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DATE DUE DATE DUE DATE DUE 5/08 KzlProilecsPreleIRCIDaIeDueindd THE PHOTORHABDUS TEMPERA TA SSPAB LOCUS IS REQUIRED FOR SYMBIONT TRANSMISSION IN HETERORHABDITIS BACTERIOPHORA By Katherine Marie Higginbotham A THESIS Submitted to Michigan State University in partial fuifillment of the requirements for the degree of MASTER OF SCIENCE Department of Biochemistry and Molecular Biology 2008 Abstract THE PHOTORHABDUS TEMPERA TA SSPAB LOCUS IS REQUIRED FOR SYMBIONT TRANSMISSION IN HETERORHABDITIS BACTERIOPHORA By Katherine Marie Higginbotham The entomopathogenic nematode, Heterorhabditis bacteriophora, and the Gram-negative, insect pathogen enteric bacterium, Photorhabdus Iuminescens, coexist in a mutualistic symbiotic relationship. As symbiont transmission is essential for the pair’s insect parasitic lifestyle, the bacteria are transmitted from the maternal nematode intestine to offspring in a sophisticated pathway that involves multiple adhesion and invasion steps. To identify genes important in symbiont transmission, a Himaer mariner transposon mutagenesis screen of ~8000 green fluorescent protein (GFP) labeled P. Iuminescens and P. temperate was conducted. A transmission defective mutant, TRN162, was identified that successfully completes the initial steps in the transmission cycle, but forms a spheroplast at 120 h post- recovery, resulting in a 0% transmission efficiency. TRN162 has a disrupted sspA gene, which is predicted to encode a homolog of the stringent starvation protein A. In Escherichia coli, SspA has been shown to be involved in survival under stressful conditions and during stationary phase, as well as being required for motility. sspAB is not essential for P. temperate virulence in insects and TRN162 survives growth under acidic conditions or in the presence of cationic microbial peptides, similar to its parent strain, NC1Tn7GFP. However, the mutant displays a growth defect, a hypen'notile twitching motility phenotype and an increased sensitivity to growth in the presence of H202. The inability of TRN162 to fully complete the symbiont transmission cycle may be explained by its increased sensitivity to oxidative stress, an inability to evade a selective stress or a yet unidentified difference in gene expression as regulated by sspAB. ACKNOWLEDGEMENTS I would like to thank my advisor, Todd A. Ciche, for the opportunity to expand my knowledge of molecular biology and microbiology. I would also like to thank Kwi Suk Kim for her assistance in construction of the cloning vector used in the complementation experiments and for her encouragement. In addition, Pooma Viswanathan provided significant scientific guidance and emotional support. Many thanks to Bettina Kaufmann-Daszczuk and all past and present members of the Ciche lab for their support and helpful suggestions. I would like to thank all my committee members, past and present, Lee Kroos, Robert Hausinger, David Amosti and Beronda Montgomery-Kaguri for their insight and guidance. Most importantly, I would also like to recognize my mother, Joan Higginbotham, my grandparents, Jack and Dorothy Zoller, and my sister, Ann Higginbotham for their endless love and encouragement, steadfast support and unyielding enthusiasm. I thank all my friends and family for always believing in me. I could not have done this without you. iii TABLE OF CONTENTS LIST OF TABLES ................................................................................... v LIST OF FIGURES ................................................................................ vi CHAPTER 1 INTRODUCTION ................................................................................... 1 Photorhabdus, Heterorhabditis bacteriophora and insect pathogenesis. . . .2 Symbiont transmission in Heterorhabditis bacteriophora ........................ 7 Roles of stringent starvation protein A .............................................. 10 Literature Cited ........................................................................... 16 CHAPTER 2 THE PHOTORHABDUS TEMPERA TA SSPAB LOCUS IS REQUIRED FOR SYMBIONT TRANSMISSION IN HETERORHABDITIS BACTERIOPHORA Abstract .................................................................................... 22 Introduction ................................................................................ 22 Material and Methods .................................................................. 25 Results ..................................................................................... 36 Discussion ................................................................................. 53 References ................................................................................ 58 CHAPTER 3 SUMMARY AND FUTURE DIRECTIONS Summary ................................................................................... 62 Future Directions ........................................................................ 64 APPENDICES RT-PCR of H-NS and RpoS .......................................................... 69 A ClpA/B type chaperone is disrupted in TRN17-96 ........................... 70 Growth rate of TRN17-96 ............................................................. 71 Phase variation of TRN17-96 ......................................................... 72 References ................................................................................ 76 Images in this thesis are presented in color. iv LIST OF TABLES Chapter 1 Table 1-1: Homologs of SspA ............................................................ 11 Chapter 2 Table 2-1: Strains and plasmids used in this study ................................. 26 Table 2-2: Growth rate of NClTn7GFP and TRN162 .............................. 42 Table 2-3: Phase variation of NC1Tn7GFP and TRN162 ......................... 44 Table 2—4: Motility of NC1Tn7GFP and TRN162 .................................... 47 Appendices Table A-1: Growth rate of NC1Tn7GFP and TRN17-96 ........................... 71 Table A-2: Phase variation of NC1Tn7GFP and TRN17-96 ...................... 74 Chapter 1 Figure 1-1: Figure 1-2: LIST OF FIGURES Life cycle of Heterorhabditis bacteriophora and Photorhabdus Iuminescens ..................................................................... 3 Photorhabdus Iuminescens transmission in Heterorhabditis bacteriophora ................................................................... 8 Figure 1-3: Synteny of sspA homologs ................................................. 13 Chapter 2 Figure 2-1: Transmission defective phenotype of TRN162 ........................ 38 Figure 2-2: Complementation of TRN162 .............................................. 39 Figure 2-3: P. temperate virulence to M. sexta ....................................... 41 Figure 2-4: Stationary phase survival of NC1Tn7GFP and TRN162 ............ 42 Figure 2-5: Growth under acidic conditions ............................................ 46 Figure 2-6: Motility of NC1Tn7GFP and TRN162 .................................... 48 Figure 2-7: Growth under oxidative stress ............................................. 50 Figure 2-8: Growth in the presence of antimicrobial peptides ..................... 52 Chapter 3 Figure 3-1: Proposed model for symbiont transmission in H. bacteriophora .............................................................. 63 Appendices Figure A-1: RT-PCR of hns and rpoS ................................................... 70 Figure A-2: Gene organization in TRN17-96 .......................................... 71 Figure A-3: Rate of phase variation switching in NC1Tn7GFP and TRN17-96 ................................................................. 