INVESTIGATING SURVIVAL MECHANISMS OF YERSINIA RUCKERI IN
RAINBOW TROUT (ONCORHYNCHUS MYKISS)
A Thesis
Presented to
The Faculty of Graduate Studies
of
The University of Guelph
by
INDERVESH
In partial fulfilment of requirements
for the degree of
Doctor of Philosophy
May, 2008
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INVESTIGATING SURVIVAL MECHANISMS OF YERSINIA RUCKERI
IN RAINBOW TROUT (ONCHORHYNCHUS MYKISS)
Indervesh Advisor: University of Guelph, 2008 Dr. R.M.W. Stevenson
Yersinia ruckeri causes hemorrhagic septicemia of salmonid fish. We hypothesized that the ability to persist inside infected fish was a key component of virulence strategy of Y. ruckeri and aimed to identify survival-essential genes of Y. ruckeri serotype 1 strain, RSI 154. After two rounds of screening of 1056 miniTn5Km2 signature-tagged mutants, 25 mutants that did not survive in kidney at 7 days post infection in immersion infected rainbow trout were selected for further study. Sequencing of interrupted genes in selected mutants identified genes homologous to znuA, which encodes a component of a zinc transporter in Escherichia coli, and uvrY, which encodes the response regulator of BarA-UvrY two-component system (TCS) in E. coli.
The uvrY mutant was hypersensitive to EkC^-mediated killing and was less invasive to Epithelioma papulosum cyprini fish cells than wild type (WT) bacteria, but was not affected in serum sensitivity or growth under iron-limiting conditions. In a competitive infection with WT, the uvrY mutant had lower infection loads in rainbow trout kidney.
When present in a low-copy plasmid, the znuACB locus of Y. ruckeri fully restored growth of a zinc-transport deficient kznuACB mutant of E. coli in Luria-Bertani (LB) medium supplemented with 2.0 mM ethylenediamine tetraacetic acid (EDTA),
indicating that znuACB locus of Y. ruckeri is likely involved in zinc transport. Unlike
AznuACB mutants of E. coli, AznuACB mutant of Y. ruckeri did not show poor growth in
zinc-deficient M9 medium and LB medium supplemented with metal chelators, EDTA
and tetrakis-(2-pyridylmethyl)-ethylenediamine, suggesting presence of additional zinc transporters in Y. ruckeri. The znuA mutant of Y. ruckeri was out-competed by WT in rainbow trout kidney.
Survival of Y. ruckeri in rainbow trout was also reduced with mutations in gene homologs encoding an O-antigen polymerase (wzy), peptidoglycan deacetylase (pdaA), protease (ptrA), bundle-forming pili (rcpA), ATPase of DNA segregation (parA) and transposase of Tn7, bacteriophage tail fiber-like protein and genes of unknown functions.
Characterization of survival-essential genes in fish helps identify new virulence genes
and contributes to understanding survival strategies and pathogenic mechanisms of Y.
ruckeri. ACKNOWLEDGMENTS
Financial support for this study was provided by the Natural Sciences and Engineering
Research Council of Canada and the Fish Culture section of Ontario Ministry of Natural
Resources through grants to Dr. R.M.W. Stevenson.
I sincerely thank my advisor Dr. R.M.W. Stevenson for her constant help, constructive criticism, and excellent mentorship throughout my thesis research. Besides learning principles of science from her, I learned so many things as a person that I will cherish throughout life.
I would like to thank members of my advisory committee, Dr. J. Wood, Dr. J. Prescott and Dr. M. Ferguson for their guidance, constructive criticism and critical evaluation of my thesis.
Thank you to members of my examination committee, Dr. J. Nash, Dr. P. Krell, Dr. J.
Wood, Dr. J. Prescott, and R. Lo for reading my thesis and suggestions.
Special thanks to Dr. L. Mutharia, Dr. Reggie Lo and Dr. Jerry King for helpful discussion on various research problems pertaining to this work.
I would like to acknowledge help of my friend, Dr. A. Singh for statistical analyses.
Many thanks go to S. Lord, V. Harper, M. Raymond, R. Quinn and R. Jones for their nice company and help with experiments. I enjoyed wonderful company and help of many friends throughout this study.
No words can express my thankfulness to my parents, grandparents and wife for everything they have done and are doing for me. Their constant help and support made this difficult task possible. You people matter the most for me.
i TABLE OF CONTENTS
Page#
TABLE OF CONTENTS i LIST OF TABLES v LIST OF FIGURES vi CHAPTER 1 INTRODUCTION 1 LITRATURE REVIEW 3 1.1. Yersinia ruckeri 3 1.2. Plasmids of Y. ruckeri 4 1.3. Serotypes of Y. ruckeri 6
1.4. Enteric redmouth disease 8
1.5. Stress and ERM disease 9
1.6. Pathogenic mechanisms of Y. ruckeri 10
1.6.1. Environmental survival of Y. ruckeri 10
1.6.2. Attachment and entry into fish 12
1.6.3. Factors involved in resistance to fish defenses 14
1.6.4. Toxins causing host damage 18
1.7. Regulation of virulence genes expression 21 1.8. Model of Y. ruckeri pathogenesis 23 1.9. Signature-tagged mutagenesis (STM) and identification 25
of virulence genes
1.9.1. Animal model for screening ST mutants 29
1.9.2. Genes identified in STM screens 32
1.10. Research objectives and approach 37
n CHAPTER 2
MATERIALS AND METHODS 38 2.1. Bacterial strains and growth conditions 38 2.2. Recombinant DNA techniques 41 2.3. Competent cell preparation 41 2.4. Polymerase chain reaction 44 2.5. Generation of signature-tagged (ST) mutants 44 2.6. Randomness of transposon insertion 49 2.7. Rainbow trout infection experiments 50 2.8. Screening of ST mutants in rainbow trout 52 2.9. Number of transposon insertions in the genome of mutants 53 2.10. Sequence characterization of mutants 53 2.11. Screening for auxotrophs 54 2.12. Identification of barA homolog of Y. ruckeri RSI 154 54 2.13. PCR amplification of barA and uvrY genes in Y. ruckeri strains 55 2.14. Growth of uvrY mutant in Luria-Bertani medium and 55 under iron-limiting conditions 2.15. Fish cell invasion by uvrY mutant 56 2.16. Serum sensitivity of uvrY mutant 57 2.17. Ultraviolet light (UV) sensitivity of uvrY mutant 57 2.18. H2O2 sensitivity of uvrY mutant 58 2.19. Sequence characterization of znuACB locus 58 2.20. PCR amplification and cloning of znuACB locus 59 2.21. Generation of AznuACB mutant 59 2.22. In vitro growth of AznuACB mutant 62 2.23. Transcomplementation of AznuACB mutant of E. coli 64 2.24. The znuAv.lacZ and znuCBv.lacZ transcriptional fusion analysis 64 2.25. Generation of isogenic mutants of Y. ruckeri 68 2.26. Statistical analyses 68
iii CHAPTER 3 GENERATION AND SCREENING OF SIGNATURE-TAGGED 69 MUTANTS RESULTS 69 3.1. Generation of ST mutants 69 3.2. Primary screening of ST mutants in rainbow trout 69 3.3. Secondary screening of ST mutants in rainbow trout 79 3.4. One-to-one competitive challenge of mutants with WT strain 81 3.5. Effect of miniTn5Km2 cassette on survival of mutants 85 3.6. Number of transposon insertions in ST mutants 85 DISCUSSION 85 CHAPTER 4 CHARACTERIZATION OF MUTANTS UNABLE TO 92 SURVIVE IN FISH RESULTS AND DISCUSSION 92 4.1. Screening for auxotrophy 92 4.2. Identification of mutated genes in ST mutants 92 4.3. Gene encoding bundle-forming pili (Bfp) 99 4.4. O-antigen polymerase 100 4.5. Polysaccharide deacetylase 101 4.6. PtrA protease 103 4.7. Genes involved in cellular transport 106 4.8. Insertion sequence (IS) /transposon proteins 106 4.9. Bacteriophage-like genes 109 4.10. ATPase for DNA segregation 111 CHAPTER 5 CHARACTERIZATION OF THE BarA-UvrY 113 TWO-COMPONENT SYSTEM RESULTS 114 5.1. In silico analysis of UvrY response regulator 114 5.2. Non-polarity of the uvrY mutation 119
IV 5.3. Growth of uvrY mutant in Luria-Bertani medium 123 5.4. H2O2 sensitivity of uvrY mutant 123 5.5. Serum sensitivity of uvrY mutant 126 5.6. Growth of uvrY mutant under iron-limiting conditions 126 5.7. Fish cell invasion by uvrY mutant 126 5.8. The uvrY was required for survival in rainbow trout 130 5.9. Identification of barA homolog of Y. ruckeri 130 5.10. Identification of BarA-UvrY TCS in different serotypes of Y. ruckeri 139 DISCUSSION 139 CHAPTER 6 CHARACTERIZATION OF THE ZnuACB TRANSPORTER 149 RESULTS 149 6.1. Identification of ZnuACB transporter 149 6.2. Generation of AznuACB mutant of Y. ruckeri RS1154 154 6.3. Phenotypic characterization of AznuACB mutant 155 6.4. ZnuACB transporter of Y. ruckeri was functionally active in E. coli 160 6.5. znuAv.lacZ and znuCBv.lacZ transcriptional fusions analyses 170 6.6. ZnuACB transporter was required for survival in rainbow trout 172 DISCUSSION 172 CHAPTER 7 GENERAL DISCUSSION 180 REFERENCES 186 APPENDIX 1: Genetic location of miniTn5Km2 insertion in the genome 207 of 25 signature-tagged mutants of Y. ruckeri APPENDIX 2: Construction of isogenic mutants of Y. ruckeri 210 APPENDIX 3: Strategy for construction of an aroA isogenic mutant 215 APPENDIX 4: Strategy for construction of an isogenic znuA mutant 217 APPENDIX 5: Strategy for construction of an isogenic B3-2 mutant 219 APPENDIX 6: Strategy for construction of an isogenic A6-11 iptrA) mutant 221 APPENDIX 7: Strategy for construction of an isogenic uvrY mutant 223
v LIST OF TABLES
Table Page # 1. Bacterial strains used in this study 39 2. Plasmids used in this study 42 3. Sequences of DNA-signature tags used for making and 45 screening signature-tagged mutants 4. List of oligonucleotide primers used in this study 46 5. Screening of signature-tagged mutants in rainbow trout 72 6. Primary screening of signature-tagged mutants in rainbow trout 74 7. Effect of infection route on mutant recovery from rainbow trout 80 8. Secondary screening of signature-tagged mutants in rainbow trout 82 9. Competitive challenge of Fl-2, A5-4, and B1-5 mutants with 84 WT strain in rainbow trout 10. Effect of the miniTn5Km2 cassette on survival of 86 signature-tagged mutants in rainbow trout 11. Accession numbers of sequences of genes interrupted in 94 signature-tagged mutants of Y. ruckeri identified in this study 12. Y. ruckeri genes identified by STM screens in rainbow trout 95 13. Competitive challenge of mutant A6-11 (protease III) 105 and WT strain in rainbow trout 14. Competitive challenge of mutant uvrY and WT strain 115 in rainbow trout 15. The uvrY mutant was hypersensitive to 1 mM and 10 mM H2O2 116 16. Ultravoilet light sensitivity of uvrY mutant and WT strain 122 17. Effects of uvrY mutation on serum sensitivity of Y. ruckeri 127 18. znuAv.lacZ and znuACB::lacZ transcriptional fusion assays 171 19. Competitive challenge of mutant C6-1 and WT strain 173 in rainbow trout
VI LIST OF FIGURES
Page# Construction of signature-tagged mutants and screening 27 of libraries for identifying survival-defective mutants in rainbow trout Strategy for making the DNA construct used for generating 60 the AznuACB mutant Gene organization of znuACB locus and strategy for 63 making the AznuACB mutant of Y. ruckeri RSI 154 Strategy for making the znuAwlacZ and znuCBv.lacZ 66 transcriptional fusions of Y. ruckeri Randomness of transposon insertion in signature-tagged mutants 70 Number of transposon insertions in the genomic DNA of 87 signature-tagged mutants that were missing in rainbow trout Growth of signature-tagged mutants in M9 minimal medium 93 Genetic organization of F5-8 locus of Y. ruckeri 108 Genetic organization of the uvrY locus of Y. ruckeri RS1154 117 Sequence alignments of UvrY homologs 120 Growth of uvrY mutant in LB medium 124 Growth of Y. ruckeri WT and uvrY mutant in LB medium 128 with and without 200 uM 2,2' dipyridyl iron chelator PCR amplification of bar A homolog of Y. ruckeri 131 Sequence alignments of BarA homologs 132 PCR amplification of bar A and uvrY genes in different 140 serotypes of Y. ruckeri Sequence alignment of Zn/Mn-binding proteins of C9 class 151 of ABC transporter family Growth of AznuACB mutant of Y. ruckeri in M9 minimal medium 156
vn 18. Growth of AznuACB mutant of Y. ruckeri in M9 minimal medium 158 supplemented with 1 uM TPEN 19. The AznuACB mutant was not defective in growth in LB medium 161 20. Effect of EDTA on growth of AznuACB mutant of Y. ruckeri 163 21. Effect of TPEN on growth of AznuACB mutant of Y. ruckeri 165 22. PCR amplification of znuACB locus of Y. ruckeri RS1154 167 23. znuACB locus of Y. ruckeri complemented 168 E. coli AznuACB mutant
viii CHAPTER 1
INTRODUCTION
Yersinia ruckeri is a Gram-negative bacterium that causes enteric redmouth
(ERM) disease in salmonid fish (Ross et al, 1966). The disease occurs in two forms: an acute hemorrhagic septicemia and a chronic carrier state (Rucker 1966, Busch and
Lingg 1975). The fish may carry bacteria without overt disease, and disease outbreaks occur under stressful conditions of high water temperature, poor water quality, transportation, handling, and overcrowding of fish (Lesel et al, 1983, Roberts 1983).
Thus, infection by Y. ruckeri does not always result in disease unless fish are stressed.
Earlier studies on Y. ruckeri failed to identify major chromosomal or plasmid-encoded virulence factors similar to those characteristic of other pathogenic Yersiniae, or of other fish pathogenic bacteria (Guilvout et al, 1988, Kawula et al, 1996, Gunasena et al, 2003).
In our proposed model of ERM disease, a host-pathogen balance is established in fish after infection by Y. ruckeri, and disease occurs when stressors tilt the host- pathogen balance. Infections by Y. ruckeri are most commonly found as an asymptomatic carrier state, in which survival and persistence are key parts of the virulence strategy. Thus, identification of genes required for survival and persistence may help identify virulence factors and aid understanding of the pathogenesis of Y. ruckeri in fish.
The objectives of this study were to identify survival-essential genes of Y. ruckeri in rainbow trout, and to further evaluate their roles in pathogenesis. To do this, I used
1 signature-tagged mutagenesis (STM), a technique in which mutants generated by a transposon insertion are screened in an animal host to select survival-defective mutants
(Hensel et al, 1995). The STM-based mutant screens allow identification of genes that, when mutated, decrease survival of the pathogen in the host (Hensel et al, 1995).
To ensure that genes identified in this study affect a natural disease process, fish infection studies were done using a water-borne bath immersion challenge method.
Rainbow trout, Oncorhynchus mykiss, were used as an animal host, since most disease outbreaks have occurred in this species. To understand the roles of gene products in pathogenesis, genes identified by STM screens were characterized by conducting various in vitro functional assays (fish cell invasion, serum-sensitivity, oxidative stress, siderophore production) and competitive time-course fish infection experiments.
Identification and characterization of various virulence and virulence-associated genes contributed in understanding survival strategies and pathogenesis of Y. ruckeri in rainbow trout. Genetic manipulation techniques for creating isogenic mutants and the rainbow trout infection model established in the study will help others to evaluate the roles of other genes in virulence mechanisms of Y. ruckeri.
2 LITERATURE REVIEW
1.1. Yersinia ruckeri
Yersinia ruckeri is a Gram-negative, rod-shaped bacterium belonging to the family Enterobacteriaceae (Ewing et al, 1978). It was first isolated in the 1950's from hatchery reared rainbow trout in the Hagerman Valley, Idaho and was initially called
Enteric Redmouth bacterium (Rucker, 1966). In Canada, Y. ruckeri was first isolated in
Saskatchewan in fish imported from the Hagerman Valley (Wobeser, 1973).
At the time of initial classification, the Enteric Redmouth bacterium showed similar DNA:DNA hybridization values (up to 30 %) with both Yersinia and Serratia
(Ewing et al, 1978). The biochemical properties and mole % guanine+cytosine content of Enteric Redmouth bacteria (47.5 % - 48.5 %) were more similar to those of Yersinia
(46 % - 50 %) than Serratia (58 %). Thus, the Enteric Redmouth bacterium was placed in the genus Yersinia (Ewing et al, 1978). However, strains of Y. ruckeri can be differentiated from other Yersiniae based on plasmid profiles, multilocus sequence typing
(MLST), and 16S RNA sequences (De Grandis and Stevenson 1982, De Grandis et al,
1988, Kotetishvili et al, 2005). By MLST, using 16S RNA and four housekeeping genes
(glnA, gyrB, recA and Y-HSP60), Kotetishvili et al (2005) reported that four strains of Y. ruckeri examined were phylogenetically most distantly related, showing the largest genetic distances from strains of 10 other environmental and human pathogenic Yersinia species.
3 The virulence mechanisms of Y. ruckeri may be significantly different from those of human pathogenic Yersiniae. The key virulence factors of Y. pestis, Y. enterocolitica and Y. pseudotuberculosis such as adhesin, Ail, virulence plasmid, pYV and Ysc-Yop type III secretion system (TTSS) are not present in Y. ruckeri (Guilvout et al, 1988,
Kawula et al, 1996, Gunasena et al, 2003). The Ail is a 17 kDa outer membrane protein required for adhesion, invasion of host cells and resistance to serum-mediated killing
(Miller and Falkow 1988, Bliska and Falkow 1992, Biedzka-Sarek et al, 2005). No signals were obtained when the chromosomal DNA of Y. ruckeri was hybridized under moderate stringency conditions with an ail probe from Y. enterocolitica (Kawula et al,
1996). The Ysc-Yop TTSS is a virulence determinant present in all human pathogenic
Yersinia that is required for invasion of host tissue and subversion of the immune defense by suppressing phagocytosis, proinflammatory cytokines, chemokines and adhesion molecules (reviewed by Cornells, 2002). This key virulence determinant is also absent from Y. ruckeri, since no PCR product was obtained using degenerate primers designed for yscN, encoding an ATPase component of the Ysc TTSS (Gunasena et al, 2003).
1.2. Plasmids of Y. ruckeri
Yersinia pestis, Y. enterocolitica, and Y. pseudotuberculosis carry a 40 - 50 MDa plasmid, pYV, shown to be involved in host invasiveness, survival in lymphoid tissues, serum resistance, apoptosis of host cells and subversion of phagocytic defenses (reviewed by Cornells et al, 1998, Cornells 2002). Ten of the 12 serotype 1 strains of Y. ruckeri examined by De Grandis and Stevenson (1982) contained a 40 - 50 MDa plasmid and eight of these 10 strains also contained an additional plasmid of 20 - 30 MDa, whereas
4 two strains did not contain any plasmid. Among five serotype 2 strains tested, two contained plasmids of 5.5 MDa or less, whereas no plasmid was present in the remaining strains. Other whole cell-antigen-based serotypes either lacked any plasmid or had plasmids of very small sizes (De Grandis and Stevenson, 1982). Similar plasmid profiles were reported by Romalde et ah (1990); all 20 serotype 1 strains contained plasmids of
40 -50 MDa and 20 - 30 MDa, whereas three serotype 2 and one serotype 4 strains contained no plasmid or had plasmids of small sizes. Thus, the majority of serotype 1 strains of Y. ruckeri contained a 40 - 50 MDa plasmid that was similar in size to pYV plasmid. The plasmids of Y. ruckeri were found to be different from that of other Yersinia based on restriction enzyme digestion patterns and DNA hybridization (De Grandis 1987,
Guilvout et ah, 1988). The BamHI, HindUI and EcoRl digestion patterns obtained on plasmid DNA of 15 serotype 1 strains of Y. ruckeri were different from that of Y. enterocolitica strains WA and CDC 2383 (De Grandis, 1987). Guilvout et ah (1988) reported that BamHI restriction patterns of plasmid DNA from three serotype 1 and one serotype 2 strains of Y. ruckeri were different from patterns obtained on plasmids of Y. pestis, Y. enterocolitica, Y. pseudotuberculosis strains. The 40 - 50 MDa plasmid of serovar 1 strains of Y. ruckeri was different from the pYV plasmid of other Yersiniae, since no hybridization signal was obtained on BamRl restricted plasmid DNA of Y. ruckeri strains when probed with the 40 - 50 MDa plasmid of Y. enterocolitica (Guilvout etah, 1988).
In other Yersinia, the presence of a 40 - 50 MDa plasmid has been correlated with a temperature-dependent calcium requirement in which bacteria need calcium to grow at
37 °C but not at 25 °C. This phenotype was displayed by pathogenic strains of Yersinia
5 (Gemski et al, 1980, Portnoy et al, 1981). None of the 10 serotype 1 strains of Y.
ruckeri that had the 40 - 50 MDa plasmid was able to grow at 37 °C either in the presence
of calcium or in calcium-deficient medium, suggesting that the presence of the 40 - 50
MDa plasmid in Y. ruckeri was not associated with a calcium requirement (De Grandis
and Stevenson 1982). The role of the 40 - 50 MDa plasmid for other virulence-associated properties of serotype 1 strains of Y. ruckeri could not be evaluated since this plasmid could not be cured (De Grandis, 1987).
1.3. Serotypes of Y. ruckeri
Initially, two serotypes were reported for Y. ruckeri, with serotype 1 strains being unable to ferment sorbitol whereas serotype 2 strains fermented sorbitol (O'Leary 1977).
However, on testing more strains, Flett (1989) observed that sorbitol fermentation was not exclusively restricted to serotype 2 strains. Based on formalin-killed whole cell antigens, six serotypes were designated (De Grandis et al, 1988, Flett 1989).
Serotype 1 strains are most often associated with ERM disease, and the majority of serotype 1 isolates were from rainbow trout, probably due to extensive farming of this species. The serotype 2 strains were mainly associated with chinook salmon and other
Pacific salmon (O' Leary 1977, Cipriano et al, 1986). However, further studies reported that both serotypes were capable of causing disease in other species of fish as well
(Bullock et al, 1976, Knittel 1981, Flett 1989).
A question of interest was whether pathogenic mechanisms used by different
serotypes might be significantly different (Flett 1989, Kim and Stevenson 1998).
Serotype 1 strains use the gastrointestinal tract for entry into rainbow trout, whereas
6 serotype 2 strains do not (Kim and Stevenson, 1998). When serotype 2 strains were introduced into the stomach of rainbow trout by oral intubation, the bacteria were eliminated from the gut without establishing systemic disease. In contrast, most of the serotype 1 strains were able to set up a systemic infection after entry through this route
(Kim and Stevenson 1998). Serotype 1 and serotype 2 strains produced high mortality in rainbow trout (60 % - 90 %) after either injection or bath immersion infection, whereas strains of two other minor serotypes caused no mortality under similar conditions (Flett
1989). Serotype 1 strains were able to suppress the oxidative burst response of striped bass macrophages, whereas there was a strong oxidative burst against serotype 2 strains
(Stave et al, 1987). More importantly, strains with varying degrees of virulence exist within a serotype (Flett, 1989). Strains RS26, RS27 (serotype 1) and RS91 (serotype 2) caused high mortality in rainbow trout, whereas strains RS7 (serotype 1) and RS3
(serotype 2) did not cause any mortality (Flett, 1989).
Past studies on Y. ruckeri have compared strains belonging to different serotypes to identify common virulence factors but without success. In the current study, we focused on identifying virulence genes of a virulent serotype 1 strain, RSI 154. The serotype 1 strain was chosen as most ERM disease outbreaks were associated with this serovar. The strain RSI 154 was originally isolated from diseased rainbow trout from
Idaho Valley, USA and had been fish-passaged in our laboratory. We previously verified virulence of strain RSI 154, as it caused up to 80 % mortality in rainbow trout infected by bath immersion (Flett, 1989). Within serotype 1, a significant phenotypic diversity exists among strains. Strains can be differentiated by plasmid profiles, ability to grow at 37 °C, outer membrane protein profiles, and polymyxin B and bacteriophage sensitivity (De
7 Grandis and Stevenson 1982, Stevenson and Airdrie 1984, De Grandis and Stevenson
1985, Davies 1991a, 1991b).
1.4. Enteric redmouth disease
ERM disease affects primarily fresh water salmonids and most cases of disease have been reported in rainbow trout. The disease occurs in two forms. The acute hemorrhagic septicemia form is characterized by hemorrhage around the mouth, jaw, operculum, lateral line, in the liver, spleen, kidney, muscles and other parts of the body.
The intestine becomes inflamed and filled with thick purulent fluid (Rucker 1966, Miller
1983, reviewed by Tobback et al, 2007). The acute disease is most common in small fish, up to fingerling size.
Infections by Y. ruckeri commonly occur as long-lasting asymptomatic covert infections (Rucker 1966, Busch and Lingg 1975). In this form, immediate mortalities are not a significant problem, but stressful conditions for carrier fish can lead to outbreaks of acute septicemic disease (Rucker 1966, Busch and Lingg 1975, Furones et al, 1993,
Hietala et al, 1995). Rucker (1966) reported a covert infection state for Y. ruckeri after isolating bacteria from the lower intestine of diseased rainbow trout two months after the experimental infections. Busch and Lingg (1975) also demonstrated a clinically asymptomatic covert state in which Y. ruckeri was found to localize in the lower intestine of 50 % - 75 % of survivor rainbow trout at 60 - 65 days post-infection. The bacteria were isolated from the lower small intestine up to 102 days after the experiment ended.
There was a 30 - 40 day cycle of intestinal shedding of the bacteria by covertly infected fish, and bacterial shedding preceded the recurrence of the systemic disease and mortality
8 by 3 - 7 days. The kidney can be another organ infected in fish, since nearly 50 % of 34 brown trout infected by bath immersion carried Y. ruckeri in the kidney without showing any signs of disease (Hietala et al, 1995). As covertly infected fish act as a constant source of reinfection for a fish population, Y. ruckeri infection may become enzootic in hatcheries (Busch and Lingg, 1975).
1.5. Stress and ERM disease
Whereas Y. ruckeri are enzootic in many hatcheries, disease outbreaks do not occur frequently. ERM disease is highly stress-associated and outbreaks have been linked to stressful conditions to fish such as fluctuations in water temperature, poor water quality, overcrowding, and transportation or handling of fish (Busch 1978, Hunter et al.,
1980, Lesel et al, 1983, Roberts 1983). An ERM disease outbreak in the UK was reported in a hatchery whose water was supplied from a highly eutrophic lake receiving effluent from many fish farms. The water had very low dissolved oxygen (Roberts,
1983). An ERM disease outbreak in rainbow trout in France was associated with transportation, handling of fish and higher water temperature (Lesel et al., 1983).
Caldwell and Hinshaw (1995) reported that under hypoxic and super-saturated levels of dissolved O2, cumulative mortality in rainbow trout increased from 10 % to 12.8 % and
17.9 %, respectively. Stress also induces shedding of Y. ruckeri by covertly infected fish
(Hunter et al., 1980). Stress of a water temperature of 25 °C induced Y. ruckeri shedding by carrier steelhead trout (Salmo gairdneri Richardson), and subsequently led to infection of healthy fish that were receiving effluent water from the tank of covertly infected fish.
9 No bacterial shedding was detected in unstressed fish (Hunter et al, 1980). The water temperature for unstressed fish was not reported.
Because of the strong association of ERM disease with stress, classical septicemia and mortality is hard to reproduce reliably. Under good animal care conditions in the laboratory, infected fish can harbor large numbers of bacteria without showing disease
(Flett 1989, Kim and Stevenson 1998, Kim 2000). This can influence criteria chosen for measuring virulence of Y. ruckeri in fish under laboratory conditions. The bacterial infection load in organs and persistence of bacteria in fish, rather than 50 % lethal dose
(LD50) determinations, were used for comparing virulence of Y. ruckeri strains in the current study.
1.6. Pathogenic mechanisms of Y. ruckeri
As noted, the pathogenic mechanisms of Y. ruckeri are not yet clearly defined. In this section, genes and phenotypic characteristics that have been evaluated for their possible role in the pathogenic lifestyle of Y. ruckeri are described based on their role at different stages of pathogenesis, including: (1) survival of Y. ruckeri in the aquatic environment, (2) attachment and colonization, (3) countering host defenses, and (4) host damage caused by toxins and enzymes.
1.6.1. Environmental survival of Y. ruckeri
The ability of Y. ruckeri to survive in the aquatic environment is important in maintaining a reservoir for future infection and spread of the disease. The fish-to-fish transmission of Y. ruckeri occurs mainly through water (Busch, 1978). After shedding from a covertly infected or a diseased fish, Y. ruckeri must survive and maintain
10 infectivity until it has an opportunity to infect another fish. In a laboratory test, three pathogenic strains of Y. ruckeri, one isolated from Atlantic salmon raised in seawater, and two isolated from salmon raised in fresh water, were able to survive in lake water and seawater of up to 20 % salinity for four months at 8 °C to 10 °C (Thorsen et al, 1992).
The environmental survival of Y. ruckeri may be aided by its ability to exist in a dormant viable but non-culturable state (VBNC) (Romalde et al, 1994). All three pathogenic strains of Y. ruckeri entered into the state of dormancy that is showing viable cells as measured by acridine orange direct counts but no culturable cells on tryptone soy agar, after 1 to 2 months of incubation in both lake water and estuary water of up to 15 % salinity (Romalde et al, 1994). A reduction of 85 % - 90 % in metabolic activity and a significant reduction in cell size were noticed in the VBNC state that might aid in survival under nutrient-limiting conditions. No changes were observed in the outer membrane protein and plasmid profiles, except that there was a slight increase in high- molecular-weight O-antigens in the LPS profiles of cells in the VBNC state. More importantly, after 100 days in the VBNC state, all three strains retained their original virulence, as measured by LD50 values from intraperitoneal infection of fingerling rainbow trout (Romalde et al, 1994).
Another adaptation that may aid in environmental survival of Y. ruckeri strains is the ability to form biofilms. Environmental strains, isolated from algae and sediments of fish farm, were 14- to 100-times more adherent (measured by bacteria/cm2 of surface) to polyvinyl and wooden surfaces than a laboratory strain (Coquet et al, 2002a, 2002b).
Yersinia ruckeri cells undergoing adaptation to a sessile mode (biofilm) had a different protein expression pattern than the planktonic cells (Coquet et al, 2002b, 2005).
11 Environmental isolates had higher expression of flagellum genes than the laboratory
strain, and displayed a 4-times larger zone of motility on 0.3 % Columbia agar.
Flagellum-based adhesion interactions and motility play a role in initial interactions with
the surfaces and in the formation of microcolonies during biofilm formation (Pratt and
Kolter, 1998). The higher flagellum-based motility of environmental strains may also aid
in better transmission of bacteria (Coquet et al., 2002b).
1.6.2. Attachment and entry into fish
For fish pathogens, the epithelia of the gills, gastrointestinal tract and skin are
possible routes of entry into the host (Evelyn, 1996) but the specific routes used by Y.
ruckeri are still to be identified. Using a bath immersion challenge with killed bacterial
cells, Zapata et al. (1987) demonstrated uptake of Y. ruckeri cells by the epithelial cells of
Atlantic salmon gills. The epithelial cells containing Y. ruckeri were later engulfed by underlying mononuclear phagocytic cells, which might be responsible for systemic
spread/killing of live bacteria. Kim and Stevenson (1998) evaluated the oral route as a possible mechanism of entry to rainbow trout by serotype 1 and serotype 2 strains. After
infection by oral intubation, serotype 1 strains colonized the gastrointestinal tract and
subsequently established a systemic infection in fish. In contrast, the serotype 2 strains were cleared from the gastrointestinal tract without establishing in the internal organs of rainbow trout.
The ability to attach to and invade fish tissues would be an important prerequisite for entry into fish from an aquatic environment and successful establishment of an
infection (Evelyn, 1996). Fish cell lines have been used to study adhesive (ability to
attach fish cells) and invasive (ability to enter into fish cells) properties of Y. ruckeri. A
12 hydrophobic cell surface enables pathogens to attach to host tissues. Yersinia ruckeri strains from different serotypes had an adhesive capability that ranged from none to moderate (10 - 50 bacteria attached/cell), dependent on the fish cell line used (Romalde and Toranzo 1993, Santos et al, 1991). Five of seven strains were moderately adhesive to chinook salmon embryo cells (CHSE-214), compared to one strain which attached to
Epithelioma papulosum cyprini cells (EPC) (Santos et ah, 1991). Adhesiveness to fish cell lines was not related to the virulence of strains for fish, since strains with low LD50 in fingerling rainbow trout were found to be nonadhesive to cell lines and vice versa
(Santos et al, 1991, Romalde and Toranzo 1993). This suggested that cell surface hydrophobic interactions may not be crucial for initial attachment of Y. ruckeri to fish.
All 12 strains of Y. ruckeri from different serotypes that were tested by Romalde and
Toranzo (1993) were capable of invasion into CHSE-214 cells. The invasive capability of strains, measured by counting intracellular bacteria surviving gentamicin treatment, ranged between approximately 10 to 10 intracellular bacteria/25cm tissue culture flask.
All invasive strains were found to be virulent in fingerling rainbow trout after intraperitoneal injections (Romalde and Toranzo 1993). The invasion of fish cells by Y. ruckeri was cell line-dependent, as Y. ruckeri strains were most successful in invasion of the fathead minnow epithelial cell line (FHM), as compared with rainbow trout gonad, and rainbow trout kidney cell lines (Kawula et al, 1996). These differences may be attributed to availability of cell surface receptors and differences in pathogen uptake pathways of cell lines. Adherence and invasion could be mediated by different mechanisms in Y. ruckeri, as strains having no to low adherence were able to efficiently invade CHSE-214 cells (Romalde and Toranzo, 1993).
13 No adhesins and invasins of Y. ruckeri have been functionally characterized, but genes for some potential adhesins/invasins have been identified. The genes inv and ail are required for adhesion and invasion by human pathogenic Yersiniae (Miller and Falkow
1988, Guntram et al., 2003). Fernandez et al. (2007a) were successful in PCR-amplifying a homolog of inv from Y. ruckeri but the encoded protein has not been further characterized. However, Kawula et al. (1996) did not obtain hybridization signals from chromosomal DNA of Y. ruckeri when using ail of Y. enterocolitica as a probe. The bfp genetic locus that encodes for type IV bundle-forming pili, another potential adhesin/invasin, was identified in Y. ruckeri by Fernandez et al. (2004), using promoter- probe based in vivo expression technology screens in rainbow trout. Thus, the bfp locus was transcriptionally active during infection in rainbow trout and may be required in pathogenesis (Fernandez et al, 2004). In other pathogens, the bfp locus was involved in adhesion, invasion, colonization, motility, autoaggregation, and biofilm formation (Wang and Chen 2005, Perez et al, 2006, Tomich et al, 2007).
The flagellum contributes to pathogenicity by acting as an adhesin as well as other functions (Ottemann and Miller, 1997). A flagellum-deficient mutant of Y. ruckeri had lower infection loads in fish organs than did a WT strain during initial infection stages. The mutant was defective in transmission from diseased to healthy fish in a cohabitation challenge, suggesting that the flagellum might be required for chemotaxis, initial attachment and entry into fish (Kim, 2000).
1.6.3. Factors involved in resistance to fish defenses
Like mammals, fish have innate immune defenses such as complement pathways, phagocytes, C-reactive proteins, interferons, lysozyme, lectins, protease inhibitors and
14 other antimicrobial peptides (reviewed by Ellis, 2001). In order to successfully colonize and produce disease, the pathogen must overcome these host defenses. An attribute of successful fish pathogens such as Vibrio anguillarum, Edwardsiella tarda, Aeromonas hydrophila, and A. salmonicida is in the ability to defend against killing by the fish serum
(Leung et ai, 1994, Merino et al, 1994, Boesen et al, 1999, Mathew et ai, 2001). From flow-cytometry analysis, Welch and Wiens (2005) concluded that Y. ruckeri was predominantly an extracellular pathogen, since very few green fluorescent protein (GFP)- labeled Y. ruckeri were found inside the cells of the immune system of rainbow trout infected by bath immersion and intraperitoneal injection. However, a small population of
GFP-positive leukocytes (neutrophils and macrophages) was detected in the blood (1.6
%), spleen (1.1 %), and the kidney (0.4 %). Being mainly an extracellular bacterium, Y. ruckeri should be able to survive and replicate in the presence of serum. All six virulent serotype 1 strains of Y. ruckeri tested by Davies (1991b) were able to grow in the presence of non-immune rainbow trout serum during three hour incubation at 22 °C, suggesting they were resistant to serum-mediated killing. Killing by serum was complement-mediated or mediated by another heat-sensitive factor as all strains were resistant to serum that was heated at 46 °C for 20 minutes. The resistance to serum might be associated with virulence in Y. ruckeri, since sixteen strains belonging to different serotypes that were effectively killed by rainbow trout were found to be non-virulent in rainbow trout after bath infection (Davies 1991a, 1991b). However, some non-virulent strains were also serum-resistant, suggesting that other factors besides serum resistance were required for virulence.
