes 15 sigma factors (Haldenwang, 1995; Kunst et al., 1997), four of which are sporulation-specific (σ E, σ F, σ G and σ K ) and two additional ones that have moderate roles in early sporulation (σ A and σ H ). In the mother cell, gene expression is 17

30 mediated by σ E and σ K, while forespore gene expression is governed by σ F and σ G (Losick et al., 1986, Stragier and Lisick, 1996; Piggott and Losick, 2002; Errington, 2003). Starvation and increased cell density (Losick et al., 1986; Errington, 1993; Stephenson and Hoch, 2002) trigger mechanisms for survival which commence by activating the σ H -holoenzyme, which is responsible for the transcription of early sporulation genes kina, Spo0F and Spo0A (Predich et al., 1992). Activation of σ H and Spo0A in stage 0, predivisional cells leads to asymmetric division (Piggot and Hilbert, 2004), a process involving condensation of the chromosome into an axial filament formation (an event also referred to as stage I ), formation of the polar septum and asymmetric division, and chromosomal partitioning (stage II, Errington, 2003) between the daughter cells; a notably larger mother cell and the smaller prespore. Stage III is characterized the engulfment of the prespore, now referred to as the forespore and is the result of the actions of σ E in the mother cell (Hofmeister, 1998). The latter stages (IV to VII) of sporulation involve σ G directed-endospore maturation (Driks, 1999) and subsequent lysis of the mother cell controlled by σ K (Errington, 2003). In some bacterial species such as P. penetrans and B. anthracis, the endospore is encased in an exosporium (Todd et al., 2003). Sigma G is responsible for the expression of small, acidsoluble spore proteins which bind spore DNA and are degraded for use as amino acid source in germinating spores (Setlow, 1988). As mentioned, the initiation of sporulation is not the result of a single nutritional effect, but rather a complex process resulting from the integration of several signals from a sophisticated network of regulatory systems (Perego and Hoch 2002, Piggot and Losick, 2002). Genomic analysis of a partial genome sequence of P. penetrans has resulted in the 18

31 identification sporulation gene candidates with well-characterized homologues in B. subtilis. To understand the evolutionary aspects of sporulation between P. penetrans, B. subtilis and other closely related Bacillus species, phylogenetic analyses (consisting of maximum likelihood and Bayesian Analysis) were performed with individual and concatenated sequences. Methods and results of these analyses are detailed in Chapter 5 of this dissertation. This information will be used to as a basis to develop functional studies to better understand sporulation in this parasite. NMR AND PROTEIN FUNCTIONAL STUDIES Nuclear magnetic resonance is a phenomenon that occurs when nuclei are immersed in a static magnetic field and exposed to a second oscillating magnetic field. All nuclei do not experience this phenomenon, only those with a property called spin (Cavanagh et al., 1996; Hornak, 2006). Spin refers to a small, innate magnetic field of nuclei that will produce an NMR signal. That is, since a nucleus is a charged particle in motion, it will naturally develop a magnetic field. In general, nuclei with odd mass numbers posses this property; for example 13 C but not 12 C. For NMR analysis, nuclei with half integral spin (1/2 nuclei), specifically hydrogen, carbon and nitrogen, are important. This means these nuclei only have two possible nuclear orientations or energy states and transitions between these two states can occur by absorption of a photon. In order for energy state transition to occur, the energy of the photon must exactly match the energy difference between the two states (Cavanagh et al., 1996; Hornak, 2006). The absorption of energy causes an atom or molecule to go from an initial energy state (the ground state) to another higher energy state (an excited state). The energy states are said to be quantized because there are only certain values that are possible, there is not a continuous spread of energy levels available (Carey, 2006). 19

32 In NMR experiments, radiolabeled proteins are exposed to radio waves in a strong magnetic field and their response or oscillations are recorded with sensitive coils connected to the sample vessel. In multidimensional nuclear resonance experiments (2D or 3D) each peak represents individual atoms, more precisely, the interactions between specific atoms depending on the type of NMR experiment (through space, COESY or through bond, NOESY interactions) (Cavanagh et al., 1996). The positions and sizes of the peaks are used to determine the distances between atoms and these constraints are fitted to generate a model. By looking at combinations of atoms, such 1 H, 13 C, and 15 N (and/or associated C α, C β, carbonyl carbons) known as spin sets, aides in determination the protein model. Once the nascent protein structure has been determined, more interesting NMR experiments can be performed. For example ligands and/or metals may be mixed with protein samples and the experiments repeated to determine protein functionality (for examples see Kojetin et al., 2005; Bobay et al., 2006; Kojetin et al., 2006). Comparison of spectra allows the determination of affected residues, and ultimately, the ligand affect on the topology of the protein. The 15 N-backbone structure for P. penetrans Spo0F, a key sporulation response regulator in endospore-forming bacteria including the model bacterium B. subtilis, was determined using NMR techniques (Kojetin et al., 2005) and the results of these experiments are discussed in Chapter 6 of this dissertation. This is the first such application of NMR techniques to study this obligate parasite. 20

33 CONCLUSIONS It is hoped that Pasteuria penetrans will eventually be used as a biological agent to control rook knot nematode. However, mass production of the infective endospore remains a challenge. In an attempt to gain more insight into the molecular aspects of sporulation in Pasteuria penetrans and the tritrophic relationship between P. penetrans, Meloidogyne species and various plant hosts, we have undertaken a comparative genomic approach. Comparative genomics involves the use of computer programs that can line up multiple genomes and look for regions of similarity among them. By comparing P. penetrans to wellcharacterized model organisms we can unravel its mysteries. Comparative genomic analysis revealed numerous putative sporulation gene candidates in P. penetrans. With this information phylogenetic analysis of sporulation-specific genes was undertaken to determine the relationship among members of this critical pathway. Utilizing this information, structural and functional studies were performed on a key sporulation response regulator that is central for the integrating of these stress signals. 21

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52 Chapter 2: 40

53 Charles H. Opperman Box 7253, NCSU Raleigh, NC (office) (fax) A Method for Isolation of Pasteuria penetrans Endospores for Bioassay and Genomic Studies Jenora T. Waterman, David McK. Bird, Charles H. Opperman Received for publication 17 November Center for the Biology of Nematode Parasitism, NC State University, Raleigh, NC Supported in part by the North Carolina Agricultural Research Service and the National Science Foundation. A portion of a Ph.D. dissertation by the first author. warthog@ncsu.edu This paper was edited by J. A. LaMondia RH: Pasteuria penetrans endospore isolation: Waterman et al. 41

54 Abstract: A rapid method for collection of Pasteuria penetrans endospores was developed. Roots containing P. penetrans-infected root-knot nematode females were softened by pectinase digestion, mechanically processed and filtered to collect large numbers of viable endospores. This method obviates laborious hand picking of Pasteuria-infected females and yields endospores competent to attach to and infect nematodes. Endospores are suitable for morphology studies and DNA preparations. Key words: endospores, method, Pasteuria penetrans 42

55 INTRODUCTION Pasteuria penetrans is a gram positive hyperparasite of root-knot nematodes (RKN: Meloidogyne spp.) and other nematodes. This bacterium is a member of the Bacillus- Clostridium clade, and it is most closely related to Bacillus halodurans and B. subtilis (Charles et al., 2005). In nature, endospores of P. penetrans attach to the cuticle of J2 migrating through soil. Pasteuria penetrans endospores germinate when the nematode establishes a feeding site in the host vasculature (Sayer, 1993). A germ tube is extended into the pseudocoelom of the nematode, which gives rise to microcolonies, thalli, and, eventually, endospores. Proliferation of the endoparasite within the pseudocoelomic cavity of the nematode causes degeneration of the reproductive tissues, greatly reducing fecundity. Endospores are released into the surrounding soil upon decay of the female cadavers and root tissue. Due to its ability to control nematode populations, P. penetrans has potential as a biocontrol agent for RKN (Chen and Dickson, 1998). Although the use of P. penetrans to control RKN is promising, its fastidious nature has inhibited mass production of endospores. Current methods for collecting endospores of P. penetrans require hand picking infected females (Oostendorp et al., 1990; Chen et al., 2000). Here we present a rapid method for collecting endospores that eliminates the need for hand picking females. Our method includes mechanical processing of digested root material and filtration to collect quantities of viable P. penetrans endospores several orders of magnitude higher than previously reported methods. MATERIALS AND METHODS Pasteuria penetrans (Gainesville, Florida, Isolate; Pasteuria Bioscience, Alachua, FL) endospores were attached to Meloidogyne arenaria Race 1 J2 using a modified method of 43

56 Hewlett and Dickson (1993). Briefly, endospores of P. penetrans were attached to 10,000 J2 at a ratio of 100 endospores/j2 by centrifugation at 3,220g for 5 min at room temperature in 10 ml distilled water in a 50-ml tube. Average attachment, average number of endospores attached to nematodes, and percentage attachment were determined. Ten thousand juveniles, with 97% ± 6 (mean ± 1 standard deviation for three replicates) having endospores attached, were used to inoculate each 4-wk-old tomato plant (Lycopersicon esculentum Rutgers large red ) maintained at approximately 30 C in a greenhouse. After about 60 d, roots were harvested from the plant, rinsed with room temperature water, and soaked for 4 hr at room temperature or overnight at 4 C in a 1:5 dilution of 100X Crystalzyme (Valley Research, South Bend, IN) pectinase solution. Softened galls (with excess root material removed) were placed in a mortar and gently smashed with a pestle to crush galls and, consequently, endospore-filled females. The smashed root material was transferred to a glass jar filled with 50 ml of distilled water and then shaken for 2 min. Endospores were collected by pouring the resultant slurry over a series of stacked sieves: a 250-µm-pore sieve, a 75-µm-pore sieve, and a 25-µm-pore sieve on the bottom. The material passing through the 25-µm-pore sieve was recovered, transferred to a glass jar and the shaking step was repeated once with an additional 50 ml of distilled water. The slurry was again filtered through the series of stacked sieves and the eluate filtered through a 5-µm polycarbonate filter (GE Osmonics, Minnetonka, MN) under vacuum; the filtrate was recovered and stored at 4 C. This sample was visually inspected for the presence of endospores using a light microscope at x400. The endospore concentration was determined using a Brightline hemacytometer (Hausser Scientific, Horsham, PA). 44

57 Differential interference images were collected using a Lieca DM1RB microscope with a x100 oil immersion objective (1.3 NA). The viability of endospores collected using the present (mortar-ground-gall) method was assessed by attachment to and infection of M. arenaria Race 1 J2. For infectivity assays, 10,000 J2 with endospores attached (from the present method or hand picked females disrupted in a glass homogenizer) were used to inoculate tomato plants. After approximately 60 d, 20 females were arbitrarily hand-picked from digested root material, visually inspected for endospores and crushed in distilled water with a glass tissue grinder to release endospores. Endospore DNA was extracted after pelleting 5 x 10 7 endospores at 18,000g for 5 min. The endospores were suspended in 10 mm Tris, 1.0 mm EDTA, ph 8.0, containing 20 mg/ml lysozyme and incubated at 37 C for 30 min. Proteinase K (20 mg/ml) and sodium dodecyl sulfate (2% w/v) were added followed by additional 30 min incubation. The pretreated endospores were transferred to a 2.0-ml impact-resistant tube containing approximately 1 g glass beads in the size range 150 to 212 µm (Sigma-Aldrich, St. Louis, MO). Phenol and chloroform were added and the endospores were disrupted using a Savant FastPrep Bio101 bead beating instrument. The samples were extracted with four 30 secpulses at 4.0 m/s interspersed with 1 min incubations on ice. The phases were separated by centrifugation at 18,000g for 10 min. The aqueous phase was further purified with subsequent phenol and chloroform extractions. Endospore DNA was precipitated by adding sodium acetate, ph 4.8, to 300 mm, 2.5 volumes 95% ethanol and mg/ml glycogen, followed by centrifugation at 20,000g for 60 min at 4 C. RNase One (Promega, Madison, WI) was used to remove contaminating RNA per manufacturer recommendations. Endospore 45

58 DNA concentration was determined fluorometrically. In order to confirm that usable genomic DNA could be isolated from spores extracted with this method, PCR was performed on DNA extracted from endospores using Pasteuria 16S ribosomal DNA primers (Duan et al., 2003). Experiments were repeated twice for a total of three trials. 46

59 RESULTS AND DISCUSSION Davies et al. (1988) found that attachment of at least 5 endospores/j2 was necessary to ensure infection of the nematode by P. penetrans, but that subsequent plant root penetration was reduced by 86% when J2 were encumbered with 15 or more endospores. In this study, we inoculated plants with 10,000 J2 with an average of six endospores attached. Pasteuriainfected females produce approximately 2 x 10 6 endospores each. Therefore, a plant infected with 10,000 Pasteuria-filled females contains up to 2 x endospores. Using our method, we were able to collect an average of 5.78 x 10 9 endospores per plant, which is approximately 30% of that theoretical maximum. This is a significant improvement over an older mortar disruption method for isolating endospores (Chen et al., 1996), which did not include enzymatic digestion or filtration to remove plant and/or nematode debris, and produced 1.67 x 10 8 endospores/plant, or about 30-fold fewer than our method. By eliminating the need for hand picking endospore-filled females, our method greatly reduced endospore collection time and increased the number of viable endospores that may be collected. The present method has been optimized for collecting endospores from the roots of three plants in 1 hr. However, it can easily be used to collect endospores from several more plants with a negligible increase in time. Although uninfected and Pasteuria-filled M. arenaria females were not initially separated, passing the endospores through a 5-µm filter effectively removed most nematode and plant debris. Visual inspection of endospores collected using the present method revealed endospores with normal morphology and only a small amount of nematode and/or root debris (Fig. 1). Exosporium, spore core, and parasporal fibers are present and intact. For more 47

60 sensitive assays, such as antiserum production, subsequent purification of endospores may be necessary. Endospores collected by our method retain full biological activity and effectively attached to and infected M. arenaria J2. There were no differences in the average attachment or percentage attachment of endospores collected using the present method when compared to endospores from hand-picked females. Average attachment was 5.6 ± 3.4 and 6.0 ± 3.1 endospores (mean ± 1 standard deviation for three replicates) per J2 for crushed galls or hand-picked females, respectively. Likewise, there were no detectable differences in the infectivity of endospores collected using the present method versus those from hand-picked females. There were 95% and 97% average infectivity of J2 with endospores prepared with the present method and from 20 hand-picked crushed females, respectively. Polymerase chain reaction analysis with primers specific for Pasteuria 16S ribosomal DNA sequences yielded the anticipated 549 bp band. Genes isolated in this way are suitable for cloning and sequencing (Anderson et al., 1999; Atibalentja et al., 2000; Trotter and Bishop, 2003). The present method is ideal for rapidly collecting vast amounts of viable endospores suitable for attaching to and infecting nematodes, morphology studies, and DNA preparations. 48

61 LITERATURE CITED Anderson, J. M., Preston, J. F., Dickson, D. W., Hewlett, T. E., and Maruniak, J. E Phylogenetic analysis of Pasteuria penetrans by 16S rrna gene cloning and sequencing. Journal of Nematology 31: Atibalentja, N., Noel, G. R., and Domier, L. L Phylogenetic position of the North American isolate of Pasteuria that parasitizes the soybean cyst nematode, Heterodera glycines, as inferred from 16S rdna sequence analysis. International Journal of Systematic and Evolutionary Microbiology 50: Charles, L., Carbone, I., Davies, K. G., Bird, D., Burke, M., Kerry, B., and Opperman, C. H Phylogenetic analysis of Pasteuria penetrans by use of multiple gene loci. Journal of Bacteriology 187: Chen, S. Y., Charnecki, J., Preston, J. F., and Dickson, D. W Extraction and purification of Pasteuria spp. Endospores. Journal of Nematology 32: Chen, Z. X., and Dickson, D. W Review of Pasteuria penetrans. Biology, ecology and biological control potential. Journal of Nematology 30: Chen, Z. X., Dickson, D. W., and Hewlett, T. E Quantification of endospore concentrations of Pasteuria penetrans in tomato root material. Journal of Nematology 28: Davies, K. G., Kerry, B. R., and Flynn C. A Observations on the pathogenicity of Pasteuria penetrans, a parasite of root-knot nematodes. Annals of Applied Biology 112:

62 Duan, Y. P., Castro, H. F., Hewlett, T. E., White, J. H., and Ogram, A. V Detection and characterization of Pasteuria 16S rrna gene sequences from nematodes and soils. International Journal of Systematic and Evolutionary Microbiology. 53: Hewlett, T. E., and Dickson, D. W A centrifugation method for attaching endospores of Pasteuria spp. to nematodes. Supplement to the Journal of Nematology 25: Oostendorp, M., Dickson, D. W., and Mitchell, D. J Host range and ecology of isolates of Pasteuria spp. from the southeastern United States. Journal of Nematology 22: Sayer, R. M Pasteuria, Metchnikoff, Pp in A. L. Sonenshein, J. A. Hoch, and R. Losick, eds. Bacillus subtilis and other Gram-positive bacteria: Biochemistry, physiology, and molecular genetics. Washington, D.C.: American Society for Microbiology. Trotter, J. R., and Bishop A. H Phylogenetic analysis and confirmation of the endospore-forming nature of Pasteuria penetrans based on the spo0a gene. FEMS Microbiology Letters 29:

63 FIGURES Figure 2.1. Nomarski differential interference contrast microscopy of the Pasteuria penetrans endospore filtrate. Scale bar = 10 µm. 51