75 vii Chapter 1: Introduction This introduction is divided into three parts. Part I describes the symbiotic nematode-bacteria relationship shared by Heterorhabditis bacteriophora and Photorhabdus Iuminescens, as well as the pathogenic relationship they share towards insect larvae. In Part II, the process of bacterial symbiont transmission in the nematode intestine is explained. Part III focuses on the role of SspA homologs in regulation of host-bacterial. interactions, motility and stress response in several enteric and pathogenic microorganisms. Introduction Photorhabdus, Heterorhabditis bacteriophora and insect pathogenesis According to Anton de Bary, symbiosis is “the living together of unlike organisms,” (3). This pervasive biological phenomenon can comprise a wide spectrum of associations from the mutually beneficial to the acutely parasitic. Studying a genetically malleable symbiotic relationship provides an opportunity to not only learn more about genetic factors essential for mutualism, but also for comparison to pathogenic relationships, which results in a drastically different outcome for the host organism, disease. One such model system is that of the nematode, Heterorhabditis bacteriophora, and species of the Gram-negative bacteria, Photorhabdus, which colonize the worm’s intestine (34). Although Photorhabdus and H. bacteriophora can be cultured separately in a laboratory setting, these two partners form an obligate mutualistic association in nature. Together they infect and kill the larval stage of a variety of insects, comprising three organisms of a tripartite relationship (31). While the nematode serves as Photorhabdus’ vector between insect hosts, the bacteria are necessary for killing the insects and for nematode reproduction. This system has been used commercially for the biological control of insect pests of crops (14). The life cycle of the tripartite bacteria-nematode-insect relationship is initiated by the free-living, developmentally arrested infective juvenile (IJ) stage nematode, which acumen m2 261 u TN .2 600225: .33 2302 £099 9588.513 1 / Opt—5.3600 q.— .. . ... q. . .53 e .._r . .a #525..." a . vfi Egooom .0 _ .< €283.93; .. . . “can... .50: 885 26: m. B 553 5 8.266 new memommEEE .1 5? $908me w: 9:. AIV .wmuofiEmc .mEQmE wEmE Sana m._ o>=mEo=m cmv m2 SE 8.9% occamto 2ch mEQEmE c.2285 m_> 2:80 cozewcom EN 9: 5 8.632%. 528.3% .1 9: c_ mama 9.3m. >__m=_:_ LEE Ag .omflm :33 F can momma .98. e 53> wcozfioccm m.m 5:95 8.98 mmuonEoc of. CV .mcmzmumo 885 E 3238 on :8 cocoommEanB 8:203 EV .cozzoquo 6590:: 550 :95: 65 8:02.35 0.53.05 .umosuoa 9m 85889: Emucooow can 835 9: =2 accomcEEs .1 >9 umoanoa wExoP Ev .mmoooa >558. on. 05563 .mccomcSEE .1 955509 g was 680:5: 83$ 05 .25 m2 .60: m acteczoocm cog: Amy .50: 885 cm L2 558 ucm 3:055? .95on mm 38852:: .1 Emu $0995: 2 3395 >__Scman_m>co 91V .8220 E0: coamumvmccommsEE2.3312211 ucm 8310583 .I So .298 23 up... 2:91 harbors bacterial symbionts within its intestine (Figure 1-1). The IJ actively seeks out its insect prey and enters the insect larva either through natural openings, such as the mouth or anus, or by using its buccal tooth to penetrate the exoskeleton (8, 9). Upon entering the insect hemocoel, a yet-unidentified cue causes the nematode to regurgitate its monoculture of intestinal symbionts (6). In this pathogenic phase, toxins produced by Photorhabdus kill the insect rapidly, within 24 to 48 hours. Germ free or axenic US are unable to kill insect larvae (20), while bacterial symbionts injected into insects are sufficient for insect death (31). The bacteria also produce compounds to discourage other saphrophytic organisms from feeding on the rich nutrients provided by the insect cadaver, such as antibiotics and nematicide (6, 25, 26, 35, 37). As the worm feeds on the bacteria and insect host, it develops through two to three generations within the insect cadaver in the saprophytic phase (20). Once all nutrients have been exhausted and the reproducing worms have reached a high density, the Us reassociate with Photorhabdus and disperse in search of another insect larva to infect (20). H. bacteriophora is a soil dwelling entomopathogenic nematode found in temperate climates (16) and has particular relevance in crop pest control. Species of Heterorhabditis nematodes belong to the family Rhabditidae and are phylogenetically related to Caenorhabditis elegans (29). Indeed, some features that make C. elegans a good model organism also apply to Heterorhabditis, such as its relatively small size and transparent body. For these reasons, H. bacteriophora has been targeted for complete genome sequencing by the National Human Genome Research Institute (NHGRI). Species of Heterorhabditis are heterogonic, that is, they are able to employ both hennaphroditic and gonochoristic (sexual) modes of reproduction. At low worm densities, when more nutrients are available, the nematodes develop gonochoristically by laying eggs outside of the female’s body. These eggs will develop into either males, females or herrnephrodites, with no US being generated (10). However, as the number of nematodes grows and nutrients become sparse, both female and hennaphroditic nematodes began to produce eggs within their body cavities. Eggs produced exclusively by hen'naphrodites will develop into US in a process that kills the nematode and is termed endotokia matricida. The US are developmentally arrested, non-feeding herrnaphroditic nematodes that harbor intestinal symbionts of the genus Photorhabdus and disperse in search of new insects to infect (20). Photorhabdus are Gram-negative, rod-shaped, bioluminescent microorganisms in the family Enterobacteriaceae. Photorhabdus Iuminescens subsp. lamondiiTTO1 and P. temperate strain NC1, are both able to colonize the intestinal tract of H. bacteriophora (7). The full genome of P. Iuminescens subsp. lamondii TTO1 has been sequenced and is predicted to encode 4,839 protein- coding genes (13). The genome encodes many genes that presumably assist Photorhabdus in its roles of nematode mutualism and insect pathogenesis, including 11 fimbriel gene clusters and the largest number of predicted toxins in a bacterial genome sequenced to date. It is presumably these toxins that kill insects infected by symbiont-harboring Us. For example, there are multiple toxin-complex (tc) loci, encoding gene products that are predicted to have Tyr- Asp motifs (13). Proteins in this superfamily are predicted to be localized to bacterial surfaces and to bind carbohydrates (32), implicating them in assisting bacterial escape of the innate immune defenses of insect prey (13). Another class of toxin proteins, repeats-in-toxin (RTX), has been identified in P. Iuminescens ssp. TTO1 (13). The organization of these genes is identical to that found in Vibrio cholerae, and is again predicted to play a large role in pathogenicity to insect prey (13). Not only does Photorhabdus encode toxins that contribute to insect pathogenicity, but also factors that aid in protection from other microorganisms that may want to benefit from the rich nutrients provided by insect cadavers, such as antibiotic synthesis genes (13). While the metabolism of P. Iuminescens is fairly similar to other enteric bacteria, the genome encodes many additional metabolic genes involved in degradation pathways not found in other enterics. These genes presumably aid in the bacteria’s nematode mutualism and entomopathogenic lifestyles (13). In comparison to other sequenced genomes, the P. Iuminescens ssp. TTO1 genome is similar to another insect and human pathogenic microorganism, Yersinie pestis, with 77% of their orthologous genes being syntenic (13, 33). Photorhabdus ssp. exhibit the phenomena of phenotypic variation. Two wildtype phase variants of Photorhabdus have been identified, where the two extemes, primary and secondary phase, have been characterized. Primary variants are isolated from Us and insects, while secondary variants arise after prolonged subculturing. Primaries are characterized by their ability to produce pigments, colonize Us and biolumenesce (15, 17). Additionally, primaries produce crystalline inclusion proteins and display antibiotic, siderophore and hemolytic activities. In contrast, secondaries do not display any of the above phenotypes. Notably they are unable to colonize US, but are pathogenic to insects (19, 20). Symbiont transmission in H. bacteriophora Due to the essential function of Photorhabdus for the H. becten'ophore’s insect parasitic lifestyle, it is not surprising that bacterial colonization of the nematode intestine and transmission to the next generation of nematodes involves many selective steps that are sure to involve regulation of gene expression from both partners. The symbiosis between H. bacteriophora and species of Photorhabdus is specific, with only P. Iuminescens and P. temperate being capable of completing the transmission cycle (16). This process begins with symbiont regurgitation by recovering US, where all intestinal symbionts are released (Figure 1-2). After regurgitation, the nematode feeds on Photorhabdus, and a subpopulation of bacteria then adhere to the posterior of the intestine and form a biofilm before adherent cells invade the rectal gland cells to gain access to the Us developing in the pseudocoelom (10). These steps, occurring between 8 and 72 h post-recovery, likely require Photorhabdus to temporally regulate pong: 9m w__co 986 .98. __m .2 me 263 w__oo .286 .98. _o co_ww>c_ .; NV 2.00 956 509 an 8.039: 8.3 .5 wk 3083 EEoB .; 8 :50 con .mEofiE 5 9.629% m: .oa >9 comb wm_o:ow> .5 N: 05625 5:663 o. 3908 Co 88.23 .5 NF m=mo «26> .mc=8E_-_mmmc>._m:a 2 «2203 Lo mocmcwcom .c om? w: 8558. E0: 880.2 3.203 __m .c w m__oo m>_m> 657.25 489.293 .0 co_mm>c_ .: m9 flu 10:20 E0: umamumv $310.29an .I E co_mw_Emcm= 8080553 .1 "a; 2:91 genes involved in host attachment andinvasion processes. Only eggs that are retained within the body cavity of the nematode develop into US (10). As the pre- IJs feed on the maternal organs, the mother nematode is eventually killed in a process termed endotokia matricida. As the Us develop, at approximately 112 h post-recovery, the maternal rectal gland cells are lysed, releasing the bacterial symbionts into the pseudocoelom. This allows the bacteria to be transmitted from the mother to the feeding US via a mechanism that is selective for true symbionts. In a clonal and presumably highly selective step, the bacteria then adhere to the pharyngeal intestinal valve cells (PIVCs) of the Us. The bacteria ultimately invade these cells before exiting and fully colonizing the U intestine. Although this transmission process is symbiont specific, the molecular biology, - specifically the gene regulation involved, is yet to be extensively studied and fully understood. A forward genetics approach was applied to learn which bacterial genes play an essential role in the transmission of Photorhabdus species to Heterorhabditis. A Himeer transposon mutagenesis screen composed of 28 independent mutagenesis experiments, was conducted using GFP-Iabeled Photorhabdus to identify mutants unable to complete the transmission cycle. The use of GFP—Iabeled Photorhabdus allowed for easy visualization of the presence or absence of symbiotic bacteria within the intestine of Us (collected in the condensation on the lids of Petri dishes) under a fluorescent microscope. Broader implications of this study include gaining insight into generalized features of symbiotic relationships, in addition to uncovering genetic factors important for host-bacterial interactions in general. Of the approximately 8,000 bacterial mutants produced, 30 were isolated that were defective in symbiont transmission to Us. One of these, TRN162, was found to contain a mutation in sspA, which encodes a homolog of the stringent starvation protein A. Roles of stringent starvation protein A SspA is highly conserved throughout the Enterobacteriaceae family, where it has been implicated in the lytic development of the bacteriophage P1 in Escherichia coli (22) and in regulation of virulence factors in Yersinie entercolitica (1) (Table 1-1 and Figure 1-3). In addition, homologs have been identified in non-enteric bacteria, such as Neisserie gonorrhoeee (11) and Frenciselle tularensis (5, 18). In E. coli, it has been shown that SspA is a RNA polymerase associated protein (28) whose expression is upregulated during stationary phase and upon starvation for glucose, nitrogen, phosphate and amino acids (36). In addition, under nutrient limiting conditions, an E. coli sspA mutant had decreased survivability compared to the parental strain (36). One of the first functional roles identified for SspA was as a transcriptional activator for the expression of late genes of the E. coli bacteriophage P1 (22). SspA, along with the late promoter activator (Lpa) protein, allows the phage P1 to enter the lytic cycle through the expression of late genes (22). 10 03951130: :12. 3893 253:8 3 3:83. can €3.16: .133» 563.98 mama 3:22; So 5:232 @6553 83.8 $1..“ 1.3 .mwmmcqocome 5 _m>_>5m new 5320 113.6281 <32 25.6.1162 .133 £23.22 3......9 1.8 1.3 <52 5;. 85325 118.5521 23 5281 385.6161 2: 1.8 1.? :3 >_ 2;. s 81.38. 95.82 1.81.2 Loam 282: 88.532... :8 1.: $3 <13 .18 .m 5 85552 2cm 29:80 3.853531 >otm $5 0.33 .(Qmm Emmeae mgotm xom_m .moEBoEmEm dmm moEBoEmEm EEEm> av 95 N5. :8 mEotmcowm Amv .SC. dwm mcmommsEE mzbnmfioscl AS 5 30.050; «dam ho 2.256 "n... 959“. r .F... r... u. 5:9 39 zoouou co_mm_Emcm._.—. 382-130. 5? 355558 $sz 35.5% (new .nEOhFCOZ SmBQEE .& 023806 co_mm_Emcm..._. 9.0-2: £3 8.33 33%. 325.3 we: 23 ESE 5.3 8.33 2823 taste 25 25> was: 2 85:52.23 35.... m25 25 >33 mg .62» 8: .635 m5 6. .m ozmmofimmoowooamsgu .m. .m was 8m99.8830986988890mmumuaoauoofia b .m 8333655939 .m .m 08038889606 .m .m oomummomoatwmflmmo .m .m 88868868038 .m cozmoEaEm awn... .8 Emacs... m+om8 ”.88. .8 m3... >0. 9%? .8 9.6.8 PDOEO .5052 988.5 03.20.. 8.33.. 25528 E 2.5 27 cholesterol (chol, 10 mg/ml, Sigma Aldrich) added when appropriate. E. coli was cultured in either SOC (super optimal broth (SOB) with 20 mM glucose) or Luria Broth (LB) (3) modified to contain 5 g/L NaCI with agar (1.5% w/v), ampicillin (100 pglml), tetracycline (10 pglml), or diaminopimelic acid (DAP, 300 pg/ml) added when appropriate. Ringer’s solution (100 mM NaCl, 1.8 mM KCI, 2 mM CaClz, 1 mM M902, 5 mM HEPES, pH 6.9) and saline solution (0.85% NaCl) were used to wash and store H. bacteriophora axenic worm stock. Strains and plasmids used in this study are listed in Table 2-1. Axenic nematode stock 50 pl of overnight cultures of TRN16 (completely defective in symbiont transmission) grown in PP3S was spread onto one half of a split well Petri dish of nutrient agar (NA) + comoil. After 48 h of incubation at 28°C, 10 pl of H. bacteriophora M31e was added and plates were incubated at 28°C for an additional 11 d. Emerging US were collected in Ringer’s solution on the empty half of the plate. Nematodes were harvested by centrifugation at 1,200 rpm for 1 min, surface sterilized in 1% commercial bleach for 5 min and washed three times with Ringer’s before being stored in 10 ml of Ringer’s solution. To ensure that the Us were axenic, 50 pl of nematode stock was homogenized using a motorized tissue grinder (Kontes Glass Co., Vineland, NJ), plated on PP3S and incubated at 28°C for 48 h. Antibiotics were added to the nematode stock at the following concentrations: 100 pglml streptomycin, 100 pglml ampicillin, 30 pglml kanamycin and 10 pglml gentamicin. 28 Transposon mutagenesis and mutant screening Overnight cultures of P. temperata strain NC1Tn7GFP and BW29427 carrying pURE1O were grown in PP3S and LB+DAP+Gm, respectively. 10 ml of fresh media was inoculated with 30 pL of overnight cultures and grown to an 00500 of 0.6. Cells were then pelleted by centrifugation, washed three times with LB+DAP and resuspended in a final volume of 500 pl LB+DAP. The two strains were then combined, centrifuged, resuspended in 50 pl LB+DAP and finally plated on LB+DAP. After 8 h of incubation at 28°C, cells were then washed off the plate using LB, centrifuged, washed three times with LB and resuspended in 1.5 ml LB before plating 100 pl on PP3S+Gm. Isolated colonies were patched onto PP3$+Gm and incubated at 28°C for 48 h. Each colony was then cultured in 250 pl PP3S+Gm for an additional 48 h at 28°C. 50 pl of the liquid culture was spread on NA+comoil+Gm plates and incubated for an additional 48 h at 28°C, after which time 10 pl of axenic nematode stock (M31e grown on TRN16) that had been washed three times with saline solution was added. Plates were incubated at 28°C for 10-12 d until le formed and were isolated in the condensation that formed on the lids of the Petri dishes. Mutants were screened and identified by visualizing the lack of GFP-labeled symbionts in the intestine of H. bacteriophora US under a fluorescent stereomicroscope (Leica MZ16F, Leica Microsystems, Wetzler, Germany). Transmission efficiency was determined by scoring for the presence of bacteria in the IJ intestine, where a single GFP- labeled cell could be detected and was calculated by dividing the total number of 29 le observed (minimum 5,000 for mutants with 0% transmission efficiencies) by the number of colonized le. Characterization of transmission processes To characterize the stages in the transmission process of NC1Tn7GFP and TRN162, 10 pl of axenic nematode stock that had been washed three times in saline solution was placed on lawns of the strain to be tested grown on NA+chol plates and incubated between 8 h and 132 h at 28°C. Worms were then transferred from the GFP-Iabeled bacteria (NC1Tn7GFP or TRN162), rinsed in sterile Ringer’s solution and transferred to lawns of unlabeled P. temperata strain NC1 for 4 h at 28°C to clear the intestine of transient labeled bacteria before being imaged, with images taken at 36, 48, 72, 120 and 132 h post-recovery. Nematodes were immobilized using 10 — 20 pm sodium azide and imaged on 1% agar pads using a fluorescent compound microscope (Leica DM5000). Transposon retrieval and sequencing Genomic DNA was purified from TRN162 grown in 3 ml of Grace’s Insect Media overnight at 28°C using the DNeasy Tissue Kit (Qiagen, Valencia, CA). Direct retrieval of the transposon is possible because the Himaer transposon has an R6Ky origin of replication and can therefore be replicated in any strain expressing the ‘IT protein. Between 5 and 20 pg of genomic DNA was digested with Sphl (New England Biolabs, lpswitch, MA), diluted to 