15 The mechanisms of serum-resistance in Y. ruckeri have not been studied. By limiting binding of complement components to the cell surface, the O-antigen chains of
LPS provide serum resistance to various pathogens, including human pathogenic Yersinia and the fish pathogens A. salmonicida and V. anguillarum (Munn et al, 1982, Boesen et al, 1999, Biedzka-Sarek et al, 2005). The serum-resistance phenotype in Y. ruckeri was not specific to any O-antigen serotype, as all serotypes contained serum-sensitive as well as serum-resistant strains (Davies, 1991b). However, strains tested for serum sensitivity by Davies (1991b) had variations in their outer membrane protein (OMP) profiles so cell surface proteins may be involved in serum resistance in Y. ruckeri. The outer membrane proteins such as YadA and Ail were the most important factors in providing resistance to serum-mediated killing in Y. enterocolitica (Biedzka-Sarek et al, 2005). Various serotypes of Y. ruckeri differ in their cell surface characteristics, as was demonstrated by the fact that bacteriophage lysis was able to differentiate virulent serotype 1 strains from other serotypes (Stevenson and Airdrie, 1984). As yet, no cell surface characteristic has been associated with virulence in Y. ruckeri, but differences in surface characteristics of different serotypes suggest that cell surface structures, including OMP, might be associated with virulence-related phenotypes such as serum resistance.
After phagocytosis, successful fish pathogens such as A. salmonicida and V. anguillarum are able to resist killing by the oxidative burst of fish macrophages (Barnes et al, 1999, Boesen et al, 2001, Ellis 2001). Afonso et al. (1998a) suggested that neutrophils and macrophages are involved in the pathogenesis of ERM disease, since infections by Y. ruckeri induced a strong inflammatory response, resulting in an influx of neutrophils and macrophages into the peritoneal cavity of rainbow trout. Subsequently, Y.
16 ruckeri was detected in phagocytic cells, suggesting a role for phagocytes in pathogenesis of disease (Afonso et al, 1998b). Stave et al. (1987) tested the ability of Y. ruckeri strains to suppress the oxidative burst of striped bass macrophages by quantifying extracellular luminal-based chemiluminescent responses. All four serotype 1 strains suppressed the oxidative-burst, whereas three of four serotype 2 strains generated a very strong oxidative-burst response from macrophages (Stave et al, 1987). These authors did not assess the virulence of the strains, making it difficult to correlate this phenotype with the pathogenicity.
Withholding iron from pathogens by strong binding to carrier proteins is an effective host defense (Ratledge and Dover, 2000). Fish possess iron-binding proteins such as transferrin and lactoferrin that bind iron with very high affinity, resulting in extremely low levels of free Fe3+ (< 10"18 M) available for bacterial growth in vivo (Ellis
2001, Trust 1986). Thus, iron acquisition mechanisms are important virulence determinants. To understand iron uptake mechanisms, Davies (1991) and Romalde et al.
(1991a) grew Y. ruckeri strains under iron-limiting conditions in the presence of transferrin, 2,2'dipyridyl and EDTA. Four outer membrane proteins (72 kDa, 69.5 kDa,
68 kDa, 66 kDa) were induced under iron-limiting conditions in all the 36 isolates tested by Davies (1991), whereas Romalde et al. (1991a) identified three proteins of 77 kDa, 73 kDa, and 69 kDa. Some of the proteins identified in these studies may be the same, as suggested by their similar sizes. Addition of FeC^ repressed expression of these proteins, indicating that expression of the proteins was iron-regulated. Further characterization of these proteins may shed light on iron uptake mechanisms of Y. ruckeri, as OMPs act as iron uptake receptors in bacteria.
17 Studies by Romalde et al. (1991a) suggested that Y. ruckeri strains produced a phenolic siderophore, as measured colorimetrically by the Arnow assay of cell-free supernatant and by production of a yellow halo by Y. ruckeri when grown on Chrome- azurol S (CAS) agar plates. Using in vivo expression technology, Fernandez et al. (2004) found that transcriptional fusions of three genes with homology to the enterobactin biosynthesis gene cluster of E. coli were upregulated in Y. ruckeri-infected rainbow trout.
Sequencing identified the entire gene cluster required for biosynthesis and transport of a catechole siderophore, Ruckerbactin. When tested on CAS agar plates and by the Arnow assay, all 12 strains of Y. ruckeri that were tested produced a siderophore, Ruckerbactin.
These strains belonged to different serotypes and had different plasmid profiles, suggesting that siderophore production was a conserved feature across serotypes, and was not a plasmid-related phenotype. Ruckerbactin appears to be involved in survival and pathogenesis in the host, since a mutation in rucC, a biosynthesis enzyme for
Ruckerbactin, caused a 100-fold increase in LD50 for fingerling rainbow trout infected by intraperitoneal injection (Fernandez et al, 2004).
1.6.4. Toxins causing host damage
ERM disease pathology involves hemorrhaging and tissue damage, suggesting potential involvement of proteases, haemolysin and other toxins. Unlike other fish pathogens such as A. salmonicida and V. anguillarum, Y. ruckeri is not a copious producer of extracellular products (ECP), though it produces some extracellular enzymes such as dermatotoxin, gelatinase, amylase, lipase, phospholipase, esterase, esterase- lipase, phosphohydrolase and glucosaminidase activities (Romalde and Toranzo, 1993).
The authors demonstrated that ECP were highly toxic to fingerling rainbow trout, with
18 LD50 for different strains of Y. ruckeri ranging from 2 ug to 9.12 ug protein/g fish. Some signs of disease were evident, including hemorrhage and necrosis at the site of injection.
The ECP produced by different strains of Y. ruckeri varied in total protein and LPS content as well as the spectrum of enzymatic activities. The ECP production by strains was not related to virulence of strains determined in fingerling rainbow trout, since even strains that produced more ECP were less virulent (higher LD50 in fish) and vice versa
(Romalde and Toranzo, 1993).
Disseminated intravascular coagulation and hemorrhaging are clinical manifestations of endotoxemia in mammals caused by responses to the LPS. Unlike other animals, fish can withstand very high levels of LPS (e.g. LD50 of 21.6 mg/ml and > 714 mg/ml for mice and coho salmon, respectively) without showing signs of endotoxemia
(Kodama et al, 1987, Paterson and Fryer 1974). The ECP of Y. ruckeri purified by
Romalde and Toranzo (1993) contained 0.8 to 1.8 mg/ml of LPS, which did not produce toxic effects in fingerling rainbow trout after intraperitoneal injection.
Different components of ECP can be purified and tested for their roles in virulence in a fish model. Secades and Guijarro (1999) purified a 47 kDa protease, Yrpl, from the culture supernatant of a casein-hydrolyzing strain of Y. ruckeri. The isogenic yrpl -deficient mutant strain showed a 100-fold increase in LD50 after intraperitoneal injection into fingerling rainbow trout (Fernandez et al, 2002). The purified Yrpl protease was able to hydrolyze proteins such as laminin, fibrinogen, gelatin, fibronectin, actin and myosin that were components of the extracelluar matrix, blood vessels, and muscles (Fernandez et al, 2003). When a Y. ruckeri strain containing a yrplwlacZ transcriptional fusion was injected intraperitoneally in rainbow trout, maximum
19 expression of the fusion, measured by blue staining of tissues for p-galactosidase activity, was noticed in the gills and intestine, with little expression in the spleen and liver.
Microscopic examination of gills and intestine tissues showed that expression of yrpl fusion was concentrated mainly around the capillary system (Fernandez et al, 2003).
Although none of the substrate proteins described above to test hydrolysis activities was of fish origin, the authors speculated that Yrpl protease might be involved in tissue damage, invasion, and hemorrhaging by causing alterations and pores in the capillary vessels in fish (Fernandez et al., 2003). Also, the expression patterns oiyrplv.lacZ fusion in rainbow trout might not be specific to this fusion, since expression patterns of all randomly generated promoter-/acZ fusions were similar to that of yrpl.
Toxins of the ShlA family are involved in tissue invasiveness and host tissue damage by Serratia species and Edwardsiella tarda (Konig et al., 1987, Strauss et al,
1997). Fernandez et al. (2004) identified a homolog of ShlA haemolysin, YhlA toxin, in
Y. ruckeri by in vivo expression technology. The ShlA forms pores in the membranes of red blood cells (RBC), fibroblasts and epithelial cells (Hertle et al, 1999). When tested for hemolytic activity, the yhlA mutant of Y. ruckeri caused approximately half the amount of lysis of sheep RBC of a WT strain (Fernandez et al., 2007b). In contrast to the
WT strain, the yhlA mutant of Y. ruckeri did not cause lysis of BF-2 (blue gill fry) fish cells after 4 hour of incubation, showing the mutant was less cytotoxic. The mutant had a
4 5 nearly 10-fold increase in LD50 (WT 2.7 x 10 , mutant 3.9 x 10 ) in rainbow trout after intraperitoneal infection, suggesting a role of this hemolysin in pathogenesis (Fernandez et al, 2007b).
20 Gunasena et al. (2003) identified a homolog of ysa (yersinia secretion apparatus)
TTSS in Y. ruckeri. Homologs of four genes, ysaV, ysaK, ysaN, and CDS19, of Y.
enterocolitica were identified on the chromosomal DNA of Y. ruckeri strain, NCIMB
1315, by PCR amplification using degenerate primers. Sequences of ysaV, ysaK, ysaN,
and CDS19 genes of Y. ruckeri showed 60 %, 51 %, 67 % and 37 % identity, respectively to homologs present in Y. enterocolitica. The Ysa TTSS system is present only in pathogenic biotype 1A strains of Y. enterocolitica and is absent from Y. pestis, Y. pseudotuberculosis and non-pathogenic strains of Y. enterocolitica (Foultier et al, 2002).
Based on sequence similarity and identical genetic organization, the Ysa system of Y.
ruckeri was homologous to the TTSS encoded by Salmonella Pathogenicity Island 1
(SPI1) and Mxi-Spa TTSS of Shigella flexneri (Gunasena et al, 2003). The Ysa homologs in these bacteria are essential for pathogenesis, and are involved mainly in the intestinal stages of the infection process (Lee and Schneewind 1999, Matsumoto and
Young 2006). Pathology of ERM disease involves gastrointestinal edema and hemorrhaging (Miller, 1983) and the gastrointestinal tract of fish has been shown to be the site of colonization by serotype 1 strains of Y. ruckeri (Kim and Stevenson, 1998).
Further characterization of the Ysa system of Y. ruckeri is needed before further speculation on its role in gastrointestinal stages of pathogenesis.
1.7. Regulation of virulence gene expression
Expression of genes encoding virulence factors such as Yrpl protease, YhlA haemolysin and Ruckerbactin siderophore biosynthesis was higher at 18 °C than at 28 °C
(Fernandez et al, 2003, Fernandez et al, 2004, Fernandez et al, 2007b). The activity of a
21 yrplv.lacZ transcriptional fusion was stimulated 4-fold at 18 °C compared with at 28 °C,
and 250-fold more casein-hydrolyzing activity was obtained in culture supernatants of Y.
ruckeri strains grown at 18 °C compared with at 28 °C (Fernandez et ah, 2003). The yhlBv.lacZ (hemolysin), rupDGC.JacZ (ruckerbactin) and rucCv.lacZ (ruckerbactin)
fusions produced seven-fold and 3-fold more (3-galactosidase activity at 18 °C than at 28
°C (Fernandez et ah, 2004, Fernandez et ah, 2007b). These studies suggested that
temperature may be one of the factors for regulating expression of virulence genes in Y.
ruckeri. A similar phenomenon exists in human pathogenic Yersinia in which expression
of many virulence genes such as virF, yadA, ylpA, ure, flaABC and Yop regulon is
upregulated at host temperature of 37 °C (reviewed by Straley and Perry, 1995). Higher expression of virulence genes at 18 °C was consistent with the temperature at which disease may occur, whereas 28 °C was used for growing bacteria under laboratory conditions. However, a temperature of 28 °C may not be encountered very frequently by
Y. ruckeri in fish. All these genes were expressed in rainbow trout held in water at 18 °C,
as reported by transcriptional fusions (Fernandez et ah, 2004); however, the expression of these genes in fish held at different water temperatures has not been studied.
Bacterial population-density dependent quorum sensing systems (QS) can be
another potential regulator of virulence genes (Miller and Bassler, 2001). Temprano et
ah, (2001) identified the yruI-yruR QS system of Y. ruckeri by cross-feeding the biosensor strain, Chromobacterium violaceum CVOblu. Studies by Bruhn et ah (2005)
suggested the existence of more than one QS system in Y. ruckeri. Five virulent strains of
Y. ruckeri produced an identical and complex acylated homoserine lactone (AHL) profile,
with at least eight analogues from three different classes of AHL. The N-(3-
22 oxooctanoyl)-l-homoserine lactone was produced at a high level followed by N-(3- oxodecanoyl)-l-homoserine lactone, N-(3-oxohexanoyl)-l-homoserine lactone, N-(3- oxoheptanoyl)-l-homoserine lactone and N-(3-oxononanoyl)-l-homoserine lactone molecules (Bruhn et al, 2005). AHL similar to those produced in growth medium were detected from spleen, liver, kidney and brain extracts of Y. ruckeri-mfccted rainbow trout, suggesting that QS was active during infection. No AHLs were detected from these organs in uninfected control fish, however, the AHLs were produced in very low concentrations in infected fish (Bruhn et al, 2005). The production of Yrpl protease, a virulence factor of Y. ruckeri, was not affected by the addition to growing cultures of Y. ruckeri of AHLs (N-(3-oxohexanoyl)-l-homoserine lactone, N-octanoyl-1-homoserine lactone, N-dodecanoyl-1-homoserine lactone, N-tetradecanoyl-1-homoserine lactone),
AHL inhibitors (N-heptylsulfanyl acetyl-L-homoserine lactone or N-pentylsulfanyl acetyl-L-homoserine lactone), or 10 % filter sterilized high-density culture supernatant
(Kastbjerg et al, 2007).
1.8. Model of Y. ruckeri pathogenesis
Some factors, such as the Yrpl protease, flagellum, YhlA hemolysin and
Ruckerbactin siderophore, are involved in pathogenesis of Y. ruckeri (Fernandez et al,
2002, Fernandez et al., 2004, Fernandez et al., 2007b). The mutations in these factors had less dramatic effects (10-100 fold increase in LD50) on virulence in fish. No major, stand alone virulence determinant has been identified in Y. ruckeri. We suggest that virulence of Y. ruckeri may require expression of multiple genes that, in aggregate, confer the virulence phenotype, although individually these gene products may have minor effects
23 on virulence. Thus, a genome-based analysis is required to identify these multiple subtle virulence factors of Y. ruckeri. The current study is a step in that direction, using a transposon-based random mutagenesis approach to identify genes of Y. ruckeri that might contribute to pathogenesis.
Our proposed model of the virulence strategy of Y. ruckeri is that, after entry, the bacterium establishes itself in fish and a 'host-pathogen balance' is established. Yersinia ruckeri persists in infected fish maintaining this host-pathogen balance, and disease occurs when stressors such as changes in water temperature, poor water quality, handling and overcrowding of fish, tilt the balance towards reducing host innate immunity. Thus, persistence in infected fish is crucial in order for Y. ruckeri to cause disease, and genes required for survival in fish are therefore essential to pathogenic strategies of Y. ruckeri.
After an acute disease, Y. ruckeri can establish a long term persistent infection in 50 % -
75 % of survivor fish (Busch and Lingg 1975, Hietala et al, 1995). To understand the genetic basis of such established Y. ruckeri-Tish interactions, we aimed to identify genes that were required for survival and establishment of Y. ruckeri in rainbow trout 7 days after infection. The genes identified in this study may control functions such as initial attachment and entry into fish, spread and colonization in internal organs, metabolism and nutrient acquisition, cell signaling to modulate gene expression in response to changing host environment, adapting to stresses imposed by the host environment, and neutralizing immune defenses of fish. Subsequent functional characterization of these genes using in vitro assays for virulence-related phenotypes, and time-course fish infection experiments may identify roles of the gene products in Y. ruckeri pathogenesis.
24 A transposon-based signature-tagged mutagenesis (STM) approach was used in this study to identify genes essential for survival of bacteria in the host (Hensel et ai,
1995). Not all the genes identified by STM as essential for survival of a pathogen in the host will directly contribute to virulence, and some of these genes cannot be termed as virulence genes. Using the classification proposed by Wassenaar and Gaastra (2001), genes identified in this study will be classified into categories of true virulence genes, virulence-associated genes and housekeeping genes. Genes whose products are involved in metabolic pathways, cell division, repair and replication of DNA were considered as housekeeping genes. True virulence genes encode proteins, RNA molecules or enzymes that are involved in interactions with the host. For example, adhesins, invasins, fimbriae, and the flagella are involved in invasion of the host tissues and cytotoxins are responsible for pathological damage to host tissues (Wassenaar and Gaastra 2001). The products of the virulence-associated genes are involved in regulation of gene expression and activation and delivery of virulence factors. They may encode two-component systems, transcriptional factors proteases, proteases, methylase and type 1 to V secretion systems.
1.9. Signature-tagged mutagenesis (STM) and identification of virulence genes
STM is a screening technique in which mutants that cannot survive in the host are identified, thus identifying mutations that decrease survival of a pathogen in host. Each mutant produced in the transposon-based STM technique is 'barcoded' with a short (20-
50 nucleotides), unique DNA-signature sequence, known as the tag, which allows each individual mutant in a mixture of mutants used to infect a host to be identified. Thus,
25 multiple mutants can be screened simultaneously, reducing the number of animals used
(Chiang et al, 1999, Hensel et al, 1995).
Originally, tags were tracked by Southern hybridization, but Lehoux et al. (1999) developed a more sensitive and rapid version that used PCR to recognize the mutants recovered from the host. Twelve signature tags, each 21 nucleotides long, were synthesized and cloned into the pUTminiTn5Km2 plasmid vectors that were present in donor E. coli strains (Figure 1).
Signature-tagged (ST) mutants of Y. ruckeri were generated by filter mating with donor E. coli strains containing pUTminiTn5Km2 plasmids. The ST mutants were screened in rainbow trout in groups of 11 mutants by taking one mutant from each tag.
This represented the input pool of mutants, whereas the mutants recovered from rainbow trout represented the output pool. Instead of time-consuming hybridization, a PCR for each of the signature tags on the mutants recovered from the host was used to determine which tags/mutants were missing (Figure 1C). PCR has a higher sensitivity than hybridization, so even mutants present in very low numbers in the bacterial population recovered from the host were detected, and not included with the 'survival-defective' mutants. Using similar PCR-based STM, Lehoux et al. (2002) successfully identified 13 virulence-attenuated mutants of Pseudomonas aeruginosa by screening 1056 mutants in a chronic lung infection rat model. The identified genes encoded proteins involved in purine metabolism, protein turnover/stress and transport.
To ensure random representation of mutations at the start, the transposon should insert randomly in the bacterial genome without any 'hotspots'. In S. typhimurium,
Legionella pneumophila, P. aeruginosa and Y. pseudotuberculosis, miniTn5Km2-based
26 Figure 1. Construction of signature-tagged mutants and screening of libraries for identifying survival-defective mutants in rainbow trout.
A. Strains of E. coli S17-Rpir containing twelve different pUTminiTn5Km2 plasmids with different DNA-signature tags were filter-mated with Y. ruckeri RSI 154. The miniTn5Km2-containing kanamycin resistant signature-tagged mutants generated with each signature tag were stored individually in 96 well plates as a library.
B. To identify survival-defective mutants, a single mutant from each library was grown in vitro individually and afterwards the mutants from all libraries were mixed in equal doses before immersion infection of rainbow trout. After 7 days infection, mutants were recovered from kidney tissue homogenates and the genomic DNA was isolated.
C. The survival-defective mutants showed no amplification of corresponding tags (lanes
1, 3) in PCR reactions with a forward 'tag-specific' primer and a common reverse primer from the kanamycin resistance gene. The position of PCR primers is shown in A.
27 Forward primer Inverted Tag Inverted repeat ^s repeat MiniTn5km2 inserted into genome of Y. ruckeri mutant
ooooooooeooe oooooooooooo ooeeoooooooe oooooooooooo oooooooooooo oooooooooooo oooooooooooo oooooooooooo oooooooooooo oooooooooooo oooooooooooo oooooooooooo oooooooooooo oooooooooooo oooooooooooo oooooooooooo Library 1 Library 12
B i A « 04 I
I 6 7 8 9
28 mutagenesis was found to be random (Hensel et al, 1995, Edelstein et al, 1999, Mecsas et al, 2001, Lehoux et al, 2002). However, none of these studies had saturating mutagenesis of the genome and only a few mutants were tested to ensure randomness of transposition.
1.9.1. Animal model for screening ST mutants
The most critical factor in STM is the animal model used to screen the mutants.
First, the route of infection will influence the number of bacteria reaching the target organ, as it will determine the host defenses encountered by a mutant. Injection of the pathogen directly in a fish will bypass many innate defense mechanisms of fish, and does not reflect the natural disease process (Evelyn 1996, Nordmo 1997). In a study of 1008
ST mutants of Mycobacterium marinum by injection into goldfish, none of the 35 virulence-attenuated mutants was involved in adherence and colonization of the host
(Ruley et al, 2004). The 'water-borne' bath immersion method resembles a natural infection process and genes identified by screening mutants by bath immersion are arguably more relevant to natural disease pathogenesis. For this reason, it was chosen as the infection route for the current study.
A second consideration is the infection dose. Sufficient numbers of bacteria should be included so that all the mutants in the input pool have an opportunity to establish an infection in the host. Having a lower dose may lead to false identification of survival-defective mutants, as even a virulent mutant may not be able to set up an infection. In pigs, challenge doses between 103 - 105 CFU of Actinobacillus pleuropneumoniae gave inconsistent recovery of an output pool of mutants from groups of pigs infected with the same input pool. Increasing the challenge dose to 10 to 10 CFU
29 overcame the inconsistency (Sheehan et al, 2003). On the contrary, a very high infection dose may overwhelm the host immune system, resulting in growth of mutants that otherwise would be attenuated (Shea and Holden, 2000).
A third consideration is the time after infection at which ST mutants are selected.
This should be long enough to allow an infection to establish and also permit the host immune system to eliminate defective mutants. After a prolonged infection time, a biased clonal expansion of selected virulent strains inside the host has been reported (Chiang et al, 1999). The appropriate time for mutant selection is also dictated by the nature of the disease. It will be shorter for an acute disease than for a chronic disease or a carrier state.
Mutant selection at early stages of infection may identify genes involved in initial entry and colonization (Shivani et al, 2005). Selection of mutants at 48 hours post-infection identified 97 mutants (12.6 %) of Actinobacillus suis that were defective in colonization of the upper respiratory tract of piglets (Shivani et al, 2005). Selection of mutants at a later stage of infection may identify genes involved in the systemic stages of infection
(Hong et al, 2000). To identify genes required for chronic infection of mice with
Brucella abortus, Hong et al. (2000) screened 178 ST mutants and identified survival- defective mutants from the spleen at 2 weeks (acute stage) and 8 weeks (chronic stage) post infection. The mutants selected at 2 weeks (28 mutants) were in genes required for establishment of infection, whereas genes selected at 8 weeks (27 mutants) were required for persistence of infection. Previous time-course infection studies from our laboratory demonstrated Y. ruckeri had established in rainbow trout organs at levels of 10 - 10
CFU/g tissue by 7 days post-infection, a time that could be used for assessing pools for survival-defective mutants (Flett 1989, Kim and Stevenson 1998, Kim 2000). Day 7
30 represented an acute stage of the infection process and products of genes of Y. ruckeri identified at this time may be involved in pathogen attachment to the host, early colonization, countering the immune system, systemic spread and establishment of the pathogen in different tissues.
Significant in the use of an animal model for STM screens is the consistency in output pool of mutants recovered from groups of animals. The compositions of the output pools from animals infected with the same input pool can vary, as has been shown in animal hosts such as mouse (Mecsas et al, 2001), chicken (Shah et al, 2005), pig
(Shivani et al, 2005) and fish (Miller and Neely, 2005). Three approaches have been used to identify the consistently missing mutants. First, the mutants that are missing in all animals infected with the same input pool were selected. Second, mutants identified in the first screening were assembled in input pools and were rescreened in the animal host
(Burall et al, 2004, Shah et al, 2005, Shivani et al, 2005, Cuccui et al, 2007). On first screening of 2088 ST mutants of Proteus mirabilis, Burall et al. (2004) identified 502 mutants and rescreening of these mutants led to the identification 114 survival-defective mutants in a mouse model. Similarly, rescreening of 45 mutants of Burkholderia pseudomallei selected after initial screening of 892 mutants, led to identification of 37 mutants that were missing from mice (Cuccui et al, 2007). In most STM-based studies, rescreening of mutants significantly reduced the number of mutants finally selected
(Sheehan et al, 2003, Burall et al, 2004, Hunt et al, 2004, Cuccui et al, 2007). A third approach used to identify/confirm the consistently missing mutants was to perform one- to-one competitive challenge of selected survival-defective mutants with the WT strain.
The majority of the survival-defective mutants identified in primary screening were
31 found to be attenuated compared to the WT strain in the animal host (Harper et al, 2003,
Sheehan et al, 2003, Shah et al, 2005, Shivani et al, 2005, Su et al, 2007). However,
mutants with varying degrees of attenuation were found, some being as virulent as the
WT, despite that fact that they appeared survival-defective in primary screenings (Harper
et al, 2003, Sheehan et al, 2003, Shah et al, 2005, Shivani et al, 2005, Su et al, 2007).
To address the question of variation in output pool between animals, the current study
analyzed the effect of bacterial infection loads in fish on the recovery of mutants in
output pool.
1.9.2. Genes identified in STM screens
What kinds of genes have been identified in STM screens in bacterial pathogens
of terrestrial animals? Toxins that act extracellularly and whose phenotypes can be
transcomplemented by the presence of other mutants in a mixed pool are not identified by
STM screens. Also, genes present in multiple copies or genes with functional redundancy
(such as multiple adhesins) and genes with very little effect on mutant survival in host
cannot be identified using STM screens. More than 50 STM based screens have been reported in Gram-negative and Gram-positive bacteria using various host systems such as
mice, rat, goat, monkey, chicken, and pig (Autret and Charbit 2005, Shah et al, 2005,
Shivani et al, 2005, Cuccui et al, 2007). Genes identified in STM-based screens can be broadly categorized into four major categories. The first category contains genes
associated with cell surface structures that include genes involved in biosynthesis/modification of LPS, capsule and peptidoglycan, genes encoding membrane
transporters involved in transport of metals, sugars or other metabolites and genes for
adhesin/invasins such as flagellum and pili required for attachment and colonization of
32 host. Between 25 % and 50 % of mutations identified in STM screens were in genes encoding for cell surface structures, indicating the importance of the bacterial cell surface in host-pathogen interactions (Mecsas et al, 2001, Autret and Charbit 2005, Miller and
Neely 2005, Cuccui et al., 2007). The LPS/O-antigens, capsules, toxin-coregulated pili and invasin identified in STM screens were subsequently confirmed to be important virulence factors of multiple pathogens such as Yersinia (Zhang et al., 1997, Guntram et al., 2003), Vibrio cholerae (Chiang and Mekalanos, 1998) and Streptococcus pneumoniae
(Magee and Yother, 2001). A second category of genes commonly identified by STM screens of Gram-negative bacteria were the type three secretion systems (TTSS), often present as a part of a pathogenicity island on the genome. The SPI1 encoded TTSS, a key virulence factor of Salmonella, was first identified using STM screens (Hensel et al.,
1995). Similarly, pYV plasmid-encoded TTSS, a common virulence factor of human pathogenic Yersinia, was identified in STM screens both in Y. enterocolitica and Y. pseudotuberculosis (Darwin and Miller 1999, Mecsas et al, 2001). Genes such as two- component systems and transcriptional factors that are involved in regulation of virulence genes expression in response to changing host environment represented a third category of genes identified in STM-based screens. Although these genes do not directly contribute to virulence, and were classified as virulence-associated genes (Wassenaar and
Gaastra 2001), the transcriptional regulators and two-component system are key regulators of virulence factors in all pathogens (reviewed by Beier and Gross, 2006). The genes involved in cellular metabolism such as glycolysis, other energy generating pathways, purine and pyrimidine biosynthesis, amino acid metabolism, DNA repair and replication, cell division, different stress response proteins such as heat shock proteins,
33 and proteases involved in degradation and removal of damaged proteins, were consistently identified in all the STM-based screens. These housekeeping genes do not qualify as virulence genes, but they help pathogens to survive and adapt to complex and changing host environment. Housekeeping genes provide valuable information on in vivo host environments, pathogen growth requirements and cellular repair mechanisms needed for survival in the host (Perry, 1999).
Since the start of this study, four studies have been published using zebrafish, goldfish, channel catfish and rainbow trout for STM screening of Streptococcus iniae
(Miller and Neely, 2005), M. marinum (Ruley et ah, 2004), E. ictaluri (Thune et ah,
2007), and Lactococcus garvieae (Menendez et ah, 2007), respectively. A total of 41 mutants of S. iniae that were not recovered from the heart and brain of intramuscularly injected zebra fish at 24 h post-infection were identified by screening 1128 mutants
(Miller and Neely, 2005). The putative proteins encoded by genes identified in these mutants were predicted to be involved in capsule biosynthesis, transport, metabolism and transcriptional regulation. Significantly, eight mutations were in different genes whose products might be involved in capsule biosynthesis, suggesting the capsule is important for bacterial survival in fish. Acapsular mutants of S. iniae identified by Miller and Neely
(2005) were more readily killed by human phagocytes (13 % - 29 % survival for different
ST mutants) than the WT strain (76 % survival). On screening of 1250 ST mutants of L. garvieae, twenty-nine (2.4 %) mutants that were not recovered from the liver and spleen of two rainbow trout three days after intraperitoneal infection were identified (Menendez et ah, 2007). The deduced proteins of nine of these 29 genes had predicted functions in cellular metabolism, DNA repair and replication. The proteins encoded by three genes
34 were identified as putative transcriptional regulators, two genes might encode putative transporters and many genes were predicted to encode hypothetical proteins (Menendez et al, 2007). For reconfirmation of attenuation, thirteen mutants were tested by one-to- one competitive challenge with WT. Importantly, these mutants showed varied degrees of attenuation, measured by the number of bacteria recovered from spleen and liver, ranging from completely missing in rainbow trout to cases when mutants were recovered in WT levels.
A total of 40 mutants of M. marinum that were not recovered from the liver of goldfish at seven days post-infection were identified on screening 1008 mutants (Ruley et al, 2004). The deduced proteins encoded by many of the M. marinum genes were predicted to be involved in cell-wall related functions such as lipid biosynthesis
(polyketide synthase, methoxymycolic acid synthase, acyl transferase), suggesting that, as in human pathogenic mycobacterium, the cell wall may play a significant role in pathogenesis in fish. A second major class of genes identified was predicted to encode proteins with proline-proline-glutamic acid (PPE) or PE signature motifs. Members of the
PPE/PE family have previously been demonstrated as virulence factors, as mutations in
PPE/PE encoding genes of M. marinum reduced its survival in macrophages, reduced granuloma formation, a hallmark of mycobacterial infections and persistence in frogs
(Ramakrishnan et al, 2000). Thus, the STM screen of M. marinum was able to identify mutations in two key virulence factors of mycobacteria, cell wall lipids and the PPE/PE family of proteins. Only one STM screen of a Gram-negative fish pathogen has been reported (Thune et al, 2007). Fifty mutants (4.7 %) of E. ictaluri that were not recovered from the liver of immersion infected channel catfish fish at 6 - 7 days after infection were
35 identified (Thune et al, 2007). Eight of the 50 mutations were in genes whose products
had predicted virulence-related functions, whereas the majority of mutations were in
genes whose products had metabolic (19 mutations) and hypothetical (10 mutations)
functions. The proteins encoded by virulence-related genes were predicted to be involved
in biosynthesis of O-antigen chains, a urease, and effector proteins of TTSS. These
virulence-related mutants were highly attenuated, based on bacterial loads in the kidney
of fish, when tested in competitive challenge with the WT strain. Based on infection
loads of TTSS mutant and WT in the kidney at different times post infection, it was
demonstrated that TTSS of E. ictaluri was not required for initial entry but played an
important role in multiplication and persistence in channel catfish. The urease gene, part
of a urease-enzyme complex, identified by STM screening of E. ictaluri is a proven
virulence factor, since mutants of this locus were defective in survival in macrophages,
and were attenuated for mortality and persistence in channel catfish (Booth, 2006).
In comparison with STM screens of pathogens of terrestrial animals, the proteins
encoded by genes identified in the four STM screens of fish pathogens can broadly be
classified into the same four classes, including cell surface structures (capsule, cell wall
lipids, membrane transporters), transcriptional regulators, secretion systems, and
metabolic enzymes (Ruley et al, 2004, Miller and Neely 2005, Menendez et al, 2007,
Thune et al, 2007). Significantly, both true virulence and virulence-associated genes
were identified in all STM-based screens in fish pathogens, contributing to understanding pathogenic mechanisms.
36 1.10. Research objectives and approach
We hypothesize that survival and persistence in fish to establish and maintain host-pathogen equilibrium are essential to allow Y. ruckeri to subsequently cause disease in stressed fish. The objectives of this study were first to identify genes essential for
survival of Y. ruckeri in fish, and then to further evaluate their roles in pathogenesis in an effort to identify virulence factors of Y. ruckeri. Signature-tagged mutants of a virulent
serotype 1 strain of Y. ruckeri were generated, and screened in rainbow trout to identify
survival-defective mutants. To ensure that genes identified were directly applicable to disease pathogenesis, fish were challenged by a natural water-borne route of infection.
The kidney was the single best organ to isolate Y. ruckeri in the infected fish (Kim and
Stevenson 1998, Kim 2000), and the fish kidney is the most important organ of the immune system as well as performing haematopoitic functions (Ellis, 2001). Therefore, rainbow trout were infected under conditions described, and mutants that were unable to survive at 7 days in the kidney tissue were selected. The following sections describe the screening of 1056 mutants in rainbow trout that led to identification of 25 survival- defective mutants. Further characterizations of two of identified genes znuACB and barA- uvrY were performed using in vitro assays and time-course infection experiments in rainbow trout to understand their role in the pathogenesis of Y. ruckeri infections.
37 CHAPTER 2
MATERIALS AND METHODS
2.1. Bacterial strains and growth conditions
The bacterial strains used in this study are listed in Table 1. Y. ruckeri RSI 154, a
serotype 1 strain originally isolated from diseased rainbow trout from Idaho Valley, USA,
served as a WT strain. In laboratory challenges, this strain was able to cause up to 90 %
mortality in rainbow trout (Flett, 1989). It has three plasmids of 88 kb, 30 kb and 15 kb
(De Grandis 1987).
The bacteria were stored at -80 °C after mixing a bacterial culture 1:1 with freezing medium (Trisodium citrate 3.0 g, distilled water 60 ml, glycerol 40 ml per 100 ml of medium). Y. ruckeri strains were grown in Luria-Bertani (LB) broth containing 10 g tryptone, 10 g NaCl, 5.0 g yeast extract per liter) or Brain Heart infusion broth (BHI;
Difco) at 18 °C, unless indicated otherwise. For making LB agar plates, 1.5 % agar
(Difco) was added to broth medium. BHIA (Difco) was used for making BHI agar plates.
Escherichia coli strains were grown in LB broth or agar plates at 37 °C.
The antibiotics kanamycin (kan) (50 [xg/ml), ampicillin (amp) (100 ng/ml), and vancomycin (van) (300 ug/ml), were added to LB broth and LB agar for selection of signature-tagged mutants. Kan (50 fig/ml), amp (100 fig/ml) and chloramphenicol (25
[ig/ml) were added to growth media, as required, for selection of mutant strains generated in this study.
38 Table 1. Bacterial strains used in this study.
Strain code Relevant characteristics Reference Escherichia coli strains W3110 F AT rph-1 IN V(rraD, rrnE) a GR352 AznuACB mutant of E. coli W3110 Grass et al., (2002)b GR352 (pBAD33/6) E. coli GR352 transformed with pBAD33/6 This study GR352 (pSP86/37) E. coli GR352 transformed with pSP86/37 This study GR352 (pHSG576) E. coli GR352 transformed with pHSG576 This study S17-Rpir thipro hsdK hsdM+ recA rp4- Tc::Mu-Km::Tn7Apir Simon et al, Donor strains for making isogenic mutants (1983)c S17-apir thi pro hsdK hsdM+ recA rp4- Tc::Mu-Km::Tn7Apir, having Lehoux et al, (pUTmini-Tn5Km2) pUTrniniTn5km2 plasmids, donor strains for signature tags (1999)c
d DH5a F-cp80d/acZ IM15I (lacZYA-argF)XJ169 deoR recAl hsdRll (rk~ FHLCC mk+) phoA supEAA X thi-1 gyrA96 reZAlUsed for cloning Yersinia ruckeri strainse RS2 Serovar II a FHLCC RS3 Serovar II b FHLCC RS7 Serovar I FHLCC
39 RS20 Serovar T FHLCC RS25 SerovarV FHLCC RS77 Serovar III/1' FHLCC RS80 Serovar VI FHLCC RSI117 Serovar II FHLCC RS1118 Serovar II FHLCC RSI154 Serovar I, isolated from rainbow trout FHLCC RSI155 Serovar II, isolated from brook trout FHLCC Y.ruckeri (AznuACB) AznuACB mutant of Y. ruckeri RS1154 This study Y. ruckeri Y. ruckeri (AznuACB) transformed with pBAD33/6 This study (AznuACB)/pBAD33/6 Y. ruckeri (pRS551) Y. ruckeri RSI 154 transformed with pRS551 This study Y. ruckeri (pRS551/41) Y. ruckeri RSI 154 transformed with pRS551/41 This study Y. ruckeri (pRS551/61) Y. ruckeri RSI 154 transformed with pRS551/61 This study Y. ruckeri Y. ruckeri RSU54 having pKOBEG-sacB This study (pKOBEG-sacB) a obtained from Dr. J. Lam, Dept. MCB, University of Guelph b obtained from Dr. C. Rensing, Dept. Soil, Water & Environmental ScL, University of Arizona c obtained from Dr. R. Levesque, Faculte de Medecine, Universite Laval d Fish Health laboratory Culture Collection, Dept. MCB, University of Guelph ^hole cell-based serology of Y. ruckeri strains (Stevenson et al, 1993)
40 2.2. Recombinant DNA techniques
Standard recombinant DNA technology procedures were followed during this study (Sambrook and Russell 2001). The list of plasmids used in this study is provided in
Table 2. Restriction enzymes from New England Biolabs, Invitrogen, and Roche, DNA ligase (Roche), PCR product and gel purification kits (Qiagen), and kits for plasmid DNA
(Sigma) and genomic DNA isolation (Pharmacia Biotech) were used in this study. The kits were used according to the procedures recommended by the manufacturers.