64 CHAPTER 3: Advances in culturing methods for the endoparasitic bacterium Pasteuria penetrans Jenora T. Waterman 1, Tom E. Hewlett 2, Keith G. Davies 3, David McK. Bird 1, 3, and Charles 1, 3, H. Opperman 1 Center for the Biology of Nematode Parasitism Department of Plant Pathology, North Carolina State University, Box 7253 Raleigh, North Carolina Pasteuria Bioscience Research Drive Alachua, FL Nematode Interactions Unit Rothamsted Research, Ltd. Harpenden, Herts AL5 2JQ, United Kingdom To whom correspondence should be addressed. C.H.O. (Office: , Fax: , warthog@ncsu.edu) 52

65 ABSTRACT Pasteuria penetrans is an obligate parasite of Meloidogyne spp. (root knot nematodes). Due to its fastidious nature, in vitro culturing of this organism has proven to be challenging. To date, there are no reports of exponential growth in vitro for P. penetrans. Here we describe a genomics approach that has lead to advances in culturing this bacterium. Using genomics clues, we modified the concentration of certain key metals found in the standard P. penetrans growth and sporulation medium and observed a critical level at which copper permitted vegetative growth and sporulation. Complete elimination of copper allowed growth and sporulation several orders of magnitude over levels observed in the standard formulation (which contained copper). Copper titration studies suggest that there exists a critical level at which copper will inhibit sporulation, but allow vegetative growth; phenomena also observed in the model organism, Bacillus subtilis. 53

66 INTRODUCTION Pasteuria penetrans is an obligate, hyperparasite of root knot nematodes (Meloidogyne spp.), who themselves are obligate parasites of various plants. Root knot nematodes cause great devastation to agricultural crops, accounting for some $50 billon annually (Sasser and Freckman, 1987). Currently, one of the most effective methods of controlling nematode pests is application of chemicals such as methyl bromide which can be costly and environmentally damaging. Pasteuria penetrans holds promise for use as an environmentally-friendly alternative for controlling nematode pests. The life cycle of P. penetrans begins when it attaches to the cuticle of nematodes migrating through soil in search of a nutrient source. Endospores germinate when the nematode host has entered the root system of a plant host and established a feeding site within the host root vasculature. A germ tube is extended into the pseudocoelom of the nematode, which eventually divides into microcolonies (mycelia), that become fragmented microcolonies (also termed thalli), and finally doublets that eventually separate into individual sporangia that undergo sporogenesis. All life stages seen in nature have been observed in P. penetrans in vitro cultures in our lab and by other researchers (Hewlett, T. E., unpublished data). Many attempts have been made to culture Pasteuria spp. in vitro (Bishop and Ellar, 1991, Reise et al. 1991, Reise et al. 1988). Bishop and Ellar (1991) screened several media formulations for their ability to support growth of P. penetrans. One formulation resulted in moderate support for up to one month with a very low yield of mature spores; however, none of the other media were successful in maintaining P. penetrans. Reise and colleagues had a measure of success in their attempts at culturing P. penetrans in the late 1980s and early 1990s. In 1988, Reise et al. tested Grace s Insect Medium M199, Schnider s Drosophila Medium, Iscore s Modified 54

67 Dulbecco s Medium and H2 medium for their ability to support the growth of Pasteuria spp. isolates. Then these standard formulations were modified by adding organic and mineral supplements, which improved growth. In that study they used Pasteuria spp. isolated from Pratylenchus brachyurus, Heterodera glycines, and M. incognita. The life stages they observed closely resembled those found in diseased nematodes. In vitro growth was maintained for 3 to 5 transfers over a few months. Then Reise et al. (1991) expanded on their culturing technique by implementing a complex medium containing 111 ingredients, which stabilized Pasteuria nishizawae growth for up to six transfers over an 8-month period. Recent breakthroughs with in vitro culturing strategies by Pasteuria Bioscience LLC. (Alachua, FL) appear promising for obtaining large amounts of endospores. This group was able to successfully grow P. penetrans using a co-culture technique with Enterobacter cloacae (Gerber and White, 2001), a rhizobacterium whose interactions have been shown to be beneficial for plant growth, P. penetrans reproduction and RKN biocontrol (Duponnois et al. 1999). However, a major challenge to the mass production of endospores is the tendency and apparent preference of P. penetrans to grow as a biofilm making it especially difficult to grow liquid cultures (Hewlett, T. E., unpublished data). Current efforts at Pasteuria Bioscience include the use of commercial-grade fermentation tanks with easily available growth media to grow several strains of P. penetrans (Hewlett, T. E., unpublished data; However, the fastidious nature of the organism continues impede development of a system that will yield mass production of endospores. We decided to implement a genomic approach to study P. penetrans and use genome sequence data to determine ways to better maintain in vitro cultures and modulate 55

68 sporulation. We have begun a whole shotgun sequencing approach to obtain the complete DNA sequence of P. penetrans strain RES147, a broad host range strain. To date, we have constructed five genomic libraries and sequenced approximately 11,000 sequence reads, resulting in over 3.4 Mb of quality-trimmed genomic sequence, which has been assembled into 563 contigs, accounting for nearly 2.5 Mb of the P. penetrans genome (unpublished data datadatadatadatadata). Preliminary analysis of the P. penetrans genome yielded many genes with significant (e-value 1.0 x ) similarities to known genes in GenBank. Examples include genes involved in sporulation such as Spo0F, having 84% homology to Bacillus subtilis (Kojetin et al., 2005). The B. subtilis Spo0F protein is a well-characterized response regulator that is a key player in modulating sporulation. By using B. subtilis as a model system, we can design targeted experiments based on comparative genomics. Here we describe in vitro culturing strategies that expand upon those described by Gerber and White (2001). Our method involved growing P. penetrans as biofilms covered by a thin layer of medium to enhance gas exchange and eliminating certain metals that have been shown to inhibit growth and sporulation in closely related Bacillus spp. at critical levels (Kolodziej and Splepecky 1962, 1964; Krueger and Kolodziej 1976; Jun et al., 2003). Growing P. penetrans in this manner yielded 100-fold more spores over commercial methods available at the time. 56

69 MATERIALS AND METHODS In vivo cultures of Pasteuria penetrans Pasteuria penetrans (FL1 isolate, Gainesville, FL) strain was obtained from Pasteuria Bioscience LLC (formerly Entomos, LLC), and initially isolated from Meloidogyne arenaria Race 1-infected peanut plants. To propagate endospores, 10,000 M. arenaria J2 (~2 days old) were mixed with 1 X 10 6 P. penetrans endospores, a ratio of 100 endospores/j2, in a volume of 10 ml tap water. Endospores were attached to J2 using a modified centrifugation method of Hewlett and Dickson (1993). Samples were centrifuged for 5 minutes, at 3,220g, at room temperature in a 50-ml tube. Ten randomly selected J2 were counted to determine the percentage and average number of endospores attached. M. arenaria J2 (10,000) were used to inoculate 4-wk-old tomato plant (Lycopersicon esculentum Rutgers large red ) maintained at 30 C in a greenhouse. After about 60 d, endospores were collected using the mortar-ground-gall method of Waterman et al. (2006). Endospores were stored in distilled water at 4 o C. Establishing in vitro cultures Tomato seeds were germinated and maintained (3 seedlings/pouch) to 4-weeks of age in sterile growth in a Percival Intellus Environmental Controller with 16-8 light-dark cycles at 26 o C. Four-week old tomato plants were inoculated with approximately 5,000 Meloidogyne arenaria J2 with attached endospores in a volume of 5-10 ml of distilled water. To allow J2 to enter the root systems, the pouches were placed flat and allowed to dry out in the growth chamber for 2 days. Then plants were watered once a week with 5-10 ml of 0.25 % Hoagland s No.2 Basal Salt Mixture and regularly as needed with 5-10 ml of 0.2-μm-filtered tap water. At 216 to 300 degree-days, as based on 17 o C/d accumulating each day above a 57

70 base temperature of 10 o C (Hatz and Dickson 1992; Darban et al., 2005) or 24 to 26 calendar days, Pasteuria-infected female nematodes were excised from roots soaked in a 1:5 dilution of 100 X Crystalzyme (Valley Research, South Bend, IN) pectinase solution for 2-4 hours at room temperature. Excised females were placed in sterile 0.1 M saline. Approximately 100 Pasteuria-infected female nematodes were surface sterilized with 1 ml 0.5% hyperchlorite, ph 7.0 for exactly 5 minutes. The hyperchlorite solution was removed with a sterile transfer pipet and infected nematodes were washed five times with 1 ml 0.1 M sterile saline. Two milliliters of fresh 0.1 M saline to the nematodes and the suspension was transferred to a new petri dish. The saline was removed and 3 ml Pasteuria growth and sporulation medium (Pasteuria Bioscience, LLC.) was added and sterile toothpicks were used to lyse females. The sample was separated into 125- or 500-μl aliquots in nonpyrognic, 6- or 24-well cell culture clusters (Costar, Cambridge, MA), respectively. The samples were visually inspected at 400X with a Ziess IM inverted light microscope, using Hoffman optics, for the presence of P. penetrans life stages (microcolonies, fragmented microcolonies or thalli, and doublets). In vitro cultures were maintained at 30 o C. After 24 hours, approximately 100 µl or 400 µl of the medium was removed from each well to get rid of contaminating, floating plant and nematode tissue, which does not generally adhere to the culture dish. Fresh medium (100 µl or 400 µl) was added to each well. Cultures were visually inspected every 1-2 days for growth and medium was refreshed once a week by replacing spent medium with fresh medium. After about 2 weeks, densely growing cultures were split to establish new cultures. Differential interference contrast images were captured with a Ziess Axiovert inverted light microscope at 320X. 58

71 Varying metal concentrations in Pasteuria growth and sporulation medium In vitro cultures were maintained as previously described using Pasteuria growth and sporulation medium prepared either with or without 3.89 mm Cu 2+ /3.22 mm EDTA, or 0.11 mm Zn 2+ /3.22 mm EDTA, or 3.39 mm Cu 2+ /0.11 mm Zn 2+ M/3.22 mm EDTA. Cultures were maintained at 30 o C for 4 weeks and cell growth was measured by microscopic visual inspection as described above. In vivo and in vitro P. penetrans endospore attachment Eggs of M. arenaria Race 1 were collected from the roots of tomato plants inoculated with 20,000 eggs approximately three months earlier. The roots were shaken in 0.5% bleach solution for exactly one minute and cleaned over a 40% sucrose pad and rinsed well with tap water. The eggs were allowed to hatch at 30 o C for two days. Five thousand J2 (~2 days old) were mixed with either 5.0 X 10 5 in vivo- or 5.0 X 10 5 in vitro-generated P. penetrans endospores in 5 ml tap water in a 50 ml tube. The samples were centrifuged for 5 minutes, at 3220g, at room temperature. Twenty females were randomly selected and visually inspected to determine the percent attachment and average attachment for each treatment. These J2 were then used to inoculate 4-week old Rutgers tomato seedlings that were kept in a greenhouse maintained at 30 o C for 60 days; after which females were checked for spores. Experiments were repeated 3 times per treatment. 59

72 RESULTS In vitro cultures In vitro populations of P. penetrans were successfully maintained and endospores were produced. Cultures generally produced endospores within 2 to 4 weeks of inoculation at 30 o C and the transfer process was repeated for eight months. All previously reported life stages in the P. penetrans (Chen et al., 1997; Oostendorp et al., 1991; Hewlett et al., 2003) including vegetative cells or Bacillus-like rods, microcolonies (mycelial balls), quartets and doublets, and immature and mature endospores were observed in laboratory cultures. Endospore attachment bioassay To compare the attachment efficacy of in vitro- and in vivo-generated P. penetrans endospores, we conducted attachment bioassays (Table 1). For each sample, 20 J2 were viewed to establish if at least one endospore was attached to the cuticle. The total number of spores attached also was determined. M. arenaria J2 had an average of 7.2 ± 2.2 in vivo and 4.9 ± 2.1 in vitro spores attached with an average of 96.7 ± 2.9 and 91.7 ± 2.9 of the nematodes having at least one endospore attached, respectively (Table 1). Spores produced in-vitro were able to infect nematodes at the same level as in vivo spores (data not shown). Varying the composition of P. penetrans growth and sporulation medium In an attempt to enhance sporulation, we varied the composition of the Pasteuria growth and sporulation medium. Standard growth conditions involved maintaining in vitro cultures of P. penetrans for in standard medium contain all components of the proprietary formulation components (Pasteuria Bioscience, LLC) two weeks at 30 o C. Table 2 shows that P. penetrans cultures maintained in the standard formulation had little growth and poor sporulation as observed by visual inspection. In cultures grown in formulations without

73 mm Cu 2+ /3.22 mm EDTA there was a substantial increase in growth and sporulation over the standard formulation (Table 2). However, in cultures where a different metal, 0.11 mm Zn 2+ /3.22 mm EDTA, was removed growth as sporulation were greatly reduced to levels comparable to the standard formulation. Removal of both metal components that is cells were grown without 3.39 mm Cu 2+, without 0.11 mm Zn 2+ M and without 3.22 mm EDTA, resulted in an increase in growth and sporulation. Removal of both metal ions from culture medium was effective in enhancing cell growth and sporulation in vitro (Table 2, Fig.1). Figure 1 is an image of 14-day old in vitro cultures grown in the absence of copper. There is a marked difference in the quantity of vegetative stages compared to cultures grown with copper. Vegetative rods, microcolonies/mycelial structures, indicative of early sporulation, are present in abundance in cultures where copper is present. Fragmented microcolonies (arrows), thalli, doublets and quartets are more prevalent in copper-deficient cultures (Fig 1A, B). The presence of fragmented microcolonies and doublets and quartets in the cultures are indicative of late sporulation. Microcolonies are present in culture containing copper, however, they are more uniform than those present in cultures lacking copper (Fig. 1B) Growing P. penetrans in the absence of zinc did not have an effect on growth and sporulation. Culturing P. penetrans in the absence of copper (Fig. 1b) resulted in approximately 100-fold more endospore production over copper-containing formulations (Fig. 1b). Nutrient agar and gelatin were ineffective in maintaining in vitro cultures of P. penetrans. Effect of copper on Pasteuria penetrans growth and sporulation The complete removal of copper and EDTA had a positive effect on growth and sporulation of in vitro cultures of P. penetrans. For certain Bacillus species, it has been reported (Jun et 61

74 al., 2003; Kolodziej and Splepecky 1962; Krueger and Kolodziej 1978) that growth and sporulation are dependent on copper levels. Table 3 contains a summary of results of copper titration experiments. Copper concentrations 2.93 mm resulted in little vegetative cell growth and poor spore formation (Experiment 5), however, good vegetative cell and spore formation was observed in Experiments 0-4 (Table 3). No cell growth or sporulation was observed in P. penetrans cultures maintained in medium containing 9.97 mm copper (Experiments 6-8). 62

75 DISCUSSION Preliminary analysis of a partial genome suggest there are proteins present, such as the sporulation response regulator Spo0F, whose functionality is regulated by the presence of metals such as Cu 2+. Culturing P. penetrans in thin layers of medium and in the absence of copper ions was beneficial for growth and sporulation in vitro. By removing copper ions from media growth and sporulation was enhanced and it is apparent that there exists a critical level at which copper will allow growth of vegetative stages while inhibiting sporulation. This concentration has been determined to be 1.47x10-4 for P. penetrans. Endospores produced by in vitro culturing methods are effective for attachment to M. arenaria J2 they display the same morphology as ones produced in vivo. Importantly, there was no apparent difference in the attachment abilities of endospores produced in vivo and in vitro. Furthermore the in vitro-produced spores were also able to infect J2 at the same level as in vivo cells. The fact that these spores attach to the cuticles of and infect nematodes with the same efficacy as in vivo generated spores is promising for biological control applications. Although mass production of endospores remains a hurdle, the work done here demonstrates the potential for metalloregulation of P. penetrans, which can shed new light on new growth medium formulations and culturing strategies. Maintaining cultures in a thin layer of medium is also beneficial as it may serve to allow better gas exchange. 63

76 ACKNOWLEDGMENTS We thank Pasteuria Bioscience, LLC. for P. penetrans endospores and Pasteuria growth and sporulation medium. J. T. W. was a NSF IGERT Fellow. This work was supported by the North Carolina Agricultural Research Service, Rothamsted Research, Ltd and Syngenta. 64

77 LITERATURE CITED 1. Ahmad, R. and Gowen, S. R. (1991) Studies on the infection of Meloidogyne spp. with isolates of Pasteuria penetrans. Nematologia Mediterranae 19, Bishop, A. H. and Ellar, D. J. (1991) Attempts to culture Pasteuria penetrans in vitro. Biocontrol Science and Technology 1, Chen, Z. X. and Dickson, D. W. (1998) Review of Pasteuria penetrans: Biology, ecology and biological control potential. Journal of Nematology 30, Chen, Z. X., Dickson, D. W., Freitas, L. G., and Preston, J. F. (1997) Ultrastructure, morphology and sporogenesis of Pasteuria penetrans. Phytopathology 87, Darban, D. A., Gowen, S. R., Pembroke, B. and Mahar, A. N. (2005) Development of Pasteuria penetrans in Meloidogyne javanica females as affected by constantly high vs fluctuating temperature in an in-vivo system. Journal of Zhejiang University Science 6B, Duponnois, R., Bâ, A. M., and Mateille, T. (1999) Beneficial effects of Enterobacter cloacae and Pseudomonas mendocina for biocontrol of Meloidogyne incognita with the endospore-forming bacterium Pasteuria penetrans. Nematology 1, Gerber, J. F. and White, J. H. (2001) Materials and methods for the efficient production of Pasteuria. International patent application. WO 01/11017 A2. 8. Giblin-Davis, R. M., Williams, D. S., Bekal, S., Dickson, D. W.l, Brito, J. A., Becker, J. O., and Preston, J. F. (2003) Candidatus Pasteuria usgae sp. nov., an obligate endoparasite of the phytoparasitic nematode Belonolaimus longicaudatus. International Journal of Systematic and Evolutionary Microbiology 53,