250 pl and then ligated with T4 DNA ligase (New England Biolabs). Circularized DNA fragments were 30 precipitated with isopropanol, washed with 70% ethanol and resuspended in 10 pl of double distilled (dd) H20 before electroporating 1 pl of DNA into 10-15 pl TransforMax EC100D pir-116 electrocompetent E. coli cells (EPICENTRE Biotechnologies, Madison, WI) using a Gene Pulser Xcell Electroporation System (BioRad, Hercules, CA). Transformed cells were recovered in SOC, harvested and plated on LB+Gm and incubated ovemight at 37°C. Isolated colonies were then grown ovemight in 3 ml LB+Gm and plasmid purified (Qiagen QIAprep Spin Miniprep Kit). The presence of the Himaer was verified by plasmid digestion with Sac! (New England Biolabs) releasing a 1 kb fragment. Digestion by Sphl was used to determine size and digestion pattern of flanking DNA. Digestion products were analyzed by agarose gel electrophoresis. Plasmids verified to have a 1 kb fragment were submitted for sequencing to the Michigan State University Research Technology Support Facility with the Himaer specific primers MarOUT and GmOUT. Analysis of sequencing data was performed using coIiBLAST (http://xbase.bham.ac.uk/colibase/blast.pl) to determine what homolog of P. Iuminescens subsp. lamondii 'l‘I'O1 genes was disrupted by transposon insertion in P. temperata. Complementation The tetracycline resistance (TetR) gene was PCR amplified from pCM639 using primers TetR for and TetR rev with Pstl restriction enzymes sites designed on the 5’ ends (Table 2-1, Pstl sequences in bold). The PCR product was ligated into pCRlI (lnvitrogen) and transformed into chemically competent E. coli DH50. 31 Plasmids purified from the resulting colonies were digested with Pstl (New England Biolabs) and cloned into the Nsil site of pUC18R6KT. Genomic DNA was purified from P. Iuminescens subsp. lamondii TT01 using the DNeasy Tissue Kit. Wildtype sspAB was then PCR amplified from TT01 genomic DNA using the primers sspAB for and sspAB rev. The PCR product was ligated into pCRll and transformed into chemically competent E. coli DH5a. Plasmids purified from the resulting isolated colonies were then digested with Xhol and Kpnl cloned into pUC18-TetR at the same restriction sites. The resulting complementation vector, pTetR-sspAB, was transformed into BW29427 and mobilized into TRN162 via a triparental mating as follows: Overnight cultures of TRN162 or BW29427 carrying either pTetR-sspAB or pUX-BF13 were grown in PP3S and LB+DAP respectively. Cells were pelleted by centrifugation, washed three times with LB+DAP before being combined and plated onto LB+DAP. After an 8 h incubation at 28°C, cells were washed off the plate with LB, centrifuged and washed three times with LB. They were then plated on PP3S+Tet and incubated at 28°C. Isolated colonies were verified by patching onto PP3S+Tet before being tested in the worm. Phenotypic variation characterizations Phase variation of Photorhabdus strains were characterized as described previously (4). Media used in dye absorption assays included nutrient agar supplemented with 2,3,5 - triphenyltetrazolium and bromthymol blue at 40 pglpl and 25 pglpl respectively, Congo Red agar (nutrient agar with 0.01% [wt/vol] 32 Congo Red) and eosin y-methylene blue agar (PPBS plus eosin y and methylene blue at 400 pglpl and 65 pglpl respectively). Bioluminescence was scored by observing 48 h mutant and parental strain colonies in the dark. Hemolytic and siderophore activities were determined by observing Photorhabdus growth on blood agar (Cole-Palmer) or chrome azurol S (CAS) agar (24), respectively. To assay for the production of extracellular antibiotics, Photorhabdus was spot inoculated onto PP3S agar and grown for 48 h at 28°C. Then 0.5 ml of an overnight culture of M. Iuteus was mixed with 20 ml of soft LB agar (0.75%) at 42°C and overlayed on top of the Photorhabdus patches. Antibiotic production was determined by the presence of zones of growth inhibition of M. Iuteus. The support of nematode growth and reproduction was monitored by growing M31e Heterorhabditis bacteriophora raised on TRN16 on lawns of TRN162. Photorhabdus growth Overnight cultures of Photorhabdus strains were inoculated in quintuple to an ODeoo of 0.05 in 150 pL of Grace’s Insect Media in a standard clear 96-well microtiter dish. Growth was carried out at 28°C with 30 sec of shaking every five min. Absorbance measurements were made at five min intervals for a total of 20 h in a SpectraMax M5 plate reader (Molecular Devices, Sunnyvale, CA). Insect virulence Manduca sexta eggs were obtained from North Carolina State University lnsectary (Raleigh, NC) and raised on Gypsy Moth Wheat Germ Diet premixed 33 with agar (MP Biomedicals lnc., Solon, OH) at 25°C with a 14 h light/ 10 h dark cycle. 3 pl of a 24 h Photorhabdus culture was inoculated into 3 ml of fresh PP3S and grown for an additional 24 h at 28°C prior to injections. Fourth or fifth instar larvae of M. sexta were injected behind the 1St proleg with 10 pL of serial dilutions of liquid cultures of Photorhabdus. Insect mortality was monitored in 24 h intervals for 72 h. Acid tolerance assay Cultures of NC1Tn7GFP and TRN162 were grown overnight in PP3S. 200 pl of PP3S, pH adjusted with HCI and supplemented with 50 mM MES, was inoculated to an ODsoo of 0.05 in a 96-well microtiter dish. Cultures were grown in a shaking 28°C incubator overnight and ODeoo measurements were then taken in a SpectraMax5 plate reader. Relative ODeoo values were calculated by dividing the ODeoo value at a particular pH by the ODaoo value at pH 7. Motility assay 5 pl of Photorhabdus strains to be tested (ODsoo adjusted to 2.0) were inoculated onto PP3S onto either swarming motility agar (0.35% agar) or twitching motility agar (0.75% agar). Plates were incubated at 28°C and diameter measurements were made under a fluorescent stereomicroscope at 20 h and 37 h. 34 Oxidative stress assays Cultures of NC1Tn7GFP and TRN162 were grown overnight in PP3S. 3 ml of PP3S + H202 was inoculated to an 00600 of 0.05 and grown at 28°C for 16-18 h. Serial dilutions were plated out on PP3S to determine colony forming units per ml (CFU/ml). Methyl viologen dichloride hydrate (paraquat) was purchased from Sigma-Aldrich. 200 pl of PP3S was supplemented with paraquat to various concentrations in a 96-well microtiter dish and inoculated with NC1Tn7GFP and TRN162 to an ODsoo of 0.05. The cultures were grown in a shaking 28°C incubator for 16-18 h and growth was measured by determining the ODsoo. Relative ODeoo values were calculated by dividing the ODeoo value in the presence of a particular paraquat concentration by the ODsoo value in the absence of paraquat. Antimicrobial peptide assays Protamine sulfate, lactoferrin and polymyxin B were purchased from Sigma- Aldrich (St. Louis, MO). For growth in the presence of antimicrobial peptides, overnight cultures of Photorhabdus strains to be tested were inoculated to an ODeoo of 0.05 in 200 pL of PP3S plus appropriate antimicrobial peptide in a 96- well microtiter plate. The samples were incubated at 28°C for 16-18 h with constant shaking. ODsoo measurements were made in a SpectraMax M5 plate reader. Each concentration was inoculated in triplicate and repeated a minimum of three times. 35 Results sspA is disrupted in the transmission defective mutant TRN162. A transposon mutagenesis screen of green fluorescent protein (GFP) labeled P. Iuminescens subsp. laumondii Tl'01 and P. temperata strain NC1 was conducted with a Himaer transposon to identify genes essential for bacterial colonization of the H. bacteriophora intestine. The screen produced approximately 30 transmission (TRN) defective mutants with various genes being disrupted. A P. temperata mutant, TRN162, was isolated that is defective in transmission. Retrieval by marker rescue and sequencing of the DNA flanking the Himaer of mutant TRN162 revealed that the transposon was inserted near nucleotide 330 of plu4013, a gene predicted to encode a homolog of the stringent starvation protein A (SspA). sspA is 642 bp long and is predicted to encode a protein of 213 amino acids in length. 