2.3. Competent cell preparation
Y. ruckeri RSI 154 electro-competent cells were made by the protocol described by Derbise et al, (2003). For making isogenic mutants, Y. ruckeri RSI 154 strain was transformed with plasmid pKOBEG-sacB. This plasmid expresses the lambda phage redyfia operon, which increases the frequency of homologous recombination in Yersinia
(Derbise et al, 2003).
Y. ruckeri cells were grown in one litre LB broth at 25 °C to an OD600 of 0.2. At that point, L-arabinose (0.2 %) was added to the culture to induce expression of the redyfia operon. At an OD60o of 0.6 - 0.8, the cells were centrifuged at 2500 x g for 10 minutes at 4 °C, and pellet was washed twice with transformation buffer (15 % (v/v) glycerol). Finally, the cells were suspended in 1 ml of transformation buffer and aliquots of 40 ul cells were stored at -80 °C.
E. coli (DH5a) cells were made competent by a protocol described by Inoue et al,
(1990). One litre of SOB medium, containing 2 % tryptone (w/v), 0.5 % yeast extract
(w/v), 0.05 NaCl (w/v), 2.5 mM KC1, and 10 mM MgCl2> was inoculated 1:100 with an
41 Table 2. Plasmids used in this study.
Plasmid Relevant characteristics Reference pUCGm Used for cloning Schweizer (1993) pUCGm(kan) pUCGM with KanR of pCR®-XL-TOPO® at Xbal site This study pCR®-XL-TOPO® Source of KanR cassette Invitrogena pBluescriptll KS+ Vector, AmpR Stratagene pCVD442 Suicide plasmid (AmpR, sacB, ori R6K) Donnenberg and Kaper (1991) pKOBEG-sacB Suicide plasmid having X, phage redyPa Derbise et al, 1995b
pBAD33Cm Low copy number plasmid having PBAD promoter Guzman et al, 1995° pBAD33/6 pBAD33 with znuACB of Y. ruckeri at Xbal This study pRS551 lacZ- transcriptional fusion vector Simons et al, 1987d pRS551/41 Plasmid having znuCBv.lacZ fusion This study pRS551/61 Plasmid having znuAv.lacZ fusion This study pYRZnuA pBluescriptll KS+ with znuA cloned at Smal-EcoRl This study pYRZnuAB pBluescriptll KS+ with znuA and znuB of This study Y. ruckeri cloned at Xbal-EcoRI sites pYRZnuABkan pYRZnuAB with KanR cloned at Smal site This study pHSG576 CamR, low copy number plasmid Takeshita et al, 1987e pSP86/37 pHSG576 with znuACB locus of E. coli Patzer and Hantke 1998e
42 a obtained from Dr. Azad Kaushik, Dept. MCB, University of Guelph b obtained from Dr. A. Derbise, Institut Pasteur Paris, France c obtained from Dr. Janet Wood, Dept. MCB, University of Guelph d obtained from Dr. T. J. Silhavy, Dept. Molecular Biology, Princeton University e obtained from Dr. K. Hantke, Mikrobiologie/Membranphysiologie, Universitat Tubingen
43 overnight culture ofE. coli. The cultures were grown at 18 °C with shaking until ODgoo of
0.6. The cells were placed on ice for 30 minutes and afterwards were centrifuged at 2500 x g for 10 minutes at 4 °C. The pellet was resuspended in 400 ml of ice cold transformation buffer (10 mM PIPES, 55 mM MnCl2, 15 mM CaCl2 and 250 mM KC1) and kept on ice for 10 minutes. The cells were centrifuged as before and the pellet was resuspended in 100 ml of transformation buffer. Dimethyl sulfoxide was added to a final concentration of 7 % (v/v) by gently mixing the cells. After keeping on ice for 10 minutes, the competent cells were stored at -80 °C in aliquots of 200 ul.
2.4. Polymerase chain reaction
The PCR, usually in 50 ul volume, was carried out in a TGradient thermocycler
(Biometra®). The recombinant Taq DNA polymerase (Invitrogen) was used in PCR unless mentioned otherwise. Typically, 10 JJ,1 of PCR product was run on an agarose gel
(1 % w/v) for analysis.
Forward and reverse primers for the specific signature-tags and KanR that were used in PCR-based identification of mutants during screening of fish are listed in Table 3.
Other primers used in this study are metioned in Table 4. The PCR primers were obtained from either Sigma or Invitrogen, resuspended in distilled water and stored at -20 °C.
2.5. Generation of signature-tagged (ST) mutants
Twelve E. coli S17-lX,pir strains containing pUTminiTn5Km2 plasmids with
DNA signature tags were kindly provided by Dr. R. C. Levesque (Lehoux et al, 1999).
44 Table 3. Sequences of DNA-signature tags used for making and screening of signature-tagged mutants.
Tag/Primer Nucleotide sequence (5'-3')a
Tagl GTACCGCGCTTAAACGTTCAG
Tag 2 GTACCGCGCTTAAATAGCCTG
Tag 3 GTACCGCGCTTAAAAGTCTCG
Tag 4 GTACCGCGCTTAATAACGTGG
Tag 5 GTACCGCGCTTAAACTGGTAG
Tag 6 GTACCGCGCTTAAGCATGTTG
Tag 7 GTACCGCGCTTAATGTAACCG
Tag 8 GTACCGCGCTTAAAATCTCGG
Tag 9 GTACCGCGCTTAATAGGCAAG
Tag 10 GTACCGCGCTTAACAATCGTG
Tagil GTACCGCGCTTAATCAAGACG
Tag 12 GTACCGCGCTTAACTAGTAGG
KanR GCGGCCTCGAGCAAGACGTTT
a Tag 3 was cross-reactive with other tags and was not used for making mutants. The tag sequence and KanR served as forward and reverse primers, respectively, in the PCR- based identification of signature-tagged mutants after fish infection.
45 Table 4. List of oligonucleotide primers used in this study.
Name Sequence (5'-3'» in) a ZnAfor rCCCCCGGGGGATTAGTGACAGGTGCGGTCAATAG.Smal ZnArev CCGGAATTCCGGCGGTAAGTTGTTATACGCCAGAG.EcoRI ZnBfor GCGGCGCrCrAGAGCAAACATCTCGATAGGATTTCTTGC.X^al ZnBrev rCCCCCGGGCTCTCGGATCATTTGTGGTCTG, Smal kanfor GCGGCGCJCTAGACACGTAGAAAGCC AGTCCG, Xbal kanrev GCGGCGCTCTAGATAATTCAGAAGAACTCGTCAAG.Xfral znuABC/or GCTCTAGAGCGTTATACGCCAGAGCGATAGTTC. Xbal znuABOev GCTCTAGAGCAGTCGTCAGAGCAGTAGGTGTTG. Xbal RSI GGArCGAGAATTCCATCAGAGGGACGAAGAG^coRI RS2 A7TCCCGGGATCCCATCCCGAGTGACTGAAC, BamRl RS3 A7TCCCGGGATCCCATCAGAGGGACGAAGAG, BamRl RS4 GGArCGAGAATTCCATCCCGAGTGACTGAAC,£:coRI BarAlforward CTKCGYGCRCGSATGWTGAT BarAlreverse CCGCTAYAKSYGCAACTGCC V2-4for GATGACCACGAACTGGTGC F2-4rev CTATTAGACAACGTCTCCGC
46 aThe restriction enzyme site added to the primer sequence is underlined and the extra bases added for efficient cutting of DNA by enzyme are shown in italics. For degenerate primers: K = (G or T),
Y = (C or T), R = (A or G), S = (C or G), W = (A or T).
47 Before being used for making mutants, these 12 signature tags were tested for cross- reactivity with each other. Using pUTminiTn5Km2 plasmids as templates, 12 tags were tested in all possible combinations for cross reactivity by PCR. The forward primer was a
21 nuclotide tag specific sequence, whereas kanR served as a common reverse primer in
PCR. The PCR conditions used included: 95 °C for 5 minutes, 29 cycles at 94 °C for 30 seconds, 64 °C for 1 minute, 72 °C for 45 seconds, followed by 72 °C for 7 minutes. The
PCR was carried out in 25 ul volume with 500 ng of template DNA, 2.5 ul of IX PCR buffer, 1.6 mM MgCl2, 50 pmol of each primer, 200 uM dNTPs and 3 units of Taq DNA polymerase. The PCR conditions were optimized such that a particular tag gave amplification only with its respective host pUTminiTn5Km2 plasmid. Tag 3 was found to be cross-reactive and was not used further for making mutants. In all, 11 signature tags were used for making mutants (Table 3).
The ST mutant library preparation was the work of Wook Kim and Alicia
Gallaccio of this laboratory. A total of 1056 mutants were made that covered nearly 20 % of the predicted 4.5 MB to 4.8 MB genome of Y. ruckeri (Romalde et al, 1991b). The mutants were made using the filter-mating protocol described by Darwin and Miller
(1999). To transfer pUTminiTn5Km2 plasmids into Y. ruckeri by conjugation, E. coli
S17-lApir donor strains were filter mated with Y. ruckeri RSI 154. E. coli and Y. ruckeri strains were grown in LB broth for 18 hours at 18 °C and 37 °C, respectively. For filter mating, Y. ruckeri (200 ul) and E. coli (100 ul) cells were mixed into 3 ml of 10 mM
MgSCv, incubated at room temperature (22 - 25 °C) for 5 minutes and afterwards filtered through a 0.45 uM filter. The filters were placed onto LB agar (supplemented with 10 mM MgS04) for 4 hours at room temperature. The cells were washed from the filters
48 with 0.9 % NaCl. To select mutants, 100 ul of 10-fold dilutions of bacterial suspensions were plated on LB agar (kanso van3oo).
The E. coli S17-lA,pir strains were ampicillin and kanamycin resistant. After legitimate mutagenesis, only the kanamycin resistance gene was inserted into the genome of mutants, making final ST mutants kanamycin resistant. Y. ruckeri RSI 154 strain was vancomycin resistant. The WT Y. ruckeri RS1154 and E. coli S17-lXpir strains were killed by kanamycin and vancomycin, respectively and selecting for kanamycin resistant
ST mutants of Y. ruckeri RSI 154.
To eliminate mutants generated by illegitimate transposition in which the whole pUTminiTn5Km2 plasmid was inserted in to the genome, making an ST mutant both ampicillin and kanamycin resistant, individual colonies obtained on LB agar (kanso and van30o) were simultaneously patched on to LB agar (kanso and ampioo) and LB (kan5o) plates. Only mutants that were both kanamycin-resistant and ampicillin-sensitive were finally picked for further study.
To ensure that mutants were generated by random mutagenesis, twelve independent filter matings were performed for each of the 11 tags. Each time 8 mutants were picked for a total of 96 mutants for each tag. The mutants were stored in 96 well plates at -80 °C.
2.6. Randomness of transposon insertion
To determine that the libraries of mutants were a collection of random mutants, 22 mutants, taking two from each of the 11 tags, were tested for the locations of transposon insertions. The genomic DNA of mutants was digested with the EcoRY, and ran on 0.8 %
49 agarose gel. Southern hybridization was performed as described in the DIG labeling and hybridization manual (Roche). Briefly, the DNA with in the gels was subjected to depurination (0.25 % HCL for 15 minutes), denaturation (0.5 M NaOH + 1.5 M NaCL for 30 minutes) and neutralization (0.5 M TrisHCL + 3 M NaCL, pH 7.5 for 30 minutes) steps. After transfer to a nylon membrane by capillary action, the DNA was cross-linked to the membrane by UV light (UV Startlinker™, Stratagene) and was hybridized to the
500 bp DIG-labeled kanamycin gene probe. The bands were detected using a DIG
Nucleic acid Detection Kit (Roche). The presence of hybridization bands of different sizes for different mutants indicated the randomness of the transposition.
For Southern hybridization experiments, a 500 bp sequence of the kanamycin gene (part of miniTn5Km2 inserted in the genome of mutant) was DIG-labeled using
DIG-PCR labeling kit (Roche). The PCR was done using cycling conditions previously described under the section 'Generation of ST mutant' using 'tag-specific' and kanR primers. PCR was carried out in a 25 ui volume with 500 ng of template DNA, 2.5 ul of
IX PCR buffer with 15 mM MgCl2, 50 pmol of each primer, 100 uM DIG-labeled dNTPs and 2.6 units of Taq DNA polymerase. DIG-labeled PCR product was purified from agarose gel using Qiaquick® Gel-purification kit (Qiagen) and stored at -20 °C.
2.7. Rainbow trout infection experiments
Rainbow trout, Oncorhynchus mykiss, were obtained from Rainbow Springs
Hatchery, Thamesford, ON. The fish had no history of exposure to Y. ruckeri or other known fish pathogens. The fish were maintained in the Hagen Aqua Laboratory,
University of Guelph under continuous supply of air and fresh water at 8 °C to 10 °C and
50 were fed commercial pelleted trout feed. All infection experiments were conducted
according to the Canadian Council for Animal Care Guidelines and standard operating
procedures approved by the Animal Care Committee, University of Guelph. During
experiments, infected fish were monitored twice daily for any sign of disease or distress.
Fish with an average body weight of 30 g to 250 g were used at different stages
of this study. Fish were acclimated to holding tanks for 7 days before infection. To
produce the natural disease process, fish were infected by bath immersion for 10 minutes
with 107 - 1010 CFU/ml of bacteria. For infection, the water supply to the challenge tank
was stopped. Fifteen litres of water was retained and the bacterial inoculum was added to
the tank. A constant supply of air to the tank was maintained and fish were monitored
during the challenge. At the end of the challenge, a water sample was taken and serial
dilutions were plated on BHI and BHI (kan) plates to calculate the infection dose. The
water supply to challenge tanks was restarted and fish were closely monitored until end
of the experiment. For sampling, fish were euthanized by submersion in water with an
overdose (>100 parts per million) of MS-222 (Tricane methane sulphonate, SIGMA).
After dissection, the kidney tissue was removed aseptically.
The kidney tissue was homogenized in 1:5 (w/v) of 0.9 % sterile saline in a
stomacher (Lab-Blender 80). To calculate bacterial loads, serial dilutions of the kidney tissue homogenates were plated on BHI and BHI (kanso) agar plates. The counts on BHI
(kan) plates represented the kanamycin-resistant mutant strain. The CFUs of WT strain were calculated by subtracting colony counts obtained on BHI plates from that obtained
on BHI (kan) plates.
51 2.8. Screening of ST mutants in rainbow trout
The 1056 mutants were screened in groups of 11 mutants each, by taking one mutant from each of 11 tags. For screening in fish, the mutants were grown on BHI
(kanso) agar from 96 well plates stored at -80 °C. In two steps of 5 ml and 50 ml, each mutant was grown individually for 20 hours at 18 °C with shaking in BHI broth. The
OD600 of cultures were calculated to ensure that each mutant grew to the same level
(Spectronic 70 Bausch and Lomb, SmartSpec™ Plus Biorad). To calculate the CFU of each mutant, cultures were plated on BHI (kan5o) agar and incubated at 18 °C.
The 11 cultures were mixed so that the challenge dose had about 108 CFU/ml of each mutant. Two rainbow trout were infected by bath immersion for 10 minutes. At 7 days post-infection, fish were euthanized and the kidney tissue was taken out aseptically.
The kidney tissue was homogenized in 1:5 (w/v) of sterile saline in a stomacher (Lab-
Blender 80). A 100 ul of serial 5-fold dilutions was plated in duplicate on BHI (kanso) agar plates. All the colonies were washed from the plates with sterile saline and the bacterial suspensions obtained from different plates for a particular fish were mixed. The genomic DNA was isolated from these bacterial suspensions using a Genomic DNA isolation kit (Pharmacia Biotech.). The mutants missing in the kidney tissue were identified by running 11 PCR using one of the 'tag-specific' primers and a common
KanR primer, using the conditions described previously (PAGE ???). Only mutants that were missing in both the fish were selected for further characterization.
For the primary screening of 96 pools (1056 mutants), 34 pools had been screened by Alicia Gallaccio in this laboratory (unpublished data) and the remaining pools were completed in this study. On the first screening of all 1056 mutants in fish, 130 mutants
52 were not recovered from the kidney of fish. To identify consistently missing mutants, a second screening was carried out on the 130 mutants in groups of 7 to 8 mutants each by challenge in rainbow trout. The second screening was done with the same procedures as the first. After this second screen, 25 mutants that had not survived in the kidney of rainbow trout 7 days post-infection were selected for further characterizations.
2.9. Number of transposon insertions in the genome of mutants
The 25 mutants that were chosen after two rounds of screening in rainbow trout were tested for the number of miniTn5km2 insertions in the genome. This was done using Southern hybridization as described for determination of randomness of transposon insertion. The number of hybridization bands on Southern blot represented the number of miniTn5km2 insertions in the mutant.
2.10. Sequence characterization of mutants
The sequences of selected 25 mutants were determined by cloning the mutated genes. The genomic DNA was digested with either one of the enzymes EcoRY, Pst\,
Kpnl, EcoRI and the digested fragments were cloned into a pBluescriptll KS (+) vector digested with the same enzyme. Recombinant clones were selected based on kanamycin resistance. The sequences of both the DNA strands were determined on an automatic sequencer (ABI PRISM model 377, version 3.4) in the Department of Molecular and
Cellular Biology, University of Guelph, Canada. The sequence analyses were done by homology search and multiple sequence alignments using BlastP, BlastX and
CLUSTALW programs available at www.ncbi.nlm.nih.gov and http://align.genome.jp.
53 For Blast sequence analyses, word size 3, gap costs existence 11, gap extension cost 1 and BLOSUM 62 matrix were used. The BLOSUM matrix, gap open penalty 10 and gap extension penalty 0.05 were used for ClustalW alignments.
2.11. Screening for auxotrophs
The 25 selected mutants were tested for auxotrophy, as a potential live-attenuated vaccine, by growing on M9 medium (6.81 g Na2HP04, 3.0 g KH2P04, 500 mg NaCI, 1.0 g NH4C1 per liter, 0.4% glucose, ImM CaCl2, 1 mM MgS04, pH 7.2). The mutants were grown in BHI (Kan50) broth for 18 hour at 18 °C. The cells were centrifuged at 2,000 x g for 10 minutes and the pellet was washed with 0.9 % saline. This was repeated three times. The cells were suspended to an OD600 of 2.0 in 0.9 % saline. A 200 fil of inoculum was spread on M9 agar plates and incubated at 18 °C for 7 days. To eliminate nutrient carry-over, cultures that grew on M9 plates were transferred twice more on fresh M9 plates.
2.12. Identification of bar A homolog of Y. ruckeri RS1154
The barA, a histidine kinase of BarA-UvrY two-component system, homolog of
Y. ruckeri RSI 154 was amplified using degenerate PCR primers that were designed from conserved sequences of other hybrid histidine kinases. The BavAlforward and
BarA2rever.se primers were used with the following conditions: 95 °C for 2 minutes, 32 cycles at 95 °C for 15 seconds, 65 °C for 30 seconds, 72 °C for 2.50 minutes followed by
72 °C for 10 minutes. The PCR was carried out in a volume of 50 ul with 5 ul of IX PCR buffer, 2.5 mM MgCl2, 250 pmol of each primer, 500 uM dNTPs and 3 units of Taq
54 DNA polymerase (Invitrogen). The 2596 bp PCR product obtained was purified from agarose gel and cloned into pCR®4-TOPO® vector (Invitrogen). Using overlapping primers, nucleotide sequences of both DNA strands were determined as described for ST mutants. The multiple sequence alignment of various bar A homologs were performed by
ClustalW program at http://align.genome.jp.
2.13. PCR amplification of bar A and uvrY genes in Y. ruckeri strains
The bar A PCR was performed as described previously. The 629 bp uvrY sequence was amplified using F2-4for and F2-4rev primers using the following conditions: 94 °C for 2 minutes, 35 cycles at 94 °C for 20 seconds, 52 °C for 30 seconds, 72 °C for 50 seconds followed by 72 °C for 5 minutes. The PCR was carried out in a 50 ^1 volume with 200 ng of template DNA, 5 ul of IX PCR buffer, 2.0 mM MgCl2, 40 pmol of each primer, 200 uM dNTPs and 3 units of Taq DNA polymerase (Invitrogen).
2.14. Growth of uvrY mutant in Luria-Bertani medium and under iron-limiting conditions
Five ml of LB medium was inoculated with single colony of WT RSI 154 and the uvrY mutant and incubated at 18 °C for 18 hours with shaking. For both strains, three flasks containing 50 ml LB were inoculated (1:100) with overnight grown cultures and incubated at 18 °C with shaking. The OD6oo and CFU were calculated at different time points. A similar protocol was used to test growth of both strains under iron limiting conditions, except that 200 \iM of 2,2' dipyridyl was added to LB medium.
55 2.15. Fish cell invasion by uvrY mutant
Cell invasion studies were performed with the Epithelioma papulosum cyprini
(EPC) and chinook salmon embryo cells-214 (CHSE) fish cell lines. The cell lines, obtained from the Fish Health Laboratory, Department of Molecular and Cellular
Biology, was grown in minimal essential medium (MEM) supplemented with 10 % fetal calf serum (FCS) at 15 °C. The 24 hours old monolayers used for infection had approximately 9.0 x 106 cells in each 25 cm2 flasks. The mutant and WT bacteria were grown to an OD600 of 1.0 in LB medium at 18 °C. After centrifugation at 2000 x g for 10 minutes, bacterial pellets were resuspended in MEM. This was repeated three times. The
EPC/CHSE cells were washed three times with MEM before being exposed to bacterial cells at a multiplicity of 100:1 (bacteriaxells). After incubation at 18 °C for 30 minutes, the bacterial suspension was poured off and the monolayers were washed three times with MEM.
To kill extracellular bacteria, MEM containing gentamicin (250 fig/ml) was added to the monolayers which were then incubated at 18 °C for 2 hours. The monolayers were then washed three times with phosphate buffered saline (PBS) (137 mM NaCl, 3 raM
KC1, 13 mM Na2HP04, 1 mM KH2P04, pH 7.4) and the cells were lysed with 0.2 %
Triton X-100 in the tissue culture flasks to release intracellular bacteria. Dilutions of the lysed EPC cells were plated on LB (for WT) and LB (kan) (for mutant) agar plates. The invasion index was calculated using the formula: (number of intracellular bacteria/ original number of EPC/CHSE cells) x 100.
56 2.16. Serum sensitivity of uvrY mutant
The WT strain RSI 154 and uvrY mutant were compared for sensitivity to non
immune rainbow trout serum. To collect serum, rainbow trout were sedated with MS-222
and blood was taken from the tail vein and maxillary sinus. The blood was kept at 4 °C
and the serum was collected from the top of the blood clot after 2 days. The serum was used at 25 %, 50 % and 100 % concentration, with dilutions made in 0.9 % saline as required.
Bacterial cultures were grown in tubes of 5 ml LB medium at 18 °C for 18 hours.
After centrifugation at 2000 x g for 10 minutes at 22 °C in polypropylene tubes
(Corning®), pellets were washed three times with 0.9 % saline and resuspended in 0.9 %
saline to an OD600 of 0.6. A 250 ul volume of the bacterial suspension and 250 u.1 of
serum (25 %, 50 % and 100 % concentration) were mixed and the mixture present in 15 ml polypropylene tubes was incubated at 18 °C with shaking. At 90 minutes and 180 minutes, samples were taken and serial dilutions were plated on LB agar plates. The
CFU/ml of cultures were determined and the percent survival was calculated using the formula: (CFU in presence of serum/CFU in absence of serum) x 100.
2.17. Ultraviolet light (UV) sensitivity of uvrY mutant
The bacterial sensitivity to UV light was tested using a slight modification of the protocol described by Croteau et al. (2006). Bacteria were grown in LB broth at 18 °C to an OD600 of 1.0. Volumes containing approximately 300 - 500 bacteria were spread on
LB agar plates and incubated at 18 °C for 30 minutes. The LB plates containing bacteria
were exposed to 1.5 J/m2 and 5.0 J/m2 of UV light (254 nm), using a hand-held UV lamp
57 (Spectroline, Westbury, NY, USA). The UV intensity was measured by UVX digital radiometer (UVP Inc. CA, USA). After UV exposure, the plates were wrapped in aluminum foil, incubated at 18 °C and the colonies were counted. The percent survival was calculated using the formula: (CFU of UV exposed cultures/CFU of unexposed cultures) x 100.
2.18. H2O2 sensitivity of uvrY mutant
The WT strain RSI 154 and uvrY mutant strains were grown in flasks of 25 ml
LB medium at 18 °C to an OD600 of 0.8. After adding 1 mM or 10 raM of H2O2, bacteria were further incubated at 18 °C with shaking. To compare the CFU/ml of the exposed and control cultures, samples were taken at 30 minutes, 60 minutes, 90 minutes, 150 minutes,
210 minutes and 270 minutes after incubation, diluted and plated on LB agar. The percent survival was calculated using the formula: (CFU of H2O2 exposed cultures/CFU of control cultures) x 100.
2.19. Sequence characterization of znuACB locus
The znuACB locus of the C6-1 mutant was cloned at the EcoRV site of pBluescriptll KS+ plasmid. Using overlapping primers, sequences of both DNA strands were determined as described for other mutants. The sequence analyses were performed using BlastN, BlastP and ClustalW (1.82) programs available at www.ncbi.nlm.nih.gov and http://align.genome.jp.
58 2.20. PCR amplification and cloning of znuACB locus
The znuACB locus of Y. ruckeri RSI 154 was amplified by Expand Long
Template PCR system (Roche). The 2766 bp PCR product was amplified using
znuABC/or and znuABCrev primers under following conditions: 94 °C for 2 minutes, 35 cycles at 94 °C for 15 seconds, 68 °C for 30 seconds, 68 °C for 2 minutes followed by 68
°C for 7 minutes. The PCR was carried out in a 50 ul volume with 200 ng of template
DNA, 5 ul of IX PCR buffer, 100 pmol of each primer, 350 uM dNTPs and 3.75 units of
DNA polymerase mixture. After digestion with Xbal enzyme, the PCR product was
cloned at the Xbal site of the pBAD33Cm vector. The resulting plasmid, pBAD33/6, was
transformed into E. coli GR352 (AznuACB) for transcomplementation studies.
2.21. Generation of AznuACB mutant
We made a znuACB deletion mutant by homologous recombination (Figure 2).
The 972 bp znuA and 993 bp znuB sequences were amplified by ZnAfor and ZnArev,
ZnBfor and ZnBrev primers, respectively. The znuA sequences were amplified using
following conditions: 94 °C for 2 minutes, 35 cycles at 94 °C for 20 seconds, 62.5 °C for
30 seconds, 72 °C for 1 minute followed by 72 °C for 7 minutes. The PCR was carried
out in a 50 ul volume with 200 ng of template DNA, 5 ul of IX PCR buffer, 2.5 mM
MgCl2, 100 pmol of each primer, 500 uM dNTPs and 3 units of Taq DNA polymerase
(Invitrogen). Similarly, the znuB sequences were amplified using the PCR conditions
described for znuA, except that the annealing temperature was 58 °C.
These PCR products were cloned into pBluescriptll KS+ at Xbal and EcoRl sites,
resulting in the plasmid, pYRZnuAB (Figure 2). After digesting pUCGm(kan) plasmid
59 Figure 2. Strategy for making the DNA construct used for generating the SznuACB mutant.
A. The 972 bp znuA and 993 bp znuB sequences were amplified by ZnAfor and ZnArev,
ZnBfor and ZnBrev primers, respectively.
B. The znuA PCR product was digested with EcoRl and Smal enzymes and cloned into pBluescriptll KS+ vector, resulting in the plasmid, pYRZnuA.
C. The znuB PCR product was digested with Xbal and Smal and cloned into pYRZnuA digested with the same enzymes, resulting in the plasmid, pYRZnuAB.
D. After digesting the pUCGm(kan) plasmid with Hindi, a 1.1 kb kanamycin resistance gene was released that was cloned at the Smal site of pYRZnuAB. The resulting plasmid was called pYRZnuABkan.
E. Using pYRZnuABkan as a template, a 3.1 kb PCR product was amplified using znuABC/br and znuABCrev primers. For homologous recombination, 500 ng of this PCR product was electroporated into Y. ruckeri (pKOBEG-sacB).
60 Smal - ZnAfor ZnArev - EcoRI Smal - ZnBrev ZnBfor - Xbal • M • <« znuA PCR znuB PCR
EcoRI znuA ^ Smal B liiii HIIIIIIIIIIII i mnmrn Xbal
znuA Smal znuB EcoRI Xbal
znuABC/or znuABCrev D znuA Km znuB
znuABC/or znuABCrev E m%m znuA Km znuB
61 with HincU, a 1.1 kb kanamycin resistance gene was released that was cloned at the Smal site of pYRZnuAB. The resulting plasmid was named pYRZnuABkan (Figure 2D).
Using pYRZnuABkan as a template, a 3.1 kb product was amplified by znuABC/or and znuABOev primers using PCR conditions as described for amplification of the znuACB locus.
The 500 ng of kit-purified PCR product was transformed into electro-competent
Y. ruckeri (pKOBEG-sacB) cells (single pulse at 2.5 V, 400 Q, 25 uP). After adding SOC medium (2 % tryptone (w/v), 0.5 % yeast extract (w/v), 0.05 NaCl (w/v), 2.5 mM KC1,
10 mM MgCi2 and 20 mM glucose), the transformed cells were incubated at 25 °C for 2 hours. To select mutants, transformed cells were plated on LB agar (kan) plates at 25 °C for 2 days. The mutants were identified by three different PCRs (Figure 3C) and the gene deletion was further confirmed by sequencing.
2.22. In vitro growth of AznuACB mutant
The growth kinetics of WT RSI 154 and the AznuACB mutant strains were examined in the LB and M9 media. For M9 studies, to remove trace minerals, glassware and plasticware were treated with 1 % hydrochloric acid and rinsed with Chelex-100 treated distilled water (DW). Five gram of Chelex-100 resin (Biorad) was added to 1 litre of DW and stirred overnight to remove traces of minerals. The M9 medium was made with Chelex-100 treated water. The zinc content of M9 medium was tested by atomic absorption spectrometer with a detection limit of 1 ng/ml (AAnalyst 800, Perkin Elmer
Instruments).
62 znuA znuC znuB
Figure 3. Gene organization of znuACB locus and strategy for making the AznuACB mutant of Y. ruckeri RS1154.
A. The znuACB locus of Y. ruckeri RSI 154 had three predicted genes, znuA, znuC and znuB. The znuA and znuCB might be divergently transcribed, and were separated by a 96 bp intergenic region (solid line connecting znuA and znuC).
B. The AznuACB mutation was made by homologous recombination using nearly 1 kb flanking sequences of znuA and znuB. The DNA sequence encoding first 23 amino acids of ZnuA, the intergenic spacer, the complete znuC gene and first 22 amino acids of ZnuB, were replaced by the kanamycin resistance gene.
C. The mutation was confirmed by PCR using the genomic DNA of AznuACB mutant as temlate with the four primers indicated in (B). Lane 2 (PCR with primers 2 (kanrev) and
3 (kan/or) predicted amplicon size 1126 bp), lane 4 (PCR with primers 2 (kanrev) and 4
(ZnArev) predicted amplicon size 2098 bp), lane 6 (PCR with primers 1 (ZnBfor) and 3
(kan/or) predicted amplicon size 2119 bp). The lanes 3, 5 and 7 were negative controls for these PCRs on the genomic DNA of WT strain, RS1154.
63 The bacteria were grown at 18 °C in 25 ml of LB medium supplemented with different concentrations of ethylenediamine tetraacetic acid (EDTA) (0.5 mM, 2.0 mM,
5.0 mM) and tetrakis-(2-pyridylmethyl)-ethylenediamine (TPEN) (2.0 uM, 12.5 uM).
The EDTA and TPEN were added to chelate metals. Similarly, bacteria were grown in
M9 medium or M9 supplemented with 1 uM TPEN. Growth was monitored by taking
OD600 of samples at different points using a spectrophotometer (SmartSpec™ Plus,
Biorad).
2.23. Transcomplementation of AznuACB mutant of E. coli
We tested functionality of the znuACB locus of Y. ruckeri by transcomplementation of the AznuACB mutant of E. coli. To create zinc-deficient conditions, various E. coli strains including W3110, GR352, GR352 (pBAD33/6),
GR352 (pSP86/37) and GR352 (pHSG576) (Table 1) were grown in LB medium supplemented with 2.0 mM of EDTA. The complementation was assessed by monitoring growth of strains over time by reading ODgoo- The AznuACB mutant of E. coli was kindly provided by Dr. C. Rensing (Grass et ah, 2002).
2.24. The znuAnlacZ and znuCB::lacZ transcriptional fusion analysis
The 500 bp sequence that included 96 bp of the intergenic region along with flanking sequences of znuA and znuC was amplified by two separate PCRs, using (RS1 and RS2) and (RS3 and RS4) primer pairs (Figure 4). Both PCRs were done under the following conditions: 94 °C for 2 minutes, 35 cycles at 94 °C for 15 seconds, 58 °C for 30
64 seconds, 72 °C for 25 seconds followed by 72 °C for 10 minutes. The PCR was carried out in a 50 ul volume with reagent concentrations as described for znuA PCR.
The PCR products were cloned in both orientations at EcoRI and BamHI sites of lacZ transcriptional fusion vector, pRS551, resulting in znuAv.lacZ and znuCB::lacZ fusions (Figure 4). These transcriptional fusion plasmids were put into WT Y. ruckeri
RSI 154 by electroporation, generating Y. ruckeri (pRS551/41), Y. ruckeri (pRS551/61) and Y. ruckeri (pRS551) strains (Table 1).
To assay transcriptional fusion activity, the p-galactosidase assay was performed as described by Zheng and Bremer (1995), with modifications described below. The Y. ruckeri strains containing transcriptional fusion plasmids were grown until mid exponential stage at 18 °C in LB medium or LB supplemented with 0.5 mM EDTA or 20 uM TPEN or 20 uM TPEN with 1 mM ZnS04 or 0.5 mM EDTA with 1 mM ZnS04 or
0.5 mM EDTA with 1 mM MnCl2.
A 50 ul aliquot of bacterial culture was mixed with 50 ul of permeabilization solution (100 mM Na2HP04, 20 mM KCl, 2 mM MgS04, 0.8 mg/ml hexadecyltrimethylammonium bromide, 0.4 mg/ml sodium deoxycholate and 5.4 ul/ml of
P-mercaptoethanol) and incubated at 25 °C for 15 minutes. The 600 ul of prewarmed (30
°C) substrate solution (60 mM Na2HP04, 40 mM NaH2P04, 1 mg/ml o-nitrophenyl-p-D- galactoside and 2.7 ul/ml of P-mercaptoethanol) was added to the lysed cells and the mixture was incubated at 30 °C until sufficient color was developed. The reaction was stopped by adding 700 ul of 1M Na2CC"3 solution.
65 Figure 4. Strategy for making znuAr.lacZ and znuCB::lacZ transcriptional fusions of Y. ruckeri.
A. The predicted znuA and znuCB transcriptional units of znuACB locus may be divergently transcribed as indicated by arrows. They were separated by a 96 bp intergenic region shown by a solid line connecting znuA and znuC. Putative translational start site for znuCB and znuA (coded on opposite strand) were shown in bold. Shown in lower case was the 9 bp imperfect palindromic sequence (AgcGTaATA T TATaACatT, center of symmetry underlined, non complementary bases shown in lower case), a putative binding site for zinc-uptake regulator (Zur) that regulated expression of znuACB locus in other bacteria (Patzer and Hantke 2000, Panina et ah, 2003). The 9 bp sequence was identified by homology with Zur-binding sequences identified in Patzer and Hantke (2000) and
Panina et ah (2003).
The 500 bp sequence that included 211 bp of znuA, intergenic spacer (underlined) and
193 bp of znuC was cloned between EcoRI and BamHl sites of pRS551 vector.
B. The diagrammatic representation of relevant features of promoterless-lacZ transcriptional fusion vector, pRS551. Tet: tetracycline, bla: ampicillin, kan: kanamycin,
TU: transcriptional terminators, lacZYA: the promoterless-lac operon. Figure 4B was adapted from Hand and Silhavy (2000).
66 znuA |—• znuc znuB
CATCAGAGGGACGAAGAGCATAATCGTGTGGCGAAGCGCCATCG GGCAGTAATACTTCCGTCGGCAGGACGCCATCGGCAATAGCCGA GGCGATAAATCCCAAAGGACGAACTGAGGTTACTACTGCGGCAG AAGCACTATTGACCGCACCTGTCACTAAAATGGTGCCGATCAGCA AAGTGCTTTTCAGCCACTTGTTTTTTTGTAACATAAAGTTATTTCT CGTTAACATGATAAGTTATCTCGTCCTTTTCGCTGGAAAagcgtaatatt ataacaTTCACTTAAATCTGCAAACGTAATCATCATGTCCACATTGAT TAGTCTTGACAAAATATCCGTCAGCTTTGGTACACGGCGGGTACT TTCTGATATTTCCCTTTCCTTGCAAGCGGGAAGAATACTGACTCTG CTTGGGCCTAACGGTGCCGGTAAGTCAACGCTGGTTCGCGTGGTG CTGGGACTCATTCCACCAACCAGTGGTTCAGTCACTCGGGATG
"tet ori bla kan TI4 lacZYA
67 To remove particulate material, reaction mixture was centrifuged at 18,000 x g for
5 minutes. The 200 ul of supernatant was put in triplicate into 96 well plates (Greiner bio- one®) and absorbance was read at 420 nm using kinetic microplate reader (Molecular
Devices). The transcriptional activity was expressed as Miller units that were calculated using the formula: 1000 x (Abs42o)/(reaction time x reaction volume x Abs6oo of culture).
2.25. Generation of isogenic mutants of Y. ruckeri
To understand role of genes in pathogenesis in fish, we attempted to generate isogenic mutants in five different genes of Y. ruckeri. The details are provided in appendices 2 to 7.