78 9. Hatz, B. and Dickson, D. W. (1992) Effects of temperature on attachment, development, and interactions of Pasteuria penetrans on Meloidogyne arenaria. Journal of Nematology 24, Hewlett, T. E. and Dickson, D. W. (1993) A centrifugation method for attaching endospores of Pasteuria spp. to nematodes. Journal of Nematology 25, Hewlett, T. E., Griswold, S. and Smith, K. S. (2004) Production of the nematode biological control agent Pasteuria ssp. the hard way. Annual International Research Conference on Methyl Bromide Alternatives and Emissons Reductions. Orlando, Florida. 12. Hewlett, T. E., Huang, Y., Hammer, W., Scheurger, A., Ogram, A., and Duan, Y. (2002) Control of root-knot nematodes at the Epcot Center using in vivo produced Pasteuria penetrans. Annual International Research Conference on Methyl Bromide Alternatives and Emissons Reductions. Orlando, Florida. 13. Hewlett, T. E., Schuerger, A. C., and Dickson, D. W. (1997) Biological control of Meloidogyne arenaria at EPCOT, Disney World. Journal of Nematology 29, Hewlett, T. E., Smith, K. S., Griswold, S. T. and Crow, T. W. (2003) Comparison of the efficacy of Pasteuria penetrans endospores produced in vivo and in vitro for the control of Meloidogyne arenaria. Annual International Research Conference on Methyl Bromide Alternatives and Emissons Reductions. San Diego, California. 15. Jun, Y., Yi, L., Yong, T., Jianben, L., Xiong, C., Qin, Z., Juaxin, D., Songsheng, Q., and Ziniu, Y. (2003) The action of Cu 2+ on Bacillus thuringiensis growth investigated by microcalorimetry. Prikladnaia biokhimiia I mikrobiologiia 39,

79 16. Kojetin, D.J., Thompson, R. J., Benson, L. M., Naylor, S., Waterman, J., Davies, K. G., Opperman, C. H., Stephenson, K., Hoch, J. A., and Cavanagh, J. (2005) Structural analysis of divalent metals binding to the Bacillus subtilis response regulator Spo0F: the possibility for in vitro metalloregulation in the initiation of sporulation. Biometals 18, Kolodziej, B. J. and Splepecky, R. A. (1962) A copper requirement for the sporulation of Bacillus megaterium. Nature 194, Kolodziej, B. J. and Splepecky, R. A. (1964) Trace metal requirements for sporulation of Bacillus megaterium. Journal of Bacteriology 88, Krueger, W.B. and Kolodziej, B. J. (1976) Measurement of cellular copper levels in Bacillus megaterium during exponential growth and sporulation. Microbios 17, Krueger W. B. and Kolodziej, B. J. (1978) Divalent cation mobility throughout exponential growth and sporulation of Bacillus megaterium. Microbios 18, Kushner, D. J. (1971) Influences of solutes and ions on microorganisms. In: Hugo, W. B. (Ed.), Inhibition and Destruction of the Microbial Cell, Academic Press, London, pp Mankau, R. (1980) Biological control of nematode pests by natural enemies. Annual Reviews of Phytopathology 18, Oostendorp, M., Dickson, D. W., and Mitchell, D. J. (1991) Population development of Pasteuria penetrans on Meloidogyne arenaria. Journal of Nematology 23, Reise, R. W., Hackett, K. L., and Huettel, R. N. (1991) Limited in vitro cultivation of Pasteuria nishizawae. Journal of Nematology 23,

80 25. Reise, R. W., Hackett, K. L., Sayer, R. M., and Huettel, R. N. (1988) Factors affecting cultivation of three isolates of Pasteuria spp. Journal of Nematology 20, Smith, K. S., Hewlett, T. E. and White, J. H. (2002) Pasteuria species for nematode control: current developments and future prospects. Annual International Research Conference on Methyl Bromide Alternatives and Emissons Reductions. Orlando, Florida. 27. Sayer, R. M., Wergin, W. P., Schmidt, J. M., and Starr, M. P. (1991) Pasteuria nishizawae sp. nov., mycelial and endospores-forming bacterium parasitic on cyst nematodes of genera Heterodera and Globodera. Research in microbiology 142, Sayre, R.M., and Starr, M.P. (1985) Pasteuria penetrans (ex Thorne, 1940) nom. rev., comb. n., sp. n., a mycelial and endospore-forming bacterium parasitic in plantparasitic nematodes. Proceedings of the Helminthological Society of Washington 52, Smith, K. S., Hewlett, T. E. and Griswold, S. (2004) Pasteuria for nematode control: development of a commercial production process. Annual International Research Conference on Methyl Bromide Alternatives and Emissons Reductions. Orlando, Florida. 30. Starr, M.P., and Sayre, R.M. (1988) Pasteuria thornei sp. nov. and Pasteuria penetrans sensu stricto emend., mycelial and endospore-forming bacteria parasitic, respectively, on plant-parasitic nematodes of the genera Pratylenchus and Meloidogyne. Annales de l'institut Pasteur Microbiology 139,

81 31. Stirling, G. R. and Wachtel, M. F. (1980) Mass production of Bacillus penetrans for the biological control of root-knot nematodes. Nematologica 26, Williams, A. B., Stirling, G. R., Hayward, A. C., and Perry, J. (1989) Properties and attempted culture of Pasteuria penetrans, a bacterial parasite of root-knot nematode (Meloidogyne javanica). Journal of Applied Bacteriology 67, Waterman, J. T., Bird, D. McK. and Opperman, C. H. (2006) A method for isolation of Pasteuria penetrans endospores for bioassay and genomic studies. Journal of Nematology 38,

82 TABLES Table 3.1. Average attachments of P. penetrans endospores attached to 20 M. arenaria J2s. In vivo Endospores In vitro Endospores Average Spores % Attachment Average Attachment % Attachment 7.2 ± ± ± ± 2.9 Table 3.2. Summary of varying Pasteuria penetrans growth conditions. Experiment Cell Growth? Spore Formation? Standard Medium* Little Poor Without 3.89 x 10-3 Cu 2+ /3.22 x 10-3 M EDTA Yes Yes Without 1.11 x 10-2 C /3.22 x 10-3 M EDTA Little Poor Without 3.39 x 10-3 M Cu 2+ /1.11 x 10-2 Zn 2+ M/ 3.22 x 10-3 M EDTA Yes Yes 5% Gelatin No N/A Nutrient Agar No N/A *Standard medium contains Cu +2, Zn 2+ and EDTA. 70

83 Table 3.3. Summary of the effect of copper on Pasteuria penetrans growth and spore formation. Experiment # Cu 2+ Added (M) Cell Growth Spore Formation 1 0 Yes Yes x10-5 Yes Yes x10-4 Yes Yes x10-4 Yes Little x10-3 Little Poor x10-3 No N/A x10-3 No N/A x10-1 No N/A 71

84 FIGURES Figure 3.1. Nomarski differential interference contrast microscopy on 14-day-old in vitro cultures of Pasteuria penetrans. Cultures of Pasteuria penetrans were grown in standard growth and sporulation with (A) and without (B) 3.89 mm Cu 2+ for 14 days at 30 o C. Microcolonies (arrows) and branched thalli (arrowheads) can be seen in both images. More growth and sporulation was observed in cultures maintained in the absence of copper. Scale bars = 10 μm. 72

85 CHAPTER 4: Comparative genomic analysis reveals microsynteny between Pasteuria penetrans and closely related Bacillus species Jenora T. Waterman 1, Keith G. Davies 2, Andrew Warry 2, Brian Kerry 2, Jeffrey L. Thorne 3, Elizabeth H. Scholl 1, Mark Burke 1, David McK. Bird 1, 2 1, 2, and Charles H. Opperman 1 Center for the Biology of Nematode Parasitism Department of Plant Pathology, North Carolina State University, Box 7253 Raleigh, North Carolina Nematode Interactions Unit Rothamsted Research, Ltd. Harpenden, Herts AL5 2JQ, United Kingdom 3 Bioinformatics Research Center North Carolina State University, Box 7566 Raleigh, North Carolina To whom correspondence should be addressed. C.H.O. (Office: , Fax: , warthog@ncsu.edu) 73

86 ABSTRACT The hyperparasite Pasteuria penetrans is a Gram-positive endospore-forming bacterium that complete is life cycle within the pseudocoelom of its nematode host. Recent phylogenetic studies of housekeeping genes have confidently placed P. penetrans in the Clostridium-Bacillus clade of Gram-positive, low-g+c-content bacteria. Here we discuss genomic comparisons between Pasteuria penetrans and several closely related Bacillus species: B. subtilis, B. anthracis, B. halodurans, B. cereus and B. thuringiensis. Pairwise and multiple genome comparisons revealed a high level of colinearity and microsynteny between P. penetrans and Bacillus species at the gene level. The abundance of conserved sequence regions and gene order between P. penetrans and the Bacilli spp. suggests that the processes controlled by these genes in model organisms, such as B. subtilis, may be the same in P. penetrans. Studying similar gene regions in related organisms, specifically model organisms, will give insight into understanding the evolution of these genomes over time and the understanding of the basic biology of Pasteuria. 74

87 INTRODUCTION Pasteuria penetrans is a Gram-positive, endospore forming bacterium that is an obligate parasite of several species of root-knot nematodes, (Meloidogyne spp.). A recent phylogenetic analysis on 40 concatenated house keeping genes from 33 bacteria established that P. penetrans is part of the Clostridium-Bacillus clade of Gram-positive bacteria (Charles et al., 2005). Endospores attach, through a poorly understood process, to the cuticle of nematode hosts migrating through the soil. When the nematode host has established a feeding site, the endospore germinates and extends a germination tube into the pseudocoelom. The germination tube then gives rise to spherical microcolonies also called septate mycelia. The microcolonies separate into vegetative cells that divide into thalli which may be doublets or quartets of cells (unpublished data datadatadatadataresults Davies, K. D. and Hewlett, T. E.). Doublets eventually separate and undergo sporogenesis and give rise to endospores. These endospores are resistant to environmental extremes and can persist in the soil for many years (Jatala, 1986; Rodrigues et al., 2003). As a result of the infection process, nematodes lose their ability to produce eggs and eventually die. Endospores of Pasteuria spp. generally have a narrow host range, infecting species only from which they have arisen. This makes P. penetrans an enticing system for environmentally-safe and effective control of nematode pests. However, two major factors have impeded the use of Pasteuria spp. as biocontrol agents; the fastidious nature of the organism has precluded mass production of in vitro endospores and the narrow host range specificity. 75

88 As P. penetrans is a pathogen, we included pathogenic Bacillus species in our comparative studies. The pathogenic Bacillus spp. considered in this study, Bacillus cereus, Bacillus thuringiensis, and Bacillus anthracis, are soil-borne species referred to as group I Bacillus spp. or the Bacillus cereus group (Ash et al., 1991). Genetically, group I Bacillus spp. are very closely related, a fact which has led to the proposal that they should be considered one species (Helgason et al., 2000a). Bacillus anthracis is a virulent pathogen of animals and humans and is the causative agent of anthrax. Endospores of B. anthracis persist for long periods of time in the environment, but little evidence suggests that saprophytic growth in soil (Ticknor et al., 2001) Either its obligate pathogenic nature or a recent genetic bottleneck may be partially responsible for it high level of molecular monomorphism (Keim et al., 1997; Keim et al., 1999; Keim et al., 2000). Bacillus cereus is an opportunistic human pathogen associated with food poisoning (Drobniewski, 1993; Helgason et al. 2000a) and periodontal disease (Helgason et al., 2000). Bacillus thuringiensis has been used extensively in agriculture as a biological pesticide because it possesses crystal toxin genes that encode proteins which are toxic to many insect larvae (Schnepf et al., 1993). Spore and crystal toxin preparations from B. thuringiensis are used as commercial insecticides and the plasmidencoded crystal proteins produced by B. thuringiensis are lethal to coleopteran, dipteran, and lepidopteran insect pests (Schnepf et al., 1993; Crickmore et al., 1998). In general, conservation of gene order does not seem to be conserved in microbial (bacterial and archaeal) genomes beyond the level of operons (Wolf et al., 2001). However, there tends to be more conservation between closely related species, especially members and/or strains of the same bacterial genus. Here we describe genome comparisons between 76

89 P. penetrans and B. subtilis with some additional comparisons between P. penetrans and B. halodurans, B. cereus, B. thuringiensis and B. anthracis. METHODS Genome sequence and annotation data for B. subtilis (AL009126), B. anthracis (AE016879), B. halodurans (BA000004), B. cereus (AE07194) and B. thuringiensis (AE017355) were obtained from EMBL-EBI (Table A.2.1.) ( Genome sequence data were extracted from annotation files using Artemis version 5). A partial Pasteuria penetrans genome (unpublished data) was compared to each Bacillus genome by pair-wise alignments using MegaBLAST and BLASTX from the NCBI standalone BLAST package version (Altschul et al., 1990). Comparative genomic analysis was achieved using the Artemis Comparison Tool (ACT; For MegaBLAST comparisons, completed genomes containing annotation data for B. subtilis, B. halodurans, B. cereus, B. anthracis and B. thuringiensis were downloaded from the EBI Database. Artemis version 5 was used to create sequence only Fasta files for each genome, including the P. penetrans partial genome. Before BLAST comparisons were preformed, the Bacillus spp. genomes were used to generate databases to be used as targets for comparisons with the partial P. penetrans genome. MegaBLAST is a greedy local sequence alignment algorithm designed to align sequences with significant sequence similarity (Zhang et al., 2000). The following parameter settings were used for MegaBLAST analyses: word size was 12, gap open and extension penalties were set at 0.0. Good alignments (or matches) were classified as those having 80% identity over a window of 12 nucleotides with a cut-off threshold (e-value) of 1e - 77

90 4. Genome comparison files BLAST output files readable by the Artemis Comparison Tool, ACT were generated using the m command flag, incorporated during the pairwise comparison step. Once the comparison file was generated, ACT (Carver et al., 2005) version 4 was used to view the comparison files. To visualize pairwise comparison files with ACT, fasta genomic sequence files for the two genomes and their corresponding comparison files were loaded into ACT. This generated a graphical interface displaying the two aligned genomes (one at the top and the other at the bottom of the screen) with lines connecting regions of identity connected by lines. The red and blue lines represent matches or regions of identity which occur in forward and reverse directions, respectively. These matches are based on user-defined pairwise search parameters mentioned above. The zoom feature (scroll bars) was used to obtain global views of the pairwise comparison. Once comparison files were displayed graphically, annotation files were overlaid onto the matching Bacillus spp. genome using the Read An Entry feature to determine the genes corresponding to these syntenic regions. To compare multiple genomes, fasta files with their associated comparison files were sequentially loaded into ACT. For protein level comparisons, the partial P. penetrans genome (unpublished data) and the Bacillus spp. were compared using BLASTX and Blosum62 substitution matris with the minimum e-value set to 1e -4. A perl script was used to parse the BLASTX results files for significant hits, which were subsequently analyzed using Artemis as described above for nucleotide level comparisons. 78

91 RESULTS AND DISCUSSION Currently, we have sequenced approximately 11,000 sequence lanes from five genomic libraries resulting in over 3.5 Mb of quality-trimmed genomic sequence, which has been assembled into 563 contigs, accounting for about 2.5 Mb of the P. penetrans genome, with a G+C content of 48.3% (unpublished data datadatadatadatadata). Preliminary analysis of the P. penetrans genome yielded many genes with significant (e-values 1.0 x ) similarities to known genes in GenBank. Comparative analysis of the incomplete P. penetrans RES147 genome has revealed a high level of genome synteny with B. subtilis, B. anthracis, B. cereus, B. halodurans and B. thuringiensis. Pasteuria penetrans is a member of the Bacillus-Clostridium clade of Grampositive bacteria (Charles et al., 2005) and it shares a high degree of sequence similarity with these bacteria especially the Bacillus spp. (e.g. BLASTP evalues < 1e -10 ). Genome comparisons revealed that inversions have occurred in the genomes of the Bacillus species and P. penetrans, indicated by intersecting regions (i.e. where an X is formed) on the colinearity graphs (Fig. 1A and 2). Figure 1A is an Artemis Comparison Tool (Carver et al., 2005) generated image depicting a BLAST comparison of B. subtilis and P. penetrans. Pairwise comparison of the two genomes revealed a high conservation of synteny or gene order between P. penetrans and B. subtilis (Fig.1A and 1B). Lines on the figure represent sequence homology matches and the white space accounts for regions which either i) did not satify search criteria and therefore did not report a match, ii) regions within the P. penetrans genome that have not yet been sequenced, or iii) regions that are unique to each organism and therefore are not present in other organisms. Red and blue lines represent forward and reverse matches, respectively. A match is a genomic region (Fig. 1B), coding or non-coding, 79