3 bp downstream of sspA is the predicted homolog of the stringent starvation protein B (sspB), which is 516 bp long, encoding a 171 amino acid protein. TRN162 has a 0% transmission efficiency, where zero cells were observed in 5,000 US examined by fluorescent stereomicroscopy. Specifically, it is able to complete the initial stages in Photorhabdus transmission in H. bacteriophora, but is unable to fully colonize the resulting infective juvenile (Figure 2-1). The mutant is able to successfully form a biofilm near the rectal gland cells at 36 h post-recovery, indistinguishable from NC1Tn7GFP. At 48 h it can be observed that the rectal gland cells at the posterior of the intestine in the 36 maternal nematode are invaded, again analogously to the parental strain. By 72 h, both TRN162 and NC1Tn7GFP have entered the vacuoles of the rectal gland cells. However, at 120 h, a weakly-fluorescent spheroplast is seen near the PlVCs of the IJ raised on TRN162. This is in direct contrast to NC1Tn7GFP, which adheres to and ultimately invades the PlVCs. The spheroplast is indicative of a dying bacterium that has lost its structural integrity. The mutant is subsequently unable to completely colonize the intestine of the U and therefore fails to complete the transmission cycle. Upon prolonged culturing of TRN162, the transmission defective phenotype changes in that the mutant is no longer able to complete even the initial steps of the transmission cycle. Therefore, samples of TRN162 were not cultured any longer than ten days to ensure that only the variant able to successfully adhere to and invade the rectal gland cells before forming a spheroplast at 120h was used for all experiments. 37 .I-m :_ 56% a ~9sz .o-< s 53% m_ 9.9.502 «925. so oSocofi 982% 53523 ”TN 059“. Log 38 The disruption of sspAB is responsible for the transmission defective phenotype of TRN 162. Several unsuccessful attempts were made to complement the transmission defective phenotype of TRN162 by expressing wildtype sspAB from a plasmid construct. As the defective stage of symbiont transmission occurs at 120 h in this particular mutant, it is difficult to maintain antibiotic selection of a complementation plasmid in the intestine of the nematode for this length of time. Therefore, wildtype genes were stably inserted into the genome in order to Figure 2-2: Complementation of TRN162. All pictures are 120h post-recovery. (A) NC1Tn7GFP (B) TRN162 (C) TRN162Tn7sspAB 39 complement TRN162. This was accomplished by using a mini-Tn7-TetR transposon. To restore the transmission defective phenotype, wildtype sspAB genes were mated into TRN162, resulting in strain TRN162Tn7sspAB. The resulting complemented mutant displays a full restoration of the transmission cycle (Figure 2-2). TRN162Tn7sspAB is able to successfully survive adherence to and invasion of the PlVCs after 120 h like NC1Tn7GFP, culminating in the colonization of Us. The transmission efficiency of TRN162Tn7sspAB is 26.0% i 5.3%. Although this transmission efficiency is not as high as NC1Tn7GFP, it still represents a significant increase over the 0% transmission efficiency of TRN162. These data support previous results indicating the defective transmission phenotype of TRN162 is due to the disruption of the sspAB operon. sspAB is not essential for insect virulence. Some factors required for symbiont transmission may also be required for insect pathogenesis. Additionally, there are known functions of SspA involvement in virulence. To determine if TRN162 has an altered pathogenicity to insect larvae, serial dilutions of NC1Tn7GFP and TRN162 were injected into 4th or 5th instar larvae of M. sexta. Virulence was assayed every 24 h for 72 h. TRN162 is able to cause significant death within 72 h after injections, similarly to NC1Tn7GFP (Figure 2-3). These results suggest that sspAB is not essential for insect vimlence. 4O Figure 2-3: P. temperata virulence to M. sexta. Insect mortality was monitored after 24 h (black bars), 48 h (gray bars) and 72 h (white bars). 100 To 80 .2 g 60 (D °\° 40 i 20 ‘ I 0 . .. PP3S 202.5 21.4 2.35 357 35.7 5 cells cells cells cells cells cells NC1Tn7GFP TRN162 TRN162 has a growth defect compared to the parental strain NC1 Tn 7GFP. It was possible that TRN162’s inability to complete the symbiont transmission process was due to a growth defect where the mutant would be unable to completely colonize the IJ intestine after adhering to the PlVCs within the time assayed. The growth of TRN162 and its parental strain was assessed by measuring the ODsoo in 5 min intervals for a period of 20 h. Both Photorhabdus strains reached similar final ODsoo. From the portion of the growth curve corresponding to exponential phase, the growth rate (p) and doubling time (T2) was calculated (Table 2-3). With a growth rate of 0.210 :r: 0.017 h", TRN162 41 exhibits a growth defect compared to the 0.273 t 0.013 h'1 growth rate of NC1Tn7GFP. As it has been reported that sspA mutants in E. coli and the mIgA mutant of F. novicida have defects in stationary phase survival, the ability of TRN162 to survive prolonged culturing was evaluated in comparison to NC1Tn7GFP. After 18 d of prolonged stationary phase culturing in liquid media, no significant difference was observed between NC1Tn7GFP and TRN162 (Figure 2-4). Table 2-2: Growth Rates of NC1Tn7GFP and TRN162 Growth Rate Doubling Time NC1Tn7GFP 0.273 t 0.013 h"I 2.54 i 0.12 h TRN162 0.210 i 0.017 h'1 3.32 :l: 0.28 h Figure 2-4: Stationary Phase Survival of NC1Tn7GFP (black) and TRN162 (gray) 1.00E+09 ‘ 1.00808“ 1.00907‘ 1.00E+06 Erooews :3 ll. 0 1.00904 l 1.00303 1.00902 1.009014 1.00300 -3 ._ ~- - 3 5 7 9 11 13 Days 42 TRN162 displays characteristics of primary phase variants. Photorhabdus species exhibit phase variation phenotypes. As only primary phase variants are transmitted by US, it was essential to determine the phase variation state of TRN162. This mutant displays primary phase characteristics, corresponding to those of NC1Tn7GFP (Table 2-3). P. temperata strain NC1 cells range in size between 3 and 6 pm (12). The cell length of TRN162 is comparable, with an average of 3.41 :l: 0.91 pm. TRN162 absorbs pigments from its media supplemented with the dyes neutral red, eosin y- methylene blue, bromthymol blue or Congo red, which is a classic characteristic of primary phase variants. Furthermore, it displayed hemolytic activity, as well as siderophore and antibiotic activity, as do primary cells. Colonies of TRN162 are convex in shape, mucoid, and pigmented with an orange color. This pigmentation is in contrast to the yellow pigmentation of NC1Tn7GFP. Individual cells produced cellular inclusion proteins, as seen by light microscopy. No obvious difference in nematode growth was observed between US grown on NC1Tn7GFP or TRN162. These results suggest that TRN162’s inability to complete the transmission cycle is not due to a secondary phase variant phenotype. 43 + 09.0.0 0.8:... £02.00 + +++ +++++ + 3o..0> 28:: £02.00 + +++ +++++ 23.5 28:55: 42.... + 30_.0> 283:. .x0>coo + +++ +++++ 20.830050. 0:0 23061002080: .0 E895 cozafimeai 30.9.99: 2.0.00 05085 5.020... 5.2.00 3.2.8 2.29.5. €2.00 0.9.3.006 .5350 0.3.050: 0.0390 3.2.0092”... 00c0om0c.E:_o_m 00. 09.00 0:5 655.590 0:3 0c0.>£0E-> Emom. 00. .2502 c8889.. 0.6 ~22”... muprFpuz Eavcoocm 32 3255 52 $0.932 252.25 wwwzm... new nEOhFCUZ 509.800 v02 508.5 902 .0 cozoNtmfiofizo coast? 0095 ”Wm 030.... 44 TRN162 does not display a significant difference in the bacteria ’8 ability to grow under acidic conditions. An E. coli sspA mutant displayed an increased sensitivity to growth under acidic conditions due to negative regulation of genes conferring acid resistance (18). Likewise, the nematode may produce some sort of stress, such as lowering the pH, to clear its intestine of nonsymbiotic bacteria at 120 h in the transmission cycle. Both TRN162 and NC1Tn7GFP grow optimally at a pH of 6.5, which is close to the unaltered pH of PPBS. Growth at pH 5.5 and above is similar between NC1Tn7GFP and TRN162, with growth being inhibited by 49.4 i 5.1% and 31.2 i 12.4% at pH 5.5, respectively. For both strains, growth is severely inhibited at or below pH 5. At this pH, NC1Tn7GFP displayed a 92.3 :1: 3.3% growth inhibition and TRN162's growth was inhibited by 89.7 :r: 2.6%. 45 Figure 2-5: Growth of NC1Tn7GFP (black) and TRN162 (gray) under acidic conditions. 1 .4007 1 .200‘ i 0.600‘ Relative OD (600nm) § 0.400‘ o.2ooi 0.000‘ 4.5 4 Motility of TRN162 In addition to an increased sensitivity to growth under acidic conditions, the E. coli sspA mutant presents a hypermotile phenotype when grown on 0.3% semi-solid agar media (18). A similar phenotype has also been observed in the Y. enterocolitica sspA mutant (1). To determine if TRN162 displayed a motility phenotype different than NC1Tn7GFP, both strains (adjusted to ODsoo = 0.2) were inoculated onto PP3S semi-solid agar plates (0.35% agar for swarming motility and 0.75% agar for twitching motility) and the diameter of the motility ring was measured after 20 h and 37 h. On 0.35% swarming motility agar, there was no significant difference in swarming ring diameter between the strains (Table 2- 4). At 37 h, the swarming ring diameter of NC1Tn7GFP was 26.1 i 3.8 mm and 46 the swarming ring diameter of TRN162 was 22.7 i 7.5 mm. Interestingly, the boundary of the TRN162 swarming ring was smooth in comparison to the scalloped edges of the NC1Tn7GFP swarming ring (Figure 2-5). In comparison to NC1Tn7GFP, the sspA mutant twitched more than four times farther on 0.75% agar media (Table 2-4) and exhibited elaborate spiked outgrowth along the twitching ring boundary (Figure 2-5, C and D). Table 2-4: Motility of NC1Tn7GFP and TRN162 Swan'ning — 0.35% agar Twitching — 0.75% agar Time NC1Tn7GFP TRN162 NC1Tn7GFP TRN162 20 h 9.5 :l: 1.3 mm 8.7 :l: 1.7 mm 6.0 :l: 0.3 mm 9.0 :l: 1.7 mm 37 h 26.1 i 3.8 mm 22.7 i 7.5 mm 8.4 i 0.6 mm 34.9 i 3.1 mm 47 o\omm.o No FZNE. $mnd No Fzmh .03 $36 nEOnFCOZ .mmm $9.0 100.2502 $sz .o. 9.0 89.502 .0. .o 2.38. 