2.26. Statistical analyses
The statistical analyses of data from different experiments were performed using two-tailed T-test (Microsoft Excel 2007). P values <0.05 were considered significant.
68 CHAPTER 3
GENERATION AND SCREENING OF SIGNATURE-TAGGED MUTANTS
RESULTS
3.1. Generation of ST mutants
The 1056 mutants of Y. ruckeri RSI 154 made using 11 signature-tags (Lehoux et al, 1999) could cover nearly 20 % of the predicted 4.5 - 4.8 MB genome of Y. ruckeri
(Romalde et al, 1991b). The 20 % coverage of the genome was deduced assuming average gene size of 1 kb, predicting a total of 4500 - 4800 genes in Y. ruckeri. The
Southern hybridization patterns obtained on the genomic DNA of 22 selected mutants (2 per tag) with a DIG-labelled Tn5-Km2 probe were random, indicating that miniTn5Km2 did not appear to have mutation hot spots in the genomic DNA of Y. ruckeri (Figure 5).
The mutant A1-2 had two miniTn5Km2 insertions (Figure 5, lane F). There were three hybridization bands present in mutant Al-1 (Figure 5, lane E); the bottom two bands were probably non-specific hybridization. Only one band was obtained when Southern hybridization was performed on Al-1 genomic DNA digested with PstI and Sail enzymes
(data not shown). Thus, a single insertion was present in mutant Al-1.
3.2. Primary screening of ST mutants in rainbow trout
For immersion challenges of rainbow trout, each of the eleven mutants in the
Q input pool was present at a concentration of 10 CFU/ml in a total challenge dose of approximately 109 CFU/ml. This was confirmed by measuring the cell density of the
69 ABCDEFGH I J KLMNOPQABCDEFG - - i , *;. . . *...'..• • • . •.••}- !•.•.•.:•" - • %
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mmm • ,.— '-„ * -o, • r » - , •• • ... set i. •• "''
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Figure 5. A Figure 5. B
Figure 5. Randomness of transposon insertion in signature-tagged mutants.
Genomic DNA of twenty two randomly selected mutants was digested with EcoRY enzyme, electrophoresed on a 0.8 % agarose gel and transferred onto a nylon membrane for hybridization with a DIG-labeled 500 bp kanamycin gene probe.
5 A. Lanes: A: DIG-labeled DNA molecular weight marker (MWM), size in kb, B: D5-
12, C: Bl-5, D: A5-4, E: AM, F: Al-2, G: F5-8, H: Hl-11,1: A2-11, J: C3-12, K: Dl-
7, L: G4-8, M: C2-9, N: Gl-9, O: F4-10, P: Fl-2, Q: H5-10.
5 B. Lanes A: DIG-labeled DNA MWM. B: D2-1, C: D2-5, D: E5-6, E: Gl-6, F: B3-7,
G: CI-4.
70 broth culture of each of the mutants by reading the OD600 and adjusting the amount of
mutant culture added to the challenge inoculum accordingly in cases of minor
differences. None of the mutants showed dramatic growth defects in vitro, as indicated by their OD600 nearly similar to WT strain when grown in BHI medium at 18 °C for 18 hours.
The challenge dose for each mutant was confirmed by plating dilutions and counting colonies.
In a pilot study, either four or five rainbow trout were challenged per input pool for seven pools of mutants (Table 5). Some mutants were consistently missing from all or most of the individual fish. Some individual fish had many missing mutants (e.g. fish
1 for pools D5 and E5) whereas others had few or none missing. As the aim was to select mutants that would consistently be missing, only mutants that were missing from more than one fish were considered. In cases where fish died prior to the 7-day endpoint, that fish was not used because of potential post-mortem changes in the dominant mutants. It is
desirable to reduce the total number of fish used for screening purposes, but initially we had concerns about fish-to-fish variability in the selection of mutants. The results for each
multi-fish pool in Table 5 were reviewed for all possible pairings of fish. The final
selection of mutants could be affected, such as in the case of the F5 pool where fish 2 and
4 indicate that four mutants should be selected (1, 2, 8, 11) whereas only two mutants (8,
11) would be selected with other combinations. However, it appeared that challenging
two fish per pool gave adequate opportunity to select the consistently-missing mutants.
The remaining mutants were screened by challenging two fish with each input pool and
selecting mutants missing from the kidney of both fish.
71 Table 5. Screening of signature-tagged mutants in rainbow trout.
Pool Mutants (signature tags 1-12) missing from the kidney offish Mutants No. selected Fishl Fish 2 Fish 3 Fish 4 Fish 5
Al none none none none - none
Bl 5 5 5 5 - 5
CI 4,8,9 11 none 5 - none C5 1,6,7,8,9 1,9 mortality mortality mortality 1,9 D5 1,4,5,6,11,12 1,4,9,12 1,4,12 12 4,12 12 E5 1,6,7,8,10,12 6,12 6,8 9,11,12 mortality 6 F5 8,11 1,2,8,11 8,11 1,2,8,11 8,11 8,11
Four or five rainbow trout were infected with each mutant pool by bath immersion (dose
108 CFU/ml of each mutant) for 10-15 minutes. Mutants missing from the kidneys after 7 days of infection were identified by tag-specific PCRs using DNA isolated from recovered bacteria as a template. For C5 and E5 mutant pools, some fish died before day
7 and were not included in analysis.
72 The 1056 mutants were distributed into 96 input pools, and the first 34 pools (Al to B5) were screened by A. Gallaccio (unpublished results), who identified 49 mutants that were missing from fish. I screened the remaining 62 input pools (C5 to HI2) and identified 81 missing mutants. Thus, a total of 12.3 % mutants (49 + 81 = 130 mutants) were missing from the kidney of the challenged rainbow trout. All 11 tags were represented among the missing mutants, ranging from a high of 30 mutants with tag 1 to a low of 4 mutants with tag 6. Results of screening of 58 input pools (G5 to H12) were
summarized in Table 6, with the final bacterial load in the kidney at 7 days post infection indicated for each fish. Four of 62 pools screened by me (C5, D5, E5 and F5) are not included in Table 6, as the infection loads in fish were not calculated and the remaining 58 pools were discussed.
In five input pools (G8, B9, D10, F10, and E12), one of the two fish did not have any bacteria in the kidney tissue at first challenge, so challenge for these input pools were repeated. Data from second challenge for these five input pools are included in Table 6.
In most infection challenges, 54 of 58 input pools, individual fish were infected to >103
CFU/g kidney, and there were only four pools (H9, CIO, H10 and Gil) in which the infection load in fish was <103 CFU/g kidney (Table 6), suggesting consistency in the challenge protocol. The median infection load across input pools was 1.1 x 106 CFU/g kidney, ranging from 4.1 x 102 (H10 pool fish 1) to >109 CFU/g kidney (D6 and H6 pools), reported as TNTC (Table 6).
The bacterial loads were compared to see if there was a correlation between infection load in the kidney tissue and the number of mutants missing from fish. For 13 of 58 input pools, two fish infected with the same input pool had approximately the same
73 Table 6. Primary screening of signature-tagged mutants in rainbow trout.
Mutant pool, CFU/g kidney Mutants missing Mutants fish number from the kidney selected G5 fishl 7.0 x 10" 1,6 6 G5 fish 2 4.7 x 10s 6 H5 fish 1 3.6 x 10" 10 10 H5 fish 2 1.5 x 107 10 A6 fishl 3.1 x 106 1,2,11 1,2,11 A6 fish2 TNTC1 1,2,11 B6 fish 1 2.1 x 10' None - 136 fish.2 4.0 x 10' None C6 fishl 1.2 xlO4 1,5,8 1,8 C6 fish2 TNTC 1,2,7,8 D6 fishl TNTC 1,2,4.5,6 1.4,5,6 D6 fish2 TNTC 1,4.5.6.8,10,11 E6 fishl 8.0 x 105 1,4,5,7,10,11 - E6 fish2 9.3 x 10b none F6 fishl 6.2 x lO* 1,2 1 F6 fish2 7.0 x Kf 1,4,9 G6 fishl 1.6 xlO4 1,9 1,9 G6 fish2 3.1x10' 1,6,9 H6 fishl TNTC 1,4,5.11,12 1,4.5,11.12 H6 fish2 TNTC 1,4,5.11,12 A7 fishl 1.9 xlO7 2,11 2,11 A7fish2 9.1 x 10' 2,11 B7 fishl 1.1 x Kf 1.2.4,5.6,7.8,9 1.2,5.7,8,9 B7 fish2 1.0 x 10° 1.2.5,7.8.9 C7 fish 1 2.4 x 10' 1.2.5.6.8 1,2.8 C7 fish2 1.1 x I04 1.2,4.8 D7 fishl 2.4 x 10-" 1,6.10,11 - D7 fish2 2.2 x 10" All E7 fishl 1.7 x 10' 1,4,5,6,8,9 1,5,8 E7fish2 2.0 x 10" 1,5,7,8,10,12 F7 fishl 5.3 x 10' 1,11,12 1 F7 fish2 1.7 x Hf 1 G7 fishl 4.1 x 107 7,9,12 12 G7 fish2 2.1 x 105 12 H7 fishl 5.9 x Hf 1,7.9,10 1.7.9,10 H7 fish2 2.2 x 10° 1,7.9.10 A8 fishl 5.9 x 10s 6,7,8,9,10,11 6,9 A8 fish2 2.4 x 10b 6,9
74 Table 6 continuec B8fishl 1.9 x 10' None - B8 fish.2 6.5 x 103 None C8 fishl 1.1 x 10" 7,10,11,12 7,10,11 C8 fish2 6.2 x 10" 5,6,7,10,11 D8 fishl 4.0 x ltf1 1.2 1 D8 fish2 8.7 x Hf 1.5 E8 fish 1 2.3 x 10" 1.2 1 E8 fish2 2.4 x 10" 1 F8 fishl 6.4 x 10' 1,2,4,5.6,7,8,10,11 - F8 fish2 NC' Died G8 fishl 3.0 x 10° 1,2,9,10 - G8 fish2 4.3 x 10' None f 18 fish 1 3.6 x 10" 1,2.5,11 2.5.11 H8 fish2 1.9 x 10" 2,4,5.11 A9 fishl 5.1 x 10* 2,4,6,10,12 - A9 fish2 1.1 x 10v None B9 fish 1 4.6 x 10s None - B9 fish2 2.9 x 10' None C9 fishl 1.3 x 10' 5.".II - C9 fish2 5.8 x 104 •> D9 fishl NC 2.11 2 D9 fish2 NC 2,7.9,12 E9 fishl NC 2,7,12 - E9 fish2 7.0 x 10" All F9 fishl 6.6 x 10" 2,5,7.12 5,7.12 F9 fish2 6.8 x 10" 5.7,12 G9 fishl NC None - G9 fish2 NC 6,8,10 H9 fishl 7.5 x U? 4.6.7.8.9,11.12 11 H9 fish2 9.0x10' 1.11 AlO fishl TNTC 1,4,6,7, 1,4,7 A10fish2 1.1 x 107 1,4,7 BIO fishl 4.8 x 10' 1,11 1,11 B10fish2 3.9 x lO"1 1.11 CIO fishl 8.0 x 102 1,4,5,6,8,10,11 1,4,11 C10fish2 1.3 x 10s 1,4,11 D10 fishl 4.1 x 10" 1 1 D10fish2 6.4 x I04 1.5.6 E10 fishl TNTC 4 - E10 fish2 2.4 x 10" 2 F10 fishl 9.3 x 10' 7,12 - F10fish2 6.3 x 10" None
75 Table 6 continued GlOfishl 4.1 x 10° 8,11 - G10fish2 1.7x10' None H10 fish 1 4.1 x 10- 9 9 H10fish2 1.0 x 10" 4,7,8,9.11 Allfishl 2.5 x 103 1,9 1 Allfish2 1.5x10" 1,6 Bll l'ishl 9.2 x 104 1,5 1,5 BM fish2 3.1 x I0-" 1,5 Cllfishl 1.6 x10s 1 1 Cllfish2 3.2 x lO^ 1,5 Dll fish! 3.2 x ](? 1,9 1,9 Dllfish2 1.3 x 10" 1,4.9,11 EllFishl 4.8 x 105 None - EllFish2 7.7 x 10° None FllFishl TNTC 10 10 FllFish2 1.5 x10s 1,10 GllFishl 8.5 x 10z 7 7 GllFish2 1.3 xlO5 7 HI lFishl 2.4 x 10" 11 - HllFish2 3.2 x 104 None A12Fishl 2.2 x 104 1 - A12Fish2 4.1 x 10" None B12Fishl 4.1 x 10-' None - B12Fish2 1.7 x 104 8 C12Fishl 3.8 x 10" None C12Fish2 1.8x10" 7 D12Fishl 6.9 x 10' 8 8 D12Fish2 2.0 x 10" 8 E12Fishl 4.3 x 10" None - E12Fish2 3.1 \ Hf 8 —•- - - F12Fishl 1.6 x 10" None FI2Fish2 3.3 x 10"* 9,10 G12Fishl 2.7 x 10s 2 2 G12Fish2 2.5 x 105 2 U12Fishl 5.3 x 10fi 1 1 H12Fish2 4.9 x 10' 1.6,7.12
76 1TNTC, Too numerous to count, more than 300 colonies in 100 ul of 1:5,000 dilution of the kidney tissue homogenate. These values were not used in calculations.
2 NC, not calculated, the CFU was not calculated as fish was found dead on day 7 before euthanasia.
The table contains data for 58 of the total 96 input pools of mutants. For each input pool, two rainbow trout were infected by bath immersion for 10 to 15 minutes with a dose containing approximately 10 CFU/ml of each mutant. On day 7 post infection, kidney was dissected out from fish and five-fold dilutions were plated on BHI (kan) agar plates to determine the CFU in the kidney. Genomic DNA was isolated from bacterial colonies on plates and mutants unable to survive in the fish were identified by 11 signature-tag specific PCRs on the genomic DNA. Mutants found missing in both fish were selected for secondary screening.
77 infection loads in the kidney (Table 6). In 28 pools, two fish differed by 10- to 100-fold in infection loads in kidney. A 103 - to 105-fold difference in infection loads between two fish was observed in another 13 input pools. The bacterial loads for four pools (F8, D9,
E9 and G9) were not calculated, as the fish were found dead a few hours before euthanasia on day 7. Thus, in the majority (13 + 28 = 41 pools) of pools, two fish either had similar infection loads or there was a < 100-fold difference in infection load.
Differences in the infection loads between fish challenged under similar conditions, however, suggested that fish varied in their susceptibility to Y. ruckeri infection.
In 14 of 58 input pools, the same mutants were missing in both fish (Table 6). In the remaining 44 input pools, the specific mutants missing and the number of mutants missing varied between two fish (Table 6). Of 44 input pools, in 11 input pools the two fish differed by 1 mutant only; in 14 pools, by 2 mutants; in 6 pools, by 3 mutants; in 7 pools, by 4 mutants; in 1 pool (A9), by 5 mutants; in 2 pools (E6 and E7), by 6 mutants; in 1 pool (D7), by 7 mutants; and in two pools (E9 and H9) by 8 mutants (Table 6). Of 14 input pools in which both fish had the same mutants missing, there were only three pools
(B6, H6 and G12) in which both fish had nearly the same bacterial loads in the kidney tissue. In the remaining 11 input pools, two fish had a 10- to 1000-fold difference in bacterial loads and still had the same mutants missing in the kidney (Table 6).
Conversely, there were 9 input pools in which both fish infected with the same input pool had nearly the same bacterial loads in the kidney and still had different mutants missing.
In many input pools, such as E6, D7, A9, fish with very low difference in infection load
(nearly 10-fold) differed by as many as 7 mutants in output pool. Since fish with the same infection loads (9 pools), as well as fish with different bacterial loads (11 input pools) had
78 the same mutants missing, this suggested that the fish-to-fish difference in output pools was not solely due to differences in bacterial loads in kidney tissue.
There were 13 input pools in which a fish, within a pair, with a high bacterial load
(difference of a 10- to 10 -fold), had fewer mutants missing than from the second fish with lower infection load (Table 6). Conversely, there were more input pools (16) in which a fish with a higher bacterial load (10- to 105-fold difference) had more number of mutants missing than the second fish with lower bacterial loads in the kidney. Fish with infection levels of <10 CFU/g kidney had fewer mutants missing than the second fish which had infection levels of >105 CFU/g kidney (H9, H10 and Gil input pools). This suggested that higher bacterial load did not relate to fewer mutants missing in the kidney tissue.
To test whether the route of infection influenced outcomes, the F5 input pool was screened by bath immersion as well as by an intraperitoneal injection route. Of five fish infected by bath immersion, three fish (1,3,5) had two mutants (8, 11) missing, and four mutants (1, 2, 8, 11) were missing in the other two fish, demonstrating differences in mutant missing in five fish (Table 7). In comparison, fish injected intraperitoneally had differences in the number and specific mutants missing in kidney, regardless of whether dose was 102, 103 or 104 CFU. Thus, fish-to-fish difference in missing mutants was evident after the injection route as well.
3.3. Secondary screening of ST mutants in rainbow trout
To identify consistently missing mutants, 130 mutants identified from the primary screening were used to reinfect rainbow trout as components of new 11 tag-libraries.
79 Table 7. Effect of infection route on mutant recovery from rainbow trout.
Dose Mutants missing from the kidney of fish Mutants Fishl Fish 2 Fish 3 Fish 4 Fish 5 Selected
Bath immersion 108 8,11 1,2,8,11 8,11 1,2,8,11 8,11 8,11 CFU/ml Intraperitoneal injection 1,8,11 8,11 1,5,7, 1,5,6, mortality 8,11 CFU 8,11 7,8,11 103 1,6,8,11 1,2,4,7,8,9, 1,6,8,11 1,8,11 1,8,11 8,11 CFU 10,11,12 104 1,8,10,11 1,8,11 none 1,8,10,11 1,8,11 8,11 CFU
Five rainbow trout were challenged with F5 input pool either by bath immersion (dose
108 cfu/ml of each mutant) for 15 minutes or by intraperitoneal injection (IP) with three doses of 102,103, and 104CFU. Mutants missing in kidney were identified by tag-specific
PCR. Fish 5 infected with 102 CFU by IP route died before day 7 and was not included in analysis.
80 Tag 1 had the highest number of first-screen mutants (30), so 15 mutants from tag 1 (C7-
1, E7-1, F7-1, H7-1, D8-1, E8-1, A10-1, B10-1, C10-1, D10-1, All-1, Bll-1, Cll-1,
Dll-1 and HI2-1) were not included in the secondary testing. The other 115 mutants were arranged in 15 input pools consisting of 7 to 8 mutants each, and were screened by challenging two rainbow trout by an immersion route, as described for primary screening. Rescreening of 115 mutants identified 25 mutants (2.4 %) that were unable to survive in both the fish (Table 8), which were chosen for further characterization.
3.4. One-to-one competitive challenge of mutants with WT strain
To further confirm that 25 mutants selected after two screenings were truly unable to survive in fish, we performed one-to-one competitive challenges of three selected mutants, Fl-2, A5-4 and Bl-5, with the WT strain by bath-infecting rainbow trout. The
Bl-5 mutant was completely missing from three of the four fish that were co-infected by the WT strain (Table 9). Mutant Bl-5 was recovered from fish 2, but in approximately
103-fold lower numbers than the WT strain. Mutant A5-4 was unable to survive in four of the five fish, whereas the WT strain was recovered in 103 - 104 CFU/g kidney from these fish (Table 9). Mutant Fl-2 was completely missing from two fish, whereas it was recovered in very low numbers in comparison to WT strain in the other three fish. Thus, all three mutants examined were found to be survival-defective, validating our selection process for ST mutants in rainbow trout. All three mutants grew as well as the WT in LB medium at 18 °C, as shown by similar OD600 of cultures after 18 hours, eliminating in vitro growth defects as a source of the difference.
81 Table 8. Secondary screening of signature-tagged mutants in rainbow trout.
Mutant pool, CFU/g kidney Mutants missing Mutants selected fish number from the kidney Pool 1 Fish 1 1.4 x10s A2-l,F2-4,B3-7,D2-8, A2-l,F2-4,B3-7, C2-9.A2-11 D2-8.A2-11 Pool 1 Fish 2 4.6 x 104 A2-l,F2-4,E5-6,B3-7, D2-8.A2-11 l\.ol 2 l-i.sh 1 2.2 \ l(f ~ none Pool 1 l-Ui 2 2.7 \ M>~ c:-2. ii 1-5 ~ _ "" ; Pool }| Mi T ' 2.S\ lo" HOIK.' " pi..»m:isii: 2." \ lo" 1)4-4. IT 7. 1-2-1 1 " Pool 4 Fish 1 1.3 \ 111' 1.2-1. 153-2. 1-4-4 1-2-1. M3-2.14-4 P.., 82 1TNTC, Too numerous to count, more than 300 colonies in 100 ul of 1:5,000 dilution of the kidney tissue homogenate. These values were not used in calculations. Of 130 mutants that were selected based on primary screening, 115 mutants were rescreened in rainbow trout. These mutants were arranged in groups of 7 to 8 mutants, and screened in rainbow trout like the primary mutant screening. 83 Table 9. Competitive challenge of Fl-2, A5-4, and Bl-5 mutants with WT strain in rainbow trout. Fish No. CFU/g kidney tissue3 WT Fl-2 WT A5-4 WT Bl-5 1 106 101 103 0 NCb NC 2 10a 0 103 0 10s 10' 3 10s 101 104 0 10' 0 4 105 0 10" 0 105 0 5 10b 101 104 0 106 0 a The CFUs were measured to the nearest power 10 values bNC , not calculated, no colonies were obtained on plating 100 ul of 1:5 dilution (lowest dilution used) of the kidney tissue. The table presents data from three separate experiments. In each experiment, five rainbow trout (average weight 246 g) were infected by immersion for 10 to 15 minutes in equal amounts of the WT strain (RSI 154) and one of the three ST mutants, Fl-2, A5-4, and Bl-5. The infection doses (CFU/ml) for three experiments included: (WT, 3.2 x 109, Fl-2 mutant 3.3 x 109), (WT, 8.5 x 109, A5-4 mutant 5.6 x 109), (WT, 1.0 x 1010, Bl-5 mutant 9.0 x 109). The infection loads in the kidney were calculated by dilution plating at 7 days post-infection. 84 3.5. Effect of miniTn5Km2 cassette on survival of mutants The mutant Al-1 that had survived in rainbow trout in a primary screening was used to test if the presence of miniTn5Km2 cassette itself had any effect on mutant survival. After a competitive challenge with WT strain, the Al-1 mutant was able to infect at least as well as the WT strain in three fish that were infected to good levels (10 - 105 CFU/g kidney) by both strains (Table 10). The mutant had an even higher infection load than WT strain in fish 5 (Table 10). 3.6. Number of transposon insertions in ST mutants All 25 mutants had single miniTn5km2 insertion in their genomic DNA, as indicated by a single band on Southern blot when probed with kanamycin gene (Figure 6). Initially, two hybridization bands were obtained in the genomic DNA of B3-7 mutant (Figure 6, lane A). The top band of approximately 20 kb was due to incomplete DNA digestion, as it disappeared in repeat Southern hybridization done with DNA digested with increased concentration of EcoRW restriction enzyme (data not shown). DISCUSSION On two rounds of screening of 1056 ST mutants, 25 mutants or 2.4 % were missing from the kidney of rainbow trout at 7 days post-infection. This was within the range of 0.5 % - 13 % that has been reported for STM-based screens in other animal hosts (Autret and Charbit 2005, Shivani et al, 2005). The number of genes identified varies widely. This may be partly be due to the criteria used for the selection of mutants, such as 85 Table 10. Effect of the miniTn5Km2 cassette on survival of signature-tagged mutants in rainbow trout. Bacterial load in kidney, 7days post-infection Strain CFU/g kidney b Fishl Fish 2 Fish 3 Fish 4 Fish 5 WT 104 10s TFTC TFTC 10" AM 104 10s TFTC TFTC 104 a Mutant A1-1 that was able to survive in fish during primary screening was used to evaluate effect of miniTn5Km2 cassette on survival of mutants. Five rainbow trout (average weight 253.0 g) were infected by 15 minutes of bath immersion with equal doses (2.0 x 1010 cfu/ml of each) of WT strain, RSI 154 and AM mutant. On day 7 post infection, fish were euthanized and dilutions of kidney tissue were plated on BHI and BHI (kan) agar plates. b The CFUs were measured to the nearest power 10 values; TFTC, Too few to count, less than 20 colonies in 100 (xl of 1:5 dilution of the kidney tissue. 86 1 ABCDEFGHI JKLMNOPQRS TUVWXY 23 9.5 ->#- **-V • - *j|«. 6.6 •••*» •,» •* • n' '• * \*« ••ft***. t 4.4 Vtotvt ... •.•••a.-fjjihi • 2.4 2.0 * Figure 6. Number of transposon insertions in the genomic DNA of signature-tagged mutants that were missing in rainbow trout. The genomic DNA of mutants was digested with EcoRW enzyme and electrophoresed on a 0.8 % agarose gel. After transfer onto a nylon membrane, the DNA was hybridized with DIG-labeled 500 bp kanamycin gene probe. The number of hybridizing bands represented the number of transposon insertions. Lanes: 1: DIG- labeled DNA marker, size in kb (Roche), A: B3- 7, B: C6-1, C: A6-11, D: F4-1, E: A10-7, F: F4-4, G: H6-4, H: A6-1,1: B3-2, J: E2-1, K: A2-11, L: F5-8, M: H5-10, N: G6-1, O: A7-2, P: D5-12, Q: RS1154strain (a negative control), R: A5-4, S: Fl-2, T: Fll-10, U: D2-8, V: A2-1, W: C5-1, X: F5-11, Y: Bl-5 87 the single/double screens in host and the number of animals used to screen an input pool. More stringent selection criteria, such as double screening in the host used in this study, reduced the number of mutants finally selected. STM screening of fish pathogens S. iniae, M. marinum, E. ictaluri and L. garvieae identified 3.6 %, 4 %, 4.7 % and 2.4 % of mutants, respectively, to be survival-defective in zebrafish, goldfish, channel catfish and rainbow trout infections (Ruley et al, 2004, Miller and Neely 2005, Menendez et al, 2007, Thune et al, 2007). Our identification of 2.4 % survival-defective mutants and four STM screens of fish pathogens suggested that a similar fraction of genes may be involved in survival of a pathogen in fish and terrestrial animal hosts. The miniTn5km2 insertions in the genome of Y. ruckeri appeared to be random, as indicated by random hybridization patterns of 22 selected mutants (Figure 5). Sequencing of mutants (described in next section), however revealed that three mutant pairs, A2-1/F4-4, F11-10/F1-2 and H5-10/A5-4, had miniTn5km2 insertions in the same genes but at different locations. The miniTn5Km2-based mutagenesis in other bacteria such as Y. pseudotuberculosis, S. gallinarum, S. typhimurium, L. pneumophila, P. aeruginosa was found to be random (Hensel et al, 1995, Edelstein et al, 1999, Mecsas et al, 2001, Lehoux et al, 2002, Shah et al, 2005). The survival defects of 25 mutants identified in this study are most likely due to single miniTn5km2 transposon mutation, as each mutant had single transposon insertion in the genomic DNA demonstrated by Southern hybridization (Figure 6). However, even a single transposon mutation can affect the expression of flanking genes when present as part of an operon and can cause polar effects. Thus, polar effects of mutations need to be checked at the time of characterization of individual mutants. Mutant Al-2 appeared to 88 have two miniTn5km2 insertions in the genome, as indicated by two bands on Southern hybridization (Figure 5, Lane F). This mutant was not defective in survival in rainbow trout and thus was not included in 25 mutants. We eliminated the possibility that the miniTn5Km2 cassette, inserted into the genome of each mutant as part of a mutation, itself had any effect on the survival of mutants in rainbow trout. Mutant Al-1 which was able to survive in fish in the first screen, was recovered in the same numbers as the WT strain from kidney of five rainbow trout, indicating that miniTn5km2 cassette did not affect the survival of mutants in fish (Table 10). We could not determine whether any of the miniTn5km2 mutations in the mutants was present on the plasmids. The WT strain, RSI 154 used for making mutants contains three plasmids of 88 kb, 30 kb and 15 kb (De Grandis 1987). Plasmid DNA could not be isolated from mutants without contaminating genomic DNA, using different isolation protocols and plasmid isolation kits. A very low yield of plasmid DNA was obtained, possibly because of a low copy number of plasmids of Y. ruckeri. o The challenge protocol consisting of 10 minutes immersion of fish in 10 CFU/ml of each of the 11 mutants consistently produced infection in fish with >103 CFU/g kidney, allowing identification of mutants under conditions that can closely mimic natural infection process. When animal hosts are used for screening mutants, often there is a problem of animal-to-animal variation in missing mutants in the output pool. We observed that in 76 % of input pools the two fish infected with the same input pool differed in the number and specific mutants missing in the kidney (Table 6). This fish-to- fish variation in output pool was not solely the result of differences in the infection loads in the kidney, since fish having the same infection loads (16 % of 58 pools) lacked 89 different mutants and fish with different infection loads (17.5 % of 58 pools) lacked the same mutants (Table 6). In only three input pools did the pair of fish have equal infection loads in the kidney and also the same specific mutants missing. The infection data also demonstrated that the number of mutants missing in a fish was not related to the infection loads in the kidney tissue, since there were 16 input pools in which a fish with higher infection levels had more mutants missing than a fish with lower infection levels (Table 6). Variation in the output pool between fish was not associated with route of infection, as fish injected with the same dose into the peritoneal cavity showed differences in the output pool, as was observed in bath-infected fish (Table 7). However, only one input pool, F5, was compared by both infection routes. Fish-to-fish differences in output pool may result from the differences in the genetics, physiological and immunological status of fish. A social hierarchy exists in a fish population that can result in fish with varied physiological and immunological status and varying susceptibility to diseases (Wedemeyer 1996). A fish with a fully functional immune system may eliminate mutants more successfully than an immuno-compromised fish in which weakly attenuated mutants may survive successfully. To minimize the effects of fish-to-fish differences in output pool and identify consistently missing mutants, the 115 mutants from the first screen were rescreened in rainbow trout. Rescreening these mutants decreased the number of selected mutants to 25, despite the fact that all 115 mutants failed to survive in the first screening. A similar phenomenon was noted in several other STM studies that performed rescreening of mutants (Burall et ah, 2004, Flashner et al., 2004). Significant reduction in the numbers of mutants selected after second screen in rainbow trout could possibly result from two 90 causes. First, two fish infected with the same input pool displayed differences in the number and specific mutants missing in the output pool (Table 8). For example, in input pools 5 and 6, two fish differed by 4 and 5 mutants in the output pool, and similarly in most of other input pools the two fish differed in missing mutants. Since mutants missing 'commonly' in both fish, not either one, were selected, this significantly reduced the number of mutants selected after second screen. The second possible cause could be that the outcome of STM screens is influenced by interactions of mutants that are combined in an input pool. Some of the mutant phenotypes, such as those affected in toxins that act extracellularly, could be transcomplemented by other mutants in a group (Autret and Charbit 2005), resulting in rescue of a mutant that otherwise would be survival-defective in the host. Thus, different combinations of mutants in an input pool could influence the outcome of STM screens. From the 25 mutants selected on two screens in rainbow trout, three mutants, A5- 4, Fl-2, Bl-5, were further tested in competitive challenges with WT strain. All mutants were completely eliminated or were recovered in very low numbers from the kidney of rainbow trout, whereas WT strain was recovered in high numbers, confirming that mutants were survival-defective relative to the parental strain (Table 9). Three other mutants, A6-11, C6-1 and F2-4, failed to survive a further passage in rainbow trout that are discussed in next sections. Thus, after two-rounds of screening, we were able to identify 25 survival-defective mutants that were further characterized, as described in the sections that follow. 91 CHAPTER 4 CHARACTERIZATION OF MUTANTS UNABLE TO SURVIVE IN FISH RESULTS AND DISCUSSION 4.1. Screening for auxotrophy All 25 mutants grew well in M9 minimal medium supplemented with glucose as the carbon source. A representative data for three mutants is shown in Figure 7. The testing included 3 sequential passages on M9 agar medium to eliminate nutrient carryover. These results suggested that none of the mutants was an auxotroph. 4.2. Identification of mutated genes in ST mutants The ST interrupted genes of the 25 mutants were identified by cloning mutated genes in pBluescriptll KS+ as described and sequences of both DNA strands were determined. The accession numbers of all sequences determined in this study are provided in Table 11. The list of genes and the putative functions assigned by bioinformatics analysis are provided in Table 12. The genes of eight mutants, specifically A5-4, A10-7, D2-8, E2-1, F4-1, G6-1, H5-10 and H6-4 did not have homologs in databases, as no homolog was identified by Blast using algorithm settings described in Materials and Methods (Table 12). No putative functions could be assigned to these genes based on sequence homology; nonetheless they are required for survival of this pathogen in fish and represent an interesting set of genes for future characterization. The genes identified in two mutants, A6-1 and D5-12 had homology with hypothetical genes 92 A2-11 mutant A5-4 mutant Bl-5 mutant WT, RSI 154 Figure 7. Growth of signature-tagged mutants in M9 minimal medium. Initially, the ST mutants were grown in BHI (kanso) broth at 18 °C. After centrifugation of bacteria, the cell pellet was washed three times with 0.9 % saline and resuspended to an OD600 of 2.0 in 0.9 % saline. A 200 jixl of inoculum was spread on M9 agar plates and incubated at 18 °C for 7 days. The mutants were given three consecutive passages on M9 plates to eliminate any nutrient carryover. The representative data for only three ST mutants (A5-4, A2-11, Bl-5) and WT strain, RSI 154 is shown. 93 Table 11. Accession numbers of sequences of genes interrupted in signature-tagged mutants of Y. ruckeri identified in this study. ST mutant Accession number ST mutant Accession number A5-4 EU169583 Fl-2 EU 169597 A6-1 EU169584 F4-1 EU169598 A2-1 EU169585 F4-4 EU 169585 A2-11 EU169586 F5-8 EU 169599 A6-11 EU169587 F5-11 EU169600 A7-2 EU 169588 Fll-10 EU 169597 A10-7 EU 169589 G6-1 EU 169596 Bl-5 EU 169590 H6-4 EU169595 B3-2 EU169601 H5-10 EU169583 B3-7 EU 169591 C6-1 (znuA) EU 139251 C5-1 EU 169592 C6-1 (znuB) EU 139253 D2-8 EU 169593 C6-1 (znuQ EU 139252 D5-12 EU 169594 F2-4 (uvrY) EU170243 E2-1 EU169602 F2-4 ibarA) EU170244 Sequences of 25 genes mutated in ST mutants of Y. ruckeri that did not survive in rainbow trout were submitted to the database at the National Center for Biotechnology Information under the accession numbers provided in the table. For two mutants, C6-1 and F2-4 that were characterized further, sequence of additional genes, the gene name mentioned in brackets, was determined. 