92 with significant sequence similarity as determined by the user-defined e-value threshold, 1e -4 for pairwise genomic analyses presented here. Put another way, matches are hits from similarity searches resulting in high-scoring sequence pairs (HSP) smaller regions of the two genomes with e-values smaller than or equal to the user-defined threshold. Therefore, the red and blue lines (Fig. 1 and 2) represent HSP between the aligned genomes of P. penetrans and the Bacillus spp. In essence, the lines represent regions of colinearity in both forward and reverse directions as they go across the entire genome. However, because there are a number of lines that cross there has been substantial rearrangement in the Pasteuria genome compared to the Bacilli. This interpretation is supported by the results shown in Figure 2, where there is clearly much less rearrangement between the Bacillus species than between the Bacillus spp. and Pasteuria. Figure 1B is a closer view of match regions from Fig. 1A. Figure 1C is a graphical representation of BLASTP results from B. subtilis (blue) and P. penetrans (pink). This region highlights the conservation of sequence (e-value < 1.0 e- 10 ), order, and spacing. Figure 2 is also an ACT generated graph showing MegaBLAST results for P. penetrans comparison with B. subtilis, B. anthracis, B. halodurans, B. cereus and B. thuringiensis. Figure 2A shows an ACT comparison of the genomes of P. penetrans (Ppen), B. subtilis (Bsub) and B. halodurans (Bhal). As indicated by the prevalence of red and blue lines, there are many regions of similarity and colinearity between the genome of P. penetrans and the two Bacillus species. Although there is an abundance of white space between the Bacillus spp. and P. penetrans, there is a trend that seems to be shared between arrangement of their genomes. For example Figures 2B to 2D show simililar patterns for ACT comparisons between P. penetrans, B. subtilis and the other three Bacillus spp.; B. 80

93 anthracis (Bant, 2B); B. cereus (Bcer, 2C); and B. thuringiensis (Bthu, 2D). The partial P. penetrans genome showed greater homology with nonparasitic Bacillus species, B. subtilis and B. halodurans, than parasitic ones, B. anthracis, B. cereus and B. thuringiensis (Fig. 2A- D) as evidenced by the presence of more lines spread across the genomes. Comparisons among the complete genomes of the Bacillus spp. (Fig. 2A-D, middle portion of panels) show a tremendous degree of colinearity supported by the intense concentration of red lines, especially near the beginning of the genomes (Fig. 2A-D) and clear evidence of chromosomal rearrangements indicated by the intensity of crossed blue lines present in Fig. 2. Once the P. penetrans genome is completely sequenced we expect a similar pattern to the Bacilli to immerge. There is conservation of gene order at the operon level and the genomes of P. penetrans and the Bacillus species show a high degree of colinearity and microsynteny at the gene level (Fig. 1 and 2). Figure 3 is a schematic diagram showing an example of the conservation of genes at the operon level between P. penetrans and Bacillus species using the B. subtilis spoiiia operon. In B. subtilis eight spoiiia genes (spoiiiaa, spoiiiab, spoiiiac, spoiiiad, spoiiiae, spoiiiaf, spoiiiag, spoiiiah) are present as a single operon (Fig. 3). However, in other Bacillus members these genes may be spread over as many as three operons (Fig. 3). For simplicity the P. penetrans spoiiia genes are illustrated as a single operon, however, it is not known whether these genes are present as a single operon (Fig. 3). This type of synteny can be seen in numerous regions throughout the genomes (Fig. 3 and 4). Figure 4 is an illustration of a 4-Kb region of P. penetrans genome depicting conservation of gene orientation, size and order with B. subtilis. 81

94 General Features As of yet, we have not found evidence of plasmids in P. penetrans, but as more genome sequence data becomes available this issue is sure to be resolved. We have, however, identified eight of the more than 14 characterized B. subtilis sigma factors in Pasteuria penetrans (Table 1). In B. subtilis, it was shown by systematic gene knock-out studies (Kobayashi et al., 2003) that only 6.6 % of its genome (271 of its 4100 genes) was indispensable for growth in Luria Bertani (LB) medium at 37 o C when singly inactivated. Table A.2.2 is a list of 122 P. penetrans homologues to the essential B. subtilis genes described in that study. The essential genes of B. subtilis have been divided into several functional categories including DNA and RNA metabolism, protein synthesis, cell envelope, cell division, glycolysis and respiratory pathways. Genes from these and other functional categories are present in P. penetrans. The identification of members of essential metabolic pathways in P. penetrans (e.g., glycolysis) is critical to unraveling the metabolic habits and nutritional needs of this obligate parasite. Competence and sporulation Quorum sensing is a type of cell-cell signaling mechanism exhibited by bacterial cells to monitor their population density (Dunny and Winans, 1999; Lazzazera, 2000). In response to nutritional stress and high cell density B. subtilis produce peptide antibiotics, including surfactins which play a role in competence development and efficient sporulation. A putative srfaa gene, which encodes surfactin in B. subtilis, has been identified in the P. penetrans. The presence of this gene may also indicate that P. penetrans utilizes a similar pathway to monitor its population density and therefore, integrate similar types of signals necessary for the development of competence and the initiation of sporulation. 82

95 Competence is a cellular differentiation process which allows B. subtilis vegetative cells to take-up DNA in a macromolecular form (Dubnau, 1991). Certain bacterial strains can become naturally competent, including Bacillus species. Of the seven proteins used in B. subtilis (Hamoen et al., 2003) specifically for the binding, uptake and integration of DNA into the genome (ComC, ComE, ComF, ComG, NucA, RecA and AddAB); four have been identified in the partial genome of P. penetrans, ComC and ComE, RecA and AddAB. Genetic competence is a physiological state which is regulated by several pathways, which form an intricate web for the control of ComK (present in P. penetrans), competence transcription factor that is the final autoregulatory control switch prior to competence development (Turgay et al., 1998) it is involved in the activation of late competence genes involved in DNA-binding, uptake and recombination. The synthesis of ComS (currently not identified in P. penetrans) in response to the quorum-sensing signal transduction pathway destabilizes the ternary ComK/MecA/ClpC complex in which ComK is held inactive (Turgay et al., 1997). Member of the repressive complex and the ClpP protein, an ATP-dependent protease that becomes associated with the ternary complex and promotes competence, have matches in the partial P. penetrans genome. The presence of these competence-specific proteins in the Pasteuria penetrans genome could suggest that this bacterial parasite, like its close Bacillus relatives, may have natural competence ability. Furthermore, Pasteuria may utilize natural competence during stress conditions to increase genetic diversity by integrating extracellular DNA into its genome or via recombination. Pasteuria penetrans has homologues for 35% (49/139) of the sporulation genes in B. subtilis (Kunst et al., 1997). A detailed phylogenetic analysis of these sporulation genes was the focus of Chapter 5 of this dissertation. One gene of importance is Spo0A, the master 83

96 regulator for entry into sporulation in endospores-forming Bacillus spp. and other bacteria. The Spo0A protein is reported to directly or indirectly influence the expression of over 500 genes (Fawcett et al., 2000). The Spo0A regulon consists of 121 genes whose activities are directly under the control of this transcription regulator (Molle et al., 2003) 47 (39%) of which have been identified in P. penetrans. Sporulation phosphorelay members all present in the well-characterized bacterium B. subtilis include sensory kinases (Kin A/B/C/D/E), Spo0F (response regulator), Spo0B (phosphotransferase, presently not identified in P. penetrans partial genome), and Spo0A (transcription factor) are also present in P. penetrans. Presently, we are unable to study the molecular aspects of sporulation in this fastidious parasite. Virulence Currently, we have not identified homologues to virulence factors unique to the parasitic Bacillus species, such as the lethal toxin of B. anthracis and the Cry toxins of B. thuringiensis. However, we have identified sinr, a DNA binding protein active in the regulation of post-exponential-phase responses genes in B. subtilis (positive regulation of comk; negative regulation of apre, kinb, sigd, spo0a, spoiia, spoiie, spoiig). The SinR protein has been shown to regulate the expression of the B. thuringiensis gene InhA, whose product is an immune inhibitor that has been shown to specifically cleave antimicrobial proteins produced by insects (Fedhila et al., 2002). We have also identified a putative membrane-associated zinc metalloprotease unique to the Bacillus cereus group, which may have a role in virulence in P. penetrans. 84

97 Transposable elements Evidence of transposon activity exists within the genome of P. penetrans. Transposable elements have been identified in the partial P. penetrans genomes that are also present in B. subtilis, including ydcp and yddh, as well as putative excisionase and integrase genes. The presence of these elements within the genomes can explain some of the diversity amongst the closely related Bacillus species and with P. penetrans. In conclusion, genomic comparisons between Pasteuria penetrans and the model bacterium B. subtilis, the alkaliphilic Bacillus, B. halodurans and the B. cereus group has provided a wealth of basic information regarding gene conservation and diversity in Bacillus spp. and systematic information that would be extremely difficult to obtain by any other approach. Although the P. penetrans genome is incomplete, comparative genomics has revealed some basic information relating to the catabolic nature of this bacterium. One intriguing matter is to finally determine how closely the P. penetrans genome will match genomes of parasitic versus nonparasitic Bacilli species. We anticipate that as the P. penetrans genome is completely sequenced application of these tools will provide more insight for understanding of the basic biology of this organism and will help clarifying the mechanisms of host recognition and attachment, germination and proliferation, and sporulation. 85

98 ACKNOWLEGEMENTS We thank Stella Chang for technical support, D. Eric Windham and T. D. Houfek for Bioinformatic support. J. T. W. was a NSF IGERT Fellow. This work was supported by the North Carolina Agricultural Research Service and by Rothamsted Research, Ltd and Syngenta. 86

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105 Table 4.1. Pasteuria penetrans Sigma Factors TABLES Sigma Factor Class Function σ A Vegetative Cell Housekeeping; early sporulation σ B Vegetative Cell General stress response σ D Vegetative Cell Chemotaxis; autolysin; flagellar gene expression σ H Vegetative Cell Postexponential gene expression; Competence; early sporulation genes σ E Sporulation Early mother cell gene expression σ F Sporulation Early forespore gene expression σ G Sporulation Late mother cell gene expression σ K Sporulation Late forespore gene expression 93

106 FIGURES Figure 4.1. BLAST comparison of Bacillus subtilis and Pasteuria penetrans genomes. (A) An Artemis Comparison Tool (ACT) representation of MegaBLAST-generated comparison results showing the relationship between the B. subtilis genome and the partial P. penetrans genome. Matches are coding or non-coding regions with significant sequence homology as determined by a user-defined e-value threshold of 1e -4. White space represents regions that did not satisfy the search criteria. Whereas lines are used to graphically link regions of homology between a pair of sequences with red and blue lines represent forward and reverse sequence matches, respectively. Regions where lines intersect to form an X indicate chromosomal rearrangements. (B) MegaBLAST comparison of part of the B. subtilis and P. penetrans sequences generated by ACT. In this case, the red lines represent sequence homology between portions of the partial P. penetrans genome that are similar to the B. subtilis rrne-16s gene. (C) Graphical representation of a portion of BLASTP results of B. subtilis and P. penetrans genome sequences. In this region there is conservation of sequence (e-value < 1.0 e-10 ), order and spacing between B. subtilis proteins (blue) and P. penetrans contigs (pink). The scales represent bases along each genome. 94

107 95

108 96

109 Figure 4.2. Comparison of the genomes of Pasteuria penetrans and Bacillus species. Comparison of three genomes showing regions sequence identity between P. penetrans (Ppen), B. subtilis (Bsub), B. halodurans (Bhal) (A); P. penetrans, B. subtilis, B. anthracis (Bant) (B); P. penetrans; B. subtilis, B. cereus (Bcer) (C); and P. penetrans, B. subtilis, B. thuringiensis (Bthu) (D). The red and blue lines represent forward and reverse sequence homology matches (BLASTP, e-value < 1.0 e-10 ), respectively. The white spaces represent regions that did not satisfy search criteria. Not surprisingly the lines between the B. subtilis and the other Bacilli are extremely dense, representing nearly identical genomes. Regions of insertions are easily identified by the X pattern formed by intersecting lines. The scales represent bases along each genome. 97

110 Figure 4.3. Microsynteny between Bacillus subtilis, Pasteuria penetrans and closely related Bacillus spp. Schematic diagram of the eight-gene SpoIIIA operon of Bacillus subtilis showing genome colinearity with P. penetrans, and other closely related Bacillus species. Protein-coding genes (boxed arrows) and non-translated regions (solid lines) are indicated. These eight genes are present in B. subtilis as a single operon, but span two or three operons in the other Bacillus spp., indicated by the different colors. The P. penetrans SpoIIIA genes are depicted as a single operon due to the lack of transcript data. Dashed lines in putative P. penetrans operon indicate regions that are not yet sequenced. 98

111 Figure 4.4. Genome synteny between Bacillus subtilis and Pasteuria penetrans. Five predicted genes in P. penetrans (dark grey) show conservation of gene order, size and sequence similarity (e-values < 1.0 e -10 ) with genes present in B. subtilis (light grey). 99

112 CHAPTER 5: Maximum Likelihood and Bayesian Analysis of Pasteuria penetrans Sporulation Genes Jenora T. Waterman 1, Elizabeth H. Scholl 1, Jeffrey L. Thorne 2, Keith G. Davies 3, Brian R. Kerry 3, David McK. Bird 1, 3 1, 3,, and Charles H. Opperman 1 Center for the Biology of Nematode Parasitism Department of Plant Pathology, North Carolina State University, Box 7253 Raleigh, North Carolina Bioinformatics Research Center North Carolina State University, Box 7566 Raleigh, North Carolina Nematode Interactions Unit Rothamsted Research, Ltd. Harpenden, Herts AL5 2JQ, United Kingdom To whom correspondence should be addressed. C.H.O. (Office: , Fax: , warthog@ncsu.edu) 100

113 ABSTRACT Pasteuria penetrans is a bacterial hyperparasite of root-knot nematodes (RKN, Meloidogyne spp.) and is a member of the Bacillus-Clostridium clade. Members of the genus Bacillus are ubiquitous, Gram-positive, endospore-forming bacteria that include both parasitic and nonparasitic species. Pasteuria penetrans control RKN populations by attacking the reproductive system of female nematodes, thereby reducing fecundity. The inherent ability of P. penetrans to control nematode pests makes it an ideal candidate for use as a biological nematicide. We are in the process of obtaining the genome sequence of P. penetrans and have performed analyses of 46 sporulation-specific genes from five Bacilli spp. and P. penetrans. Codon- and amino acid-level analyses were performed using each gene and protein separately; and concatenated sets of each. Outgroups were included in single sequence analyses so that the phylogenetic relationship between P. penetrans and Bacilli spp. could be accurately determined. Maximum likelihood and Bayesian analysis for all data sets revealed a tendency of P. penetrans to cluster with nonparasitic Bacilli, B. subtilis and B. halodurans, rather than parasitic relatives, B. anthracis, B. cereus, and B. thuringiensis with strong support. Codon models which allowed the nonsynonomous/synonomous rate ratio (ω =d N /d S ) to vary among lineages and sites were implemented to determine whether any sites were under diversifying selection. Likelihood ratio tests were applied to determine whether the d N /d S ratio varied among lineages and sites and whether the d N /d S ratio for sites of interests is greater than one, indicating positive Darwinian selection. Not surprisingly, lineages seemed to be under purifying selection pressure with synonymous substitutions occurring more often than nonsynonymous ones. The species in question have spent a majority of time undergoing negative selection with 101

114 respect to sporulation genes. Likelihood ratio tests and codon-sites models predicted two membrane proteins with sites under diversifying selection, SpoIIIAA and SpoIIIAB. Taken together, our results support the conclusion that P. penetrans is a basal member of the Bacillus group. Sporulation genes in P. penetrans and the Bacilli spp. studied here show an overall tendency toward purifying selection resulting in maintenance of conserved genes although certain key residues within proteins have been shown to be under positive diversifying selection. We propose that these key residues may be involved in facilitating host-parasite interactions between P. penetrans and RKN. 102

115 INTRODUCTION The Gram-positive bacterium Pasteuria penetrans is an obligate endoparasite of Meloidogyne species (root knot nematodes, RKN), which in turn are agriculturally-important pests that cause over $100 billion of crop losses annually (Sasser and Freckman, 1987). Pasteuria penetrans has been shown to be efficient in controlling M. arenaria populations (Hewlett et al., 1997) as well as multiple other RKN species (Chen and Dickson, 1998; Atibalentja et al., 2000; Davies et al., 1990; Davies, 2005). Endospores are the infectious stage in the P. penetrans life cycle, which begins with spore attachment to the cuticle of second stage juveniles (J2) migrating through soil in search of host roots. Once J2 enter plant roots and begin to feed, endospores germinate, via a process that remains unclear. Germination is complete when the mycelia emanating from the germ tube begin to divide into vegetative rods and microcolonies. As the nematode continues to feed, molt and grow P. penetrans cells proliferate within the females, attacking the reproductive system and reducing fecundity and ultimately killing the female. Endospores are released into the surrounding soil upon root and cadaver decay. Although advances have been made with respect to in vitro culturing of P. penetrans (Opperman unpublished results; Kojetin et al., 2005; Smith et al., 2004; Gerber and White, 1997), exponential growth has yet to be reported. An understanding of the sporulation process of Pasteuria will help in understanding the basic biology of the organism. Sequence comparisons with a well characterized organism such as B. subtilis, may give clues to the developmental processes leading to spore development in P. penetrans. Single gene phylogenetic studies using rrna (Anderson et al., 1999) and 16S rrna genes (Atibalentja et al., 2000) indicated that members of the genus Pasteuria belong to the Clostridium- 103