9.22.5 00.25 .0. 0:0 04.9th02 2. .o £39: 9.5.020 .8.sz 0:0 8.9562 .0 2....22 new 050.". 48 TRN162 shows an increased sensitivity to H202 There is evidence that the F. novicida sspA homolog, mglA, has decreased resistance to oxidative stresses (14). To determine if sspA plays a role in Photorhabdus’ response to oxidative stress, both TRN162 and NC1Tn7GFP were grown in media treated with up to 100 mM H2O2 or 10 mM paraquat, a compound that reacts with O2 to produce superoxide and H202. While NC1Tn7GFP grew to a comparable CFU/ml in the presence of 100 mM H202 and the PPSS negative control, TRN162 displayed a two-order of magnitude decrease in CFU/ml between 0 mM H202 and 100 mM H2O2 (Figure 2-6). In media inoculated with a dose as low as 0.1 mM paraquat, both NC1Tn7GFP and TRN162 showed a significant reduction in growth with relative ODeoo values of 0.174 :t 0.122 and 0.405 1: 0.243, respectively. At the larger dose concentration of 10 mM paraquat, there was no significant difference between the two strains’ ability to grow, with relative ODeoo values of 0.110 i 0.072 and 0.094 :I: 0.030 for NC1Tn7GFP and TRN162, respectively. 49 Figure 2-7: Growth under oxidative stress. NC1Tn7GFP (black), TRN162 (gray) (A) Growth in the presence of H202. (B) Growth in the presence of paraquat. (A) 1.800‘ 1.600 ~ 1400‘ 1.200 ' 1.000‘ 0.800 1 Relative CFUImI 0.600 ‘ 0.400 ‘ 0.200 ‘ 0.000 ‘ 0 1 10 H202 (mM) 100 50 Figure 2-7 continued (B) 1000‘ 0.900 ‘ 0.800 ~ 0.700 1 0.600‘ 0.500 ‘ Relative OD (600nm) 0.400‘ 0.300 ‘ 0.200 ‘ 0.1001 0.000‘ o 0.1 1 Peraquat (mM) sspAB is not essential for Photorhabdus ability to grow in presence of cationic antimicrobial peptides. One mechanism shown to be employed by the innate immune systems of organisms commonly encountering microorganisms is the production of antimicrobial peptides (APs) (5). APs commonly are cationic in nature, and thus have a negative impact on the negatively charged bacterial capsule that leads to membrane permeabilization and eventually cell death. It is possible that H. bacteriophora produces one or more APs to clear its intestine of non-symbiotic bacteria, imposing a selection process to ensure that only true symbionts are transmitted to Us. To determine if the outer membrane or bacterial capsule of the sspAB mutant was defective, TRN162 and NC1Tn7GFP were grown in the presence of 51 the APs polymyxin B and lactoferrin (Figure 2-8). In the presence of 75 pg/ml polymyxin B, the growth of both strains was inhibited approximately 50%. When polymyxin B was added to the growth media at 300 pglml, growth was inhibited by 53.2 i 9.3% in NC1Tn7GFP and 50.2 3: 8.5% in TRN162. In comparison, the highest concentration of lactoferrin tested in this study (450 pglml), produced no more than a 4.6% inhibition of growth for both strains. Figure 2-8: Growth in the presence of antimicrobial peptides. NC1Tn7GFP (black), TRN162 (gray). (A) Polymyxin B, (B) Lactoferrin (A) Relative 0D (600nm) o 75 150 300 derrmdn B (point) 52 Figure 2-8 continued (B) 120). [Ill Relative OD (600nm) . . .0 :0 .-‘ - - - -3 -31, j- E E 011D" Lactiurin (pdn'l) Discussion Photorhabdus exists in an obligate symbiotic relationship with the IJ stage of H. bacteriophora and together, these two organisms are pathogenic to a variety of insect larvas. As symbiont transmission in H. bacteriophora nematodes is essential in nature, it is imperative that symbionts are selectively transmitted to the IJ. The aim of this study was to identify essential symbiosis factors employed during Photorhabdus transmission in H. bacteriophora in order to gain insight into the molecular cues behind this selective process and, in particular, the gene regulation involved in the establishment of host-bacterial interactions. To this end, a transposon mutagenesis screen was conducted to isolate bacterial mutants unable to complete the transmission cycle, resulting in axenic nematode le. One mutant, TRN162, was determined to carry a 53 transposon disruption in the gene encoding a homolog of the stringent starvation protein A (sspA). It is also probable that there are polar effects on the downstream sspB. Unlike all other mutants isolated during this genetic screen, this mutant is defective at a very late stage in the transmission process; survival after adherence to the IJ intestine. At 120 h post-recovery, this mutant has been observed to form a spheroplast near the nematode PlVCs, suggesting that the death of the bacterium might be due to an inability of sspAB to positively regulate stress response gene(s) required for evasion of a stress. In particular, this study aimed to demonstrate that the transmission defect of TRN162 is due to the disruption of sspAB and to determine the underlying cause(s) of why TRN162 is unable to successfully complete the transmission cycle in H. bacteriophora nematodes. Our research has shown that the sspAB genes are essential for bacterial colonization of the nematode intestine and that TRN162 displays a slight growth defect compared to the parental strain NC1Tn7GFP. In the absence of competition from the parental strain, it is unlikely that this growth defect explains the mutant’s inability to complete the transmission cycle. In addition, TRN162 displays characteristics that identify it as primary phase variant and not a secondary phase variant that may have arisen spontaneously upon prolonged culturing in the laboratory. Insect virulence assays demonstrated that sspAB is not essential for insect virulence as TRN162 is still able to cause significant death within 72 h of 54 injection into M. sexta. However, comparing the calculated lethal dose or time required to kill 50% of the insects, L050 and LT50 respectively, for NC1Tn7GFP and TRN162 would answer whether or not sspAB plays a role in pathogenicity. As it has been shown that some factors implicated in symbiont transmission are also involved in insect virulence (2, 13, 20, 25), it is interesting that there is no essential function for sspAB in insect pathogenicity. These results suggest that sspAB is a novel symbiosis factor in Photorhabdus. Under competitive conditions, where both NC1Tn7GFP and TRN162 were present, it may be that the sspAB mutant would have a competitive disadvantage in insect pathogenicity. The weakly fluorescent spheroplast observed near the PlVCs of Us raised on TRN162 at 120 h is indicative of a bacterium that has lost its structural integrity and is dying. We propose that the nematode produces a stress to clear its intestinal lumen of non-symbiotic bacteria and moreover, that TRN162’s inability to complete the transmission process results from a defect in surviving or evading this particular stress. There was no significant difference in either of the strains’ ability to grow in acidic media, suggesting that in Photorhabdus, sspAB does not play a role in the regulation of acid resistance genes as it does in E. coli. In addition, it is unlikely that the inability of TRN162 to survive a potential reduction in intestinal pH is the cause of the transmission defective phenotype. Furthermore, the ability of these strains to grow in the presence of APs was evaluated. Again, no significant difference in growth in the presence of the APs tested was found between TRN162 and NC1Tn7GFP, indicating that either 55 sspAB does not play a role in resistance to this particular type of stress or that the transmission defect is specific to a Heterorhabditis AP not tested in this study. Conversely, when grown in the presence of H202, TRN162 displayed a two-order of magnitude decrease in survival compared to NC1Tn7GFP at 100 mM H202. Although there was no significant difference in the strains’ ability to grow in the presence of another oxidative stressor, paraquat, this study has provided evidence that suggests an oxidative stress may be introduced into the nematode intestine at 120 h post-recovery and that resistance to this stress is mediated through sspAB. While it would be expected that TRN162 would respond similarly when exposed to paraquat and H202, the method of paraquat exposure in this study likely contributed to the