94 Table 12. Y. ruckeri genes identified by STM screens in rainbow trout. Mutant Homolog in sequence database3 % identity/ Bit score/ Reference (Accession number) similarity (span) E-value Cell Wall biogenesis Fl-2 O-antigen polymerase, 19.6/34.7 (443) Zhang et al, Y. enterocolitica (AAC60768) (1997) A2-11 PDAA, Peptidoglycan deacetylase, 18/32.1 (323) Fukushima et al, B. subtilis (NP_388679) (2005) Fl 1-10 Same as Fl-2 mutant Transport B3-2 TsgA permease, Major Facilitator Superfamily, 74.9/84.8 (393) Guzzo and Dubow E. coli (NP_417823) (2000) C6-1 ZnuA, Zn-binding periplasmic protein, 61.9/75(344) Patzer and Hantke £.co//(NP_416371) (1998) Protein turnouT, l)N\ replication, metabolism A6-11 Protease III, 62.6/78.4 (962) Dykstra and Kushner E. coli (NP_417298) (1985) 95 Table 12 continued Bl-5 Soj ATPase, DNA-partitioning system, 19.8/36.8 (133) Lee and Grossman B. subtilis (NP_391977) (2006) C5-1 Quinol-monooxygenease 22.8/30.1 (123) Adams and Jia E. coli (AAC76065) (2005) Cell signaling F2-4 UvrY response regulator, 84/94.5 (219) Pernestig et al, E. coli (NP_416424) (2001) Adhesion/secretion F5-11 RcpA pilus protein of bundle-forming pili, 31.7/53.9 (347) Clock et al, A. actinomycetemcomitans (AAD29695) (2008) Insertion scquence/transposon/bacteriophage-like proteinfins A2-1 ORF23, structural protein of 50/65 (344) Yang et al, bacteriophage-like bacteriocin (2006) P. luminescens (AAN64227) A7-2 TnsB transposase of Tn7, 26.2/42.4 (465) Floresefa/., (1990) E. coli (X17693) Arciszewska et al, (1991) 96 Table 12 continued B3-7 ORF22, tail-fiber assembly protein of 33.3/44.7 (150) Allison et al, bacteriophage SFV (2002) S.flexneri (AAL89429) F4-4 Same as A2-1 mutant F5-8 ORFA of transposase of IS3 91/95 (92) 175/ S. proteamaculans (YP_001476624) 8e-43 Hypothetical genes of unknown (unction A6-1 lMmcl_0151 hypothetical protein, 29/50 (230) 119/ Magnetococcus sp. (YP_864084) 2e-25 D5-12 NAS141_01456 hypothetical protein, 43/59 (323) 272/ Sulfitobacter sp. (ZP_00964694) 2e-71 No putative function assigned due to lack of homolog in database, but required for survival in the kidney A5-4 No homologue A10-7 No homologue D2-8 No homologue E2-1 No homologue F4-1 No homologue G6-1 No homologue H5-10 Same as A5-4 mutant H6-4 No homologue 97 a Approximately 500 bp to 1 kb sequences of genes interrupted by miniTn5Km2 were used for homology searches using default settings of BlastX and BlastP programs at www.ncbi.nlm.nih.gov. When none of the homologs identified by homology search was functionally characterized, the top most homolog identified by Blast search was listed in the table. Homology searches with a bit score of >100 were considered significant. When any of the homolog identified by Blast search was functionally characterized (might not be the top hit), its protein sequence was aligned with corresponding sequence of Y. ruckeri by pairwise alignment algorithm (ClustalW program) using default settings at http://www.ebi.ac.uk/emboss/align/. When results of the Blast search are reported, the corresponding bit score and e value are mentioned. bPercent amino acid identity/similarity over the length of the span (number of amino acids) obtained with ClustalW program is shown. 98 of unknown functions and no conserved domains or motifs could be identified in these sequences. Interestingly, three mutant pairs, A2-1/F4-4, F11-10/F1-2 and H5-10/A5-4, had mutations in the same genes but at different positions. Because of the potential role in virulence of other pathogens, two genes, znuA (C6-1 mutant) encoding a homolog of the zinc transporter and uvrY (F2-4 mutant) encoding a homolog of the response regulator of the BarA-UvrY two component system, were selected for further characterization of roles of their products in pathogenesis of Y. ruckeri in rainbow trout. The possible functions of proteins encoded by other genes identified in this study are described in the following section. 4.3. Gene encoding bundle-forming pili (Bfp) The F5-11 protein sequence showed 36.6 %, and 21 % amino acid identity respectively, with the proteins encoded by rcpA of Actinobacillus actinomycetemcomitans and cpaC of Caulobacter crescentus. These genes are part of a tight adherence (tad) locus that encodes outer membrane protein of the type 4 bundle- forming pili (Kachlany et al, 2000, Skerker and Shapiro 2000, Tomich et al, 2007). Thus, mutation in an rcpA homolog might have affected function of the Bfp in F5-11 mutant. The homologs of other 14 tad locus genes exist in Y. ruckeri (accession number AY576541, Fernandez et al, 2004). The tad locus was crucial for colonization and pathogenesis of A. actinomycetemcomitans (Schreiner et al, 2003) and Pasturella multocida (Fuller et al, 2000) in rat and mice models. The Bfp might act as a potential adhesin/invasin of Y. ruckeri. The attenuation of F5-11 mutant in rainbow trout might have been due to its reduced ability to attach to and invade fish tissues, limiting entry in 99 fish and decreasing colonization of internal organs. Fernandez et al. (2004) suggested involvement of the tad locus in the systemic stages of disease caused by Y. ruckeri, as the rcpAy.lacZ fusion was active in the liver, spleen and intestine of rainbow trout after intraperitoneal injection of bacteria. 4.4.0-antigen polymerase The deduced 443 amino acids sequence of F1-2/F11-10 mutants showed 19.6 % and 11.6 % identity to O-antigen polymerases (Wzy) of Y. enterocolitica (Zhang et al, 1997) and P. aeruginosa (De Kievit et al, 1995). The Wzy proteins are involved in the polymerization of individual O-antigen units into long chain O-antigens and the wzy mutants of Y. enterocolitica 08 (Bengoechea et al, 2004) and P. aeruginosa (De Kievit et al., 1995) had semi-rough LPS phenotypes. The Wzy homologs shared low sequence identity (Morona et al, 1994), but had common features that were identified in Fl-2 sequence. The Fl-2 sequence was highly hydrophobic with 10 predicted transmembrane segments, had 35.5 % G + C content (48 % for Y. ruckeri genome), five rare codons present in first 30 amino acids and leucine, isoleucine and phenylalanine together made up to 33 % of all amino acids. Another feature of wzy homologs is their genetic linkage with genes such as wzx (O-antigen flippase) and wzz (O-antigen chain length determinant) involved in O-antigen biosynthesis (Samuel and Reeves 2003). Downstream of the Fl-2 mutation, we identified a putative ORF that showed similarity to wzx (accession number AAV74529) of E. coli. Thus, in silico analysis suggested that the Fl-2 sequence was a wzy homolog. In a one-to-one competitive challenge, mutant Fl-2 was 100 unable to compete with WT and was completely eliminated from rainbow trout kidney by day 7 (Table 9). Similar to the Fl-2 gene homologs, genes required for the biosynthesis of LPS were identified in STM screens of Y. pseudotuberculosis, Y. enterocolitica and B. pseudomallei, suggesting their key role in pathogenesis in the host (Darwin and Miller 1999, Mecsas and Falkow 2001, Cuccui et al, 2007). An O-antigen-deficient mutant of E. ictaluri caused no mortality as compared to 61 % mortality caused by the WT in channel catfish (Lawrence et al, 2001) and mutant had increased susceptibility to complement from catfish serum (Lawrence et al, 2003). Similarly, the wzy mutants of Y. enterocolitica (Najdenski et al, 2003, Bengoechea et al, 2004) and Vibrio vulnificus (Nakhamchik et al, 2007) had increased susceptibility to complement components, phagocytes and antimicrobial peptides and were poor in colonization of internal organs. Thus, mutant Fl-2 might have increased its susceptibility to immune system components and poor colonization of internal organs of fish, but these hypotheses need experimental confirmation. 4.5. Polysaccharide deacetylase The A2-11 mutation was in an open-reading frame (ORF) encoding a protein of 323 amino acid. A polysaccharide deacetylase domain (PF01522) was identified in A2-11 sequence (http://pfam.sanger.ac.uk/). On tertiary-structure-based predictions, the A2-11 sequence was homologous to nodulation factor B (NodB), a rhizobial chitooligosacchride deacetylase (accession number dlnyla) and PDAA, a peptidoglycan deacetylase of Bacillus subtilis (accession number clwl7a) (QuickPhyre program, 101 http://www.sbg.bio.ic.ac.uk/~phyre/, Kelley et al, 2000). The sequence A2-11 had a 'TFDDG' motif (71 - 75 amino acid) characteristic of polysaccharide deacetylases. Sequence analysis suggested that A2-11 sequence may encode a putative polysaccharide deacetylase of Y. ruckeri. Homologs of A2-11 sequence belong to family 4 of carbohydrate-active enzymes (www.Cazy.org/fam/CE4.html) that remove N-linked or O- linked acetyl group from N-acetyl glucosamine (NAG) and N-acetyl muramic acid (NAM) containing polysaccharides substrates such as the peptidoglycan (PG), exopolysaccharides/capsules, chitin and xylan (Caufrier et al, 2003, Davies et al, 2005). The PDAA of B. subtilis (Fukushima et al, 2005) and PGDA of Listeria monocytogenes (Boneca et al, 2007) catalyze the N-deacetylation of PG, whereas the IcaB of Staphylococcus epidermidis (Vuong et al, 2004) and HmsF of Y. pestis (Forman et al, 2006) catalyze N-deacetylation of a cell surface exopolysaccharide, a homopolymer of f>- 1,6 NAG molecules. As the homologs of sequence A2-11 have varied substrate specificity, in silico analysis alone may not accurately identify the substrate for the putative A2-11 polysaccharide deacetylase of Y. ruckeri. Further sequence analysis of the A2-11 locus did not provide additional information, as no homolog was identified in the database for the ORF next to the A2-11 mutation. I suggest that PG and previously described surface exopolysaccharide may not be the substrate for the putative A2-11 polysaccharide deacetylase, as the N-deacetylation of these substrates has been suggested to occur in the periplasm or at cell surface by the periplasmic or membrane anchored enzymes (Vuong et al, 2004, Vollmer 2007). No signal sequences were identified in the A2-11 sequence that were characteristic of membrane-anchored lipoproteins, proteins secreted by general secretory pathway or twin-arginine pathway (Signal P, Signal L and 102 Signal T programs, http://www.cbs.dtu.dk/services/). The A2-11 protein was predicted to be a cytoplasmic protein (http://www.psort.org/psortb/index.html), suggesting that putative A2-11 polysaccharide deacetylase might be acting on some cytoplasmic substrate that need to be identified. 4.6. PtrA protease The mutated gene in the A6-11 mutant was predicted to be 2886 bp long coding for 962 amino acids. The A6-11 sequence showed identity with zinc-dependent metalloendopeptidases such as the PtrA of E. coli (62.6 %) and insulin-degrading enzyme (DDE) of humans (26.4 %) that belong to subfamily M16A of enzymes (Gottesman 1996, Barrett and Rawlings 2004). The first 25 amino acids of the A6-11 sequence (iMRKQLTRLAGVVLLLGLWMPGSWAA25) have positively charged residues (lysine and arginine) followed by a strong hydrophobic core that are features of a signal sequence, suggesting that PtrA homolog of Y. ruckeri may be a secreted periplasmic protein. The PtrA of E. coli is a periplasmic protein (Dykstra and Kushner 1985, Baneyx and Georgiou 1991). The A6-11 sequence possesses a catalytically and structurally important motif (HXXEHM69EX6E) that is characteristic of the M16A subfamily (Barrett and Rawlings 2004). Similar to E. coli and other Yersiniae, ptrA homolog of Y. ruckeri was flanked by recB and recC genes that encode beta- and gamma-subunits of exonuclease V (RecBCD) involved in homologous recombination and DNA damage repair. The last codon of ptrA homolog of Y. ruckeri was predicted to overlap with first codon of recB gene, raising the possibility of a polar effect due to the ptrA mutation. In E. coli, the ptrA mutation did not have a polar effect on adjoining recB and recC genes, as 103 determined by unaltered sensitivity of mutant to the UV light (Baneyx and Georgiou 1991). The non-polarity of A6-11 mutation needs to be experimentally confirmed. In a competitive infection in which bacterial loads of WT and mutant A6-11 were compared in the kidney of each individual fish, the mutant was able to infect as well or even slightly better than the WT strain in most of the fish on day 1 (Table 13). This suggested that mutant A6-11 was not affected in gaining entry into fish. During subsequent infection on days 3 and 5, bacterial load of mutant in the kidney dropped by nearly 10- to 100-fold relative to the WT strain in different fish. By days 7 and 9, the mutant was recovered in 104- to 108-fold lower numbers than WT in most of the fish, indicating that mutant was survival-defective (Table 13). Although required for survival in fish, the A6-11 gene was not essential in vitro, as the CFU/ml of mutant A6-11 were similar to WT strain when grown LB medium at 18 °C for 18 h. Similarly, the PtrA protease in E. coli was not essential for survival in vitro, as no phenotypic differences were noticed in cells lacking or over-expressing this protease, except for a slightly slower growth (Dykstra and Kushner 1985, Baneyx and Georgiou 1991). The exact functions of A6-11 homologs are unknown, though data on the PtrA of E. coli (Baneyx and Georgiou 1991), IDE of mammals (Farris et al, 2003), Ax lip and Ste23p of yeast (Adams et al, 1995) suggest that these enzymes may be involved in protein turnover (clearance of polypeptides) and protein processing. I speculate that the PtrA homolog of Y. ruckeri might be involved in clearance of damaged polypeptides in the periplasm. The PtrA homolog was essential for survival in fish, but not in vitro, could possibly be because of excessive protein damage that might occur in 104 Table 13. Competitive challenge of mutant A6-11 (protease III) and WT strain in rainbow trout. Fish# CFU/g kidney (WT) CFU/g kidney (MT) Ratio (WT/MT) 4 Dayl J. 1.8xl0 1 2 6.2xl02 1.2xl03 0.5 3 7.5xl03 1.2xl04 0.6 4 4.7xl03 6.7xl02 7 5 1.3xl05 1.2xl05 1 Day 3 1 1.2xl06 2.2xl04 58 2 7.4X101 7.4X101 1 3 l.lxlO7 4.6xl05 23 4 1.4xl05 2.4xl02 5.9xl02 5 1.3xl07 1.9xl06 7 Day 5 1 1.5xl04 9.5xl02 15 2 5.7xl06 5.7xl05 10 3 3.2xl05 7.7xl04 4 4 1.7xl02 8.5X101 2 5 l.lxlO5 1.9xl02 5.8xl02 Day 7 1 4.2xl06 4.0xl(T lxlO4 2 4.0xl04 1.0x10° 4xl04 3 2.6xl05 4.2xl04 6 4 8.9xl06 7.2xl02 1.3xl04 5 5.0xl06 1.3xl02 3.7xl04 Day 9 1 8.7X106 4.6xl0z 1.9xl04 2 9.2xl05 5.7xl04 16 3 1.2xl07 8.4xl05 14 4 3.0xl08 1.0x10,'o 3.0xl08 Total 25 rainbow trout (average weight 94 g) were bath infected with 5.0 x 108 CFU/ml each of WT RSI 154 and mutant A6-11. The infection loads of mutant (MT) and WT were calculated in the kidney at different time post-infection. To compare both strains, ratio (WT/MT) of infection load in each individual fish was calculated. Since one fish died few hours after challenge, only four fish were left for sampling on day 9. 105 the hostile environment in fish. The excessive accumulation of damaged polypeptides in the periplasm could cause toxicity that can affect cell survival. 4.7. Genes involved in cellular transport Mutant B3-2 sequence showed 74.9 % identity to a 43 kDA, TsgA cytoplasmic membrane protein of E. coli K-12 that belongs to major facilitator superfamily of transporters (Pao et al, 1998). We predicted an ORF encoding a protein of nearly 43 kDa (393 amino acids) in B3-2 sequence. Twelve transmembrane segments were identified in the B3-2 sequence (http://www.sbg.bio.ic.ac.uk/~phyre/), suggesting it to be a membrane protein. Guzzo and Dubow (2000) reported that luminescence of a tsgAv.luxAB transcriptional fusion increased several-fold in the presence of increasing concentrations of sodium selenite or sodium tellurite. They reconfirmed upregulation of the tagA in response to selenite and tellurite by quantitative Northern-blotting. However, these authors did not determine if TsgA was involved in the transport of these substances. Neither the substrate specificity nor the tertiary structure of other primary sequence-based homologs of B3-2 sequence has been determined. 4.8. Insertion sequence (IS)/transposon proteins Mutation F5-8 was in a homolog of a transposase of the IS3 family, which catalyzes transposition of \§3 in its target DNA molecules (Mahillon and Chandler 1998, Chen and Hu 2006). In general, the IS3 contains two partially overlapping ORFs, ORFA and ORFAB (Polard et al, 1991, Chen and Hu 2006). The ORFA encodes the N-terminal domain of transposase, whereas ORFAB encodes the complete transposase in which the 106 N-terminal domain is fused to the C-terminal catalytic domain by translational frame shifting (Polard et ai, 1991, Chen and Hu 2006). Mutation F5-8 was in the homolog of ORFA of IS3 (Figure 8). The IS3 homolog identified in mutant F5-8 lacked full length ORFB, suggesting that this IS might be non-functional. The locus was sequenced further to determine the genetic location of IS3 homolog in mutant F5-8. The ISJ homolog in Y. ruckeri was located in a homolog of iron-binding periplasmic (IBP) protein of iron ABC transporters of Y. pestis and Y. pseudotuberculosis (accession numbers, NP_670175 and ZP_01497262). The IBP proteins of Y. pestis and Y. pseudotuberculosis are 389 amino acids long. In comparison, Y. ruckeri homolog was truncated at amino acid position 224 due to insertion of IS3 homolog and sequence between 224-389 amino acid of Y. pestis and Y. pseudotuberculosis was not present in Y. ruckeri. Thus, the IBP homologue of Y. ruckeri might be non-functional. On the other side of IS3 homolog about 250 bp downstream, we identified a homolog of UDP-hexose epimerases/4-6 dehydratase in Y. ruckeri (Figure 8). The UDP-hexose epimerases/dehydratases are members of short chain dehydrogenases/reductases superfamily (SDR) (Jornvall et al., 1995). As the IS3 identified in mutant F5-8 may be non-functional due to an incomplete ORFB, mutation F5-8 may not affect the function of IS3 itself. I hypothesize that the ORFA homolog of IS3 in mutant F5-8 has promoter/gene regulatory sequences that might control the expression of the UDP-hexose epimerases/dehydratase homolog in Y. ruckeri. Due to insertion of the miniTn5km2 cassette in ORFA, the expression of UDP- hexose epimerases/dehydratase homolog might have been affected in mutant F5-8. The likely substrate for UDP-hexose epimerases/dehydratase homolog of Y. ruckeri could not be predicted based on sequence homology, as sequence identity among members of SDR 107 UDP-hexose epimerase/dehydratase Insertion sequence 3 Iron-binding periplasmic protein E T Tn5 Figure 8. Genetic organization of F5-8 locus of Y. ruckeri. The Blast search was performed on F5-8 sequence and homologs of three genes encoding putative insertion sequence 3, UDP-hexose epimerase/dehydratase, and an iron-binding periplasmic protein were identified in Y. ruckeri. The miniTn5Km2 cassette was inserted in a homolog ORFA of insertion sequence 3. The direction of arrows indicates predicted direction of transcription. The figure is not to scale. 108 family did not imply conservation of catalytic chemistry or substrate specificity (King et al, 2007). Mutations in truncated ORFA that contained regulatory sequences for leukotoxin operon ItxCABD affected the expression and amount of leukotoxin produced by mutants of A. actinomycetemcomitans (Lyndsay et al, 2008). Using STM-based screens, Thune et al, (2007) reported that a mutation in insA homolog, a transposase of ISi, led to poor survival of E. ictaluri in channel catfish. The authors suggested that ins A mutation might have effected expression of flanking essential genes. Mutant A7-2 had a mutation in gene encoding a homolog of TnsB protein, a component of the transposase core complex, TnsABC, of the Tn7 transposon (Sarnovsky et al, 1996). The TnsB protein recognizes cis-acting ends of the Tn7 transposon, and along with TnsA, is involved in cutting and joining Tn7 transposon at its DNA integration sites (Arciszewska et al, 1989, Sarnovsky et al, 1996). The poor survival of mutant A7-2 in rainbow trout at 7 days of infection might be because of polar effects of the mutation on genes present on the putative Tn7 locus of Y. ruckeri. The complete genetic composition of this locus could not be determined due to limited sequence available. In other bacteria, the Tn7 transposons are part of pathogenicity islands that contain genes for secretion systems, bacteriocin transporter, DNA restriction and modification system, bacteriophages, metal uptake and detoxification (Parks and Peters 2007). 4.9. Bacteriophage-like genes Mutant A2-1 sequence showed 49.7 % and 42 % identity to orf23 and afpl2 of Photorhabdus luminescens and Serratia entomophila, respectively. Upstream of orf23 109 and afpl2 homologue in mutant A2-1, we identified sequences that were homologous to afpl3 and orf24 of S. entomophila and P. luminescens, respectively. The homologs of mutant A2-1 in S. entomophila and P. luminescens are part of loci that produce phage tail-like bacteriocins (PTB) (Hurst et al, 2004, Yang et al, 2006, Hurst et al, 2007). Mutation A2-1 was in a gene homolog encoding structural protein of PTB. It has been suggested that the PTBs, by injecting tail-like structures into host, may be involved in delivery of toxins that are encoded by genes associated with PTB locus (Strauch et al, 2001, Yang et al, 2006). The PTB loci of S. entomophila and P. luminescens possess toxin encoding genes such as homologs of insecticidal toxin sepC of Y. pestis, Yop T effector of Y. enterocolitica, and Halovobrin toxin of V. fischeri(Waterfiel d et al, 2004, Yang et al, 2006). Significantly, similar to mutation A2-1 of Y. ruckeri, mutations in structural genes of PTB in S. entomophila and P. luminescens resulted in attenuated virulence of mutants in insect models (Hurst et al, 2004, Yang et al, 2006, Hurst et al, 2007). Further sequencing of A2-1 locus may reveal if we have identified a PTB- encoding locus associated with toxin gene in Y. ruckeri. The gene of mutant B3-7 was homologous to genes encoding putative bacteriophage tail fiber assembly proteins such as yfdK of KplEl prophage of E. coli K12 and orfll of Shigella flexneri bacteriophage V (SfV). Further sequencing of mutant B3-7 identified homologs of subsequent genes (yfdL and orfll) of KplEl and SfV bacteriophages. The SfV bacteriophage contains genes responsible for glycosylation of O-antigens, affecting serotype conversion and virulence in S. flexneri (Huan et al, 1997, Allison et al, 2002). The KplEl bacteriophage also has serotype-conversion genes, but their role in serotype conversion has not been evaluated. In general, many bacteriophages 110 are part of the pathogenicity islands that contribute to pathogenesis by encoding virulence factors (Wagner and Waldor 2002, reviewed by Brussow 2007). We speculate that, through polar effects, mutation B3-7 may have affected the expression of virulence- related genes present on this locus of Y. ruckeri. As yet, there are no reports on roles of the bacteriophages in pathogenesis of Y. ruckeri; however Stevenson and Airdrie (1984) were successful in isolation of bacteriophages that differentiated strains based on their ability to grow at 37 °C, a virulence-associated phenotype in human pathogenic Yersinia. 4.10. ATPase for DNA segregation The mutated gene in mutant Bl-5 was predicted to encode 294 amino acid long protein. The ParA, SopA and Soj proteins were identified as the closet homolog of Bl-5 sequence, using primary sequence and predicted tertiary structure (QuickPhyre) based comparisons. These proteins function as ATPases in the DNA-partitioning systems (DPS) that are involved in segregation of plasmids and chromosomes to daughter cells at the time of cell division (Gerdes et al, 2000). Sequence Bl-5 had deviant Walker-A (KGGVGKTT, amino acid 11 to 18) and Walker-B motifs (DYILID, amino acid 144 to 149) that were characteristic of this class of ATPases (Koonin 1993, Gerdes et al., 2000). Downstream of the ParA homolog in mutant Bl-5, we identified an ORF of 133 amino acid that was homologous to ParB proteins, a second component of the DPS. Sequence of another ParA protein of Y. ruckeri was present in the database (accession number YP_001101748). However, Bl-5 sequence showed only 26 % identity with it, indicating that we have identified a second DPS of Y. ruckeri. The ParA and ParB homologs identified in mutant Bl-5 showed maximum sequence identity to the ParA and ParB 111 (ParA, 74.1 %, ParB, 45.6 %) of Acidovorax sp. JS42 (accession number YP_987160). The ParAB DPS of Acidovorax sp. JS42 was located on the chromosome. There was less than 25 % sequence identity between Bl-5 sequence and other homologs identified by Blast analysis. It is likely that ParAB homologs identified in mutant Bl-5 are located on the chromosome, but further sequencing of Bl-5 mutant is required to identify flanking genes that would confirm chromosomal or plasmid origin of these genes. The chromosomal location of ParAB homologs of mutant Bl-5 was also suggested by the fact that chromosomal DNA was used for cloning and sequencing of mutant, however the possibility that the chromosomal DNA preparations contained traces of plasmid DNA was not ruled out. The ParAB systems are widely conserved in bacteria, however in several members of Gammaproteobacteria, including E. coli, Salmonella and Yersinia, no chromosomal par homologs were identified (Linvy et ah, 2007), suggesting differences (assuming Bl-5 DPS on chromosome) between Y. ruckeri and human pathogenic Yersinia. Mutation Bl-5 of Y. ruckeri was not lethal, and the mutant grew as well as the WT strain in vitro in BHI medium, suggesting that ParA homolog of Y. ruckeri was not essential for growth under these conditions. An STM screen of Francisella tularensis identified the parB gene of ParAB DPS as essential for bacterial survival in mice (Su et al, 2007). Similar to mutant Bl-5, F. tularensis mutant was not growth defective in vitro. I speculate that mutation Bl-5 might cause production of survival-defective anucleate cells due to failure of daughter cells to receive a copy of chromosome, as has been reported in V. cholerae (Saint-Die et al, 2006) and P. aeruginosa (Lasocki et al, 2007). 112 CHAPTER 5 CHARACTERIZATION OF THE BarA-UvrY TWO-COMPONENT SYSTEM In pathogenic bacteria, TCSs act to control expression of virulence and survival- essential genes in response to the host environment (reviewed by Beier and Gross 2006). The two-components of these systems are a histidine kinase (HK) and a response regulator (RR). Many RR are transcription factors that contain two domains, the receiver domain and a DNA-binding regulatory domain. In response to an environmental signal, the HK autophosphorylates at a conserved histidine residue and phosphorylates the receiver domain of RR at a conserved aspartate residue. The phosphorylation of RR leads to conformational changes in the DNA-binding domain that affects its binding to regulatory sequences, resulting in altered gene expression (Hoch and Varughese, 2001, West and Stock 2001). The Bar A is a hybrid HK having four domain architecture (Nagasawa et al, 1992, Pernestig et al, 2001). BarA was identified as the HK for the RR UvrY of E. coli, as in vitro it specifically phosphorylated UvrY protein but not other RRs, ArcA, PhoB and CpxR (Pernestig et al, 2001). Mutations in homologs of bar A or uvrY impaired bacterial virulence in every bacterial-host system examined, including S. typhimurium (BarA-SirA in cattle; Ahmer et al., 1999), Erwinia carotovora (ExpS-ExpA in potato tubers; Eriksson et al, 1998), V. cholera (VacS-VacA in mice; Wong et al, 1998), E. coli (BarA-UvrY in chicken; Herren et al, 2006) and P. aeruginosa (GacS-GacA in Caenorhabditis elegans; Tan et al, 1999). The reduced virulence of bar Al uvrY mutants in other pathogens was associated with multiple phenotypes such as sensitivity to H202-mediated killing/oxidative stress 113 (Herren et al, 2006), reduced siderophore production/ iron acquisition (Pernestig et al, 2000), decreased flagellum-mediated motility (Goodier and Ahmer 2001, Teplitski et al, 2003) poor invasion of host cells, and regulation of SPI1 encoded TTSS (Johnston et al, 1996). I identified that a mutation in a homolog of uvrY decreased survival of Y. ruckeri in fish and the uvrY mutant was unable to compete with the WT strain in rainbow trout (Table 14). Hypersensitivity to oxidative stress (Table 15) and poor invasion offish cells were identified as the possible reasons for poor survival of the uvrY mutant of Y. ruckeri. A homolog of the cognate hybrid histidine kinase, BarA was identified in Y. ruckeri. RESULTS 5.1. In silico analysis of UvrY response regulator Mutant F2-4 did not survive in kidney at 7 days post-infection in two independent screens each using two rainbow trout. In this mutant, the miniTn5km2 had inserted in a gene homologous to uvrY, which encodes a response regulator of a TCS. From the 4.0 kb sequence determined for the uvrY mutant, the gene organization of the uvrY locus in Y. ruckeri RSI 154 was predicted as shown in Figure 9. No sequence homolog was identified in the database for the 1.4 kb sequence on one side of the putative uvrY gene, but a gene encoding a putative oxidoreductase was identified on the other side. At the 3' end of uvrY, we identified a homolog of a uvrC gene. In other bacteria, uvrC encodes a subunit of the UvrABC enzyme complex required for repair of the UV-induced DNA damage (Sancar, 1996). 114 Table 14. Competitive challenge of mutant uvrY and WT strain in rainbow trout. Fish # CFU/g kidney (WT) g kidney (MT) Ratio (WT/MT) Dayl 1 1.9xl04 3.6xl03 5 2 8.0xl05 8.9xl04 9 3 7.6xl02 1.9xl02 4 4 3.5xl04 6.5xl03 5 5 4.4xl04 2.8xl03 16 Day 3 1 2.6xl09 2.6xl09 1 2 1.2xl05 4.7xl03 25 3 2.1xl06 4.9xl04 42 4 7.4xl03 6.3X101 1.1 xlO2 5 8.1xl05 1.2xl04 69 Day 5 1 1.4xl03 9.6X101 15 2 6.8xl04 1.0x10° 6.8xl04 3 3.5xl04 l.lxlO3 31 4 3.6xl02 1.0x10,'0 3.6xl02 5 3.0xl06 2.1xl03 1.4xl03 Day 7 1 7.2xl08 5.0x10° 1.4xlOz 2 3.3xl07 7.4xl04 4.5xl02 3 4.2xl02 1.4xl02 3 4 1.5xl06 4.3xl02 3.4xl03 5 6.6xl06 5.7xl03 1.2xl03 Day 9 1 3.2xl05 1.0x10,'o ?>2x\tf 2 1.2xl07 1.3xl03 9.5xl03 3 3.9xl07 4.8xl03 8.2xl03 4 7.3xl06 1.9xl04 3.2xl02 5 7.2xl06 2.4xl03 3.0xl03 Total 25 rainbow trout (average weight 102 g) were bath infected with 1.8 x 108 CFU/ml each of WT RSI 154 and uvrY mutant. The infection loads of mutant (MT) and WT were calculated in the kidney at different time post-infection. To compare both strains, ratio (WT/MT) of infection load in each individual fish was calculated. 115 Table 15. The uvrY mutant was hypersensitive to 1 mM and 10 mM H2O2. Percent survival of bacteria measured as CFU/ml 1 mM H202 10 mM H202 Minutes Wild type uvrY mutant Wild type uvrY mutant 30 69 ±7 49 ±2* 37 ±4 26 ±2* 60 62 ±11 51 ±5* 39 ±2 12 ±3* 90 44 ±4 40 ±4 41 ±5 15 ±2* 150 37 ±3 41 ±5 41±1 11±1* The WT RSI 154 and uvrY mutant cultures were grown to an OD600 of 0.6 in 25 ml LB medium at 18 °C with shaking. Five ml of the culture was put into four test tubes and cultures in three test tubes (one kept as a control) were exposed to 1 mM or 10 mm H2O2, and further incubated at 18 °C with shaking. Aliquots of each suspension were spread onto LB agar plates after 30 - 150 minutes and colonies counted after 24 hours to calculate CFU in H2O2 exposed and control cultures. Control cultures had approximately 108 - 109 CFU/ml, which was used to calculate percent survival of H2O2 exposed cultures using formula: (CFU in H2O2 exposed cultures/CFU in control unexposed cultures) x 100. Values represent mean ± SD of three independent experiments in which cultures in three tubes were sampled in each experiment. Statistically significant values (p < 0.05) are marked with (*). 116 Figure 9. Genetic organization of the uvrY locus of Y. ruckeri RSI 154. The genetic organization of a 3987 bp sequence of uvrY mutant was determined by finding putative ORFs. Different ORFs along with their names, nucleotide positions and direction of transcription (shown by arrows) are shown. The uvrY and uvrC were predicted to encode a response regulator and subunit of a UvrABC enzyme, respectively. The uvrY ORF was predicted to start at 1576 bp and end at 919 bp. The upward facing arrow on uvrY indicates point of Tn5 insertion between 1382 bp - 1383 bp. The circle over the 3' end of uvrY indicates overlap of uvrY and uvrC ORFs in the 3' end of uvrY. The uvrC ORF was predicted to start in the 3' end of uvrY at 948 bp by a frame shift shown by a box on the sequence. The sequence between 901 bp - 1655 bp is shown with a break indicated by..., with the positions of putative ribosomal-binding site (RBS), promoter sequences and start codons for uvrY and uvrC genes. The RBS and promoter sequences were identified using BPROM program at http://www.softberry.com/berry.phtml. 