116 Bacillus-Streptococcus clade of Gram-positive bacteria. Recent studies have been conducted on sporulation-specific sigma factors, σ E, σ F (Preston et al., 2003) and the master regulator for sporulation initiation, Spo0A (Trotter and Bishop, 2003) which support this finding. Preliminary analysis of a partial P. penetrans genome confirmed that placement of Pasteuria spp. in the Clostridium-Bacillus branch of Gram-positive bacteria (Charles et al., 2005). That work further indicated that Pasteuria is a basal member of the Bacillus spp., and P. penetrans is more closely related to B. halodurans and B. subtilis than B. anthracis, B. cereus (Charles et al., 2005). With this knowledge we undertook phylogenetic studies on P. penetrans sporulationspecific genes. An understanding of the molecular events controlling sporulation in P. penetrans may come through comparisons with its closest relatives. The model organism B. subtilis, for which extensive genetic and molecular data exist (Stragier and Losick, 1996, Eichenberger et al., 2004, Piggott and Hilbert, 2004) has been shown to be closely related to P. penetrans serves as the reference model. The alkaliphilic bacterium B. halodurans, the source of many industrially useful alkaliphilic enzymes such as protease, cellulase and amylase (all additives of laundry detergents), is apparently the closest of the Bacillus groups surveyed thus far (Charles et al., 2005). Parasitic Bacillus species have also been included in this study, all of which belong to the Bacillus cereus group, consisting of six members: Bacillus anthracis, Bacillus cereus, Bacillus mycoides, Bacillus pseudomycoides, Bacillus thuringiensis and Bacillus weihenstephanensis. These species are closely related and it has been suggested that they should be placed within one species (Helgason et al., 2000). However, recent work on a set of seven concatenated housekeeping genes from 105 members of this group by Priest et al. (2004) revealed that there is clear genetic heterogeneity among 104

117 these members and that the lineages studies were sufficiently distinct enough to warrant a separate label. Three animal-pathogenic members of the B. cereus group were included in our study: B. anthracis, the causative agent of anthrax, B. cereus, an opportunistic human pathogen associated with food poisoning and diseases of the mouth, and B. thuringiensis, the agriculturally-important bacterium whose toxic proteins are used as pesticides. Recent studies by Kojetin et al. (2005) surveyed the effect of copper ions on sporulation in B. subtilis and P. penetrans and reported that in the presence of copper, sporulation was similarly disrupted in both species. This finding supports the premise that sporulation in P. penetrans may be under control mechanisms similar to the model organism B. subtilis. To examine the sporulation phylogeny of P. penetrans and closely related Bacillus we used maximum likelihood Bayesian approaches to analyze 46 single and a concatenated set of sporulation genes and proteins from the six previously mention bacterial species. Pairwise and multiple sequence comparisons, coupled with maximum likelihood, Bayesian, and Bayesian approaches methods were used in this analysis. There has been much debate over models used to infer sites under diversifying selection (Suzuki and Nei, 2004) due to the high false-positive rate in small data sets. We implemented an Empirical Bayes approach for maximum likelihood estimations (MLEs) of parameters in the ω distribution that uses numerical integration (Yang et al., 2005). The new Empirical Bayes procedure has addressed uncertainties in previous methods (Anisimova et al., 2002) and corrections have been implemented for previous branch-site models (Yang et al., 2005; Yang and Nielsen, 2002). Thus the maximum likelihood-based empirical Bayes 105

118 approach is a powerful tool for calculating MLEs of parameters and inferring sites under diversifying selection in small data sets. 106

119 METHODS Bacillus subtilis sporulation genes described by Stragier and Losick (1996) and Kunst et al. (1997) were obtained from the National Center for Biotechnology Information (NCBI) (Wheeler et al., 2002) and used to construct a local database of ~140 deduced proteins. Pairwise comparisons were performed between a P. penetrans partial genome (Opperman, C. H. unpublished data) and the B. subtilis sporulation protein database using Tera-BLASTX and BLOSUM62 scoring matrix. Proteomes of B. anthracis Ames, B. cereus ATCC 10987, B. halodurans C-125 and B. thuringiensis Serovar Konkukian Str were downloaded from the NCBI (Wheeler et al., 2002) and compared to the B. subtilis sporulation protein database. Perl scripts were written to parse BLAST results files for significant hits (e-values < 1e -4 ). There were 49 matches to the B. subtilis sporulation database from a P. penetrans partial genome. Of these 49 proteins, 46 were present in at least four Bacillus species and were used for analysis in the present study (Table 1). Multiple sequence alignments were performed for the 46 proteins separately using ClustalW version 1.82 in MegAlign (DNASTAR, Inc., Madison, WI). A perl script was used to concatenate separate alignments for a total of 11,509 amino acids. Maximum likelihood analysis was performed for separate and concatenates protein sets using the CodeML (AAML) program of PAML version 3.14 (Yang, 1997). Multiple sequences were edited by hand-eye adjustment with GeneDoc (Nicholas et al., 1997). Gaps were treated as missing data. Outgroups were used to root the trees so that the true relationship between P. penetrans and Bacillus species could be determined. Outgroups were selected based on their closeness to the Bacillus spp. and P. penetrans as determined by BLASTP BIT scores and initial ClustalW alignments and distances. 107

120 For codon analysis, codon models of (Yang and Nielsen, 1998) and the Bayesian approaches models (Yang et al., 2005) were implemented to infer sites under diversifying selection. Nucleic acid sequences for all genes were loaded into MegAlign, translated, aligned and back translated to recover aligned codons. The alignments were edited manually using GeneDoc with the following criteria, (i) gaps were treated as missing data and (ii) regions where there were less than three sequences aligned were trimmed to prevent two taxa from seeming more closely related than they in fact are. Codon models were applied to 46 sporulation gene data sets to infer positively selected sites. Table 2 gives the details of sequence length and number of codons. Tree topologies were estimated using a one-ratio model with the omega held constant (ω = 0.4) for all lineages; the F3x4 model of codon frequency was used. To save on computation, we initially used model M0 (one-ratio) to estimate branch length and fixed them when fitting site model M8 (Yang et al., 2005). Phylogenies were constructed using Bayesian inference and Markov Chain Monte Carlo (MCMC) techniques, as employed by version 3.1 of the MrBayes software (Huelsenbeck and Ronquist, 2001; Ronquist and Huelsenbeck, 2003). A mixed model was chosen for the amino acid alignments of the proteins. For the codon alignments a general time reversible (GTR) model was used. In each case, four chains were run for 50,000 iterations, sampling every 50 iterations for a total of 1,000 samples. Graphs indicate the first 50 data points were sufficient to reach stationarity. These data points were discarded as burnin. Two additional runs from different random starting points were performed to confirm parameter convergence. A concatenated set of all proteins was analyzed with the same initial parameter set and run for 100,000 iterations, sampling every 50 iterations. The first 50 data 108

121 points were again discarded as burn-in. All phylogenetic trees were viewed and edited using Tree View version (Page, 1996). 109

122 RESULTS Of the phylogenetic analyses performed with 46 individual sporulation proteins and 7 bacterial species, 41 placed P. penetrans with nonparasitic Bacilli, B. subtilis and B. halodurans with strong support, which is consistent with the concatenated proteins (Fig. 1). Bayesian analysis for concatenated protein phylogenies were concordant maximum likelihood findings (Fig.1 and 2). Figure 1 is a maximum likelihood inferred evolutionary tree for 46 concatenated proteins from the five Bacillus species and P. penetrans. The parasitic species B. cereus, B. thuringiensis and B. anthracis are clustered together and are newly diverged and more closely related to B. subtilis than to P. penetrans, the basal member of this group (Charles et al., 2005). The single gene trees inferred for the SpoVAE protein by Bayesian and maximum likelihood analyses (Fig. 3) varied in the placemen of P. penetrans. These trees both placed P. penetrans between B. anthracis and B. cereus (Fig. 3). The spovae gene is a member of a six-gene operon and all of these proteins have membrane-spanning domains (Stragier and Losick, 1996). The single gene tree for this gene was incongruent in Bayesian and maximum likelihood analyses as well as for the concatenated trees. Although the DacB protein of Pasteuria penetrans remains at the basal position with respect to the all five Bacillus spp. (Fig. 4), it is apparently more similar to the parasitic species, B. cereus, B. anthracis, and B. thuringiensis; than the proteins of the nonparasitic species. dacb is a σ E -regulated gene that is expressed in the mother-cell and whose product becomes associated with the mother-cell membrane and the forespore outer membrane where it is active in regulation of the low degree of cross-linking of spore peptidoglycan. 110

123 The single gene tree for Bayesian analysis clustered P. penetrans with B. subtilis and their next closest relative was B. cereus (Fig. 5). Functionally, SpoIIAA is referred to as anti-antisigma factor or the antagonist of SpoIIAB (Stragier and Losick, 1996). In Bacillus subtilis, SpoIIAB inhibits the interaction of σ F with RNA polymerase (RNAP) by sequestering it in a ternary complex with ATP. The SpoIIAA protein binds to SpoIIAB in the presence of ADP selectively in the forespore (Stragier and Losick, 1996; Campbell et al., 2002; Iber et al., 2006) liberating σ F and allowing its interaction with RNAP. Figure 6 is the single gene tree for for the small acid soluble spore protein (SASP-B, SspB) which has P. penetrans situated between B. subtilis and B. anthracis. These proteins are localized on the nucleoid in developing forespores and to the ring-shaped nucleoid of the germinating spore (Stragier and Losick, 1996). We performed codon-level analyses on each gene individually to determine whether specific sites within lineages were under diversifying selection. Preliminary analysis of each gene using the one ratio model (M0) (Table 2) did not suggest that any lineages or branches (representing genes in this case) were under diversifying selection as determined by dn/ds (ω). However, it is known that particular sites within a gene may be under diversifying selection pressure, while the other majority of sites are under purifying selection (Yang, 2005). Models 7 and 8 of (Yang et al., 2000) were used to construct likelihood ratio tests to determine the presence of sites under diversifying selection (Table 3). Not surprisingly, results of these analyses suggest that in general sporulation proteins are not under diversifying pressure. However, certain membrane proteins, SpoIIIAA, SpoIIIAB (Fig. 7) and one DNA binding protein, SspB (Table 3) has sites that were predicted to be under diversifying selection pressure. However, we subsequently performed deep phylogenetic 111

124 analyses on these proteins with by exhaustively searching GenBank for all relevant proteins. Results of these subsequent analyses yielded predictions of different and identical codon sites potentially under diversifying selection, however none of which were well supported (data not shown). Figure 7 is a codon-level-inferred phylogenetic trees for the initial comparison between P. penetrans and the five Bacillus spp. (Fig 7A) and with a total of 22 other taxa (Fig.7B). Although P. penetrans is within the Bacillus-Clostridium group, in this evaluation it is placed between Desulfotobacterium hafniense and Dessulfotomaculum reducens, both of which are dehalogenating bacteria commonly used in industry to degrade contaminants and pollutants (Villemur et al., 2002). For consistency C. perfringens was used as the outgroup for both phylogenies (Fig. 7A, 7B). 112

125 DISCUSSION The initial phylogenetic analyses performed with 46 individual sporulation proteins and 7 bacterial species, a six-taxa ingroup and one-taxon outgroup, yielded slight differences in tree topology. These variances stem from the relative placement of P. penetrans with the nonparasitic species. Three topologies were prevalent: i) P. penetrans basal to B. subtilis or 2) B. halodurans; or 3) placed between the two of them. However, in 41 of 46 single trees, P. penetrans clustered with nonparasitic Bacillus spp., B. subtilis and B. halodurans with strong support, which is consistent with the concatenated proteins (Fig. 1) for Bayesian and maximum likelihood analyses (Fig. 2). Furthermore, P. penetrans appears to be genetically more similar to B. subtilis and B. halodurans (non-parasitic species), rather than to the pathogenic ones. This finding was consistent with both methods used. The variation between single gene trees observed here is not uncommon (Rokas et al., 2003) and the use of combined protein data helped resolve this problem (de Queiroz et al., 1995). However, differences in single gene tree topologies primarily resulted in membrane associated proteins (Figs. 3-7). The outer surface of Gram-positive bacteria is the facilitator of host parasite interactions and is responsible for mediating virulence (Mesnage et al., 1998; Navarre and Schneewind 1999; Cossart and Jonquières, 2000). The cell wall of Gram-positive bacteria contain an outer peptidoglycan layer, a large covalently linked molecule which forms a supportive net around a bacterium that resembles a chain-link fence. Similarities of P. penetrans with the membrane proteins for example with SpoVAE, DacB, SpoIIAA and SpoIIIAB (Fig. 3, 4, 5, 7, respectively) could imply mechanisms by which Pasteuria interact with their nematode hosts. 113

126 Detecting positive Darwinian selection is an area of considerable interest and resolving inconsistencies in accurately predicting sites under diversifying selection remains a hurdle (Suziki and Nei, 2004; Massingham and Goldman, 2005; Zhang et al., 2005). Studies presented here suggest that current models for selecting diversifying selection are not yet suitable for applications to small data sets. Sporulation is a key development process that allows Gram-positive cells to become dormant during times of environmental stress. This process has been well characterized in B. subtilis, which serves as a model for cell differentiation. Phylogenetic data presented here and previous experiments in our lab (Charles et al., 2005) suggest that Pasteuria diverged very early from other Bacillus spp. and P. penetrans is more closely related to B. halodurans and B. subtilis, than to pathogenic species B. thuringiensis, B. anthracis and B. cereus. The apparent homology between the sporulation genes from P. penetrans and the Bacillus spp. suggest that Pasteuria may undergo sporulation in a similar manner. Furthermore, variations in the single gene trees of select membrane-associated and surface proteins suggest a possible role in virulence. Although the fastidious nature of P. penetrans makes it difficult to carry out classical genetic experiments at this time, comparative genomics/proteomics has made is possible to infer plausible explanations about the nature of this pathogenic bacterium. 114

127 ACKNOWLEDGMENTS We thank Mark Burke for Bioinformatic support. J. T. W. was a NSF IGERT Fellow. This work was supported by the North Carolina Agricultural Research Service and by Rothamsted Research, Ltd. 115

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136 TABLES Table 5.1. Description of sporulation protein used in present study. Protein CotD CotJA Description a Spore coat protein (inner) Polypeptide composition of the spore coat; required for the assembly of CotJC DacB DacF FtsA Gdh GerM b KbaA KinA KinB KinC KinD KinE SigA SigE SigF SigG SigK D-alanyl-D-alanine carboxypeptidase (penicillin-binding protein) Penicillin binding protein with D,D-carboxypeptidase Septation protein Glucose 1-dehydrogenase Germination protein containing a lipoprotein-like signal peptide KinB signaling pathway activation protein two-component sensor histidine kinase two-component sensor histidine kinase two-component sensor histidine kinase two-component sensor histidine kinase two-component sensor histidine kinase RNA polymerase major sigma-43 factor (sigma-a) Early mother cell-specific gene expression Early forespore-specific gene expression RNA polymerase sporulation-specific sigma factor (sigma-g) RNA polymerase sporulation-specific sigma factor (sigma-k) (N-terminal half) 124

137 Spo0A Spo0F Spo0J SpoIIAA SpoIIAB SpoIIE SpoIIQ SpoIIR SpoIIIAA SpoIIIAB SpoIIIAD SpoIIIAE SpoIIIAG SpoIIIAH SpoIIIE SpoIVA SpoIVB SpoIVFA two-component response regulator two-component response regulator site-specific DNA binding protein anti-anti-sigma factor (antagonist of SpoIIAB) anti-sigma factor (antagonist of sigma-f) and serine kinase serine phosphatase Membrane associated protein, required for completion of engulfment required for processing of pro-sigma-e Membrane associated protein, mutants block sporulation after engulfment Membrane associated protein, mutants block sporulation after engulfment Membrane associated protein, mutants block sporulation after engulfment Membrane associated protein, mutants block sporulation after engulfment Membrane associated protein, mutants block sporulation after engulfment Membrane associated protein, mutants block sporulation after engulfment DNA translocase Required for proper spore cortex formation and coat assembly Serine peptidase of the SA clan Inhibition of SpoIVFB (negative regulation) and hypothesized to stabilize the thermolabile spoivfb product (positive regulation) SpoIVFB SpoVAC Membrane metalloprotease Sporulation protein VAC, mutants lead to the production of immature spores SpoVAE Sporulation protein VAE, mutants lead to the production of immature 125

138 spores SpoVB SpoVD SspA SspB SspC SspD SspI Involved in spore cortex synthesis Penicillin-binding protein involved in cortex synthesis Small acid-soluble spore protein (alpha-type SASP) small acid-soluble spore protein (beta-type SASP) small acid-soluble spore protein (alpha/beta-type SASP) small acid-soluble spore protein (alpha/beta-type SASP) small acid-soluble spore protein a Descriptions from Stragier and Losick (1996) and B. subtilis genome annotations (Kunst et al. 1997). b Germination protein. 126

139 Table 5.2. Maximum likelihood estimates of parameters for data set analyzed in this article. a Gene L c κ ω S CotD CotJA DacB DacF FtsA Gdh GerM KbaA KinA KinB KinC KinD KinE SigA SigE SigF SigG SigK Spo0A Spo0F Spo0J SpoIIAA SpoIIAB SpoIIE SpoIIQ SpoIIR SpoIIIAA SpoIIIAB SpoIIIAD SpoIIIAE SpoIIIAG SpoIIIAH SpoIIIE SpoIVA SpoIVB SpoIVA SpoIVB SpoVAC SpoVAE SpoVB SpoVD SspA