observed phenotype. Had the strains been cultured in a larger volume with greater aeration during growth, TRN162 may have displayed an increased sensitivity to paraquat. Lastly, TRN162 displayed a hypermotile twitching motility when grown on 0.75% agar plates, where it migrated over four times farther from the point of inoculation than NC1Tn7GFP. As twitching motility is mediated by type IV pill (21), it may be that this mutant is hyperpiliated. NC1Tn7GFP and TRN162 showed similar swarming motility. Therefore, while it is unlikely that sspAB plays a role in swarming motility, it is clear that it functions to negatively regulate twitching motility. These motility phenotypes may have particular significance in symbiont transmission. It may be that swarming or twitching motility is essential for the bacteria to invade the PlVCs in order to escape an intestinal-clearing 56 stress produced by developing nematodes to ensure only true symbionts are transmitted to Us. Much remains to be Ieamed about the sspAB operon in Photorhabdus and in particular, its role in symbiont transmission to H. bacteriophora le. Analysis of genes differentially regulated by the sspAB mutant vs. the parental strain by DNA microarray experiments would offer great insight into the role sspAB plays in Photorhabdus. The upstream signals of sspAB have yet to be identified in this model organism, as well as genes functioning downstream. At this time, it is unknown if the phenotypes shown in this study are mediated solely through sspA or rather through a combined effect of the non-functional sspAB operon. A prudent line of investigation would be to construct a targeted in-frame gene knockout of sspA and sspB individually to determine the answer to this question. Additionally, a closer examination of the surface of TRN162 cells, perhaps by electron microscopy, could answer questions pertaining to the piliation of the mutant as compared to the parental strain. 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MglA regulates Francisella tularensis subsp. novicida response to starvation and oxidative stress. J Bacteriol. 189:6580—6586. Han, R., and R. U. Ehlers. 2000. Pathogenicity, development, and reproduction of Heterorhabditis bacteriophora and Steinernema carpocapsae under axenic in vivo conditions. J. Invertebr. Pathol. 75:55- 58. Hansen, A.-M., Y. Gu, M. Li, M. Andrykovitch, D. S. Waugh, D. J. Jin, and X. Ji. 2005. Structural basis for the function of stringent starvation protein A as a transcription factor. J. Biol. Chem. 280:17380-17391. Hansen, A.-M., H. Lehnherr, X. Wang, V. Mobley, and D. J. Jin. 2003. Escherichia coli SspA is a transcription activator for bacteriophage P1 late genes. Mol. Microbiol. 48:1621-1631. Hansen, A. M., Y. Qiu, N. Yeh, F. R. Blattner, T. Durfee, and D. J. Jin. 2005. SspA is required for acid resistance in stationary phase by downregulation of H-NS in Escherichia coli. Mol. Microbiol. 56:719-734. lshihama, A., and T. Saitoh. 1979. Subunits of RNA polymerase in function and structure. IX. Regulation of RNA polymerase activity by stringent starvation protein (SSP). J. Mol. Biol. 129:517-530. Joyce, S. A., R. J. Watson, and D. J. Clarke. 2006. The regulation of pathogenicity and mutualism in Photorhabdus. Curr. Opin. Microbiol. 9:127-132. 59 21. 22. 23. 24. 25. 26. Mattick, J. S. 2002. Type IV pili and twitching motility. Annu. Rev. Microbiol. 56:289-314. Milstead, J. E. 1979. Heterorhabditis bacteriophora as a vector for introducing its associated bacterium into the hemocoel of Galleria mellonella larvae. J. Invertebr. Pathol. 33:324-327. Poinar, G. 0. 1975. Description and biology of a new insect parasitic rhabditoid, Heterorhabditis Bacteriophora N. Gen., N. Sp. (Rhabditida; Heterorhabditidae N. Fam.). Nematologica 21:463-470. Schwyn, B., and J. B. Neilands. 1987. Universal chemical assay for the detection and determination of siderophores. Anal. Biochem. 160:47-56. Watson, R. J., S. A. Joyce, G. V. Spencer, and D. J. Clarke. 2005. The ebe gene of Photorhabdus temperata is required for full virulence in insects and symbiosis with the nematode Heterorhabditis. Mol. Microbiol. 56:763-773. Williams, M. D., T. X. Ouyang, and M. C. Flickinger. 1994. Starvation- induced expression of SspA and $3sz the effects of a null mutation in sspA on Escherichia coli protein synthesis and survival during growth and prolonged starvation. Mol. Microbiol. 11:1029-1043. 60 Chapter 3: Summary and Future Directions This chapter is divided into two parts. Part | is a summary of the work presented in this study. Part II focuses on future research to be done on this project. 61 Summary Research presented here describes a Photorhabdus temperata ssp. NC1 transmission defective mutant, TRN162, which is unable to complete the colonization process in Heterorhabditis bacteriophora nematodes. The disrupted gene in TRN162 is sspA, encoding a putative homolog of the stringent starvation proteins A. There is also a predicted polar effect on the downstream sspB. In addition, this study provides some insight into what kind of stress may be employed by the nematode to ensure that only true bacterial symbionts complete the transmission process. An oxidative stress, such as H202, seems to be the most likely to be produced by the nematode at 120 h post-recovery as this was the only type of stress studied here that showed a difference between NC1Tn7GFP and TRN162. Moreover, the results of this study suggest that the sspAB regulon may function differently in Photorhabdus than it does in other microorganisms. For example, TRN162 does not show an increased sensitivity to acidic stresses, while the E. coli sspA homolog displayed just the opposite phenotype (2). Also, in Photorhabdus, sspAB seems to negatively regulate twitching motility. In both E. coli and Y. enterocolitica,’sspA negatively regulates swarming motility (1, 2). A proposed model for the role of sspAB in Photorhabdus transmission in H. bacteriophora is outlined here (Figure 3-1). As the nematode develops through its life cycle, its intestinal symbionts go through an elaborate selective process that ensures that only true symbionts are transmitted to the next 62 20E3_ .02_.00.2_ 2.000 :00 20.0222: 020 00.00.0200 2000003.. 20.00.... 0 .0 22.0220. 2. 02300. .9000 0202022202 0 2030.2. 0.3005 02.00.00 02. 000000 .0 0.00. 05.0 0. 0.2.0“. .0. 90.000 020.822 0 2...... 0.20.0220 03.. .3 0020380000 .220 0. 0022 02. 02.002. 020 20E3_ .02..00.2. 02. 02.2.0 .3 0020 02.00.00 02. .0 20.00>w Am. 9000 022.023. 0 2...... 0.20.0E>0 03.. .5 .220 00>.>.30 0. 020.000 0..0.02.>0-202 .0 202.3. 02:00.2. 0.. .00.0 0. 00202.02 02. .5 0003005 00020 < 2. 02.000 2030.... 00.0.002. 00 >.0>000.-.000 2 ON. .0 0.023.083 .I 2. 20.00.E020.. 20.0.20 .0. .0006 0000205 < .70 0.30.". 63 generation of Us. At 120 h post-recovery, the developing IJ produces a stress that only true symbionts will be able to survive. Survival of this stress is mediated through sspAB. Data presented in this study suggest that an oxidative stress, such as H202, may be produced at this time. Alternatively, it may be that only true symbionts are able to invade the PlVCs, thus escaping the selective stress produced in the nematode intestine at 120 h. This process may be mediated through sspAB and its effects on motility. Failure to either resist or escape this selective pressure results in the formation of a spheroplast and ultimately, death of the bacterium. Future Directions While some fundamental characterizations of TRN162 were made in this study, many questions remain unanswered. Are the phenotypes described in this study mediated solely through sspA or sspB, or are both genes involved? What are the genes regulated by sspAB in Photorhabdus and how do these genes function in the transmission process? How do H. bacteriophora nematodes ensure only true symbionts complete the transmission process? It is unknown if the phenotypes shown in this study are mediated solely through sspA or rather through a combined effect of the non-functional sspAB operon. It would be prudent to construct targeted in-frame gene knockouts of sspA and sspB individually, so that there are no polar effects on sspB. By then 64 examining the oxidative stress resistance and twitching motility of these strains, the answer to this question could be determined. Additionally, a DNA microarray study would be useful to identify genes differentially regulated between TRN162 and NC1Tn7GFP. Similar previous approaches in an E. coli sspA mutant led researchers to the acid sensitive sspA phenotype (2). In particular, using a microarray approach with the Photorhabdus-H. bacteriophora model system may provide greater insight into the stress imposed upon intestinal inhabitants near the PlVCs of pre-le. Reverse transcription PCR (RT-PCR) experiments would confirm the suggested sspAB regulation of genes involved in the resistance to oxidative stresses or the biosynthesis of type IV pili. In an effort to discover how H. bacteriophora controls the transmission of symbionts to developing le, growth in the presence of varying stresses was monitored in this study. However, an alternative approach to this would be to monitor the response of TRN162 to these stresses over a shorter amount of time. In this way, one would be monitoring survival as opposed to growth and there may be a more distinct phenotype in the presence of these stresses, which could offer greater insights into the particular stress that is hypothesized to be introduced in the nematode intestine. For example, much of the H202 may be degraded by bacteria not initially killed in a shorter exposure. When the H202 has reached a lower, more tolerable level for these bacteria, they may resume growth, resulting in a reduced CFU/ml as seen in this study. 