117 uvrY No homology Oxidoreductase 3724 3987 901GAG AAA GCG TAG TTT CGT AAG TCA GTG ATA ATC TGT TGC AGA GGC GTA )48 E KADFCETVI LCRRR M START (uvrQ 919 AGT CAG TGA TAA TCT GTT GCA GAG GCG TAA TTT GTT GGG (uvrY) STOP DSNSLTE A N F L G CAC CGC ATA ACG GTT CAC CCA ATT GAG ATG TAG TGG TGA TTA TAA TTC GAA TGA TTT HRI ALHTLEVDGSI NLK SF -10 sequence (uvrQ GTAAGCTAT CGCW •1568CGA TTA GTT, ^TCTTTTAi s^AGAGG 11Tl'l'l'lATTCCCGCA M R Y R S I L -35 sequence (uvrC) START (uvrY) RBS (uvrY) TTGGATTAifil7ATGAGAAACAAATGCCGACACTATifi^AATGAACAAAAGTTGGTTTifi^ -10 sequence (uvrY) -35 sequence (uvrC) 118 By in silico analysis (BPROM program at http://softberry.com), we identified putative promoter sequences and ribosomal-binding sites upstream of uvrY of Y. ruckeri (Figure 9). The 3' end of uvrY was predicted to overlap with the 5' end of uvrC and there was a possibility that uvrY and uvrC homologs of Y. ruckeri might be transcribed together by a putative promoter identified upstream of uvrY. Additional putative promoter sequences for uvrC homolog of Y. ruckeri were identified within the 3' region of the uvrY coding sequences (Figure 9). The predicted 218 amino acid long uvrY ORF might start with a TTG codon as predicted based on sequence alignment with the functionally characterized UvrY protein of E. coli and other uvrY homologs (Pernestig et al, 2001). The deduced UvrY protein of Y. ruckeri shared sequence identity to known UvrY proteins in E. coli (84 %), S. enterica (85.4 %), E. carotovora (85.8 %), V. cholerae (73.9 %), and P. aeruginosa (56.6 %) (Figure 10). Thus, the putative BarA of Y. ruckeri showed high sequence identity with homologs present in a diverse group of bacteria. The receiver domain and DNA-binding domain with a conserved aspartate residue that were characteristics of a response regulator were identified in the putative UvrY protein of Y. ruckeri (Figure 10). 5.2. Non-polarity of the uvrY mutation Because of the predicted genetic linkage of uvrY and uvrC genes, we examined the uvrY mutant of Y. ruckeri for polar effects, by testing its sensitivity to UV light at 254 nm. The low intensity, 1.5 J/m2, UV light was not effective in killing mutant or WT, as both strains had a high survival rate of 89 % and 92 %, respectively (Table 16). Exposure to high intensity UV light, 5.0 J/m2, reduced the survival of mutant and WT strain to 9 % 119 Figure 10. Sequence alignments of UvrY homologs. The sequence alignments were performed using ClustalW program at http://align.genome.jp. The receiver domain and DNA-binding domain are shown by solid and dotted lines on top of the sequences, respectively. The domains were predicted using http://smart.embl-heidelberg.de/. The aspartate residue that is phosphorylated by the histidine kinase is indicated by O). The (*), (:) and (.) symbols indicate identical residues, conserved substitutions and semi-conserved substitutions, respectively. The accession numbers of sequences used include: UvrYYR, Yersinia ruckeri (EU139251); UvrYEC, Escherichia coli K12 (NP_416424); SirAST, Salmonella typhimurium (AAC08300); UvrYYPE, Yersinia pestis (NP_405429); UvrYYE, Yersinia enterocolitica (YP_001006098); UvrYYPS, Yersinia pseudotuberculosis (YP_070261); ExpAECR, Erwinia carotovora (YP_050973); GacAPA, Pseudomonas aeruginosa (NP_251276); VarAVC, Vibrio cholerae (AAC82663); UvrYAS, Aeromonas salmonicida subspecies salmonicida (YP_001141391). 120 UvrYEC (84, 0%) MINVLLVDDHELVRAGIRRILEDIKGIKWGEASCGEDAVKWCRTNAVDWLMDMSMPGI 60 UvrYYE (92 .2%)MISVLLVDDHELVRAGIRRILEDIKGIKVADEMQCGEDAVKWCRNHIVDIVLMDMNMPG I 60 UvrYYPS(92, 7%)MISVLLVDDHELVRAGIRRILDDIKGIKVAGEMQCGEDAVKWCRSHWDIVLMDMNMPGI 60 UvrYYPE(92. 7%)MISVLLVDDHELVRAGIRRILDDIKGIKVAGEMQCGEDAVKWCRSHWDIVLMDMNMPGI 60 SirAST (85. 4%) MINVLLVDDHELVRAGIRRILEDIKGIKWGEACCGEDAVKWCRTNAVDWLMDMNMPGI 60 ExpAECR(85, 8%) MISVFLVDDHELVRAGIRRILDDIKGLKWGEACCGEDAVKWCRSNDVDWLMDMSMPGI 60 GacAPA (56. 6%) MIKVLWDDHDLVRTGITRMLADIEGLQWGQADCGEDCLKLARELKPDWLMDVKMPGI 60 VarAVC (73. 9%)MISVFLVDDHELVRTGIRRIIEDVRGMKVAGEADSGEEAVKWCRTNHADVILMDMNMPGI 60 UvrYAS (72. 5%)MINVFLVDDHELVRTGIRRILEDVRGIKWGEAQSGETAVTFCRQHHPDVILMDMNMPGI 60 . * p *..**** . ***.** *;: *:.*::* .: .** .:. .* *..***.# **** Consensus MISVLLVDDHELVRAGIRRILEDIKGIKV-GEA-CGEDAVKWCR---VD-VLMDMNMPGI UvrYYR LISVLLVDDHELVRAGIRRILEDIRGIKVLGEAQCGEDAVKWCRSNSVDIVLMDMNMPGI 60 UvrYEC GGLEATRKIARSTADVKIIMLTVHTENPLPAKVMQAGAAGYLSKGAAPQEVVSAIRSVYS 120 UvrYYE GGLEATRKILRFSPDTKVIMLTIHTENPLPAKVMQAGASGYLSKGAAPQDVVTAIRAVHS 120 UvrYYPS GGLEATRKILRFSPDTKVIMLTIHTENPLPAKVMQAGAGGYLSKGAAPQDVITAIRMVHA 120 UvrYYPE GGLEATRKILRFSPDTKVIMLTIHTENPLPAKVMQAGAGGYLSKGAAPQDVITAIRMVHA 120 SirAST GGLEATRKIARSTADIKVIMLTVHTENPLPAKVMQAGAAGYLSKGAAPQEWSAIRSVYS 120 ExpAECR GGLEATRKILRFTPDIKVIMLTIYTENPLPAKVMQAGAAGYVSKAAAPQEVISAIRAVHA 120 GacAPA GGLEATRKLLRSQPDIKWWTVCEEDPFPTRLMQAGAAGYMTKGAGLEEMVQAIRQVFA 120 VarAVC GGLEATKKLLRVNPDIKIIVLTVHTENPFPTKVMQAGAAGYLTKGAAPDEMVNAIRIVHS 120 UvrYAS GGLEATRKILRIRPDVRIIVLTIHSENPFPTKVMQAGAAGYLTKGAAPDEMIHAIRLVHS 120 ******.*. .***** **. Consensus GGLEATRKILR—PD-KVIMLTIHTENPLPAKVMQAGA-GYLSKGAAPQ-V—AIR-VH- UvrYYR GGLEATRKILRFSPDIKVIMLTIHTENPLPAKVMQAGAGGYLSKGAAPQDVINAIRAVHS 120 UvrYEC GQRYIASDIAQQMALSQIEPEKTESPFASLSERELQIMLMITKGQKVNEISEQLNLSPKT 180 UvrYYE GQRYIASDIAQQMALGQLEP-PTETPFSCLSERELQIMLMITKGQKVNEISEQLHLSPKT 17 9 UvrYYPS GQRYIASDIAQQMALSQLEP-PAETPFSCLSERELQIMIMITKGQKVNEISEQLHLSPKT 17 9 UvrYYPE GQRYIASDIAQQMALSQLEP-PAETPFSCLSERELQIMIMITKGQKVNEISEQLHLSPKT 179 SirAST GQRYIASDIAQQMALSQIEPAKTETPFASLSERELQIMLMITKGQKVNEISEQLNLSPKT 180 ExpAECR GKRYIASDIAQQMALSQLEP-QTDAPLECLSERELQIMLMITKGQKVTEISEQLNLSPKT 179 GacAPA GQRYISPQIAQQLALKSFQPQQHDSPFDSLSEREIQIALMIANCHKVQSISDKLCLSPKT 180 VarAVC GQRYISPEIAQQMALSQFSP-ASENPFADLSERELQIMLMITKGQKVTDISEQLSLSPNT 17 9 UvrYAS GQRYISPEIAQQMALSQFAS-ADENPFKSLSERELQIMMMITKGQKVTDISEQLNLSPKT 179 Consensus GQRYIASDIAQQMALSQ-EP E-PF—LSERELQIMLMITKGQKVNEISEQL-LSPKT UvrYYR GQRYIASDIAQQMALSQLEP-QVDTPFACLSERELQIMLMITKGQKVNEISEQLHLSPKT 179 UvrYEC VNSYRYRMFSKLNIHGDVELTHLAIRHGLCNAETLSSQ- 218 UvrYYE VNSYRYRMFSKLNISGDVELTHLAIRHGLFNAETLSNSD 218 UvrYYPS VNSYRYRMFSKLNISGDVELTHLAIRHGLFNAETLSNSD 218 UvrYYPE VNSYRYRMFSKLNISGDVELTHLAIRHGLFNAETLSNSD 218 SirAST VNSYRYRMFSKLNIHGDVELTHLAIRHGLCNAETLTSQ- 218 ExpAECR VNSYRYRMFSKLNINGDVELTHLAIKHGLFTAETLLSSE 218 GacAPA VNTYRYRIFEKLSITSDVELALLAVRHGMVDAAS 214 VarAVC VNSYRYRLFAKLNINGDVELTHLAIRHGILDTEKL 214 UvrYAS VNSYRYRLFSKLDINGDVELTHLAIRYGMLDADTL 214 Consensus VNSYRYRMFSKLNI-GDVELTHLAIRHGL-NAETL UvrYYR VNSYRYRMFSKLNISGDVELTHLAIRHGLFNAETLSNSD 218 121 Table 16. Ultravoilet light sensitivity of uvrY mutant and WT strain. Percent survival, measured as CFU Dose (J/m2) Wild type uvrY mutant P value 1.5 92 ±5 89 ±7 0.60 5.0 11 ±2 9±2 0.32 Total 300 - 500 cells of uvrY mutant or wild type strain RSI 154, taken from cultures grown in 5ml of LB at 18 °C to OD600 of 1.0, were plated on LB agar and incubated for 30 minutes at 18 °C. The bacteria on plates were exposed to UV light and the percent survival was calculated by comparing to unexposed cultures. The values represent mean ± SD of three experiments and in each experiment ten agar plates of each strain were exposed to UV light. 122 and 11 %, respectively. There was no statistically significant difference (p values, 0.60, 0.32) in susceptibility of the mutant and WT strain at either intensity of UV light, indicating that the uvrY mutation did not have a polar effect on uvrC. 5.3. Growth of uvrY mutant in Luria-Bertani medium In a time-course of growth in LB medium at 18 °C, the uvrY mutant and WT strains had similar growth patterns indicated by similar optical density and CFU/ml (Figure 11), showing that there was no observable growth defect in the uvrY mutant of Y. ruckeri in LB medium. 5.4. H2O2 sensitivity of uvrY mutant The WT and uvrY mutant were susceptible to both 1 mM and 10 mM H2O2, but the mutant was more readily killed than the WT strain. At 1 mM H2O2, the most significant difference between strains was observed within 30 minutes in which the mutant had 49 % survival in comparison to 69 % survival of WT (Table 15). Mutant and WT had nearly same susceptibility (37 % - 44 % survival) after 90 minutes in 1 mM H2O2 that did not change afterwards, suggesting development of an adaptive response. Both strains were more susceptible to a higher concentration (10 mM) of H2O2, but the mutant had significantly lower survival (11 % - 26 %) than WT (37 % to 41 %) at all times. The WT strain developed the adaptive response quickly within 30 minutes and its survival did not change much afterwards; however the mutant showed the adaptive response after 60 minutes as there was a 14 % decrease in survival of mutant between 30 minutes and 60 minutes in 10 mM H2O2 (Table 15). 123 Figure 11. Growth of uvrY mutant in LB medium. The WT RSI 154 and mutant strains were grown in LB medium at 18 °C for 18 hours. LB medium (50 ml) was inoculated 1:100 with these cultures and incubated at 18 °C with shaking. The OD600 (HA, arithmetic scale; 11 B semi-log scale) and CFU (11 C) were plotted at various time points. The values represented the mean ± SD of three independent culture flasks. The SD bars are small and not visible. WT RSI 154 (A), uvrY mutant (•). 124 Figure 11. A 10 a a i B B 1 H S s o o O 0.01 ~\ r "i r 5 10 15 20 25 30 35 40 Hours Figure 11. B 10.0 n 9.0 - __ &es "•—^ » 8.0 - Ofe Di) O 7.0 - 6.0 0 5 10 15 20 25 30 35 40 Hours Figure 11. C 125 5.5. Serum sensitivity of uvrY mutant Neither the WT strain nor the uvrY mutant was killed by non-immune rainbow trout serum during incubation for 1.5 hours or 3.0 hours at any tested concentration (Table 17). Both strains were able to grow in presence of the serum. The serum sensitive strain, RS12, had 32 % survival at 3.0 hours and served as a positive control for killing activity of the serum. 5.6. Growth of uvrY mutant under iron-limiting conditions Growth of both the uvrY mutant of Y. ruckeri and the WT strain were inhibited by the addition of an iron-chelating agent 2, 2' dipyridyl, to the LB medium at 200 uM (Figure 12). The mutant strain was not significantly different from the WT in the degree of inhibition. 5.7. Fish cell invasion by uvrY mutant The invasion indices, which indicate the number of intracellular bacteria per 100 EPC cells, of uvrY mutant (9.6 ± 0.7) and WT strain (14.1 ± 2.1) were significantly different (p value 0.027), indicating that the uvrY mutation decreased the ability of Y. ruckeri to invade the epithelial EPC cells. Invasion studies on CHSE cells, a cell line of salmonid origin, did not give consistent bacterial recovery after infection, as there was a high difference among different replicates even within a strain (data not shown). 126 Table 17. Effects of uvrY mutation on serum sensitivity of Y. ruckeri. Strain Survival (% ) in non-immune rainbow trout serum3 90 minutes incubation 3.0 hours incubation 25% serum 50% serum 100% serum 25% serum 50% serum 100% serum Y. ruckeri 216 ±9 250 ±7 269 ±7 338 ±7 372 ±11 435 ± 12 RS1154,WT uvrY mutant 211 ±10 259 ±28 296 ±18 287 ±8 356±33 333 ±9 a The LB grown overnight bacteria were washed three times with 0.9 % saline, and the pellet was resuspended in saline to an ODgoo of 0.6. A 250 ul volume of bacterial suspension and 250 ul of serum (25 %, 50 % and 100 %) were mixed and mixture was incubated at 18 °C with shaking. At 90 minutes and 180 minutes, CFUs of cultures were determined, and bacterial survival was calculated using the formula: (CFU in presence of serum/CFU in absence of serum) x 100. The control cultures in saline used for calculating bacterial survival had approximately 108 CFU/ml. The values represent the means ± SD of three independent experiments. 127 Figure 12. Growth of Y. ruckeri WT and uvrY mutant in LB medium with and without 200 uM 2,2' dipyridyl iron chelator. The overnight grown cultures of WT RSI 154 and uvrY mutant strains were used for 1:100 inoculation of three culture flasks each containing 25 ml of LB medium only or LB supplemented with 200 |^M of 2,2' dipyridyl. Cultures were incubated at 18 °C with shaking. The bars represent standard deviation around mean of three independent experiments. Y. ruckeri RSI 154 WT (A) and uvrY mutant (•) in LB or LB supplemented with 2,2' dipyridyl, Y. ruckeri RSI 154 WT (A) and uvrY mutant (•). 128 0 10 15 20 25 30 Hours Figure 12. A 10.0 „i.o = e © A 0 1 -I 0 1 o.o -i— o 10 15 20 25 30 Hours Figure 12. B 129 5.8. The uvrY was required for survival in rainbow trout After an immersion challenge of rainbow trout with approximately 10 CFU/ml each of the uvrY mutant and WT, bacterial loads of both strains in the kidney of each individual fish were compared and are presented in Table 14. Both strains were able to infect all fish on day 1, but the mutant had 5- to 15-fold lower bacterial loads in the kidney, suggesting that the mutant might be defective for invasion of the fish (Table 14). The differences in bacterial loads of WT and mutant increased during subsequent stages of infection on days 3 and 5, indicting that the mutant was unable to successfully compete with WT strain. By day 9, the uvrY mutant was recovered in nearly 10 - to 10 -fold lower numbers than the WT strain, though the mutant was not completely eliminated (Table 14). Thus, the infection data demonstrated that the uvrY mutant was unable to compete with WT strain, showing that the uvrY mutation affected survival of Y. ruckeri in rainbow trout. 5.9. Identification of bar A homolog of Y. ruckeri Using degenerate primers designed from conserved sequences of the barA homologs, we amplified the expected 2596 bp sequence from Y. ruckeri RSI 154 (Figure 13). The positions of the PCR primers are shown in Figure 14. The DNA sequences encoding the first 5 and last 48 amino acids (positions as per BarA of E. coli) of BarA of Y. ruckeri were not amplified, as there were no conserved regions in homologs for designing primers at the extreme ends. The putative BarA sequence of Y. ruckeri showed substantial identity with the sequences of known BarA proteins in E. coli (59.3 %), S. enterica (58.8 %), 130 1 2 &m rat •^fiV"'"* ^•M", _ •* *f J« 3.1 •:' • , ImmI Figure 13. PCR amplification of bar A homolog of Y. ruckeri. A 2596 bp DNA fragment was PCR-amplified from genomic DNA of Y. ruckeri RSI 154 using BarAlforword and BarA2reverse primers. Lanes 1: 1 kb DNA MWM (Invitrogen). 2: 2596 bp barA PCR product. 131 Figure 14. Sequence alignments of Bar A homologs. Sequence alignments were performed using the CLUSTALW program at http//align.genome.jp. The amino acid sequence used to design degenerate BarAfor and BarArev primers for amplification of bar A of Y. ruckeri RSI 154 are shown in boxes. Conserved histidine and aspartate residues involved in the phosphorelay are indicated by symbol (T).The sequence numbering was as per BarA protein of E. coli. The positions of different domains including transmembrane domain 1 (TM1) (amino acid 10 - 32), TM 2 (amino acid 177 - 199), HAMP (amino acid 200-252), HK domain (amino acid 299 - 520), RR domain (amino acid 669 - 785) and HPT domain (amino acid 822 - 918) were indicated on top of the sequences. The domains were predicted using http://expasy.org/prosite/. Symbols indicate identical residues (*), conserved substitutions (:) and semi-conserved substitutions (.). The accession numbers of sequences included are: BarAYR, Yersinia ruckeri (EU170244); BarAEC, Escherichia coli K12 (NP_417266); BarAAST, Salmonella typhimurium (NP_461879); BarAYPE, Yersinia pestis (NP_991702); BarAYE, Yersinia enterocolitica (YP_001005086); BarAYPS, Yersinia pseudotuberculosis (YP_069291); ExpSECR, Erwinia carotovora (YP_051659); GacSPA, Pseudomonas aeruginosa (NP_249619); VarSVC, Vibrio cholerae (ZP_01681975); BarAAS, Aeromonas salmonicida subspecies salmonicida (YP_001143196). 132 BarA/or primer TM1 (10-32) BarAEC (59.3%) -YITNYSLR&RNMILILAPTVLIGLLLSIFFWHRYNDLQRQLEDAGASIIEPLAVSTEYG 59 BarAYE (72.5%) - V1TKYSLRARMMILILAPTLLLGLLLSTTFMVNRYNELQKQLVNAGTNIIEPLAVASEYG 59 BarAYPS(70.4%) -VITKYSLRkRMMILILAPTLLLGLLLSTSFMVNRYNELQNQIVSAGTNIIEPLAVASEYG 59 BarAYPE(71.8%) - <1TKYSLRARMMILILAPTLLLGLLLSTSFMVNRYNELQNQIVSAGTNIIEPLAVASEYG 59 BarAST (58.8%) - YITNYSLR&RMMILILAPTVLIGLLLSIFFVVHRYNDLQRQLEDAGASIIEPLAVSSEYG 59 ExpSECR(59.3%) - vlTKYSLR A.RMLILILAPTLMIGLLLSSFFVIHRYNQLQAQLADSGTSIIEPLATISAYA 59 GacSPA (32%) M FKDLGIK 3RVLLLTLLPTSLLAMVLGGYFTWVQLSDMRAQLIERGQLIAEQLAPLAATA 6 0 VarSVC (49.3%) MrQRYGLRkRVMTLTLAPTLIIGLLLSALFSFNRYHDLETQVINSGASIIEPLAIASEDA 60 BarAAS (45.2%) - YITKYGLRkRVLAFTILPTLIIGGLMAGYFTFHRYQQLENNLIDQGINIIEPLAIASEYG 59 Consensus T7RARMMILILAPTLL-GLLLS-F-V-RYN-LQ-QL G--IIEPLAVASEYG BarAYR LRARMMILILAPTLLLGLLLSTSFMVHRYNELQNQWNAGTSIIEPLAVASEYG 54 BarAEC MSLQNRESIGQLISVLHRRHSDIVRAISVYDENNRLFVTSNFHLDPSSMQLGSNVPFPRQ 119 BarAYE MTFRNRDTVRQLINLLHRRHSNIVRSISVFDADNNLFVTSNYNYNSSQLRLPKDDAIPSS 119 BarAYPS MSFRNRDTVRQLINLLHRRHSNIVRSISVFDVDNKLFVTSNYNYNSSQLRLPPGEPVPDS 119 BarAYPE MSFRNRDTVRQLINLLHRRHSNIVRSISVFDVDNKLFVTSNYNYNSSQLRLPPGEPVPDS 119 BarAST MNLQNRESIGQLISVLHRRHSDIVRAISVYDDHNRLFVTSNFHLDPSQMQLPAGAPFPRR 119 ExpSECR ITHRQMDTIPALINTLHRRHSAIIRTISVFDARNQLLATTNVRNSVTLQPISSDALSYNR 119 GacSPA LARKDTAVLNRIANEALDQPD--VRAVTFLDARQERLAHAG PSMLTVAPAGDASH 113 VarSVC LRMQSRESVRRIISYAHRKNSKLVRSIAVFDANHELFVTSNFHPNFEKLMYPKDKPIPFL 12 0 BarAAS MTQHSRESLKRLIGLTHRKNSPLIKSIAVFTQDNQLFVTSNYHRDFTRLRLPDGSQIPEL 119 Consensus M NR QLIN-LHRRHS-IVRSISVFD--N-LFVTSN L-LP P-- BarAYR MTFRNRGTVRQLINLLHRRHSYIVRSIAVFDTDNNLFVTSNYNYNSSQLRLPNNIPIPNS 114 BarAEC LTVTRDGDIMILRTPIISESYSPDESPSSDAKNSQNMLGYIALELDLKSVRLQQYKEIFI 179 BarAYR LMLTYRGDSLILRMPIVSEYNLSSDKPPEDNI-NNRPLGYISIDLDLQSVRLQQYKEVFI 173 BarAYE LMLSYRGDSLILRMPIVSESNLSSDRSSDSDI-TNRPLGYIAMDLDLQSVRLQQYKEIFI 178 BarAYPS LMLSYRGDSLILRMPIVSESNLSSEQASGQGE-SDQILGYIAIDLDLQSVRLKQYKEIFI 178 BarAYPE LMLSYRGDSLILRMPIVSESNLSSEQASGQGE-SDQILGYIAIDLDLQSVRLKQYKEIFI 178 BarAST LSVDRHGDIMILRTPIISESYSPDESAIADAKNTKNMLGYVALELDLKSVRLQQYKEIFI 17 9 ExpSECR LHLHHTNDALILHMPIVNDSEFMPGGATPVLPGTATPLGYLVIELDTSTIRLQQYQEIFI 179 GacSPA LSMSTELDTTHFLLPVLGRHHSLSGATEPDDE RVLGWVELELSHHGTLLRGYRSLFT 170 VarSVC SSSAIDENTLILRTPIISERTVLDNGDANPAT PVMGYIAIELDLSSLRLQQYQEIFS 177 BarAAS TSVTLYGDDIILRTPIQAETS--MDGFPLPSDVEPPMIGYISMQMTTDRAMILQYRDTFF 177 Consensus L GD-LILR- PI -SES LGYIA—LDL-SVRLQQYKEIFI BarAYR LMLTYRGDSLILRMPIVSEYNLSSDKPPEDNI-NNRPLGYISIDLDLQSVRLQQYKEVFI 173 133 TM 2 (177-199) HAMP (200-252) BarAEC SSVMMLFCIGIALIFGWRLMRDVTGPIRNMVNTVDRIRRGQLDSRVEGFMLGELDMLKNG 239 BarAYR STLLLLFSLCIAILFAYRLMRDVTGPIRNMVNTVDRIRRGQLDSRVEGHMLGELNILKNG 233 BarAYE STLLLLFCMCVAILFAYRLMRDVTGPIRNMVNAVDRIRRGQLDSRVEGHMLGELNILKNG 238 BarAYPS STLLLLFCMCVAILFAYRLMRDVTGPIRNMVNTVDRIRRGQLDSRVEGHMLGELNILKNG 238 BarAYPE STLLLLFCMCVAILFAYRLMRDVTGPIRNMVNTVDRIRRGQLDSRVEGHMLGELDILKNG 238 BarAST SSVMMLFCIGIALIFGWRLMRDVTGPIRNMVNTVDRIRRGQLDSRVEGFMLGELDMLKNG 23 9 ExpSECR AALLLLLSLGAAVLFAYRLMRDVTTPIRNMVDTVDRIRRGQLDSRVEGYMLGELDMLKNG 23 9 GacSPA SLLLIAAGLGVTALLALRMSRAINAPLELISQGVAQLKEGRMETRLPPMGSNELDELASG 230 VarSVC AGLVLVIGILLSGVFASRLMYDVTRPITHMKNMVDRIRRGHLDVRIEGKMHGELDTLKKG 237 BarAAS AVIMVLIGIAFSTLFGFRLVKSVIQPITDMVQAVHKIREGRLDTRVSGQLTGELDMLKNG 23 7 : ::: : : ::.*: : *: : :*:::.*:::*: .**:*.* Consensus S-LLLLF A-LFA-RLMRDVTGPIRNMVNTVDRIRRGQLDSRVEG-MLGELD-LKNG BarAYR STLLLLFSLCIAILFAYRLMRDVTGPIRNMVNTVDRIRRGQLDSRVEGHMLGELNILKNG 233 HAMP. BarAEC INSMAMSLAAYHEEMQHNIDQATSDLRETLEQMEIQNVELDLAKKRAQEAARIKSEFLAN 299 BarAYR INSMAMSLAAYHEEMQQNIDQATSDLRETLEQMEIQNVELGLAKKRAQEAARIKSEFLAN 293 BarAYE INSMAMSLAAYHEEMQQNIDQATSDLRETLEQMEIQNVELGLAKKRAQEAARIKSEFLAN 2 98 BarAYPS INSMAMSLAAYHEEMQQNIDQATSDLRETLEQMEIQNVELGLAKKRAQEASRIKSEFLAN 2 98 BarAYPE INSMAMSLAAYHEEMQQNIDQATSDLRETLEQMEIQNVELGLAKKRAQEASRIKSEFLAN 2 98 BarAST INSMAMSLAAYHEEMQHNIDQATSDLRETLEQMEIQNVELDLAKKRAQEAARIKSEFLAN 299 ExpSECR INSMAMSLTAYHEEMQQNIDQATYDLRETLEQMEIQNVELDLAKKRAQEAARIKSEFLAN 2 99 GacSPA INRMAETLQSAQEEMQHNIDQATEDVRQNLETIEIQNIELDLARKEALEASRIKSEFLAN 290 VarSVC INAMAVSLSEYHVEMQHSIDQATSDLRETLEQLEIQNVELDIAKKRAQEAARVKSEFLAN 2 97 BarAAS INAMAKALSEYHEEMQQNIDQATSDLRETLEQIEIQNVELDMAKKRAQEAARVKSEFLAN 297 Consensus INSMAMSL-AYHEEMQQNIDQATSDLRETLEQMEIQNVELDLAKKRAQEAARIKSEFLAN BarAYR INSMAMSLAAYHEEMQQNIDQATSDLRETLEQMEIQNVELGLAKKRAQEAARIKSEFLAN 293 HK (299-520) BarAEC MSHELRTPLNGVIGFTRLTLKTELTPTQRDHLNTIERSANNLLAI INDVLDFSKLEAGKL 359 BarAYR MSHELRTPLNGVIGFTRQTLKTSLTPTQTDYLQTIQRSANNLLSI INDVLDFSKLEAEKL 353 BarAYE MSHELRTPLNGVIGFTRQTLKTSLTPTQTDYLQTIQRSANNLLCI INDVLDFSKLEAEKL 358 BarAYPS MSHELRTPLNGVIGFTRQTLKTSLTPTQTDYLQTIQRSANNLLCI INDVLDFSKLEADKL 358 BarAYPE MSHELRTPLNGVIGFTRQTLKTSLTPTQTDYLQTIQRSANNLLCI INDVLDFSKLEADKL 358 BarAST MSHELRTPLNGVIGFTRLTLKTELNPTQRDHLNTIERSANNLLAI INDVLDFSKLEAGKL 359 ExpSECR MSHELRTPLNGVIGFTRQTLKTPLNTTQTDYLLTIERSANNLLNI INDVLDFSKLEAGKL 359 GacSPA MSHEIRTPLNGILGFTNLLQKSELSPRQQDYLTTIQKSAESLLGI INEILDFSKIEAGKL 350 VarSVC MSHELRTPLNGVIGFTRQMLKTQLTNSQADYLQTIEKSANNLLTI INDILDFSKLEAGKL 357 BarAAS MSHELRTPLNGVIGFTRQLLKTALTPSQTDYMQTIEKSARNLLGI INDILDFSKLEAGKL 357 **..*****.** ** Consensus MSHELRTPLNGVIGFTRQTLKT-LTPTQTDYLQTI-RSANNLL-I INDVLDFSKLEAGKL BarAYR MSHELRTPLNGVIGFTRQTLKTSLTPTQTDYLQTIQRSANNLLSI INDVLDFSKLEAEKL 353 134 HK BarAEC ILESIPFPLRSTLDEWTLLAHSSHDKGLELTLNIKSDVPDNVIGDPLRLQQIITNLVGN 419 BarAYR VLEHIPFPLRETLDEWILLAHTAHEKGLELTMQVNNDVPEQWGDSMRLQQVMTNLLGN 413 BarAYE ILEHIPFSLRGALDEVIILLAHTAHEKGLELTLHVNKDVPEQVIGDAMRLQQIVTNLLGN 418 BarAYPS ILEHIPFSLRGTLDEVIILLAHTAHEKGLELTLHINNDVPDQVLGDAMRLQQIVTNLLGN 418 BarAYPE ILEHIPFSLRETLDEVIILLAHTAHEKGLELTLHINNDVPDQVLGDAMRLQQIVTNLLGN 418 BarAST ILESIPFPLRNTLDEWTLLAHSSHDKGLELTLNIKNDVPDNVIGDPLRLQQVITNLVGN 419 ExpSECR VLEDIPFSLHSTLDDWMLLAHTAHEKGLELTLSIQNDVPEQFVGDPLRIQQIITNLLGN 419 GacSPA VLENLPFNLRDLIQDALTMLAPAAHEKQLELVSLVYRDTPIQLQGDPQRLKQILTNLVGN 410 VarSVC ALENIPFEFQEVLEEWNLQATSAHEKGLEITLKIDPKIPRGWGDPLRIQQVLTNLVGN 417 BarAAS QLEHIPFSLRDTLNETMHLLGPSAHDKQLELSLQVDAEVPDYLTGDPMRLQQVLTNLAGN 417 . * . * **. *..*..*** ** Consensus -LE-IPF-LR-TLDEV-LLAH- AHEKGLELTL-I—DVP-QV-GDP-RLQQI-TNL-GN BarAYR VLEHIPFPLRETLDEWILLAHTAHEKGLELTMQVNNDVPEQWGDSMRLQQVMTNLLGN 413 HK. BarAEC AIKFTENGNIDILVEKRALSN-TKVQIEVQIRDTGIGIPERDQSRLFQAFRQADASISRR 4 78 BarAYR AIKFTENGNVDIHVSLRSHTR-RQIKLAVEIQDTGIGISEQQQSQLFQAFRQADASISRR 4 72 BarAYE AIKFTEQGNIDILVAVRAIAS-QQVTLWEIHDTGIGISESQQAQLFQAFRQADASISRR 4 77 BarAYPS AIKFTEEGNIDILVWKAIAS-QQVTLMVEIHDTGIGISESQQAQLFQAFRQADASTSRR 4 77 BarAYPE AIKFTEEGNIDILVWKAIAS-QQVTLMVEIHDTGIGISESQQAQLFQAFRQADASTSRR 4 77 BarAST AIKFTESGNIDILVEKRALSN-TKVQIEVQIRDTGIGIPERDQSRLFQAFRQADASISRR 4 78 ExpSECR AIKFTEQGNIDIRVEKRRQEH-HQVQLEVQIRDTGIGIAELQQSQLFQAFRQADTSISRR 478 GacSPA AIKFTQGGTVAVRAMLEDESD-DRAQLRISVQDTGIGLSEEDQQALFKAFSQADNSLSRQ 469 VarSVC SIKFTERGNIDVSVEMRALRD-DVIDLQFMVRDTGIGISERQQAQLFQAFSQADASISRR 4 76 BarAAS AIKFTERGNVDVHIEQTGGSNGNKVRLNVLIRDTGIGISEEQQRQLFQAFNQADSSISRR 477 Consensus AIKFTE-GNIDI -V-R V-L-V-1 -DTGIGISE-QQ-QLFQAFRQADASISRR BarAYR AIKFTENGNVDIHVSLRSHTR-RQIKLAVEIQDTGIGISEQQQSQLFQAFRQADASISRR 4 72 HK (299-520) BarAEC HGGTGLGLVITQKLVNEMGGDISFHSQPNRGSTFWFHINLDLNPNIIIEGPSTQCLAGKR 53 8 BarAYR HGGTGLGLVITERLVKEMDGDISFHSELNRGSTFRFHITLDLHEGMVYRQPTMESLAGKH 532 BarAYE HGGTGLGLVITERLVKEMGGDISFHSQVDRGSTFRFHLTLDLNEAIPSRRPDMSHLEGKT 537 BarAYPS HGGTGLGLVITERLVKEMGGNISFQSQMSCGSIFRFHLTLELNESIPYRQPDMAHLEGKQ 537 BarAYPE HGGTGLGLVITERLVKEMGGNISFQSQMSCGSIFRFHLTLELNESIPYRQPDMAHLEGKQ 537 BarAST HGGTGLGLVITQKLVNEMGGDISFHSQPNRGSTFWFHINLDLNPNVIIDGPSTACLAGKR 538 ExpSECR HGGTGLGLVITQRLVKEMGGDISFQSQLNKGSTFWFHITLPLNPHAMPTEPAYTMLQGKH 538 GacSPA AGGTGLGLVISKRLIEQMGGEIGVDSTPGEGAEFWISLSLPKSRDD-NEEPGASWAAGQR 52 8 VarSVC YGGTGLGLVITQKLVSHMGGEISLTSRLHQGSTFWFTLRLHTTELPLNDGYNADSLNHRH 536 BarAAS YGGTGLGLVITQKLVQQMGGQIRFESELGKGSIFSFSLDMDVSPLPQTEKLPLDRIQGKR 53 7 *********:;:*; 135 BarAEC LAYVEPNSAAAQCTLDILSETPLEWYSPTFSALPPAHYD MMLLGIAVTFR 589 BarAYR LAYVERNPAAAQATLDMLSITPLRVTHSPTLAQLADKHYD ILLLGVPIPFR 583 BarAYE LAYIERNAAAARATLDILSITPLQVTHSLTLAQLPIQHYD FLLVGVPIPYR 588 BarAYPS LAYIERNSTAARATINILNTTPLQVTHRQSLAQLPEQHYD FLLVGVPIPFR 588 BarAYPE LAYIERNSTAARATINILNTTPLQVTHRQSLAQLPEQHYD FLLVGVPIPFR 588 BarAST LAYVEPNATAAQCTLDLLSDTPVEWYSPTFSALPLAHYD IMILSVPVTFR 589 ExpSECR LAYVEYHPIAAQATLDILSQTPLMVSYSPTFEQLPEGDFD ILLLGIPVQYR 589 GacSPA VALLEPQELTRRSLHHQLTDFGLEVSEFADLDSLQESLRNPPPGQLPISLAVLGVSAAIH 588 VarSVC LLLIEPNMQAAAIVQQTLVQSGLEVTYRSAIPEE-QHVYD YVLLNLAPSKE 586 BarAAS VWLLEPDPFSHSSLCALLAEWQLDVQSLAIDTIWPEMSNQ DMVIIGSSTLH 588 ::*.: *:* : ::.. Consensus LAY-E-N—AA—T L— TPL-V LP- -HYD LLGVP R BarAYR LAYVERNPAAAQATLDMLSITPLRVTHSPTLAQLADKHYD ILLLGVPIPFR 583 BarAEC EPLTMQHERLAKAVSMTDFLMLALPCHAQVNAEKLKQDGIGACLLKPLTPTRLLPALTEF 64 9 BarAYR TNMAGHQDKLIAALKMADQVILALPSQSQIDAEQLKQLGAKACLAKPVSANRLMPLLLKE 643 BarAYE NNNAVHQDKLIAAMKISEQVILALPSQSQVEAEQLKQIGAKACLVKPISANRLIPLLSYE 648 BarAYPS NNMAIHQDKLLTALKIADHVILALPSQSQVEAEQLKQIGAKGCLLKPISSIRLIPLLLAE 64 8 BarAYPE NNMAIHQDKLLTALKIADHVILALPSQSQVEAEQLKQIGAKDCLLKPISSIRLIPLLLAE 64 8 BarAST EPLTMQHERLAKAASMTDFLLLALPCHAQINAEKLKQGGAAACLLKPLTSTRLLPALTEY 64 9 ExpSECR NMLLDYTPRLRDICRRAPCVILALPSLAQMDAEQLKTFGVHACLSKPLAASRLLPLLQDS 649 GacSPA PPEELSQSFWEFERLGCKTLVLCPTTEQAQYHATLPDEQVEAKPACTRKLQRKLQELLQV 64 8 VarSVC TNPTLVELWVQRALAMTHNVIVGVPSTELALADQLMQRYPVKCISKPLSRKKLLQTLAAQ 646 BarAAS TPQQVIAR--LDALDGQQNTIVLLSSHEPSLYEAMLAHGAQHCLSKPTNHRKLLHALLSP 646 • : : : : * Consensus L--A VILALPS—Q—AEQLKQ-GA-ACL-KP RLLP-L BarAYR TNMAGHQDKLIAALKMADQVILALPSQSQIDAEQLKQLGAKACLAKPVSANRLMPLLLKE 643 RR (669-785) BarAEC C HHKQNTLLPVTDESKLAMTVMAVDDNPANLKLIGALLEDMVQHVELCDSGH 701 BarAYR T PLALPAPVEPVTKGSRLPLRVMAVDDNPANLKLIGTLLEEQVEEIILCESGE 696 BarAYE K AHSELPWEQAHKQPKLPFKVMAVDDNPANLKLMGTLLEEQVEETVLCDSGA 701 BarAYPS D THSKVPSEEQPRKQPKLPFRVMAVDDNPANLKLIGTLLEEQIEETVLCDSGA 701 BarAYPE D THSKVPSEEQPRKQPKLPFRVMAVDDNPANLKLIGTLLEEQIEETVLCDSGA 7 01 BarAST C QLNHHPEPLLMDTSKITMTVMAVDDNPANLKLIGALLEDKVQHVELCDSGH 701 ExpSECR TLFQLSFLPDATTSHQSWRHPARLPLSVMAVDDNPANLKLIGTLLEEQVDTIILCESGT 709 GacSPA R PTRSDKPHAMVSGRPPRLLCVDDNPANLLLVQTLLSDLGAQVTAVDSGY 698 VarSVC Q PQLANTSLPKPQADKLPLCVMAVDDNPANLKLITALLQERVEYWSCTSGQ 698 BarAAS E ATKSAPLPTPSPRSVQAIKVLAVDDNAANLKLIAAMLKEMVSQWVCKNGK 698 Consensus KLP—VMAVDDNPANLKLIGTLLEE-V LCDSG- BarAYR T PLALPAPVEPVTKGSRLPLRVMAVDDNPANLKLIGTLLEEQVEEIILCESGE 696 136 RR (669-785) BarAEC QAVERAKQMPFDLILMDIQMPDMDGIRACELIHQLPHQQQT PVIAVTAHAMAGQKEK 58 BarAYR AAIAIARDHTLDVILMDIQMPEVDGIRASELIHQLPNHTNT PIIAVTAHAISGQQEQ 53 BarAYE KAIAYARENTLDIILMDIQMPEIDGIRASEVIHQMSHHQDT PIIAVTAHAVRGQQEQ 58 BarAYPS MAITYAREHTLDIILMDIQMPEIDGIRASEIIHQIPHHQET PIIAVTAHAVKGQQEQ 58 BarAYPE MAITYAREHTLDIILMDIQMPEIDGIRASEIIHQIPHHQET PIIAVTAHAVKGQQEQ 58 BarAST QAVDRAKQMQFDLILMDIQMPDMDGIRACELIHQLPHQQQT PVIAVTAHAMAGQKEK 58 ExpSECR DAISQAKMHQIDIILMDIQMPGMDGICASELIRQIPHHATT PIIAVTAQTMTGEREH 66 GacSPA AALEWQRERFDLVFMDVQMPGMDGRQATEAIRRWEAEREVSPVPVIALTAHALSNEKRA 58 VarSVC EAIEQAQSRQFDIILMDIQMPHMDGVTACKAIKQLKGYRDT---PVIAVTAHAMAGERDR 55 BarAAS EAVRLAQSQPFDIIFMDIQMPIMDGISATQAIRSQSLNTET PIVAVTAHAIPGERER 55 *..*.**. Consensus -AI—A DIILMDIQMP-MDGI-A-E-I-Q-PH T-- -PIIAVTAHA--GQ-E- BarAYR AAIAIARDHTLDVILMDIQMPEVDGIRASELIHQLPNHTNT-- -PIIAVTAHAISGQQEQ 753 RR (669-785) BarAEC LLGAGMSDYLAKPIEEERLHNLLLRYKPGSGISSRWTP E 7 98 BarAYR FLKIGMADYLAKPIDETRLTQALARFYQHEPAHQQKT 790 BarAYE LLKLGMADYLAKPIDEARLIQALSRYLPDSASQLHDEQPHALQQSHVLQQSHTLQQQGES 818 BarAYPS LLRLGMADYLAKPIDEARLLQVLSRYQSDNPPLSTASTASTASTASTASTASTASTASTA 818 BarAYPE LLRLGMADYLAKPIDEARLLQVLSRYQSDNPPLSTAS TA 797 BarAST LLSAGMNDYLAKPIEEEKLHNLLLRYKPGANVAARLMAP E 798 ExpSECR LLRSGMDDYLAKPIDEQMLKSVLTRHARKDPLKRDRG NI 805 GacSPA LLQAGMDDYLTKPIDEQQLAQWLKWTGLSLGQSLAS 795 VarSVC LLKAGMDDYLTKPIEEHILQQVLVHWSPHTRSKQVAKVTPP DGA 7 99 BarAAS LIRQGMDDYLAKPIDESMLAQLITDFAHRRHQNHGDQ 792 Consensus LL—GM-DYLAKPIDE-LQ-L-R BarAYR FLKIGMADYLAKPIDETRLTQALARFYQHEPAHQQKT- 790 HPT (669-785) BarAEC VNEIWNPNA TLDWQLALRQAAGKTDLARDMLQMLLDFLPEVRNKVEEQLVGE- 851 BarAYR VSVINALPNA---WEGSINWPLAIRQAANKEDLAQELLSMLVAFLPQVIQRVQAIIDGA- 846 BarAYE EAHIQSVPQSE--CKESIDWPLAVRQAANKEALAKDLLTMLLEFTPQVIQRVQAILAGS- 875 BarAYPS ESLPLPVPKG AGVIDWAQAVRQAANKEDLARDLLTMLLEFMPQITARVQAILAGA- 873 BarAYPE ESLPLPVPKG AGVIDWAQAVRQAANKEDLARDLLTMLLEFMPQITARVQAILAGA- 852 BarAST PAEFIFNPNA TLDWQLALRQAAGKPDLARDMLQMLIDFLPEVRNKIEEQLVGE- 851 ExpSECR AGLLNEHDDS QLSLDWALAQQQAANKPELARDLLQMLLDFLPEVRQKIENVLNGQ- 860 GacSPA MSRAPQLG--QLSVLDPEEGLRLAAGKADLAADMLAMLLASLAADRQAIRQARDND- 84 9 VarSVC AVISNALPSSPPAEEAIIDWPVALRQSANKEDLAKEMLGMLVDYLREVETVVNTALEDEE 85 9 BarAAS QIDWSLAVRQAAGKLDLAKEMLTMLMASFDEVDPVLEAAFAGQ- 835 Consensus P IDW-LA-RQAANK-DLA-D-LQMLL-FLP-V V L-G- BarAYR VSVINALPNA WEGSINWPLAIRQAANKEDLAQELLSMLVAFLPQVIQRVQAIIDGA- 846 137 7. BarAEC -NPEGLVDLIHKU GSCGYi GVPRM: BarAYR -EDGHIVDLIHKU :GSCAY£. BarAYE -HDDDILNLIHKFI GSCACJ GVPRL: BarAYPS -DDGDILNLVHKFt GSCACS GVPRL: BarAYPE -DDGDILNLVHKFI GSCACJGVPRL: BarAST -NPNGLVDLVHKLJ :GSCGY£.GVPRM: ExpSECR -TDDNIIELVHKIi GSCSY£ GVPRL GacSPA -DRTALLERVHRU GATRYCGVPQL: VarSVC YPASDLLHHIHKLI- GSCSYE GVPRL: BarAAS VEDAEVLAQLHRM GGCAY3GVPGL' Consensus --D L-L-HKLHGSC-YS BarAYR -EDGHIVDLIHKLHGSCAYS BarAEC MDNVAREASKILG 918 BarAYR BarAYE IDNVRFAARNYLGAA 944 BarAYPS IENVSEAAKTYLRTE 942 BarAYPE IENVSEAAKTYLRTE 921 BarAST MDNVAREAKKILG 918 ExpSECR IDNVNKAAQPHIKPLHSSL 933 GacSPA ASATTHLSSTSLDSSEL-- 925 VarSVC MAKVLEASRDYLN 927 BarAAS MEQVRREAPLYLA 903 138 E. carotovora (59.3 %), V. cholerae (49.3 %) and P. aeruginosa (32 %) (Figure 14). The histidine 296, aspartate 708, and histidine 856 recognized as essential for the phosphorelay for hybrid HKs were identified in the deduced Bar A protein sequence of Y. ruckeri (Figure 14). In silico analyses of the barA sequence identified four domains, including Histidine kinase Adenyl cyclase Methyl accepting proteins Phosphatase (HAMP), HK, RR, and phosphotransfer domain (Hpt) (Figure 14), which were characteristic of hybrid HKs (Nagasawa et al., 1992, Pernestig et al., 2001). 5.10. Identification of BarA-UvrY TCS in different serotypes of Y. ruckeri PCR amplification confirmed the presence of barA and uvrY genes in 11 strains of Y. ruckeri, as the expected amplicons of 629 bp for uvrY and 2.6 kb for barA were obtained (Figure 15). Eleven strains were representative for all six serotypes recognized based on whole-cell antigens (Table 1, Stevenson et al, 1993). DISCUSSION A uvrY miniTn5Km2 mutation impaired the survival of Y. ruckeri in fish. It was found missing at 7 days in the kidney of four rainbow trout in two independent screens (Tables 6 and 8). A time course one-to-one competitive challenge with WT strain also demonstrated that mutant uvrY was unable to compete with the WT (Table 14). From the beginning of infection on day 1, the mutant was recovered in 5- to 15-fold lower numbers than WT strain, suggesting that mutant might be defective for fish invasion (Table 14). 139 12 3 4 5 6 7 8 9 10 11 12 13 «a...vv-j,jy 3.1 •j^^-^-^SE^SKHiSioirs—'*T* *""'j«iei' m%w 2.0 •*Sfe* :? Figure 15. A 10 11 12 13 0.6 • Figure 15. B Figure 15. PCR amplification of bar A and uvrY genes in different serotypes of Y. ruckeri. A. The PCR amplification of 2.6 kb barA using genomic DNA of various strains as a template. Lane 1: 1 kb DNA MWM (Invitrogen), size approximately in kb. B. The PCR amplification of 629 bp uvrY using genomic DNA of various strains as a template. Lane 1: 100 bp DNA MWM (Invitrogen), size in kb. Figures 15 A and 15 B, Lane 2: No template DNA added, negative control, 3: RS22a, 4: RS32b, 5: RS71, 6: RS201', 7: RS254, 8: RS771', 9: RS806,10: RS11172,11: RS11182,12: RS11541, 13: RS11552. Superscript numbers denote serotype of Y. ruckeri strains based on whole cell-antigens (Stevenson et al, 1993). 