140 SspB SspC SspD SspI a Basic statistics for the data set analyzed in this article estimated under the simple model (M0) of one ω ratio for all sites (Goldman and Yang, 1994). L c, number of codons in the sequence; κ, transition/transversion (mutation) rate ratio; ω, Nonsynonymous/synonymous rate ratio, d N/ d S ; S, tree length, measured by the number of nucleotide substitutions along the tree per codon. 128

141 Table 5.3. Log-likelihood values for M7 (H 0 ) vs. M8 (H A ) LRT for Sporulation Genes and positively selected sites. Gene l 0 (M7) l 1 (M8) 2Δl P value Positively Selected Sites a SpoIIIAA H, 163E SpoIIIAB M SspB Y a Model 8 (beta & omega) was used to infer sites under diversifying selection. Sites inferred under selection at the 95% level are listed in italic and those reaching 99% are shown in bold. 129

142 FIGURES Figure 5.1. Maximum likelihood analysis of the 46 concatenated proteins for six bacterial species. Phylogenetic trees were inferred for 46 concatenate sporulation proteins for six taxa (Bacillus subtilis, Pasteuria penetrans, Bacillus halodurans, Bacillus anthracis, Bacillus cereus and Bacillus thuringiensis) using a maximum likelihood. The Bootstrap values are shown. 130

143 Figure 5.2. Bayesian analysis for the 46 concatenated sporulation proteins from six bacterial species. The concatenated set of Pasteuria penetrans sporulation proteins were placed in an ancestral position relative to the Bacillus spp.. The alkaliphilic bacterium, B. halodurans is its closest relative as inferred by Bayesian analysis. Markov Chain Monte Carlo analysis was ran for 100,000 iterations and resulted in a single tree with 100% support. 131

144 Figure 5.3. SpoVAE single gene trees for seven bacterial species. The single gene tree for SpoVAE differed from the concatenated proteins dataset in it placement of P. penetrans. (A) Evolutionary tree inferred from Bayesian analysis placed P. penetrans between B. anthracis and B. cereus with good support. Markov Chain Monte Carlo (MCMC) analysis consisted of a 50,000-generation run. (B) The SpoVAE protein of P. penetrans was more closely related to that of B.anthracis and B.halodurans as inferred by maximum likelihood analysis. Clostridium perfringens was used as an outgroup to root the tree. 132

145 Figure 5.4. Bayesian analysis of DacB. Phylogenetic analysis of the DacB protein revealed a tendency for the parasitic Bacillus species, B. cereus, B. anthracis, and B. thuringiensis to cluster near P. penetrans with good support as shown by posterior probability values. The B. cereus DacB protein is more closely related to P. penetrans than proteins from the nonparasitic species, B. subtilis and B. halodurans. Number indicate percent support from 1,000 iterations. 133

146 Fig SpoIIAA single gene cladogram inferred by Maximum likelihood analysis. The maximum likelihood for the single gene tree placed P. penetrans and B. subtilis and B. cereus with 99% confidence. Bootstrap values represent 1,000 generations. Streptococcus coelicolor was used as an outgroup. 134

147 Fig Small acid soluble spore protein B single gene tree. The small acid soluble spore protein B (SspB) of P. penetrans is positioned between B. subtilis and B. anthracis as inferred by Bayesian analysis. These evolutionary distances suggest that P. penetrans should be placed basal to B. halodurans. Letters at above branches represent Bayesian estimated branch lengths as follows: a, 0.746; b, 0.054; c, 0.166; d, 0.559; e, 0.074; f, 0.119; g, 0.82; h, 0.013; i, Clostridium perfringens is the outgroup. Markov Chain Monte Carol (MCMC) analysis was run for 50,000 iterations. Scale bar represents 0.1 codon substitutions per site along a given branch. 135

148 Fig.5.7. Bayesian Analysis of SpoIIIAB codons. Analyses of the codons of the SpoIIIAB membrane-associated protein inferred diversifying selection using the single gene analyses at the codon level using Clostridium perfringens as the outgroup. (A) The maximum likelihood inferred phylogram for seven species. Pasteuria penetrans is basal to the Bacillus species. (B) A deep phylogeny of the SpoIIIAB gene using 23 taxa. Pasteuria penetrans is positioned between Desulfitobacterium hafniesens and Desulfotomaculum reducens to the Bacillus spp. species, which cluster tightly near the bottom of the figure. Scale bar represents 1 codon substitutions per site along a given branch. 136

149 137

150 CHAPTER 6: Structural and Functional Analysis of the Pasteuria penetrans Sporulation Response Regulator Spo0F Patrick D. McLaughlin 1,*, Jenora T. Waterman 2,*, Richele J. Thompson 1, Keith G. Davies 3, Brian R. Kerry 3, David McK. Bird 2,3, Charles H. Opperman 2,3 and John Cavanagh 1 1 Department of Molecular and Structural Biochemistry North Carolina State University, Box 7622 Raleigh, North Carolina Center for the Biology of Nematode Parasitism Department of Plant Pathology, North Carolina State University, Box 7253 Raleigh, North Carolina Nematode Interactions Unit Rothamsted Research, Ltd. Harpenden, Herts AL5 2JQ, United Kingdom * These authors contributed equally to this work. To whom correspondence should be addressed. C.H.O. (Office: , Fax: , warthog@ncsu.edu) and J.C. (Office: , Fax: , john_cavanagh@ncsu.edu). 138

151 ABSTRACT Pasteuria penetrans is an endospore forming hyperparasite of root-knot nematodes (Meloidogyne spp.) and is a member of the Bacillus-Clostridium clade of Gram-positive bacteria. Bacillus subtilis, the reference model endospore-forming bacteria including P. penetrans, utilizes an intricate phosphorelay signal transduction system to internalize the unfavorable growth signal(s). Spo0F is the secondary messenger in this phosphorelay and its three dimensional structure allows it to function effectively in this capacity. This chapter details the results of structural and functional analysis of P. penetrans Spo0F with gene and protein level comparisons with the model organism, B. subtilis. Protein alignments and phylogenic studies revealed that P. penetrans Spo0F is at least 73% similar to the Bacillus Spo0F proteins and it is more closely related to B. subtilis and B. halodurans proteins than to parasitic ones, such as B. anthracis. In silico analysis of P. penetrans Spo0F revealed that this protein is significantly more hydrophobic than other Bacillus spp. Results of 15 N heteronuclear single quantum correlation (HSQC) experiments show that P. penetrans Spo0F adopts a similar three-dimensional conformation to B. subtilis Spo0F and homology modeling studies revealed that the conserved active site residues are presented in a slightly different arrangement in P. penetrans compared to B. subtilis Spo0F. Despite subtle diffenences in these reponse regulators they are functionally the same. Therefore, it is likely that sporulation in P. penetrans involves the same response regulator, Spo0F, and this secondary messenger may be under similar regulatory control as other Bacillus species. 139

152 INTRODUCTION Pasteuria penetrans is an obligate endoparasite of Meloidogyne species (root-knot nematodes: RKN), which are agriculturally-important pests because they cost the industry billions of dollars annually. Pasteuria penetrans has been shown to be efficient in controlling M. arenaria populations (Hewlett et al., 1997). Endospore are the infectious stage in the P. penetrans life cycle, which begins with spores attached to the cuticle of second stage juveniles (J2) migrating through soil in search of a nutrient source. Once J2 enter host root vasculature and begin to feed on specialized, nematodeinduced feeding cells (Bird, 1961), endospore germinate, in a process that remains unclear. A germ tube penetrates the nematode cuticle and enters that pseudocoelom and begins to proliferate in the female reproductive tract. Germination is complete when mycelia originating from the germ tube begin to divide into vegetative rods and microcolonies (Davies, K. G. and Hewlett, T. E. unpublished data). As the nematode continues to feed, molt and grow, P. penetrans cells proliferate within the females, reducing fecundity and ultimately killing the female. Approximately two million endospore are released into the surrounding soil upon root and female cadaver decay (Sayre et al., 1991). Although advances have been made with respect to in vitro culturing of P. penetrans (Smith et al., 2004; Gerber and White, 1997), exponential growth has yet to be reported. This caveat has made Pasteuria unamenable to traditional molecular manipulations such as mutagenesis and gene knock-out studies because it is difficult to grow adequate amounts of vegetative cells. Therefore comparative genomic tools were employed for this study. Sequencing has begun to obtain the entire genome sequence of 140

153 P. penetrans. Preliminary analysis of the partial P. penetrans genome has revealed that P. penetrans is closely related to the Bacillus species. Recent phylogenetic studies on 40 housekeeping genes from 33 bacterial species (including P. penetrans) confirmed the placement of P. penetrans within the Bacillus-Clostridium clade of Gram-positive bacteria (Charles et al., 2005). Furthermore, P. penetrans was determined to diverge prior to expansion of the Bacillus clade, and surprisingly, to be more closely related to nonparasitic species B. halodurans and B. subtilis than parasitic relatives B. anthracis, B. cereus (Charles et al., 2005). The fact that P. penetrans is a close relative of Bacillus subtilis, a model organism whose level of attention from researchers is only rivaled by Escherichia coli, makes studying the less pliable Pasteuria more feasible. Using the well-characterized B. subtilis sporulation model will aid in elucidating the sporulation process in Pasteuria; thus providing the framework necessary for understanding the basic biology of this bacterium as well as provide insight for designing functional experiments. Under poor growth conditions such as lack of nutrients or increased external cell density (Stragier and Losick, 1996), B. subtilis will abandon the binary fission mode of cell division and undergo sporulation. There are diverse metabolic pathways that modulate this high adaptability in microbes and lower eukaryotes, one of which is the two-component system and, the more complex, phosphorelay signal transduction system (Burbulys et al., 1991; Hoch, 2000; Hoch and Varughese, 2001). The sporulation phosphorelay involves the coordinated actions KinA/B, Spo0F, Spo0B and Spo0A (Fig. 1) which results in the expression of sporulation-specific genes. KinA has been observed as the preferred kinase for phosphorylating Spo0F in laboratory settings (Jiang et al., 2000). The kinases sense internal (KinA) and external (KinB) sporulation signals (Fig. 1, 141

154 Feher et al., 1998) and transfer such signals to Spo0F a response regulator by phosphorylation Asp54 of B. subtilis Spo0F following autophosphorylation (of the sensor kinase) (Hoch and Varughese, 2001). Spo0F~P then becomes a substrate for Spo0B, which is a phosphotransferase that catalyzes the transfer of the phosphate from Spo0F to Spo0A, a transcription factor that activates sporulation essential genes and represses those which inhibit sporulation (e.g., abrb) (Burbulys et al., 1991; Hoch and Varughese, 2001). The structure of B. subtilis Spo0F was solved by x-ray crystallography (Madhusudan et al., 1996, 1997) and nuclear magnetic resonance, NMR (Feher et al., 1997). Spo0F is a single domain response regulator, composed of 5 parallel β-strands, 5 α-helices and 5 loops. The three-dimensional (3D) arrangement of the secondary structural elements of Spo0F consists of a (α/ β) 5 fold with a centrally-located 5-strand-βsheet surrounded by the 5 α-helices α1-helix and α5-helix on one side and α 2, 3, 4- helices on the other (Hoch and Varughese, 2001). The loops, α -helices and β3-strand form the active site, which contains five conserved residues, three aspartates, D10, D11, D54, a threonine, T82, and one lysine, K104. Aspartate-54 is located at the end of β3- strand and is the residue that becomes phosphorylated by Kinase A or Kinase B (Feher et al., 1997). The other active site residues are located on loops, D10 and D11 are on loop 2, and T82 is on loop 4 and K104 is part of loop 5 (Feher et al., 1997). The acidic aspartyl residues of the active site form a negatively charged pocket that is good for binding cations. It has been reported that Spo0F binds magnesium, which is believed to play a role in stabilizing the phosphorylated state of Spo0F (Madhusudan et 142

155 al., 1996; Zapf et al., 1996; Feher et al., 1998; Hoch and Varughese, 2001) and phosphorylation reactions. Magnesium binding by the aspartyl pocket has local and to some degree, global effects on the structure of Spo0F. There is rigidity in the active site of the apo form of Spo0F (Madhusudan et al., 1997). For example in the apo form of Spo0F, this rigidity can be attributed to the hydrogen-bonding network established by the Nζ of K104, which interacts strongly with carboxylates of D10, D54 and the carboxyamide of E21, which have N O distances of 2.83, 2.65, and 3.19Å, respectively. When the aspartyl pocket binds a divalent cation, such as Ca 2+, two of these interactions are disrupted (D54 and E12) and only the interaction between K104 and D10 remains intact (Madhusudan et al., 1997). Another interesting characteristic of the active site is that the side chain has a positively charged arginine residue, R47, which protrudes into the aspartyl pocket and forms salt bridge interactions with the carboxylate of D54. In order for proteins to become active there must be a conformation change and phosphorylation is a common way in which proteins can become activated (Creighton, 1993). Phosphorylation induces conformational changes because it causes the formation of new hydrogen bonding between the negatively charged phosphoryl group and hydrogen (proton) donors within proteins. As previously mentioned, Spo0F interacts with several classes of proteins (Fig. 1) such as sensor kinases, a phosphotransferase, other response regulators and phosphatases (RapA, RapB, and Spo0E) (Stragier and Losick, 1996; Feher et al., 1998; Hoch and Varughese, 2001). The interactions with KinA/B, Spo0B and Spo0A, which have a positive influence on sporulation, will be discussed. 143

156 Although Spo0F is a relatively weak cation-binding protein compared to other response regulator family members, magnesium is required for its phosphorylation by KinA. Spo0F has a lower affinity for Mg 2+ than CheY (a homologue found in E. coli), binding affinities are respectively 20mM and 0.5mM, while phosphorylation half-lives are 12 hours and 20 seconds, respectively (Feher et al., 1995). However, the fact remains that KinA/B phosphorylates Spo0F in a magnesium-dependent manner. Therefore, in the presence of Mg 2+ Spo0F will be phosphorylated by KinA/B at residue D54 and the resulting product, Spo0F~P, becomes a substrate for Spo0B. It has been reported that Spo0B functions as a dimer (Varughese et al., 1998) and thus has two active sites and therefore, two histidine (H30) residues that can interact with two molecules of Spo0F~P. Co-crystallization of Spo0F and Spo0B (Zapf et al., 2000) revealed that the 3Darrangement of the α1-helix of Spo0F puts D54, of Spo0F, and H30 of Spo0B in close proximity, thus facilitating phosphoryl transfer from Spo0F to Spo0B (Varughese, 2002) which is then transferred to Spo0A in a reversible reaction. The α1-helix of each Spo0F molecule actually becomes associated with the α1-helix of one subunit of the Spo0B dimer. There are also interactions between the residues of loop 4 of Spo0F and the α2- helix of the second subunit of the Spo0B dimer. Zapf et al. (2000) presented a model that explains the transition-state intermediate of Spo0F during phosphotransfer to Spo0B. The formation of the Spo0F- Spo0B complex puts D54 and H30 in the necessary geometric arrangement ideal for phosphate transfer from the D54 to H30. Also other properties of the active site facilitate this reaction. Several hydrophobic residues strengthen interactions with the phosphate group as it rearranges for transfer to H30 surround the active site (Zapf et al., 2000). 144

157 Lysine 104 and Mg 2+ neutralize its charge during the rearrangement process and the active site is tightly sealed to prevent hydrolysis of the phosphate group during the transition-state (Zapf et al., 2000). In keeping with the theme of phosphorylation-induced conformational changes, there are other key residues that transmit the conformational change to remote parts of the protein by directly or indirectly interacting with the acyl phosphate. Feher et al. (1997, 1998) reported that R16, T82 and K104 directly interact with the acyl phosphate and that M81, L53 and L66 propagate the conformational change at the active site (acyl phosphate) to more remote parts of Spo0F. Coincidently, these same sites have conformational flexibility and are also the sites of protein-protein interactions (Feher et al., 1997). The side chains of R16, T82 and K104 have been shown to be important in post-phosphorylational conformation changes in Spo0F. According to Feher et al. (1998), the side chain of R16 is oriented such that it directly interacts with the D54 acyl phosphate and impacts the local region, which happens to be β1- α1-loop, a region previously identified as a site of protein-protein interactions (Feher et al., 1997). The T82 residue has a γ-hydroxyl group that can adopt multiple conformations, one of which allows it interacts directly with the D54 acyl phosphate. Lysine 104, which is an essential residue of the active site, can also assume a conformation such that it also interacts with the acyl phosphate from its position on the β5-α5 loop. Other residues such as M81 (and to some degree L53 and L66) cause the conformational change to spread to other residues in more remote parts of the protein. Due to the diversity of interactions by Spo0F, it must provide an active site that can selectively accommodate docking of these different proteins. Feher et al. (1997) 145