65 It was shown here that TRN162 has an increased twitching motility phenotype compared to the parental strain. As twitching motility is mediated by type IV pili, electron microscopy of individual cells of TRN162 or fluorescent microscopy using antibodies specific for type IV pili would conflrrn if this strain is hyperpiliated. Additionally, targeted deletions of type IV pili biosynthetic genes would confirm that Photorhabdus symbionts need to have functional twitching motility in order to invade the PlVCs of developing Us. 66 References Badger, J. L., and V. L. Miller. 1998. Expression of invasin and motility are coordinately regulated in Yersinia enterocolitica. J. Bacteriol. 180:793- 800. Hansen, A. M., Y. Qiu, N. Yeh, F. R. Blattner, T. Durfee, and D. J. Jin. 2005. SspA is required for acid resistance in stationary phase by downregulation of H-NS in Escherichia coli. Mol. Microbiol. 56:719-734. 67 Appendices 68 RT-PCR of H-NS and RpoS As it has been shown in an E. coli sspA mutant, there are altered levels of the H-NS and RpoS proteins (3), RT-PCR was performed on hns and rpoS to determine if these genes were being expressed in TRN162. RNA was isolated using Trizol (lnvitrogen) from TRN162 and NC1Tn7GFP grown in Grace’s Insect Media at 28°C for 24 h, 12 h and 4 h. RNA was DNasel (lnvitrogen) treated and converted to cDNA using Therrnoscript reverse transcriptase (lnvitrogen) at 53°C using the primers below. cDNA was then used as a template for amplification PCR. hns RT for: 5’ cgtactttacgagcccaagc 3’ hns RT rev: 5’ gcacgacgttttgctttacc 3’ rpoS RT for: 5’ ttcggttgatacccccatta 3’ rpoS RT rev: 5’ ttccaaaagaccaaaacgac 3' In both NC1Tn7GFP and TRN162, hns is expressed at all time points tested here (Figure 3-2). In the rpoS samples, the gene is being expressed by both strains at 12 h and 24 h of growth. However, at 4 h growth, neither strain showed strong expression of rpoS. This could be due to an un-optimized annealing temperature for these primers, as amplification was not as robust as in the hns samples. Alternatively, the absence of rpoS mRNA at 4 h could be due to the early growth stages of these cultures, as the expression of rpoS is increased under stressful or starvation conditions (2). While this experiment examined the presence or absence of hns and rpoS mRNA, quantitative RT-PCR 69 could be used to determine if there are altered mRNA levels corresponding to these genes in TRN162 compared to the parental strain. Figure A-1: RT-PCR of hns and rpoS. (A) hns. 1, 24h; 2, 12h; 3, 4h. (B) rpoS. 1, 24h; 2, 12h. T, TRN162. N, NC1Tn7GFP. (A) (B) A ClpA/B type chaperone is disrupted in TRN17-96 Another NC1Tn7GFP-derived mutant identified in the Himaer transposon mutagenesis screen was TRN17-96. This mutant is defective at the initial stage of biofilm formation in the H. bacteriophora intestine and the gene disrupted is plu2287, which is predicted to encode a homolog of a ClpAlB-like chaperone protein (Figure 3-1). Located just downstream at plu2285 and plu2284 are genes encoding putative homologs of the Bordetella virulence gene (ng) two component response regulator (ngA) and sensor kinase (ngS), respectively. However, primer extension analysis has shown that in Photorhabdus, these genes are not co-transcribed with plu2287, but in fact have their own promoter (1 ). 70 Figure A-2: Gene organization surrounding transposon insertion in TRN17-96. prA/B is disrupted by the Himaer transposon. Himaer bng ng plu2286 clpA/B W- _< Growth rate of TRN17-96 As with any mutant being studied, it is prudent to determine the growth rate and doubling time. Growth rate (p) and doubling time (T2) was measured as described in Chapter 2 (Table 3-1). Table A-1: Growth rate of NC1Tn7GFP and TRN17-96 Growth Rate Doubling Time NC1Tn7GFP 0.273 :I: 0.013 h'1 2.54 1 0.12 h TRN17-96 0.248 1 0.017 h" 2.80 :l: 0.19 h While the growth rate of TRN17-96 (0.248 i 0.017 h") is slower than NC1Tn7GFP (0.273 :t 0.013 h“), it is not likely that this causes the defective transmission phenotype. Just as with TRN162, examining this mutant at later times post-recovery does not resolve the mutant’s inability to complete the transmission process, although in a competitive situation, TRN17-96 may be at a disadvantage compared to NC1Tn7GFP. 71 Phase variation of TRN17-96 As secondary phase variants cannot colonize the nematode intestine, several phase characteristics of TRN17-96 were determined as described previously. This mutant displays mostly primary phase characteristics comparable to NC1Tn7GFP (Table 3-2). It has a yellow pigmentation, produces crystalline inclusion proteins and is positive for siderophore activity. TRN17-96 is able to absorb pigments from its media supplemented with neutral red, bromthymol blue or Congo red. However, when grown in the presence of eosin y-methylene blue, TRN17-96 did not produce the metallic green sheen observed when NC1Tn7GFP was grown on the same media, but rather exhibited purple pigmented growth. Although the majority of phase variation characteristics studied in TRN17- 96 suggest that it is a primary phase variant, the rate at which it switches to a secondary phase variant is much more rapid than NC1Tn7GFP (Figure 3-2). Interestingly, a mutation in the Photorhabdus bng homolog has been shown to increase the rate at which the strain transitions from primary to secondary phase variants (1). To monitor this phase switching, NC1Tn7GFP and the mutant were inoculated to the same ODeoo (0.2) in 100 ml of PP3S and grown at 28°C. At 24 h intervals, 10-fold serial dilutions were made and plated on PP3S. Colonies were scored for phase variation on the basis of their translucence observed under a dissecting microscope. The production of crystalline inclusion proteins, a hallmark of primary phase variants, makes colonies opaque under this microscope. Even after only 24 h of growth, more than 10% of the colonies of 72 TRN17-96 exhibited translucence. Between 7 to 8 d of growth, approximately 50% of the colonies of TRN17-96 were scored as secondaries. In comparison, NC1Tn7GFP did not exhibit any translucence until 8 d of growth. However, there is doubt that this increased rate of switching to a secondary phase variant is the cause of the transmission defect in TRN17-96 because even at 72 h of growth, more than 80% of the cells can be classified as primaries. 73 30=0> 0.0022 202.00 + + I+++ + >>0=0> 2003:. 3.0.2.00 + + +++++ 0..2>> 2832.202 ..0.... .so..0> 2832. 20200 + + +++++ 2030.20Efl 80.02902. .8200 02.0.0.2 20.0202. 0.2.00 2.2.00 0.0222020 0.03.8.2 0.2.82.3 0020000222220 00. 00200 03.2 .oE..2.Eo.m 022 02032.05; 2.00m 00. .8502 2022.002... 0.6 3.2.25. 0002502 b00283 p02 E0522 ’02 00>000< 02.302020 .om-.....zm._. 020 nEOnchwoz 50020000 .02 $202.20 .02 .0 20..0N..0.00.020 20..0..0> 0002a .N..< 030... 74 Figure A-3: Rate of phase variation switching of NC1Tn7GFP (black) and TRN1 7-96 (gray) 100‘ m1 3%; %Socondary B 9% é 8? 8 _L C — O 11 12 13 75 References Derzelle, S., S. Ngo, E. Turlin, E. Duchaud, A. Namane, F. Kunst, A. Danchin, P. Bertin, and J. F. Charles. 2004. AstR-AstS, a new two- component signal transduction system, mediates swarming, adaptation to stationary phase and phenotypic variation in Photorhabdus Iuminescens. Microbiol. 150:897-910. Gentry, D. R., V. J. Hernandez, L. H. Nguyen, D. B. Jensen, and M. Cashel. 1993. Synthesis of the stationary-phase sigma factor sigma s is positively regulated by ppGpp. J. Bacteriol. 175:7982-7989. Hansen, A. M., Y. Qiu, N. Yeh, F. R. Blattner, T. Durfee, and D. J. Jin. 2005. SspA is required for acid resistance in stationary phase by downregulation of H-NS in Escherichia coli. Mol. Microbiol. 56:719-734. 76 llllllllllllllllllllllllllllll IIHIHWIIUIllillMW“!IllHllNHHIHIUIWIUIIIIHl