140 Besides being defective at the entry stage, the mutant was unable to sustain itself as well as the WT strain in majority of the fish during subsequent stages of infection. However, the mutant was recovered from the fish on days 7 and 9 and not completely missing, as was observed in first two screenings. This might be because the mutant had less competition (1:1) than in initial screens (1:10), in which 11 mutants were competing simultaneously. Similar results have been reported in other STM screens in which mutants completely missing in initial screens were able to survive in the host to different extents in competition with the WT (Sheehan et al, 2003, Shivani et al, 2005). Nonetheless, the uvrY mutant was clearly survival-defective and was unable to compete with the WT strain in rainbow trout. Based on the time-course infection data, I suggest three reasons for poor survival of the mutant in rainbow trout. First, mutant uvrY was impaired in entry into fish and fewer bacteria got into the fish to compete with the WT. Second, the mutant was impaired in invasion and colonization of internal organs and unable to achieve a high bacterial load in fish. Third, although not investigated, the mutant may have increased susceptibility to fish immune defenses such as complement components and phagocytes that caused rapid elimination of mutant from the fish. The in vitro phenotypic characterization of mutant uvrY was performed in efforts to understand possible reasons for its poor survival in rainbow trout. Mutant uvrY was more sensitive to HaCMhan the WT (Table 15). The BarA-UvrY TCSs of other bacteria such as E. coli, S. typhimurium, E. carotovora and Pseudomonas spp were also involved in regulation of the oxidative stress response, as the mutants were hypersensitive to I^CVmediated killing (Eriksson et al, 1998, Whistler et al, 1998, 141 Pernestig et al, 2001). The killing profiles of Y. ruckeri were similar to that of E. coli in which at 1 mM concentration maximum killing was observed within the first 30 to 60 minutes (Table 15) but after that cells developed an adaptive response (Pernestig et al, 2001). The WT strain of Y. ruckeri was better in dealing with the oxidative stress than Y. pestis, as it had a nearly 40 % survival at 60 minutes in 10 mM H2O2 (Table 15), whereas Y. pestis was completely killed by 2.2 mM H2O2 in the same time period (Charnetzky and Shuford, 1985). However, growth temperature and medium for Y. pestis are different from that of Y. ruckeri, making direct comparison difficult. The ability to combat oxidative stress has been suggested to contribute to Y. pestis survival in macrophages (Charnetzky and Shuford, 1985). Increased susceptibility of uvrY mutant of Y. ruckeri to oxidative stress could be due to its inability to efficiently detoxify oxidative free radicals generated from H2O2 that cause damage to DNA, lipids and proteins in the cell. Catalases are mainly responsible for detoxification of H2O2, along with some other enzymes such as alkyl hydroperoxide reductase and glutathione reductase (Demple, 1991). The reduced production of catalases could be one reason for hypersensitivity of uvrY mutant to oxidative stress. My hypothesis is based on the results of Mukhopadhyay et al. (2000) in which the barAluvrY mutants of E. coli had reduced production of catalases, measured by mRNA and protein levels, causing hypersensitivity of mutants to H2O2. Another reason for hypersensitivity of Y. ruckeri mutant to oxidative stress could be that the mutant was less efficient in repairing the damage caused by oxidative free radicals, which might be due to altered production of proteins in the uvrY mutant such as the DNA replication and repair 142 enzymes, heat shock proteins, chaperones required for protein folding and turnover that are needed to combat the oxidative stress. Y. ruckeri would encounter the oxidative stress in macrophage and neutrophils that are important contributor to fish innate immunity (Ellis, 2001). As suggested earlier, increased susceptibility to oxidative killing of phagocytic cells would cause rapid clearance of the uvrY mutant from the fish. Afonso et al (1998a, b) noted that Y. ruckeri were located inside peritoneal neutrophils and macrophages of infected rainbow trout, suggesting a role for phagocytosis in pathogenesis of Y. ruckeri. As suggested for the uvrY mutant of Y. ruckeri, the uvrY/barA mutants of E. coli were 103-104 times more susceptible to killing by the chicken macrophage cell line, HD-11 (Herren et al, 2006). Besides phagocytes, complement components and antibacterial compounds in the serum are a significant part of fish defenses. Mutant uvrY and WT Y. ruckeri were similarly resistant to killing by non-immune rainbow trout serum (Table 17). This is in contrast to the uvrY mutants of an avian pathogenic E. coli which were hypersensitive to killing by the chicken serum (Herren et al, 2006). My data suggested that BarA-UvrY TCS might not control serum resistance in Y. ruckeri and the poor survival of mutant in fish is not because of susceptibility to serum factors. The virulence of Y. ruckeri was not solely related to serum resistance, as many virulent strains were very effectively killed by the non-immune rainbow trout serum and non-virulent strains were serum resistant (Davies 1991b). The BarA-UvrY TCS controls bacterial invasion of the host cells, as the barAluvrY mutants of E. coli and S. typhimurium had 100-fold reductions in invasion of the chicken embryo fibroblasts and HEp-2 cells, respectively (Johnston et al, 1996, 143 Herren et al, 2006). We also found that the uvrY mutant of Y. ruckeri had significantly reduced invasion of EPC cells, epithelial cells of carp origin. Invasion studies were also performed using a salmonid fish cell line, CHSE-214. We did not obtain a consistent recovery of bacteria as large variations were observed among replicates even within a strain. Considering toxicity to CHSE cells as the possible reason, different multiplicity of infections (100:1, 10:1, 1:1) and incubation times (60 minutes, 30 minutes, 15 minutes), were used during protocol optimization, but similar variable results were obtained (data not shown). Y. ruckeri was able to invade CHSE-214 cells with up to 104 to 106 intracellular bacteria/25cm tissue culture flask (Romalde and Toranzo, 1993). We could not compare our results on CHSE-214 cells with those of Romalde and Toranzo (1993), since the authors did not specify the number of CHSE cells and bacteria used for invasion studies. Fish cell invasion by Y. ruckeri has previously been shown to be cell-line dependent, with maximum invasion observed in epithelial cells, FHM, of fathead minnow (Kawula et al, 1996). This property can help Y. ruckeri to invade various epithelial surfaces of fish. The poor invasion of epithelial cells by the uvrY mutant might lead to poor survival in rainbow trout for two reasons. First, the mutant was impaired in entry into fish due to reduced invasion of epithelium at the gills and gastrointestinal tract of fish, the possible entry routes for Y. ruckeri. This is supported by time-course infection data in which the mutant uvrY had lower infection loads than the WT on the first day of infection (Table 14). Second, the mutant was impaired in invasion of internal organs of fish, reducing bacterial spread in tissues that led to lower infection loads. The barAluvrY 144 mutants of E. coli were also impaired in colonization of the internal organs of chicken (Herren et al, 2006). Two possible mechanisms, including decreased production of adhesin/invasin and regulation of TTSS, have been suggested by which BarA-UvrY TCS can regulate invasion of host cells. The E. coli mutants had 2- and 3-fold reductions in mRNA levels for papA and fimA, which encode major structural components of Type P and Typel fimbriae, respectively (Herren et al, 2006). The sirA mutant (uvrY homolog) of S. typhimurium had reduced invasion of epithelial cells, which was controlled by SPI1 encoded TTSS through final regulation by BarA-SirA TCS (Johnston et al, 1996). Currently, invasion mechanisms of Y. ruckeri are not known, however genetic loci for putative adhesins/invasins such as the bundle forming pili and inv have been identified (Fernandez et al, 2004, Fernandez et al, 2007). Our STM screening also identified a putative bfp locus (mutant F5-11) to be essential for bacterial survival in rainbow trout. Y. ruckeri possesses Ysa TTSS, a homolog of SPI1 TTSS of S. typhimurium, but Ysa has not been characterized (Gunasena et al, 2003). The inv, bfp and ysa could be possible targets of BarA-UvrY TCS for regulating the cell invasion phenotype in Y. ruckeri. Fernandez et al. (2004) demonstrated that iron acquisition in fish tissue is a virulence determinant of Y. ruckeri, as mutations in the biosynthetic/secretion pathways of the siderophore Ruckerbactin attenuated virulence in rainbow trout. We tested the hypothesis that BarA-UvrY TCS controls iron-acquisition in Y. ruckeri. Mutant uvrY grew as well as the WT strain in LB supplemented with 200 uM of an iron chelator 2, 2' dipyridyl (Figure 12), suggesting that uvrY mutation did not affect iron acquisition by Y. 145 ruckeri under conditions tested. This suggested that poor iron acquisition might not be responsible for attenuation of the mutant uvrY in fish. The molecular mechanism by which the BarA-UvrY TCS regulates iron acquisition is not known, however mutants of E. coli (Pernestig et al, 2000), P. viridiflava and P. marginalis (Liao et al, 1996, Liao et al, 1997) had reduced siderophore production. Y. ruckeri produces a catecholic siderophore, Ruckerbactin and as the uvrY mutant grew as well as WT under iron- deficient conditions, the siderophore production may not be affected. The BarA-UvrY TCS has however been involved in regulation of iron acquisition and siderophore production in E. coli (Pernestig et al, 2000, Pernestig et al, 2003), Bordetella bronchiseptica (Giardina et al, 1995), P. viridiflava and P. marginalis (Liao et al, 1996, Liao et al, 1997), but strain-to-strain differences have been noted in E. coli and B. bronchiseptica. Thus, differences exist among bacterial species regarding regulation of iron-acquisition by homologs of the BarA-UvrY TCS. Diarrhea and hemorrhaging of the gastrointestinal tract (GIT) are part of Y. ruckeri-induced pathology in fish (Rucker 1966, Miller 1983). The intestine of rainbow trout served as the site of initial colonization by serotype 1 strains of Y. ruckeri, leading to establishment of a systemic infection (Kim and Stevenson 1998). The intestine of fish is a prime organ for establishment of chronic carrier state by Y. ruckeri (Busch and Lingg 1975). We hypothesize that the BarA-UvrY TCS could be involved in the colonization and invasion of the GIT of fish. The BarA-UvrY homologs in other pathogens control the intestinal stages of pathogenesis. The vacA mutant of V. cholerae and sirA mutant of S. typhimurium had significantly reduced colonization of mouse and bovine intestine, respectively (Wong et al, 1998, Ahmer et al, 1999). The BarA-SirA of S. typhimurium 146 regulated SPI1 and SPI5 encoded TTSS genes, which in turn control the intestinal stages of disease (Ahmer et al, 1999). I suggest that Ysa TTSS of Y. ruckeri, a homolog of SPI1 TTSS, might be regulated by BarA-UvrY TCS and can control the GIT stages of pathogenesis in fish. I worked on the hypothesis that BarA-UvrY TCS could contribute to varied virulence of different serotypes of Y. ruckeri. A three step approach is being undertaken that includes testing (1) whether all serotypes contained BarA-UvrY TCS, (2) if the TCS was active in all serotypes and (3) if there is a differential gene regulation by this TCS in different serotypes. Our results demonstrated that BarA-UvrY TCS was present in all serotypes, as twelve strains belonging to five different serotypes gave positive PCR amplification for bar A and uvrY (Figure 15). Currently, we are optimizing the reverse transcription-PCR for checking functional activity of BarA-UvrY TCS in different serotypes. In silico analysis predicted a genetic linkage between the uvrY and a downstream gene uvrC, a subunit of the UvrABC enzyme complex required for repairing UV-induced DNA damage (Figure 9). Due to the insertion of miniTn5Km2 cassette, a uvrY mutation could cause a polar effect on the downstream uvrC gene by blocking the transcription of uvrC from the promoter present upstream of uvrY. The transcription could be stopped by transcription terminator in the miniTn5Km2 cassette. The uvrY mutation in Y. ruckeri did not have a polar effect on uvrC, as indicated by the unaltered sensitivity of the mutant to UV light (Table 16). Two putative promoter sequences were identified in the uvrY-uvrC locus by in silico analysis, one upstream of uvrY and the second in the C-terminal coding region of uvrY (Figure 9). It is possible that both of these putative promoter sequences 147 might be regulating transcription of uvrC in Y, ruckeri. The minTn5Km2 mutation in uvrY mutant was upstream of the alternative putative promoter of uvrC (Figure 9), and would only block transcription from promoter present upstream of uvrY and not from the alternative promoter. The alternative promoter might drive transcription of uvrC and might explain unaltered sensitivity of mutant to UV light. The non-polarity of mutation suggested that the phenotypes observed in uvrY mutant of Y. ruckeri were most likely due to uvrY mutation. A similar genetic organization, uvrY-uvrC, exist in E. coli (Moolenaar et al, 1987), E. carotovora (Eriksson et al, 1998), V. cholerae (Wong et al, 1998) and Pseudomonas spp. (Laville et al, 1992, Reimmann et al, 1997). Similarly, the uvrY mutation in E. coli did not have a polar effect on uvrC due to transcription of uvrC by additional promoters present within 3' end of coding sequence of uvrY (Moolenaar et al, 1987, Shanna et al, 1986, Sluis et al, 1983). This produced sufficient UvrC protein for the activity of UvrABC enzyme complex, so that uvrY mutant was not hypersensitive to UV light. The uvrY mutations in E. carotovora (Eriksson et al, 1998), V. cholerae (Wong et al, 1998), and Pseudomonas spp (Laville et al, 1992, Reimmann et al, 1997) had polar effects, producing increased sensitivity of mutants to UV light. 148 CHAPTER 6 CHARACTERIZATION OF THE ZnuACB TRANSPORTER Pathogens face the challenge of acquiring essential nutrients in the host, where many nutrients are not freely available. The availability of Zn to bacteria is limited by its strong binding to albumin and other proteins, and by sequestration of zinc in vacuoles in the host cells (Reviewed by Blencowe and Morby 2003). Mutations in pathogen genes required for acquiring nutrients lead to poor colonization of tissues and attenuation of virulence in the host (Ammendola et al., 2007). In the current study, STM screens identified mutant C6-1 of Y. ruckeri as defective in survival in rainbow trout. A preliminary in silico analysis of the C6-1 sequence suggested that this locus might be involved in zinc transport. Zinc is an essential micronutrient needed for both structural and catalytic functions in all organisms and is a cofactor for essential enzymes in the cell. Zinc deficiency can cause pleiotropic effects and is critical for cell survival (Blencowe and Morby 2003). Further characterization of mutant C6-1 led to the identification of a high-affinity ZnuACB transporter of Y. ruckeri that might be involved in zinc transport and required for survival of Y. ruckeri in its natural host, rainbow trout. RESULTS 6.1. Identification of ZnuACB transporter The C6-1 ST mutant that did not survive in rainbow trout had a mutation in a gene homologous to a gene encoding ZnuA protein required for zinc transport in E. coli 149 (Patzer and Hantke, 1998). The mutant was found missing from the kidney at 7 days post-infection in four rainbow trout, in two independent screens using two fish each (Tables 6, 8). Further sequence analysis of the C6-1 DNA identified znuC and znuB homologs in Y. ruckeri. The homologs of znuA, znuC and znuB in Y. ruckeri were predicted to encode a putative zinc transporter, ZnuACB, which would belong to cluster 9 of ATP-binding cassette (ABC) family of transporters (Claverys, 2001). By multiple sequence alignments with homologs, we identified putative start and stop codons for znuA, znuC and znuB genes (Figure 16). The genes were arranged in two putative transcriptional units, znuA and znuCB that were predicted to be transcribed divergently on opposite DNA strands. The transcriptional units were separated by a short intergenic region of 96 bp (Figures 3A, 3B and 4). An overlap of one codon at znuC and znuB genes was predicted for the znuCB transcriptional unit. ZnuC of Y. ruckeri was predicted to be a 252 amino acid protein. The ZnuC was identified as a putative ATPase required for providing energy for zinc transport. The ATPases are characterized by presence of Walker A, Walker B and LSGQ motifs (Davidson and Chen 2004, Dawson et al, 2007). We identified the characteristic Walker A (GPNGAGKST, 37 to 45 amino acids), Walker B (LLVLD, 141 to 145 amino acids) and 'LSGGQ' (121 to 125 amino acids) motifs in the ZnuC sequence of Y. ruckeri. ZnuB of Y. ruckeri was predicted to be a 261 amino acid protein. The ZnuB was identified as a putative inner-membrane permease. The SOSUI signal database (http://bp.nuap.nagoya- u.ac.jp/sosui/sosuisignal/sosuisignal_submit.html) predicted a 29 amino acid N-terminal signal sequence in the ZnuB protein, a characteristic of membrane proteins. The 7 potential TM segments were identified in the ZnuB protein by the TMHMM program 150 Figure 16. Sequence alignment of Zn/Mn-binding proteins of C9 class of ABC transporter family. The alignment was performed using CLUSTALW program at http//.align.genome.jp. The histidine and acidic amino acid rich loop was indicated by a solid line on top of the sequences. The residues involved in substrate-binding are shown by symbol (T). The symbols indicate identical residues (*), conserved substitutions (:) and semi-conserved substitutions (.). The accession numbers of sequences used were: znuAYR, Yersinia ruckeri (EU139251); znuAEC, Escherichia coli K12 (NP_416371); znuASAL, Salmonella typhimurium (NP_460848); znuAYP, Yersinia pestis (NP_669558); znuAYE, Yersinia enterocolitica (YP_001006607); znuATP, Treponema pallidum (NP_218474); znuASYN, Synechocystis (1PQ4); troATP, Treponema pallidum (AAC45725); psaA, Streptococcus pneumoniae (AAF70668); mntC, Synechocystis (BAA17919). 151 ZnuAEC (62.3%) MKCYNITVLIFITMIGRIMLHKKTLLFAALSAALWGGATQAADAAWAS 49 znuASYN (24.1%) MFIFPAVPRFVQPLGVAFVLGLSTLGCQPAVEQVGQNGQVEDAPVADAMDITVS 54 znuAYE (82.5%) MLHKNKWLKRAMLASAILIANPFNIASAAWTS 33 znuAYP (81.2%) MLHKNKWLKQAMLASALLLANPFNLASAAWTS 33 znuATP (81.2%) MLHKNKWLKQAMLASALLLANPFNLASAAVVTS 33 znuASAL (61.7%) MIECYNITFHTFITMISRIMLQKNTLLFAALSAALWGSATQAADAAWAS 50 troATP (22.8%) MIRERICACVLALGMLTGFTHAFGSKDAAADGKPLWTT 39 psaASP (19.1%) MKKLGTLFVLFLSVIVLVACASGKKDAASGQKLKWAT 38 mntCSYN (17.9%) MATSFASRGGLLASGLAIAFWLTGCGTAEVTTSNAPSEEVTAVTTEVQGETEEKKKVLTT 60 Consensus : ML L L A AAWTS znuAYR MLQKNKWLKSTLLIGTILVTGAVNSASAAWTS 33 znuAEC LKPVGFIASAIADGVTETEVLLPDGASEHDYSLRPSDVKRLQNADLWWVGPEMEAFMQK 109 znuASYN IPPQQYFLEKIGGDLVRVSVLVPGNNDPHTYEPKPQQLAALSEAEAYVLIGLGFEQPWLE 114 znuAYE VRPLGFIASAIADGVLPTEVLLPDGASPHDYALRPSDVQRLRSADLVIWVGPEMEAFLSK 93 znuAYP IRPLGFIAAAIADGVLPTEVLLPDGASPHDYALRPSDVQRLRSAELVIWVGPEMEAFLSK 93 znuATP IRPLGFIAAAIADGVLPTEVLLPDGASPHDYALRPSDVQRLRSAELVIWVGPEMEAFLSK 93 znuASAL LKPLGFIASAIADGVTDTQVLLPDGASEHDYSLRPSDVKRLQGADLWWVGPEMEAFMEK 110 troATP IGMIADAVKNIAQGDWLKGLMGPGVDPHLYTATAGDVEWLGNADLILYNGLHLETKMGE 99 psaASP NSIIADITKNIAGDKIDLHSIVPIGQDPHEYEPLPEDVKKTSEADLIFYNGINLETGGNA 98 mntCSYN FTVLADMVQNVAGDKLWESITRIGAEIHGYEPTPSDIVKAQDADLILYNGMNLERWFEQ 120 :.. : ..** .:: *: .*:* Consensus --PLGFIA-IADGV TEVLLPDGASPHDY-LRPSDV-RL—ADLV-WVGPEME-F znuAYR VRPLGFIASAIADGVLPTEVLLPDGASPHDYALRPSDVQRLRGAELVIWVGPEMEAFLSK 93 znuAEC PVSKLPGAKQVTIAQLEDVKPLLMKSIHGDD DDHDHAEKSDED HHH 155 znuASYN KLKAANAN-MKLIDSAQGITPLEMEKHDHSHGEEEGHDDHSHDGHDHGSESEKEKAKGAL 173 znuAYE PLTQVADNKQIALAQLPSVMPLLMKGNEDDE HEGEGDGHDHDHAKDNQHDEHHH 147 znuAYP PLTQVAENKQIALSQLPSVTPLLMKSDEHDE AEEGESGHHHDHAKDNPTDDHHH 14 7 znuATP PLTQVAENKQIALSQLPSVTPLLMKSDEHDE AEEGESGHHHDHAKDNPTDDHHH 147 znuASAL SVRNIPDNKQVTIAQLADVKPLLMKGADDDE DEHAHTGADEEKGDVH HHH 160 troATP VFSKLRGSRLWAVSETIPVSQRLS LEE 12 7 psaASP WFTKLVENAKKTENKDYFAVSEGVDVIYLEG KNEK 133 mntCSYN FLGNVKDVPSWLTEGIEPIPIADGPYTDKP 151 Consensus -L NK L—V- PLLM D—E- znuAYR PLSQLADNKKIALSELKSVKPLLMKGEEHDE -HEGESVGHDHDHAKDHHSDEHHH 147 znuAEC GDFNMHLWLSPEIARATAVAIHGKLVELMPQSRAKLDANLKDFEAQLASTETQVGNELA- 214 znuASYN MVADPHIWLSPTLVKRQATTIAKELAELDPDNRDQYEANLAAFLAELERLNQELGQILQ- 232 znuAYE GEYNMHIWLSPTIAKQSAIAIHDRLLELMPQNKDKLDANLRRFEDQLAQNEKNIATMLK- 206 znuAYP GEYNMHIWLSPAIAKQAAIAIHDRLLELTPQNKDKLDANLRRFEDQLAQNEKNIVTMLK- 206 znuATP GEYNMHIWLSPAIAKQAAIAIHDRLLELTPQNKDKLDANLRRFEDQLAQNEKNIVTMLK- 206 znuASAL GEYNMHLWLSPEIARATAVAIHEKLVELMPQSRAKLDANLKDFEAQLAATDKQVGNELA- 219 troATP AEFDPHVWFDVKLWSYSVKAVYESLCKLLPGKTREFTQRYQAYQQQLDKLDAYVRRKAQS 187 psaASP GKEDPHAWLNLENGIIFAKNIAKQLSAKDPNNKEFYEKNLKEYTDKLDKLDKESKDKFNN 193 mntCSYN NPHAWMSPRNALVYVENIRQAFVELDPDNAKYYNANAAVYSEQLKAIDRQLGADLEQ. 208 Consensus -E-N-H-WLSP-IA A-AIH—L-EL-PQN LDANL—FE-QL L- znuAYR GEYNLHLWLSPDIAKLTAIAIHDRLLELMPQKRDKLDANLRQFEDQLAQSNEKIANMLQ- 206 152 znuAEC -PLKGKGYFVFHDAYGYFEKQFGLTPLGHFTVNPEIQPGAQRLHEIRTQLVEQKATCVFA 273 znuASYN -PLPQRKFIVFHPSWAYFARDYNLVQIPIEVE--GQEPSAQELKQLIDTAKENNLTMVFG 289 znuAYE -PAQGKGYFVFHDAYGYFEKHFGLSPLGYFTVNPEIQPGAQRLHQIRTQLVEHKAVCVFA 265 znuAYP -PVQGKGYFVFHDAYGYFENHFGLSPLGHFTVNPEIQPGAQRLHQIRTQLVEHKAVCVFA 265 znuATP -PVQGKGYFVFHDAYGYFENHFGLSPLGHFTVNPEIQPGAQRLHQIRTQLVEHKAVCVFA 265 znuASAL -PLKGKGYFVFHDAYGYYEKHYGLTPLGHFTVNPEIQPGAQRLHEIRTQLVEQKATCVFA 278 troATP LPAERRVLVTAHDAFGYFSRAYGFEVKGLQGVSTASEASAHDMQELAAFIAQRKLPAIFI 24 7 psaASP IPAEKKLIVTSEGAFKYFSKAYGVPSAYIWEINTEEEGTPEQIKTLVEKLRQTKVPSLFV 253 mntCSYN VPANQRFLVSCEGAFSYLARDYGMEEIYMWPINAEQQFTPKQVQTVIEEVKTNNVPTIFC 268 Consensus -P—GKGYFVFHDAYGYFE GL-PLG-FTVNPE-QPGAQ-L-I--TQLVE-KA-CVFA znuAYR -PVQGKGYFVFHDAYGYFEKYFGLTPLGHFTVNPEIQPGAQRLHQIRTQLVEQKAVCVFA 265 • znuAEC EPQFRPAWESV ARGTSVRMG TLDPLGTNIKLGKTSYSEFLSQLANQYASCL 325 znuASYN ETQFSTKSSEAI AAEIGAGVE---LLDPLAADWSSNLKAVAQKIANANSAQP--- 338 znuAYE EPQFRPAVINAV AKGTDVRSG TLDPLGSGIVLGKDSYVNFMSQLSNQYVSCL 317 znuAYP EPQFRPAVINAV AKGTNVRSG TLDPLGSGIVLDKDSYVNFLSQLSNQYVSCL 317 znuATP EPQFRPAVINAV AKGTNVRSG TLDPLGSGIVLDKDSYVNFLSQLSNQYVSCL 317 znuASAL EPQFRPAWEAV ARGTSVRMG TLDPLGTNIKLGKTSYSAFLSQLANQYASCL 330 troATP ESSIPHKNVEALRDAVQARGHWQIGGELFSDAMGDAGTSEGTYVGMVTHNIDTIVAALA 307 psaASP ESSVDDRPMKTV SQDTNIPIYAQIFTDSIAEEGKEGDSYYSMMKYNLDKIAEGLA 308 mntCSYN ESTVSDKGQKQV AQATGARFGGNLYVDSLSTEEGPVPTFLDLLEYDARVITNGLL 323 *. . . : : *. : . : Consensus EPQF-P AV A-GT-VR-G TLDPLG— I -L SY—F—QL-NQY-SCL znuAYR EPQFRPAIIDAV AKGTQVRSG TLDPLGSGIVLGKDSYVTFLTQLSNQYVSCL 317 znuAEC KGD 328 znuASYN znuAYE KQD 320 znuAYP K 318 znuATP K 318 znuASAL KGD 333 troATP R 308 psaASP K 309 mntCSYN AGTNAQQ 33 0 Consensus KGD znuAYR K 318 153 (http://www.cbs.dtu.dk/services/TMHMM/), further suggesting ZnuB to be a membrane protein. ZnuA of Y. ruckeri was predicted to be a 325 amino acid protein with high sequence identity to ZnuA proteins of zinc transporters in E. coli (62.3 %) (Patzer and Hantke, 1998) and Treponema pallidum (81.2 %) (Desrosiers et al, 2007) (Figure 16). The ZnuA protein of Y. ruckeri contained a 28 amino acid histidine and acidic amino acid-rich loop, present between positions 120 and 147 (Figure 16). This loop has previously been proposed to function as a zinc scavenger in the periplasm of bacteria (Hantke, 2005). The residues identified as important for zinc-binding in other ZnuA proteins are as His78, Hisl61, His225 and Glu77 (Lee et al„ 2002, Banerjee et al, 2003, Chandra et al, 2007, Li and Jogl 2007). These amino acids were present in the ZnuA protein of Y. ruckeri (Figure 16). 6.2. Generation of AznuACB mutant of Y. ruckeri RS1154 For functional characterization of the znuACB locus, we generated a mutant strain of Y. ruckeri that lacked znuACB. Using homologous recombination, we replaced the part of the znuACB locus that would encode the first 23 amino acids of ZnuA, the intergenic region, the complete znuC sequences, and the sequence encoding the first 22 amino acids of ZnuB, with a kanamycin resistance gene (Figure 3B). The mutation was at the intended place as confirmed by three different PCRs (Figure 3C, lanes 2, 4, 6). We obtained the expected products of 1120 bp (lane 2), 2094 bp (lane 4) and 2113 bp (lane 6), using AznuACB mutant genomic DNA as the template for PCRs. As expected, there was no amplification with any of these PCRs when using genomic DNA of the WT strain 154 as the template (Figure 3, lanes 3, 5 and 7). Sequence analysis of the AznuACB locus further confirmed that the mutation was at the correct location. 6.3. Phenotypic characterization of AznuACB mutant The AznuACB mutant was tested for its ability to grow under Zn-deficient conditions, in M9 and LB media with the added metal chelators, EDTA and TPEN. EDTA is a non-specific chelator for various divalent cations including zinc, whereas TPEN chelates Zn with very high affinity without affecting Ca2+ and Mg2+ (Arslan et ah, 1985, Patzer and Hantke 1998, Patzer and Hantke 2000). However, TPEN can chelate Cd, Co, Ni, and Cu (Sigdel et ah, 2006). Atomic absorption spectroscopic analysis showed that the M9 medium (without any chelator added) used for growth studies contained less than 1 ng/ml of zinc (the lowest detection limit of spectrophotometer), suggesting that the medium was zinc-deficient. There was no significant difference in growth of the AznuACB mutant when compared to WT strain RSI 154. The mutant had slightly slower growth between 40 and 72 hours, but was able to reach close to the WT levels in the end (Figure 17). Both strains had similar generation times of 6 hours in M9 medium. Unlike E. coli, no dramatic growth retardation was observed for the AznuACB mutant in zinc-deficient M9 medium (Figure 17). To eliminate any residual zinc, TPEN at a concentration of 1 (ig/ml was added to the M9 medium and growth of the strains was compared. TPEN decreased growth of both strains by nearly 10-fold, as OD600 of cultures were approximately 2.0 and 0.2 in M9 and M9 supplemented with TPEN (Figures 17 and 18). The generation times of AznuACB mutant (34 hours) and WT strain (44 hours) were different in presence of 155 Figure 17. Growth of AznuACB mutant of Y. ruckeri in M9 minimal medium. The WT RSI 154 (A) and the mutant (•) were grown in 20 ml of M9 minimal medium at 18 °C with shaking. The values represented averages of ODeoo ± SD of three independent culture flasks. The OD600 were plotted on arithmetic (A) and semi-log scale (B). The generation times of WT (6 hours) and mutant (6 hours) were calculated using the semi log plot. 156 2.5 i 2.0 S 1.5 c o I10 O 0.5 0.0 0 20 40 60 80 100 120 Hours Figure 17. A 10.0 -i 1.0 H s o o § 0.1 0.0 0 20 40 60 80 100 120 Hours Figure 17. B 157 Figure 18. Growth of AznuACB mutant of Y. ruckeri in M9 minimal medium supplemented with 1 uM TPEN. The WT RSI 154 (A) and the mutant (•) were grown in 20 ml of M9 minimal medium at 18 °C with shaking. The values represented averages of OD600 ± SD of three independent culture flasks. The OD600 were plotted on arithmetic (A) and semi-log scale (B). The generation times of WT (44 hours) and mutant (34 hours) were calculated using the semi log plot. 158 0.25 -J 0.2 - g 0.15 & © 1 0.1 o 0,05 1 0 1 1 1 1 1 1 1 1 1 1 1 0 20 40 60 80 100 120 140 160 180 200 220 Hours Figure 18. A 0.01 1 1 1 1 1 1 1 1 1 1 1 0 20 40 60 80 100 120 140 160 180 200 220 Hours Figure 18. B 159 TPEN. Growth of the AznuACB mutant and WT strain were not significantly different in M9 medium supplemented with TPEN (Figure 18). But, the mutant had slightly slower growth after 120 hours. The AznuACB mutant grew as well as the WT strain in zinc-sufficient LB medium (Figure 19). When grown in LB medium in presence of 0.5 mM EDTA growth of both the mutant and the WT strain were slowed to the same extent (Figure 20). Higher concentration of EDTA (2.0 mM, 5.0 mM) produced effects similar to 0.5 mM concentration for both strains (data not shown). Growth of the mutant was also tested by adding 2.0 uM and 12.5 uM of TPEN in LB medium. The mutant and WT stain had the same growth under both concentrations of TPEN (Figure 21). 6.4. ZnuACB transporter of Y. ruckeri was functionally active in E. coli The 2766 bp znuACB locus of Y. ruckeri RSI 154 was PCR amplified using a high-fidelity DNA polymerase (Figure 22). The PCR product was cloned in a low-copy number plasmid, pBAD33Cm, confirmed by restriction digestion and sequencing before using it in transcomplementation studies. Under zinc-deficient conditions in LB medium supplemented with 2.0 mM EDTA, the AznuACB mutant of E. coli had slower growth than isogenic WT (Figure 23). Growth was restored to the WT levels by complementation with the znuACB locus of Y. ruckeri RSI 154 (Figure 23). Similarly, complementation with WT znuACB locus of E. coli restored poor growth of the AznuACB mutant of E. coli (Figure 23). 160 Figure 19. The AznuACB mutant was not defective in growth in LB medium. The WT (A) and mutant (•) strains were grown in LB medium at 18 °C for 18 hours. The 50 ml of LB medium was inoculated 1:100 with these cultures and incubated at 18 °C with shaking. The OD60o were taken at various time points. The values represented the mean ± SD of three independent culture flasks. The OD600 were plotted on arithmetic (A) and semi-log scale (B). 161 3.5 i 3,0 2.5 I 2'° c» 1.5 - \oo Q 1 0 O 1U 0.5 0.0 0 3 9 12 15 18 21 24 Hours Figure 19. A 10.0 -. 1.0 s & o o 0.0 —i 1— 0 3 6 9 12 15 21 24 Hours Figure 19. B 162 Figure 20. Effect of EDTA on growth of AznuACB mutant of Y. ruckeri. The WT Y. ruckeri RSI 154 and AznuACB mutant were grown in LB medium supplemented with different concentrations of metal chelator, EDTA. The growth was monitored by OD600 at different time points. WT in LB (A), mutant in LB (•), WT in 0.5 mM EDTA (A), mutant in 0.5 mM EDTA (•). Growth curves of both strains in 2.0 mM and 5.0 mM EDTA were similar to those obtained in 0.5 mM EDTA (data not shown for clarity of the graph). Values represented averages ± SD of three independent culture flasks. The OD600 were plotted on arithmetic (A) and semi-log scale (B). 163 3.5 -| 3.0 - 2.5 - | 2.0 H o o £ 1 5 O l.o H 0.5 0.0 _, -i 1 1— T r —i— i 0 4 6 8 10 12 14 16 18 Hours Figure 20. A 10.0 1.0 = a o s© Q O 0.1 0.0 T 1 —i 1 1— i"" • •—i — i 0 2 4 6 8 10 12 14 16 18 Hours Figure 20. B 164 Figure 21. Effect of TPEN on growth of AznuACB mutant of Y. ruckeri. The WT Y. ruckeri RSI 154 and AznuACB mutant were grown in LB medium supplemented with 2.0 [iM and 12.5 \iM of TPEN. Growth was monitored by taking ODgoo at different time points. WT in LB only (•), mutant in LB only (A), WT in 2.0 uM TPEN (•), mutant in 2.0 \iM TPEN (•), WT in 12.5 ^M TPEN (A), mutant in 12.5 ^M TPEN (x). The values represented averages ± SD of three culture flasks. The OD600 were plotted on arithmetic (A) and semi-log scale (B). 165 Figure 21. A 10.0 n 1.0 s S3 © o Q o o.i -] 0.0 n 1 1 r "i r "i 1 0 2 6 8 10 12 14 16 18 20 Hours Figure 21. B 166 3.1 Figure 22. PCR amplification of znuACB locus of Y. ruckeri RSI 154. The complete znuACB locus was PCR amplified from the genomic DNA of Y. ruckeri RSI 154 using ZnuABC/br and ZnuABCrev primers. Lanes 1: 1 kb DNA MWM (Invitrogen). 2: The 2.6 kb PCR product of znuACB locus 167 Figure 23. znuACB locus of Y. ruckeri complemented E. coli AznuACB mutant. The different E. coli strains were grown at 37 °C in LB medium supplemented with 2.0 mM EDTA. The strains included: wild type W3110 (•), AznuACB mutant GR352 (•), GR352/pSP86/37 (A) (GR352 transcomplemented with znuACB locus of E. coli), GR352/pBAD33/6 (+) (GR352 transcomplemented with znuACB locus of Y. ruckeri RSI 154), GR352/pHSG576 (0) (empty vector control for GR352/pSP86/37) and GR352/pBAD33 (x) (empty vector control for GR352/pBAD33/6). Values represented averages of OD600 ± SD of three independent culture flasks. The OD600 were plotted on arithmetic (A) and semi-log scale (B). 168 2.5 i 2.0 A g 15- a o § 1.0 - o 0.5 - 0.0 0 2 4 6 8 10 12 Hours Figure 23. A 10.00 -i 1.00 H © 0.10 - © a o 0.01 - 0.00 0 2 4 6 8 10 12 Hours Figure 23. B 169 6.5. znuA::lacZ and znuCBidacZ transcriptional fusions analyses The 500 bp sequence that was thought to contain putative promoters of znuA and znuCB transcriptional units was cloned in a promoterless-/acZ transcriptional fusion vector, pRS551 (Figure 4). The P-galactosidase activities of fusions were assayed in LB medium in the presence of metal chelators EDTA and TPEN. On LB agar supplemented with IPTG and X-gal, Y. ruckeri RSI 154 (host strain for fusions) produced blue colonies, indicating it was P-galactosidase positive. Y. ruckeri RSI 154 transformed with plasmid vector (negative control) produced P-galactosidase activity between 114 to 272 Miller's units under different test conditions (Table 18). In LB medium that contains about 10 uM of Zn (Campoy et al, 2002), transcriptional fusions gave P-galactosidase activity of 4944 ± 984, 3057 ± 400 Millers units for znuAv.lacZ and znuCBv.lacZ fusions, respectively. After addition of 20 uM TPEN to LB medium, znuAv.lacZ and znuCBv.lacZ fusions were derepressed nearly 2.5-fold, showing 13237 ± 219 and 7356 ± 183 Miller units, respectively. Addition of 1 mM ZnSo4 to TPEN-containing LB medium repressed activities of the znuAv.lacZ and znuCBv.lacZ fusions, showing 2615 ±151 and 1713 ± 45 Miller units, respectively. Similar to TPEN, addition of 0.5 mM EDTA to LB medium caused 3.5- to 4-fold derepression of both transcriptional fusions (Table 18). Addition of 1 mM ZnSo4 to EDTA-containing LB medium repressed activities of znuAv.lacZ and znuCBv.lacZ fusions, showing 1611 ± 161 and 1310 ± 48 Miller units, respectively. Also, the addition of 1 mM MnCb to LB medium containing EDTA repressed znuAv.lacZ and znuCBv.lacZ fusions, showing P-galactosidase activity of 1697 ± 46, 1346 ± 67 Millers units, respectively (Table 18). 