158 discussed hydrophobic and electrostatic surfaces that would account for the diversity of protein-protein interactions observed in Spo0F. There are two such hydrophobic surfaces within the B. subtilis Spo0F protein. One such surface occurs at the interface of α1- helix and α5-helix (residues G14, I15, I17, L18, V22, P105, F105 and I108) and a second surface exists at α2-3-helices and the β4- α 4 loop (residues L37, L40, I67, R68, V71, I72, L87, M89). Hence, these surfaces provide an ideal environment for protein-protein interactions between Spo0F and Spo0B. The unphosphorylated and phosphorylated forms of Spo0F participate in proteinprotein interactions and therefore, there must be a degree of selective flexibility which allows the protein to adopt multiple conformations permitting these interactions. Studies on the peptide backbone of Spo0F (Feher et al., 1997) showed that there are surfaces that accommodate multiple conformers. For example D11, G14 and G59 comprise an area near the active site that has been speculated to adopt multiple conformations. Two more regions of high flexibility are found at α 1 and β5-α4-loop and the α4-β5-loop. Therefore, these multiple conformations allow Spo0F to adopt arrangements which facilitate protein-protein interactions and catalysis. There is extensive genomic, proteomic, genetic and molecular resources available for B. subtilis, making it an ideal model organism for Pasteuria and other Gram-positive, sporulating bacteria. Recent studies by Kojetin et al. (2005) surveyed the effect of copper ions on sporulation in B. subtilis and P. penetrans and reported that in the presence of copper sporulation was disrupted. In that study, in vitro cultures of P. penetrans were maintained in a proprietary growth and sporulation medium (Pasteuria Biosciences) in the presence or absence of Cu 2+ (copper dependence experiments) and 146

159 with or without Cu 2+, Zn 2+ and EDTA (varied composition experiments) at 30 o C for two weeks. It was determined that in the absence of Cu 2+ sporulation was increased 100-fold over the standard medium formulation, which contained 3.89 mm Cu 2+. To expand on this work, we conducted copper titration experiments were P. penetrans cells were grown in medium containing no Cu 2+ or increasing amounts of copper. The full details of these experiments can be found in Chapter 3 of this dissertation (Kojetin et al., 2005). In this chapter, the observations from comparative protein approaches between Spo0F from Pasteuria penetrans and B. subtilis, B. cereus, B. halodurans, B. anthracis and B. thuringiensis are reported. Maximum likelihood and Bayesian analyses were used to construct the Spo0F phylogeny. Chromatography, NMR, and protein modeling techniques were employed for functional and structural analyses of the P. penetrans Spo0F. 147

160 MATERIALS AND METHODS Spo0F Phylogeny The Spo0F amino acid sequences for B. subtilis, B. anthracis, B. halodurans, B. cereus, and B. thuringiensis were obtained from GenBank (Wheeler et al., 2002). To construct the spo0f protein phylogeny, multiple sequence alignments were preformed for Spo0F proteins of P. penetrans (gene sequence from Dr. Charles H. Opperman unpublished) and closely related Bacillus species using ClustalW version 1.82 in MegAlign (DNASTAR, Inc., Madison, WI). Multiple sequences were edited by handeye adjustment with GeneDoc (Nicholas et al., 1997). Gaps were treated as missing data. Outgroups were used to root the tree so that the true relationship between P. penetrans and the Bacillus species could be determined. Maximum likelihood analysis was performed for proteins using the CodeML program of PAML version 3.14 (Yang, 1997). Bootstrapping was performed using SeqBoot to (PHYLIP package, Felsenstein, 1995) generate 1000 permuted datasets and a consensus tree was generated with Consense (PHYLIP package, Felsenstein, 1995). Bayesian inference and Markov Chain Monte Carlo (MCMC) techniques were performed using MrBayes 3.1 (Huelsenbeck and Ronquist, 2001; Ronquist and Huelsenbeck, 2003). A mixed model approach was employed and four chains were run for 50,000 iterations, sampling every 50 iterations for a total of 1,000 samples. Graphs indicate the first 50 data points were sufficient to reach stationarity and they were discarded as burn-in. Confirmation of parameter convergence was established with two subsequent runs from different random starting points. Phylogenetic trees were viewed and edited using Tree View version (Page, 1996). 148

161 Pasteuria penetrans Spo0F Homology Modeling and Parameter Estimation. PSIPRED v2.5 ( McGuffin et al., 2000) and ProtParam ( Gasteiger, 2005) programs were employed to predict the secondary structure and general functionality parameters of P. penetrans Spo0F, respectively. The P. penetrans spo0f homology structure was generated using the multiple sequence alignment interface of Swiss-Prot s SWISS-MODEL software (Guex and Peitsch, 1997) using the B. subtilis Spo0F NMR solution structure (PDB Code = 2FSP_) as the reference model. Multiple sequence alignments were constructed as described above and loaded into SWISS- MODEL. Normal PDB format was designated for output files. Construction of the pet 43.1a P. penetrans Spo0F expression vector The P. penetrans Spo0F vector template was provided by Dr. Charles H. Opperman (N. C. State University, Raleigh, NC). The purified protein corresponds to the wild-type full length sequence of Spo0F fused to an N-terminal, thrombin cleavable NusA solubility enhancement tag. Two additional residues (GS) are added to the N- terminus of the protein as a consequence of the removal of the NusA-tag. The Spo0F gene sequence insert was amplified by PCR methods using the forward and backward oligonucleotide primer pair shown below: P. penetrans Spo0F-Fwd: 5 -TCCCCCGGGGCAGCATGATGAATGAAA AG -3 P. penetrans Spo0F-bkd 5 -CGGGATCCCTACAGTAGGGAGTAGGTG- 3 Primers were purchased from Integrated DNA Technologies, Inc. (Coralville, IA) and were designed to incorporate SmaI and BamHI restriction endonuclease cleavage sites at the 5 and 3 ends of the coding sequence, respectively. PCR-amplified fragments were 149

162 subsequently cleaved and ligated into the pet43.1a+ protein expression vector (Novagen, San Diego, CA). The resulting plasmid is an isopropyl- -D-thiogalactopyranoside (IPTG) inducible expression vector that confers ampicillin resistance upon its host. The expression vector was introduced into Escherichia coli BL(21)DE3 cells (Novagene, San Diego, CA) and tested for over-expression of the target protein, P. penetrans Spo0F, upon induction with IPTG. All cells transformed with the P. penetrans Spo0F-pET43.1a plasmid produced a fusion protein of approximately 75,000 Daltons; consisting of an N- terminal, cleavable NusA tag (59 KDa) joined to P. penetrans Spo0F (15.4 KDa) by a six histidine-residue (0.8 KDa) linker region. Expression and purification of P. penetrans Spo0F pet43.1a Construct DNA was isolated with a QIAprep mini-prep spin kit (Qiagen, Valencia, CA) according to manufacturer s suggestions. The purified plasmids were transformed into competent E.coli BL(21)DE3 cells (Novagene, San Diego, CA). One liter LB broth containing 100 μg/ml ampicillin was inoculated and grown at 34 C, 140 rpm to an optical density (OD) A 600nm of 0.9. The cells were pelleted by centrifugation and re-suspended in M9T-Amp Medium [40 mm Na 2 HPO 4, 20 mm KH 2 PO 4, 10 mm NaCl, [ 15 N]NH 4 Cl, 60 mm Glucose, 100 μg/ml Thiamine, 100 mm MgSO 4, 10 μm FeCl 3, 0.1 mm CaCl 2, 100 μg/ml ampicillin] and allowed to shake for 30 minutes. IPTG was added to 1 mm final concentration and the cells were grown for 4 hours. The cells were pelleted by centrifugation and re-suspended in 50 mm NaH 2 PO 4, 300 mm NaCl, 0.25 μm 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF) and 0.01% Triton X-100, ph 8.0. The cells were sonicated on ice for 20 cycles of 60-second 150

163 bursts/2 minute rests. The resulting suspension was centrifuged at 13,500 rpm for 15 minutes. The supernatant was collected and saved as the crude extract. The crude extract is applied to a Ni-NTA affinity resin (Qiagen, Valencia, CA). After the P. penetrans Spo0F pet43.1a crude lysate was incorporated into the resin, the column was washed with 5-10 column volumes of wash buffer [50 mm NaH 2 PO 4 and 300 mm NaCl, ph 8.0] and fractions were collected of the purified P. penetrans Spo0F protein using a mm Imidazole gradient in the wash buffer. The increasing concentration of Imidazole is responsible for the release of the His-tag from the Ni-NTA resin. This procedure provided > 90% pure protein after one-step of purification. The steps of protein purification were all monitored by SDS-PAGE chromatography to determine the efficiency of each step. Removal of the thrombin cleavable NusA-tag After the protein fractions were pooled the protein was concentrated to a volume of ml, and 100 units of thrombin (GE Healthcare, Piscataway, NJ) were added to the protein for cleavage of the NusA-tag. This reaction was left to occur overnight (18 hours) at room temperature and monitored by SDS-PAGE gel electrophoresis. Once the cleavage reaction was complete was stopped by the addition of 1 ml of Protease Inhibitor Cocktail (Sigma Aldrich, St. Louis, MO). To prepare the protein for structural studies the protein was extensively dialyzed against the following buffer NMR buffer [20 mm Tris, 50 mm KCl, ph 7.3] and concentrated to a final volume of 600 μl. 151

164 Alternative Expression and purification of P. penetrans Spo0F pet43.1a One liter LB broth containing 100 μg/ml ampicillin was inoculated and grown at 34 C, 140 rpm to A 600nm of The cells were pelleted by centrifugation and resuspended in M9T-Amp Medium [40 mm Na 2 HPO 4, 20 mm KH 2 PO 4, 10 mm NaCl, [ 15 N]NH 4 Cl, 60 mm Glucose, 100 μg/ml Thiamine, 100 mm MgSO 4, 10 μm FeCl 3, 0.1 mm CaCl 2, 100 μg/ml ampicillin] and allowed to shake for 30 minutes. The cells were induced by adding IPTG to a final concentration of 1 mm and grown for an additional 4 hours. Cell were harvested by centrifugation and lysed immediately or stored at -80 o C. Cell pellets were re-suspended in 35 ml of 20mM Tris (ph 8.0), 10mM NaCl, and placed on ice for 30 minutes. Cells were lysed by sonicating on ice for 12 cycles of 60-second bursts (amplitude size = μm) interspersed by 5-10 minute rests. The resulting suspension was centrifuged at 13,500 rpm for 15 minutes. The supernatant (~ 40 ml) was collected and saved as the crude lysate. Seven grams of ammonium sulfate were gradually added to the crude lysate in 0.5 gram amounts over 4 hours. After each hour one ml was collected, pelleted and analyzed by SDS-PAGE. Alternative Removal of the thrombin-cleavable NusA-tag The salt concentration of the crude lysate, which contained the 75 kda Spo0F- NusA fusion protein, was adjusted to 2M. The sample was dialyzed against 4 liters of 20mM Tris (ph 8.0) and 500 units of Thrombin (GE Healthcare, Piscataway, NJ) were added to cleave the six histidine residues linking the NusA-tag to P. penetrans Spo0F. This cleavage reaction was left at room temperature overnight (18 hours) and monitored by SDS-PAGE gel electrophoresis. Once the cleavage was complete, 1 ml of Protease 152

165 Inhibitor Cocktail (Sigma-Aldridge) was added to stop the reaction. To prepare for Hydrophobic Interaction Chromatography (HIC) the salt concentration was adjusted to 2M. Hydrophobic interaction chromatography was then carried out using a Phenyl- Sepharose resin (Sigma-Aldridge St. Lois, Mo). The cleaved lysate was incorporated into the resin, which was then washed with 125 ml wash buffer [20 mm Tris (ph 8.0), 2 M NaCl] followed by fractionation of the sample with a salt gradient from two to zero molar. This reverse salt gradient liberated P. penetrans Spo0F from the resin, which will not bind the resin under low salt conditions. Fractions containing P. penetrans Spo0F were determined by SDS-PAGE. It was discovered that fractions containing Spo0F (~ 15kDa) also had a 50 kda contaminating protein which had to be removed by Size Exclusion Chromatography (SEC). To prepare for SEC, the HIC fractions were collected, pooled and dialyzed against 4 liters of 25mM Tris (ph 7.1), 50 mm KCl for 4 hours and concentrated to a volume of 3 ml. The sample was loaded onto the sizing resin and fractionated. More than 10 constructs were generated in an effort to express and purify P. penetrans Spo0F protein. Supplemental methods for each construct are detailed in the appendix (Supplemental Methods A.5.1). The steps of protein purification were all monitored by SDS-PAGE (see figure 7 for example) chromatography to determine the efficiency of each step. To prepare the protein for heteronuclear spin quantum correlation (HSQC) experiments using NMR, the protein was extensively dialyzed against the following buffer: 20 mm Tris, 50 mm KCl ph of 7.3 and concentrated to 600 μl. 153

166 NMR 15 N HSQC Experiment The 15 N labeled P. penetrans Spo0F samples used for NMR data contained approximately 200 mm protein and were > 90% pure. NMR experiments were run at 300K on a Bruker DRX 500 equipped with 3 radiofrequency channels and a triple axis pulsed field gradient/triple resonance probe (or a Varian Inova 600 spectrometer equipped with 4 radiofrequency channels and a single axis pulsed field gradient/triple resonance probe. 15 N spectral widths were: DRX, 1343 Hz; Inova, 1900 Hz. For 15 N dimension, the carrier set at ppm and data points were collected and referenced to the published chemical shifts for B. subtilis Spo0F (Feher et al., 1997) and analyzed with NMR-View (Johnson and Blevins, 1994). 154

167 RESULTS Spo0F Sequence Homology Results of pairwise and multiple sequence alignments (Fig. 2 and Table 1) show that there is homology of Spo0F proteins structures at primary, secondary and tertiary levels. Table 1 shows the percent identity and similarity between Pasteuria penetrans, B. subtilis, B. halodurans, B. cereus, B. thuringiensis and B. anthracis. There is a high level of identical residue matches among P. penetrans and the Bacillus spp. The degree of residue conservation between these proteins ranged from 73% in the case of B. halodurans, up to 80% with B. subtilis (Table 1). A multiple sequence alignment revealed that Spo0F proteins from P. penetrans and the Bacillus species are highly conserved (Fig. 2). A closer look at the alignment (Fig. 2) revealed the conservation of several residues in the Spo0F of P. penetrans and the other Bacillus species studies, including B. anthracis. For example, it was demonstrated that K56 of B. subtilis Spo0F, conserved in all six taxa studied here, when mutated to an asparagine resulted in 23-fold increase in autophosphatase activity (Zapf et al., 1998). It is believed that the autophosphatase activity is an inherent ability to combat premature phosphorylation. It was deduced by Zapf et al. (1998) that K56 is important for ensuring efficient interactions between Spo0F and Kinase A, the first step in initiating the phosphorelay of the stress signal. Other proximal and distal residues previously reported to stabilize the 3Dstructure of the aspartate active site in B. subtilis Spo0F (Feher et al., 1997), R16, T82, M81, K104, L53, L53, E21, L66, are also present in P. penetrans Spo0F (Fig. 2). Furthermore, other residues that mediate with B. subtilis Spo0B-Spo0F interactions 155

168 (Hoch and Varughese, 2001) are also present in P. penetrans Spo0F, which include I16, E21, K18, F106, L56, in addition to the active site residues previously mentioned (Fig. 2). Inference of the Spo0F Protein Phylogeny Results of both phylogenetic analyses placed P. penetrans with non-parasitic bacteria, B. subtilis and B. halodurans with good support (Fig. 3 and 4). Streptomyces coelicolor, a medically important, Gram-positive spore-forming organism, was chosen as an outgroup since a small dataset (n = 6) was used in these analyses. The outgroup was used to root the Spo0F phylogenetic tree so that the true relationship between P. penetrans that the Bacillus species studied here could be determined. Using S. coelicolor as the phylogenetic root, it was determined that P. penetrans was more closely related to B. halodurans and B. subtilis, than B. anthracis, B. thuringiensis, and B. cereus (Fig. 3 and 4). Phylogenies inferred from maximum likelihood (Fig. 4) and Bayesian (Fig. 3) analyses presented here were consistent with the placement of P. penetrans near B. halodurans in phylogenetic studies with other genes (Charles et al., 2005). Effects of copper on growth and sporulation of B. subtilis and P. penetrans Copper titration studies were preformed by growing cultures of B. subtilis and P. penetrans in media with no copper or in formulations with steady increases in copper concentration (Table 2). Both bacteria had the highest level of growth and sporulation in media without copper (see Experiment 1, Table 2). Vegetative B. subtilis cells grew in medium containing 9.97x10-5 M and 5.01x10-4 M Cu 2+, however no spores were generated (see Experiments 1 & 2, Table 2). On the contrary, P. penetrans was able to 156

169 grow vegetatively and sporulate under those conditions (see Experiments 1 & 2, Table 2). In Experiment 3 (1.47x10-4 M, Table 2) B. subtilis had very little cell growth and no spores were produced, while Pasteuria cultures grew well and produced spores. For Pasteuria cultures, cell growth and sporulation began to fade in medium containing 2.93x10-3 M Cu 2+ (Experiment 4, Table 2). In essence, the two bacteria behaved in a similar manner with respect to copper concentrations at or above 2.93x10-3 M resulted, which in virtually no growth or sporulation (see Experiments 4-7, Table 2). P. penetrans Spo0F general properties and secondary structure prediction Primary structure (raw sequence) comparisons alone cannot determine the true relatedness of the proteins. To gain more insight into understanding the Spo0F proteins structural and functional features were studied. PSIPred was used to predict the secondary structure of P. penetrans (Fig. 5) based on its amino acid sequence. PSIPred acts by comparing the query protein sequence to a database of targets with know secondary structures. To predict secondary structure, PSIPred employs a three step process which involves the generation of a sequence profile as part the search process, prediction of initial secondary structures and finally the predicted structures are filtered to determine the best structure for the query protein. For P. penetrans Spo0F, PSIPred predicted 5 beta-strands and five alpha-helices for interspersed by loop regions, beginning at residue 6 to 134 (Fig. 5). These secondary structures are also present in the Bacillus species analyzed. The ProtParam program was utilized to compare the theoretical and calculated properties of the Spo0F proteins which included basic properties such as molecular mass 157

170 and the distribution of residues in each protein (Table 3) as well as calculated properties based on theoretical functionality predictions stemming from 3D-structure. By comparing the general properties, a striking number of variances were observed among these conserved proteins. By comparing the distribution of positively (aa + ) and negatively charged (aa - ) residues in each protein, it was noted that P. penetrans Spo0F has an equal distribution of these charged residues (see Table 3); implying an inherent net charge of zero. Whereas all of the Bacillus species had consistently more aa - than aa + resulting in overall net negative charges. Moreover, the theoretical pi values for the Bacillus spp. differ greatly compared to P. penetrans Spo0F, which has a theoretical isoelectric point of 6.73, compared to the more acidic pis of the Bacillus spp.. ProtPred was also used to calculate the grand average of hydropathicity (GRAVY) for each of the Spo0F proteins (Table 3). The GRAVY value for a peptide or protein is calculated as the sum of hydropathy values (Kyte and Doolittle, 1982) of all the amino acids, divided by the total number of residues in the sequence. This then results in the overall hydrophobicity of the protein, compared to a hydropathy plot, which gives regional hydropathy scores of the protein obtained by using a size-dependent sliding window (Kyte and Doolittle, 1982). The GRAVY score for P. penetrans Spo0F was 0.204, which was at least 2-fold (in the case of B. halodurans) to more than 10-fold higher than its Bacillus relatives (Table 3). Hydrophobicity is a major characteristic influencing the behavior of proteins (Kyte and Doolittle, 1982) and hydropathy plots are useful in predicting globular (hydrophilic) and (transmembrane) domains in proteins. When the window size is 19, peaks that cross the midline (1.8) indicate possible transmembrane regions and when the window is set to 158

171 9, strong negative peaks are representative of possible surface regions of globular proteins (Kyte and Doolittle, 1982). Currently it is accepted that a window of 7 to 9 detects hydrophobic helices in globular proteins, but a much larger window of 19 to 25 is appropriate for detecting transmembrane helices. The larger window has a) a better smoothing effect, and b) usually only transmembrane segments are that long (Kyte and Doolittle, 1982). Kyte-Doolittle hydropathy analysis of the Spo0F proteins from B. subtilis and Pasteuria penetrans (Fig. 6) indicates that both proteins are generally hydrophilic shown by peaks below the 1.8 midline (Fig. 6A, B) and there are no transmembrane regions (Fig 6C, D) with all peaks falling below 1.8. For example in the first window of fig. 6C and 6D (amino acids 1-19), the values are primarily below zero in B. subtilis and greater than zero, but lower than one in P. penetrans. This pattern can be observed by a step-by-step look at the plots (Fig. 6). A systematic look at each window along the length of the proteins reveals that P. penetrans Spo0F is overall more hydrophobic in than B. subtilis Spo0F. The stability of a protein is extremely important for laboratory investigations. The instability index provides an estimate of the stability of proteins in a test tube setting. Statistical analysis of 12 unstable and 32 stable proteins has revealed (Guruprasad et al., 1990) that there are certain dipeptides, whose occurrence differs significantly in unstable proteins compared with those in the stable ones. As a result of their analyses, Guruprasad et al. (1990) asserted that a protein whose instability index is smaller than 40 is predicted as stable while a value above 40 predicts that the protein may be unstable. P. penetrans Spo0F was predicted to be unstable in a test tube with a corresponding 159

172 instability index of Whereas all Bacillus spp. had indices within the stable range, to (Table 3). Structural Analysis of P. penetrans Spo0F Protein For NMR experiments, protein samples were prepared using column chromatography and SDS-PAGE analysis was used to monitor the purification process. Figure 7 is an example of such a gel. Samples were prepared using HIC followed by SEC and the arrow (Fig. 7) points the ~16 KDa P. penetrans Spo0F protein as the expected size. Analysis of 15 N-HSQC spectra from B. subtilis Spo0F (Fig. 8A) and P. penetrans Spo0F (Fig. 8B) reveal nearly identical peaks; which indicates retention of the three dimensional arrangement of both proteins over time. Numerous regions in the spectra can readily be observed and several have been highlighted, using rectangles, circles and pentagons (Fig. 8). For example in the upper-left quadrant of the both spectra in figure 7, nine major peaks are seen displaying the same pattern, outlined by the rectangle. Other regions have been framed to highlight similarities in the two spectra. Consistent with these finding is the homology model generated for P. penetrans Spo0F (Fig 9B, D). Comparisons with B. subtilis Spo0F NMR solution structure (Fig 9A, C) revealed consistent spatial arrangement of the secondary structures in P. penetrans (Fig. 9B, D). Both proteins have the a (α/β) 5 fold and active site residues (Asp10, Asp11, Asp54, Thr82, and Lys104) are displayed on the same surfaces of the B. subtilis Spo0F and P. penetrans Spo0F proteins as shown in figure 9C and 9D, respectively. 160

173 DISCUSSION There is significant sequence homology between the Spo0F proteins of Pasteuria penetrans, B. subtilis and other closely related Bacillus spp. analyzed in this study (Fig. 2-4). As shown by the multiple sequence alignment (Fig. 2) and secondary and tertiary structure predictions (Figs. 5, 8, 9). Amino acid comparisons revealed that all active site residues (Asp 10, 11, 54, Thr82 and Lys104) in the well-characterized B. subtilis Spo0F are present in P. penetrans Spo0F. Phosphorylation at the carboxylate of Asp54 activates Spo0F in B. subtilis, and is most likely the residue which forms an acyl phosphate in Pasteuria, which is critical for activation resulting in a conformational change of the protein which allows it to interact with the next member of the phosphorelay, Spo0B. The Pasteuria penetrans Spo0F protein also contains identical residues which are necessary for interactions with Spo0B. The in vitro culturing studies presented in this paper hint that positively charged metals, such as copper play a role in modulating sporulation by interacting with the Spo0F, the phosphorelay member whose action is central in integrating stress signals. These is supported by the previously reported Cu 2+ binding studies (Kojetin et al., 2005) and analyses from the present study. It is apparent that there exists a critical level at which copper will allow vegetative grow, but is inhibitory to sporulation. These findings are corroborated by reports of similar observations in B. megaterium the big beast endospore-former important in the biotechnology industry for manufacturing enzymes and antibiotics (Kolodziej and Splepecky, 1962, 1964; Krueger and Kolodziej, 1976; Jun et al., 2003) where certain metals, including copper, were found to impede or prevent growth and/or sporulation. Growing P. penetrans without copper in the medium is beneficial for enhanced sporulation in vitro. Results of in vitro copper titration 161

174 experiments presented here indicate that there are indeed critical levels at which Cu 2+ allow vegetative growth, but inhibit sporulation. For B. subtilis and P. penetrans these critical Cu 2+ levels are mm and 2.93 mm, respectively (Table 2, Kojetin et al., 2005). Looking at multiple sequence alignment is only a first step in determining the relatedness of proteins. Work presented here suggests that proteins may appear to be very similar in terms of sequence. However, the 3D arrangement of the atoms within residues of a protein and interactions between the side chain atoms are better indicators the function of the protein. Comparing the ProtParam program output from several Spo0F proteins from several taxa (Table 3) revealed characteristics that may explain the challenges associated with working with P. penetrans Spo0F in the laboratory. ProtParam revealed a theoretical isoelectric point for P. penetrans Spo0F that was 6.73, almost two points higher than the Spo0F from the model bacterium B. subtilis. The most surprising results from Protparam was the theoretical instability index (II) calculated for P. penetrans Spo0F (Table 3). This finding was particularly surprising and enlightening because the calculated II for P. penetrans Spo0F was 51.14, which is points over the threshold level. This is consistent with our observations of instability of the protein during technical preparations. Despite the inherent difficulties, NMR-levels of P. penetrans Spo0F protein were successfully prepared to conduct structural and functional studies (Fig. 8). The diversity of characteristics observed in P. penetrans Spo0F could be due to the niche P. penetrans occupies that allows its protein to perform similar tasks as its relatives, but under very different conditions. To date, not detailed studies on the milieu of the root-knot nematode ovary have been conducted. 162

175 However, it is plausible that the environment within the nematode, specifically the ovary, has had an evolutionary effect on P. penetrans Spo0F which allows is to function like a typical sporulation regulator, while differing significantly from its Bacillus spp. relatives. Obtaining a functional P. penetrans Spo0F protein for in vitro studies has been extremely challenging, probably due the inherent hydrophobic nature of the protein. It was determined that P. penetrans Spo0F readily flowed through several columns, even under carefully predicted buffer conditions (data not shown). Grand average of hydropathicity analysis (Table 3) and hydropathy plots (Fig. 6) showed revealed a high degree of hydrophobicity in P. penetrans Spo0F compared to its Bacillus relatives analyzed here. For example P. penetrans Spo0F is twice as hydrophobic as B. halodurans Spo0F, and its hydrophobicity exceeds the other that of the other protein by an even greater margin (Table 3). The peaks on the HSQC spectra for P. penetrans (Fig. 8B) were more diffuse than B. subtilis (Fig. 8A) because the concentration of P. penetrans Spo0F was approximately half that of the B. subtilis Spo0F samples. This is due the instability of P. penetrans Spo0F and the challenges associated with obtaining adequate amounts of the protein. However, even with a lower concentration of the P. penetrans Spo0F NMR sample, it is clearly obvious these proteins have an incredible degree of conserved 3D homology. In summary, we propose that aspects of sporulation involving Spo0F in P. penetrans proceed in a similar manner as B. subtilis. Comparative analyses of Spo0F proteins from P. penetrans and closely related Bacillus species, including the model bacterium B. subtilis, has revealed a high level of conservation in terms of phylogeny and 163

176 apparently, function. Pasteuria penetrans Spo0F displays high similarity with other members of this class of response regulators. It adopts a (β/α)-5-barrel scaffold with its negatively charged active site situated at the carboxyl end of the β-strands. It is likely that P. penetrans Spo0F behaves in the same manner as B. subtilis Spo0F. The structure of Spo0F has a significant impact on its functionality. Although proteins must maintain some rigidity to be stable in their native form, it is clear that there must be a degree of flexibility to allow conformational changes and subsequently, protein-protein interactions. Spo0F is a key player in the phosphorelay signal transduction pathway of B. subtilis and its 3D structure makes is optimal for propagating the sporulation signal. The active site residues form a negatively charged aspartyl pocket, necessary for binding magnesium, with itself is essential for phosphorylation of Spo0F. By altering the composition of the growth/sporulation medium of P. penetrans, growth and sporulation was enhanced. Through recent work by Kojetin et al. (2005) and Chapter 3 of this thesis, it is plausible that copper molecules interacting with the response regulator Spo0F is mainly responsible for these phenomena. The inherent differences between P. penetrans Spo0F and other closely related Bacillus spp. does not have any apparent effects on its ability to internalize and integrate the sporulation stress signal necessary for the initiation of sporulation. As more of the P. penetrans genome becomes available, it will undoubtedly provide even greater insight into understanding the physiology and metabolism of this fastidious organism. Furthermore, the analyses presented here show that with a combination of the right tools and approaches it is possible to study molecules from the most difficult systems, such as Spo0F from the impenetrable P. penetrans. 164

177 ACKNOWLEDGMENTS We thank Elizabeth H. Scholl for Bioinformatic support. J. T. W. was an NSF IGERT Fellow. This work was supported by the North Carolina Agricultural Research Service, Syngenta and by Rothamsted Research, Ltd. 165

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185 TABLES Table 6.1. Percent identity and similarity between Pasteuria penetrans and Bacillus species Spo0F proteins and e-values as reported by BLASTP. Species Identity Similarity Expect Bacillus anthracis 53% 78% 7e -31 Bacillus cereus 53% 78% 7e -31 Bacillus halodurans 55% 73% 7e -31 Bacillus subtilis 53% 80% 2e -32 Bacillus thuringiensis 53% 78% 7e

186 Table 6.2. Summary of the effect of copper on vegetative cell growth and spore formation in Bacillus subtilis and Pasteuria penetrans. Bacillus subtilis a Pasteuria penetrans b Experiment Cu 2+ added Spore Spore (M) Cell growth formation Cell growth formation 0 0 Yes Yes Yes Yes x10-5 Yes No Yes Yes x10-4 Yes No Yes Yes x10-4 Little No Yes Little x10-3 No N/A Little Poor x10-3 No N/A No N/A x10-3 No N/A No N/A x10-1 No N/A No N/A a Data taken from Kojetin et al., b Data taken from Chapter 3 of this thesis. 174

187 Table 6.3. ProtParam parameter predictions for Spo0F proteins. Parameter Bacillus Bacillus Bacillus Bacillus Bacillus Pasteuria anthracis cereus halodurans subtilis thuringiensis penetrans L aa MW (Da) pi aa aa AI GRAVY II 20.33/S 20.33/S 30.75/S 25.49/S 20.33/S 51.14/U L aa = Number of amino acids in protein. MW = Molecular weight in Daltons. pi = Theoretical pi. aa + = Total number of positively charged residues (Asp + Glu). aa - = Total number of negatively charged residues (Arg + Lys). AI = Aliphatic index. GRAVY = Grand average of hydropathicity. II = Instability Index, S, stable; U, unstable. 175

188 FIGURES Figure 6.1. Diagram of proteins involved in the phosphorelay required for initiating the sporulation signal transduction pathway in B. subtilis. The open arrows represent environmental signals, which initiate the transfer of the phosphoryl group via the phosphorelay. Spo0A activates sporulation and represses abrb, represented respectively by the shaded arrow and crossbar. (Diagram from Feher et al., 1998) 176

189 Figure 6.2. Spo0F amino acid sequence comparison. Amino acid sequences from Bacillus thuringiensis (Bthu_Spo0F), Bacillus cereus (Bcer_Spo0F), Bacillus anthracis (Bant_Spo0F), Bacillus subtilis (Bsub_Spo0F), Bacillus halodurans (Bhal_Spo0F), and Pasteuria penetrans (Ppen_spo0F) were aligned using ClustalW. Active site residues (D10, D11, D54, T82 and K104 in B. subtilis) are highlighted with rectangles. Residues are colored as follows: AVFPMILW = red; DE = blue; RHK = magenta and STYHCNGQ (Chenna et al., 2003). Consensus symbol at the bottom of each column denotes the degree of conservation. *, identical residues in all sequences; :, highly conserved column;., weakly conserved column (Ramu et al., 2003). 177

190 Figure 6.3. Bayesian analysis of the response regulator Spo0F. A Bayesian method was used to construct the Spo0F phylogeny using seven taxa: Bacillus thuringiensis, Bacillus cereus, Bacillus anthracis, Bacillus subtilis, Bacillus halodurans, Pasteuria penetrans and Streptomyces coelicolor (outgroup). The Markov Chain Monte Carlo analysis was run for 50,000 iterations and posterior probabilities are shown. 178

191 Figure 6.4. Maximum likelihood phylogram of Spo0F. Maximum likelihood analysis of Spo0F proteins from Bacillus thuringiensis, Bacillus cereus, Bacillus anthracis, Bacillus subtilis, Bacillus halodurans, Pasteuria penetrans and Streptomyces coelicolor (outgroup) was performed. This phylogram positioned P. penetrans near non-parasitic Bacillus spp., B. subtilis and B. halodurans. Bootstrap values above 50% are shown. Scoe, outgroup. 179

192 Figure 6.5. PSIPred secondary structure prediction for Pasteuria penetrans Spo0F protein. 180

193 Figure 6.6. Hydropathy comparison for B. subtilis and P. penetrans. Kyte-Doolittle hydropathy plots for Bacillus subtilis and Pasteuria penetrans Spo0F proteins with a window size of 9 (A, B) and 19 (C, D). Purple line indicates the midline value of 1.8 used for identifying transmembrane regions when the window size is set to

194 Figure 6.7. SDS-PAGE analysis of Pasteuria penetrans protein. Size exclusion chromatography fractions containing a 16 KDa P. penetrans Spo0F. The NusA-Spo0F fusion protein was thrombin-cleaved and samples were prepared using Hydrophobic interaction column followed by size exclusion chromatography. See text for more details. The arrow is pointing to the 16 KDa P. penetrans Spo0F protein. 182

195 Figure 6.8. Three-dimensional structure conservation in Spo0F proteins. Structural homology is observed from 15 N-HSQC spectra from Spo0F proteins from (A) Bacillus subtilis and (B) Pasteuria penetrans. Several regions have been highlighted using pentagons (green); rectangles (red), and circles (blue) to emphasize conservation of the spatial arrangement of residues in both proteins. 183

196 Figure 6.9. Structural homology between B. subtilis and P. penetrans Spo0F proteins. (a, b) Three-dimensional structures models of Spo0F from (a) Bacillus subtilis (2fsp_.pdb) and (b) Pasteuria penetrans (homology model) showing a (α/β) 5 fold. α-helices, purple; β-strands, orange and loops, gray. (c,d) Spo0F active site residues, Asp10, Asp11, Asp54, Thr82, and Lys104, are highlighted. Residues comprising the active site are highlighted as follows: Aspartate, pink; Threonine, yellow; Lysine, green. 184