170 Table 18. znuAv.lacZ and znuACBv.lacZ transcriptional fusion assays. Strain LB 0.5 mM 0.5 mM EDTA 0.5 mM EDTA 20 uM 20 uM TPEN EDTA +1 mM ZnSo4 +1 mM MnCl2 TPEN +1 mM ZnSo4 Y.ruckeri 3057 ±400 10339 ±281 1310 ±48 1346 ± 67 7356 ±183 1713 ±45 pRS551/41 Y.ruckeri 4944 ± 983 19610 ±2678 16.11 ±161 1697 ± 46 13237 ±19 2615 ±151 pRS551/61 Y.ruckeri 140 ±5 187 ±4 114 ± 11 150 ±8 288 ±6 272 ±22 pRS551 Y. ruckeri strains, pRS551/61and pRS551/41 containing znuAv.lacZ and znuCB::lacZ fusions, respectively were grown in 5 ml of medium at 18 °C until mid-exponential stage under different growth conditions and the P-galactosidase activity was assayed and expressed in Miller's unit. Y.ruckeri pRS551 containing empty vecter served as a negative control. Values represented mean ± SD of triplicate samples. 171 6.6. ZnuACB transporter was required for survival in rainbow trout To understand the role of znuACB in pathogenesis, bacterial loads of mutant and WT strains were compared in the kidney of individual fish after bath infection. During the first two days of infection, the mutant was able to infect nearly as well as the WT strain in most of the fish, but in later stages of infection the bacterial loads of mutant started to decrease in comparison to WT strain (Table 19). On days 5 and 9 of infection, mutant had nearly 102- to 103-fold lower infection loads than the WT strain in most fish, suggesting that the mutant was unable to compete with the WT strain. Although mutant demonstrated poor survival in most fish, it was not completely eliminated (Table 19). DISCUSSION The in silico analysis suggested that the gene that we identified might encode a zinc transporter. In other bacteria, the zinc transporter ZnuACB consists of three components, a zinc-binding periplasmic protein, ZnuA, a membrane permease ZnuB, and an ATPase, ZnuC. The homologs of all three components were identified in the C6-1 sequence of Y. ruckeri. The substrate specificity of ZnuACB transporter is determined by the zinc-binding protein, ZnuA that scavenges zinc in the periplasm (Hantke, 2005). A high sequence identity of up to 81 % among C6-1 sequence and ZnuA proteins of other bacteria (Figure 16), suggested that ZnuACB transporter of Y. ruckeri might be involved in zinc transport. The ZnuA homolog of Y. ruckeri contained glutamate (77), histidine (78), histidine (161), and histidine (225) residues (Figure 16) that were identified to be involved in zinc-binding, based on the crystal structures of ZnuA proteins in other 172 Table 19. Competitive challenge of mutant C6-1 and WT strain in rainbow trout. Fish# CFU/g kidney (WT) CFU/g kidney (MT) Ratio (WT/MT) Dayl 1 9.7xl04 l.lxHT 1 2 7.9xl03 1.5xl04 0.5 3 1.9xl04 l.OxlO4 2 4 3.7xl04 1.9xl04 2 5 3.6xl04 1.3xl04 3 Day 2 1 7.5xl04 3.4xl03 22 2 3.4xl04 2.9x104 1 3 2.4xl05 9.6xl04 3 4 1.4xl04 5.2xl03 3 5 1.6xl04 6.8xl03 2 Day 3 1 3.0xlCf 3.4xl03 87 2 2.8xl04 2.9xl04 1 3 2.0xl04 9.6xl04 0.2 4 1.7xl05 5.2xl03 32 5 8.3xl04 6.8xl03 12 Day 5 1 1.4xl06 4.7xl03 3 2 6.7xl04 2.6xl03 26 3 2.7xl05 1.6xl03 1.7xl02 4 l.lxlO6 2.0xl03 5.7xl02 5 1.5xl06 2.4xl02 6.3xl03 Day 9 1 4.3x10° 1.2X103 36 2 4.7xl06 1.9xl03 2.4xl03 3 6.9xl06 3.9xl04 1.7xl02 Total 23 rainbow trout (average weight 85 g) were bath infected with Y. ruckeri RSI 154 (WT) and mutant C6-1 (dose: 6.0 x 10& CFU/ml each) for 10 minutes. The infection loads of mutant (MT) and WT were calculated in the kidney at different time post infection. To compare both strains, ratio (WT/MT) of infection load in each individual fish was calculated. 173 bacteria (Banerjee et al, 2003, Chandra et al, 2007, Li and Jogl 2007, Lee et al, 2002). The ZnuA homolog of Y. ruckeri contained a 28 amino acid long histidine and acidic amino acid rich sequence (Figure 16) that has been proposed to function as a Zn chaperone in other ZnuA proteins (Hantke, 2005). The genetic organization of znuACB locus of Y. ruckeri was similar to those of znuACB loci of E. coli, S. typhimurium and T. pallidum (Patzer and Hantke 2000, Campoy et al, 2002, Desrosiers et al, 2007). The znuACB locus of Y. ruckeri was predicted to be transcribed in two transcriptional units, znuA and znuCB that were separated by a very short intergenic region of 96 bp (Figure 4). The znuACB loci of E. coli and S. typhimurium have similar genetic organizations with both transcriptional units separated by a very short intergenic region of 24 and 25 bp, respectively (Campoy et al., 2002, Patzer and Hantke 2000). In silico predictions were supported by experimental data when znuACB locus of Y. ruckeri was able to functionally complement AznuACB mutant of E. coli (Figure 23). The transcomplementation by znuACB locus of Y. ruckeri was as good as complementation provided by WT znuACB locus of E. coli (Figure 23). This indicated that ZnuACB transporter of Y. ruckeri was a functional homologue of ZnuACB transporter of E. coli. The ZnuACB transporter of E. coli had previously been shown to be involved in zinc transport using a radioactive isotope of zinc as a substrate (Patzer and Hantke, 1998). Functional complementation of AznuACB mutant of E. coli by znuACB locus of Y. ruckeri and in silico analyses suggested that ZnuACB transporter of Y. ruckeri might be involved in zinc transport. These results need to be confirmed by binding assays in which the purified ZnuA protein of Y. ruckeri should bind to Zn with high specificity and affinity. This was demonstrated, using inductively coupled plasma-optical emission 174 spectroscopy, for T. pallidum in which ZnuA protein bound 1.07 ± 0.34 of Zn and no manganese, a related transition metal (Desrosiers et al., 2007). The metal specificity of ZnuACB transporter of Y. ruckeri can also be confirmed by measuring transport of a radioactive isotope of zinc by AznuACB mutant of E. coli after complementation with the WT znuACB locus of Y. ruckeri. In vitro growth experiments under zinc-limiting conditions on AznuACB mutant suggested the existence of another high-affinity transporter in Y. ruckeri. Under zinc- limiting conditions, no dramatic growth retardation phenotype was observed in AznuACB mutant of Y. ruckeri, as the mutant grew as well as the WT strain in LB medium supplemented with up to 5 mM of EDTA (Figure 20). Similarly, in zinc-deficient M9 medium the AznuACB mutant of Y. ruckeri grew quite well (Figure 17). Although the M9 medium used in growth studies was zinc-deficient, as indicated by atomic absorption spectroscopy, growth studies with AznuACB mutant were also performed in M9 medium supplemented with 1.0 uM of TPEN to remove any traces of zinc and the mutant grew well under these conditions, but had increased generation time than the WT strain (Figure 18). Our suggestion of the existence of more than one high-affinity zinc transporter contradicts what has been reported in E. coli, S. typhimurium and P. multocida. Only one high-affinity zinc transporter, ZnuACB, has been suggested to exist in these species. This was based on the results that mutation of znuACB locus in these species nearly abolished growth of AznuACB mutants in enriched growth medium supplemented with 0.5 mM or higher concentrations of EDTA (Patzer and Hantke 1998, Campoy et al, 2002, Grass et al, 2002, Garrido et al, 2003, Ammendola et al, 2007). We also found that the AznuACB mutant of E. coli, in contrast to the mutant of Y. ruckeri, had severely impaired 175 growth under these conditions (Figure 23). Mutation in znuACB locus of E. coli and S. enterica led to very slow growth of mutants in zinc-deficient minimal medium (Patzer and Hantke 1998, Ammendola et al, 2007), which was not observed in Y. ruckeri (Figures 17 and 18). The additional transporter (s) suggested in Y. ruckeri could be either zinc specific or a general metal transporter which, along with zinc, might transport other related metals such as Mn, Fe, Ni and Cu. This has been reported for T. pallidum, which possesses ZnuACB and TroABCD transporters. The ZnuACB of T. pallidum was a zinc-specific transporter (Desrosiers et al, 2007), whereas TroABCD was involved in the transport of Zn, Fe and manganese (Hazlett et al, 2003, Desrosiers et al, 2007). To identify promoter sequences and metals to which znuACB locus of Y. ruckeri was responding, activities of znuAv.lacZ and znuCBwlacZ transcriptional fusions were studied. Since both znuAv.lacZ and znuCBwlacZ fusions produced higher P-galactosidase activity than control plasmid, it suggested that the 500 bp fragment used in analysis contained putative promoters for znuA and znuCB transcriptional units (Table 18). Besides containing 96 bp intergenic spacer that might contain putative promoters, the 500 bp sequence also contained the 5' coding sequences of znuA and znuC genes (Figure 4). Thus, primer extension assays and RT-PCR need to be performed to identify the exact promoter sequences and transcriptional start sites of znuA and znuCB of Y. ruckeri. Transcriptional fusion analyses suggested that the znuACB locus of Y. ruckeri responded to the availability of zinc as well as manganese in growth medium. Both znuAv.lacZ and znuCBv.lacZ fusions were derepressed 2.5- to 4-fold by addition of metal chelators, EDTA/TPEN and addition of zinc or manganese to growth medium repressed 176 transcriptional fusions by nearly 4- to 12-fold (Table 18). We suggest three explanations for the response of transcriptional fusions to Zn as well as Mn. First, addition of MnC^ to the growth medium would reduce the effective concentration of EDTA/TPEN that was available to chelate Zn in medium, as Zn and Mn compete for binding sites on EDTA/TPEN. This might leave some Zn free in medium that might repress the transcriptional fusions. Second, Mn might be contaminated with traces of Zn. Third, Zn and Mn may act as repressor and corepressor of the znuACB locus and both metals may be transport substrates of the ZnuACB transporter of Y. ruckeri. Patzer and Hantke (2000) reported data suggesting that Mn can act as a corepressor of znuACB locus of E. coli in which addition of 5 u,M of Mn, similar to 5 u,M of Zn, to buffer for DNase I protection assay enhanced protection of operator sequences by Zur, a regulator of ZnuACB locus. As yet, zinc is the only substrate identified for ZnuACB transporter of E. coli by transport assays using Zn (Patzer and Hantke, 1998). However, Zn was the only substrate evaluated in the study, and no transport assays have been performed using zinc- related transition metals such as Mn, Ni, Co and Cu. Metal specificity of the ZnuACB transporters of other bacteria have not been confirmed using transport assays. Multiple substrate-specificity is common among metal transporters of C9 ABC-family of transporters to which ZnuACB belongs (Claverys 2001). In fact, Patzer and Hantke (1998) and Grass et al. (2002) reported that besides zinc, growth of AznuACB mutant of E. coli was partially restored by supplying nickel and cobalt in growth medium. The authors explained this was due to contamination of nickel and cobalt salts with traces of zinc. Thus, our data and literature suggested that ZnuACB can transport multiple substrates. This needs to be tested by transport assays. 177 Experiments are in progress to further understand the regulatory mechanisms of znuACB locus of Y. ruckeri. The zinc-uptake regulator (Zur) regulates expression of znuACB locus in E. coli by binding to 29 bp long operator sequences that contained an 11 bp imperfect palindromic sequence, CTTCACACTATAATATTGTAAAG (Patzer and Hantke 2000, Outten and O'Halloran 2001). I identified a 9 bp imperfect palindromic sequence AGCGTAATA T TATAACATT in the intergenic region of znuACB locus (Figure 4) that might act as the binding site for Zur homolog of Y. ruckeri. I am optimizing PCR conditions to amplify the Zur homolog of Y. ruckeri, using degenerate primers designed from Zur proteins. Although growth of AznuACB mutant was not severely impaired in vitro under metal-limiting conditions, it did not survive in rainbow trout in both of the initial mutant screenings as well as in one-to-one competitive challenge with WT strain (Tables 6, 8, 19). Infection kinetics revealed that the mutant did not have any problem entering into fish, as evidenced by nearly equal bacterial loads of WT and mutant in initial two days of infection (Table 19). However, the mutant was unable to sustain itself as well as the WT strain during later stages of infection. The mutant might have enough zinc stored from in vitro growth in enriched LB medium that was sufficient to sustain its growth for initial few days in fish. The growth of mutant was impaired subsequently due to inability to acquire sufficient zinc in the fish tissues because of non-functional ZnuACB transporter. Similar observation was made by Ammendola et al. (2007) in which, during the initial days of infection, the znuA mutant of S. typhimurium had approximately similar infection loads to that of WT in spleen of mice. The infection loads of the znuA mutant declined sharply in later stages of infection (Ammendola et al., 2007). 178 Although AznuACB mutant was survival-defective, it was recovered in lower numbers from the fish (Table 19). This could be due to the fact that mutant was able to obtain zinc through other transporters but not in sufficient quantity to maintain high infection loads. This agrees with our in vitro growth data on AznuACB mutant that also suggested existence of additional zinc transporter (s) in Y. ruckeri. I speculate that ZnuACB transporter might be working synergistically with other transporter (s) to supply sufficient zinc to Y. ruckeri. A synergistic action of two zinc transporters, ZnuACB and TroABCD, has been reported for T. pallidum in which both transporters were concomitantly expressed in a rabbit orchitis model (Desrosiers et ah, 2007). Poor survival of AznuACB mutant in fish, but no slower growth phenotype under zinc-deficient conditions in vitro, could possibly be due to lower expression of additional transporter (s) in fish as compared to in vitro, and/or more difficulty in obtaining zinc due to tight sequestration in fish tissues. The poor availability of zinc might have affected the survival of the AznuACB mutant in two ways. First, expression of some virulence genes might have been affected in the mutant, as many transcriptional factors that control virulence genes are regulated by zinc (Coleman 1992, Akira et ah, 2002, Crane et ah, 2007). Second, activities of house-keeping enzymes such as polymerases and metabolic enzymes which require Zn for structural and catalytic functions would have been affected, causing poor survival of the mutant. Crane et ah (2007) reported that expression of genes encoding virulence- related transcriptional regulators PerA, Ler and virulence factors such as bundle-forming pilus, EspA, intimin and genes of locus for the enterocyte effacement in enteropathogenic E. coli were affected by the availability of zinc in the intestine of rabbit. 179 CHAPTER 7 GENERAL DISCUSSION The main objective of this research was to identify survival-essential genes of Y. ruckeri in rainbow trout, and further evaluate their roles in pathogenesis. In previous studies, no single major virulence factor was identified and mutations of some virulence factors such as the Ruckerbactin siderophore, YhlA hemolysin, Yrpl protease and flagellin did not produce dramatic effects on virulence of Y. ruckeri (Kim 2000, Fernandez et ah, 2002, Fernandez et ah, 2004, Fernandez et ah, 2007b). We suggested that pathogenesis of Y. ruckeri is mediated through concerted actions of multiple subtle virulence factors and a genome-wide approach would be useful in identifying multiple subtle virulence genes. We hypothesized that long-term survival in infected fish constitutes a key part of virulence of Y. ruckeri. The STM was selected over other approaches because STM is a genome-wide approach and identifies survival-essential genes in a host (Hensel et ah, 1995). Screening of 1056 ST mutants by immersion challenge of rainbow trout identified 25 survival-defective mutants, representing 22 unique genes, since three mutant pairs (A2-1/F4-4, F11-10/F1-2, H5-10/A5-4) had mutations in the same genes. This study contributed new knowledge by identifying two virulence-associated genes and characterized the role of their products in pathogenesis of Y. ruckeri in rainbow trout. We identified the BarA-UvrY two-component system and demonstrated that the uvrY mutant of Y. ruckeri was defective in invasion of epithelial fish cells and was hypersensitive to oxidative stress that led to its poor survival in rainbow trout (Table 15). The uvrY mutant 180 was impaired in entry into rainbow trout and was unable to sustain infection loads similar to the WT strain during subsequent infection (Table 14), possibly due to poor invasion of tissues and increased susceptibility to oxidative stresses of immune system cells in fish. Thus, this study is consistent with the conclusions from studies on S. typhimurium (Ahmer et al, 1999), E. carotovora (Eriksson et al, 1998), V. cholerae (Wong et al, 1998), E. coli (Herren et al, 2006) and P. aeruginosa (Tan et al, 1999) that the BarA- UvrY TCS is important for virulence in other hosts. The ZnuACB transporter of Y. ruckeri was identified and characterized. Significantly, the znuACB locus of Y. ruckeri functionally complemented the zinc transport deficient AznuACB mutant of E. coli, suggesting that znuACB locus of Y. ruckeri might be involved in zinc transport (Figure 21). The in vitro growth experiments on AznuACB mutant of Y. ruckeri suggested the existence of a second high-affinity zinc transporter, which on experimental verification would be the first report on the presence of more than one high-affinity zinc transporter in Enterobacteriaceae. Fernandez et al. (2004) demonstrated that iron acquisition is an important virulence determinant of Y. ruckeri, since mutations in Ruckerbactin siderophore locus reduced virulence in rainbow trout. Our data on time-course infection of AznuACB mutant in which mutant was unable to survive and compete with the WT strain (Table 19), demonstrated that zinc acquisition was important for pathogenesis of Y. ruckeri in rainbow trout. The O-antigen polymerase and Bfp were other two putative virulence factors identified in this study, whose roles in pathogenesis of Y. ruckeri can be evaluated further. In future, additional virulence-associated genes may be identified by characterization of eight mutants (A5-4, A10-7, D2-8, E2-1, F4-1, G6-1, H5-10 and H6- 181 4), whose sequence did not show homology to known genes in the database at present. The techniques for making isogenic mutants and the rainbow trout infection model optimized in this study can be used for evaluating role of other genes in pathogenesis of Y. ruckeri. A significant finding was that the cell surface of Y. ruckeri seems to be important in pathogenesis in rainbow trout, since five mutations including Bfp, O-antigen polymerase, ZnuACB transporter, major facilitator superfamily transporter (B3-2 mutant), and polysaccharide deacetylase were in genes whose products had predicted functions associated with cell surfaces or membranes. The products of genes identified in this study could contribute to various stages of the infection process of Y. ruckeri. These include (a) attachment to and entry into fish: for example, bfp (F5-11 mutant), and BarA-UvrY TCS; (b) colonization of internal organs and spread within tissues: for example, BarA-UvrY TCS, O-antigen polymerase (Fl-2 mutant), and Bfp; and (c) countering fish immune defenses: for example, BarA-UvrY TCS and O-antigen polymerase. All together, this study contributed in understanding survival strategies and pathogenic mechanisms of Y. ruckeri in rainbow trout. The STM screen did not identify any toxin genes of Y. ruckeri. A similar limitation has been reported by STM studies on other pathogens of fish and terrestrial animals (Ruley et al, 2004, Autret and Charbit 2005, Miller and Neely 2005, Menendez et al, 2007, Thune et al., 2007). This is not surprising for Y. ruckeri, since it does not produce the copious amount of toxins found with such fish pathogens as A. salmonicida and A. hydrophila (Leung 1987, Romalde and Toranzo 1993). 182 The fish-to-fish variations in missing mutants in output pools represent an important problem in selection of the survival-defective mutants. To identify missing mutants consistently, multiple screenings of mutants in fish were required, making STM screening both a time-consuming and labor-intensive process. Another significant limitation of STM is that more than 50 % of survival-essential genes identified through this approach do not qualify as true virulence factors and do not directly contribute to virulence. The genes such as the ParAB DPS, PtrA protease and monooxygenase identified in this study did not have virulence-related functions and may be performing housekeeping functions. However, these genes provide valuable information on in vivo host environments and cellular repair mechanisms needed for survival in the host (Perry 1999). The identification of these genes also contributes to functional annotation of the genes, since despite having complete genome sequences of many pathogens functions of up to 50 % of the genes in bacteria are unknown. It is also important to acknowledge that many genes in pathogens can be described as virulence-associated rather than virulence genes, since they are the prerequisite to delivery of virulence gene products (Wassenaar and Gaastra 2001). It is predictable that STM studies will identify more virulence- associated than true virulence genes, since there are generally more of the former than of the latter. Besides STM, various genome-based approaches such as in vivo expression technology (rVET) and differential fluorescence induction (DFI) have been used to identify virulence genes of bacterial pathogens (Mahan et al., 1993, Valdivia and Falkow 1997). The promoter-trap techniques of IVET and DFI are more time consuming than STM, since they require two steps, first identifying bacterial promoters differentially 183 upregulated in host and second, evaluating the role of upregulated genes in virulence using isogenic mutants by infecting the host. How does STM compare to other genome-based approaches such as the IVET and DFI in identifying virulence factors? Specifically, IVET has been applied to Y. ruckeri and genes such as those involved in nutrient acquisition (iron by Ruckerbactin siderophore), adhesion (Bfp), metabolite transport and host damage (YhlA, hemolysin) were identified (Fernandez et al, 2004). Importantly, STM also identified the Bfp (F5-11 mutant) and nutrient acquisition (zinc by ZnuACB transporter) to be important for virulence of Y. ruckeri in rainbow trout. In general, the majority of genes identified by IVET and DFI belonged to functional categories such as the nutrient acquisition (iron, zinc, phosphorous, sugars, amino acids) central metabolic pathways (tricarboxylic acid cycle, glycolysis, sugar and amino acid metabolic pathways), purine and pyrimidine metabolism, DNA repair and recombination, and stress responses (oxidative stress, acid tolerance, osmoregulation) (reviewed by Rediers et al, 2005). A significant number of genes identified by IVET and DFI belonged to virulence-associated genes such as the transcriptional factors and two-component systems. Similar to STM, nearly 10 % of the genes identified through IVET and DFI are true virulence factors such as the TTSS, adhesins and toxin genes (Rediers et al, 2005). Thus, in essence the genome-based approaches are complementary to each other in that taking one approach may render the other redundant, although the different methods may identify different virulence determinants. Our proposed model of enteric redmouth disease divides the pathogenesis of Y. ruckeri in two stages. The first stage includes establishment and persistence of Y. ruckeri 184 infection in fish. The second stage includes causation of acute disease symptoms by Y. ruckeri in infected fish upon stress such as high water temperature (22 °C - 25 °C), population density and handling. The current study identified genes whose products were involved in the first stages of ERM disease pathogenesis. Future research can focus on identifying genes of Y. ruckeri that are turned on/off when a specific stressor such as water temperature of 22 °C - 25 °C, is applied to Y. ruckeri carrier fish. Using IVET or DFI, genes that are differentially regulated between stressed and unstressed fish can be identified. This might identify genes whose products are involved in host damage and tilting the host-pathogen balance in favor of Y. ruckeri to cause acute disease. Multiple approaches can be undertaken to identify the virulence-associated genes of Y. ruckeri required at both stages of our proposed model of ERM disease. First, homologs of known virulence genes from fish pathogens, Yersiniae and pathogens of Enterobacteriaceae can be identified by homology searches in Y. ruckeri and their role in pathogenesis can be evaluated by making isogenic mutants using rainbow trout infection model. Second, the genes encoding immuno-dominant proteins, potential vaccine candidates, can be identified by screening genomic-expression libraries with convalescent anti-Yersinia ruckeri serum. 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Chem. 270:11181-11189. 206 APPENDIX 1 GENETIC LOCATION OF miniTn5Km2 INSERTION IN THE GENOME OF 25 SIGNATURE-TAGGED MUTANTS OF Y. ruckeri CTTTCTCGGTGACCG CTGTC GTA CGGTGTCAGCCTTAGTT A5-4 mutant CCGGTTTTTTACCCTCrGrC GrACGTCCTTGACCTTCCAA A6-1 mutant GAATAAGCAGCATCACTGrC GrACGCCTAATGGCGCTCCA A2-1 mutant ACTGCTCAATAAAACCrGrC GrACGCAATAAAACAGTCTA A2-11 mutant GGCCCTACCTTTATCCrGrC GTA CGCGCTATCGCCAGTTG A6-11 mutant GGCGTTTACATGCACCrGrC GrACGGAATTGATGAGAACA A7-2 mutant ACTCGACTCCTCACTCrGrC GTA CGC ATT ACTCCTCACTC A A10-7 mutant TCAAAGATATCTCCACrGrC GTACGGCCTGACGTAGCCTG Bl-5 mutant CGGGAAGATCATACCCTGJC GTA CGAGCC AT ACTGAAGA A B3-2 mutant 207 TATTTCTAGCGGAAGCrGrC GrACGCTCTTCTTCCTTCGT B3-7 mutant CTGTCGCGCCTACCACTGrC G7ACGACTGTGTATCGATCA C5-1 mutant GATAGAATTACAGCACrGJC G7ACGGCCTTCGGGTCAGCA D2-8 mutant TAGAAAAATAAAGCACrGrC G7ACGGTCCAGATTTGATTC D5-12 mutant ACTCGACTCCTCACT CTGTC G7ACGCATTACTCCTCACTC E2-1 mutant GTGACTATTTGTACTCJGrC G7ACGGTTAAATATCGCTCA Fl-2 mutant CGAGTGGCTTCCAACCrGrC GTA CGCCACCGATGCCCGGC F2-4 mutant CTCACTCATTACTCCCrGJC GrACGTCACTCATTACTTTA F4-1 mutant TGGCGTGGCGATTCGCrGrC GrACGCATTGTGCCAATACC F4-4 mutant 208 GCGCCTCAGGCAAGACTGrC GrACGACTTAAGCGGGTGAC F5-8 mutant CTGATACCACAAGCCCrGrC GrACGAGAAGCAGAAACAAA F5-11 mutant ATATATGGTACCAATCTG7T G7ACGTGGTATGTTATTAAG Fll-10 mutant ACCGCAACGGGGACTCTG7T G7ACGGCCTAAGGCTAAGCT G6-1 mutant TTTAACCAAGCTAGTCTGrC G7ACGCATTTATGTAAGTAA H5-10 mutant AGGCGTACTCATAGACrGrC G7ACGGGTTAAGATTGTCTG H6-4 mutant These 25 mutants were not recovered from the kidney of immersion infected rainbow trout at 7 days post-infection. The genes interrupted by miniTn5km2 insertion were identified by cloning and sequencing (section 2.10, Materials and Methods). Fifteen nucleotide sequence of Y. ruckeri from both sides of the miniTn5km2 insertion are shown. Five nucleotides of the miniTn5km2 sequence from each terminus are shown in bold and italics. There was a duplication of 9 bp sequence (underlined) at the point of miniTn5km2 insertion. I have removed 9 bp duplicated sequence from other side of transposon insertion. The complete sequences of mutants of Y. ruckeri can be accessed in the sequence database using the accession numbers provided in table 11. 209 APPENDIX 2 CONSTRUCTION OF ISOGENIC MUTANTS OF Y. ruckeri We identified 25 signature-tagged mutants that were not recovered from rainbow trout kidney at 7 days post-infection. The four of these genes, which belonged to different functional categories, were selected for making isogenic mutants. Our objective was to make isogenic mutants in these selected genes and perform time-course infection experiments in rainbow trout to study the effect of gene mutation on pathogenesis. The genes included C6-1 (znuA, encoding a periplasmic zinc-binding protein), A6-11 (ptrA, encoding a periplasmic protease), F2-4 (uvrY, encoding a response regulator of BarA- UvrY two-component system) and B3-2 (unknown function). A fifth gene aroA, encoding 5-enolpyruvylshikimate 3-phosphate synthase, involved in biosynthesis of folate, p-aminobenzoic acid and aromatic amino acids, was not a signature-tagged mutant identified in this study, but was included for its potential use as a live-attenuated vaccine. The approach of homologous recombination used in efforts to make isogenic mutants involved the following steps. a. PCR amplification of the wild type of copy gene to be mutated b. Cloning of the PCR product into pUCGm plasmid vector c. Mutation of the WT gene by inserting a gentamicin resistance gene d. Sub-cloning of the mutated gene into a donor pCVD422 plasmid e. Transfer of recombinant pCVD422 in E. coli S17-lXpir by electro-transformation f. Transfer of recombinant pCVD422 in WT Y. ruckeri strains RSI 154 and RS7 by conjugation or electro-transformation 210 g. Selection of isogenic mutant on antibiotic plates and tentative confirmation by PCR The steps (a) through (d) for different genes are described in Appendices 3 to 7. Two approaches, conjugation and electro-transformation, were used to transfer the mutated copies of genes (recombinant pCVD422) into Y. ruckeri cells. Initially, conjugation experiments were performed and no isogenic mutant was obtained using this approach. Subsequently, I performed electro-transformation of Y. ruckeri. For conjugation of donor E. coli S17-Rpir and recipient Y. ruckeri, E. coli and Y. ruckeri strains were grown in LB broth for 18 hours and 28 hours at 37 °C and 18 °C, respectively. Aliquots of 100 ul of E. coli and 200 ul of Y. ruckeri (RSI 154 or RS7) cultures were mixed in 3 ml of 10 mM MgSC>4 solution and the mixture was left at 25 °C for 5 minutes. The other ratios of E. coli and Y. ruckeri (40 ul donor : 100 ul recipient; 250 ul donor : 5 ml recipient mixed in 10 mM MgSCU; and 250 ul donor : 2 ml recipient mixed in distilled H2O instead of 10 mM MgSC>4) were used in optimization of the conjugation protocol. The bacterial mixture was passed through a 0.45 urn filter and the filter was placed (cells side up) on plates of LB agar 10 mM MgSC>4 at 25 °C for 4 hours. Incubation times of 8 hours, 12 hours and 24 hours were also used for optimization. The filter was washed with 5 ml of sterile 0.9 % (w/v) NaCl and dilutions of the wash were plated on LB agar (vancomycin2oo and gentamicin2s). The plates were incubated at 25 °C for 7 days to obtain bacterial colonies. The WT Y. ruckeri RSI 154 and RS7 strains are resistant to vancomycin but sensitive to gentamicin. The donor E. coli strain harbouring recombinant pCVD422 plasmid is gentamicin-resistant but vancomycin-sensitive. The expected isogenic mutant of Y. ruckeri would be both vancomycin and gentamicin resistant. 211 For electro-transformation, Y. ruckeri RS1154/RS7 cells were made electro- competent. The bacteria were grown in 1 litre of LB broth at 25 °C to an OD600 of 0.5 - 0.8. The cells were centrifuged at 2500 x g for 10 minutes and pellet was washed with sterile transformation buffer (15% glycerol). This step was done twice. The cells were dissolved in 1 ml of transformation buffer and stored in 40 ul aliquots at -70 °C. For electro transformation, 40 ul of Y. ruckeri RS1154/RS7 competent cells were mixed with recombinant pCVD422 plasmid DNA and incubated on ice for 1 minute. To determine the optimum DNA concentration for generating an isogenic mutant, different concentrations of pCVD422 DNA (50 ng, 100 ng, 250 ng, 500 ng and 1000 ng) were used. After incubation on ice, the cells were subjected to a single electric pulse at 2.5 V, 400 Q, 25 JAF (Bio-rad). After adding 1 ml of SOC medium (section 2.21), the transformed cells were incubated at 25 °C for 2 hours. The cells were plated on LB agar (gentamicin25), incubated at 25 °C for 7 days and observed for bacterial colonies. As plasmid pCVD422 can not replicate in Y. ruckeri, the gentamicin resistant colonies should represent mutants generated by single or double crossover homologous recombination. The single and double crossover mutants can be differentiated by sucrose sensitivity, as double crossover mutants will not grow in presence of 5 % sucrose (Donnenberg and Kaper 1991). As a positive control for electro-transformation, Y. ruckeri cells were also transformed with different concentrations (50 ng, 100 ng, 250 ng, 500 ng and 1000 ng) of pUCGm plasmid. This plasmid, unlike pCVD422 plasmid, can replicate in Y. ruckeri cells and can be used to calculate the transformation efficiency. 212 I did not obtain any bacterial colonies on LB agar (vancomycin2oo, gentamicin2s) after conjugation of Y. ruckeri strains RSI 154 and RS7 for any of the five genes tested in this thesis. Similarly, no colonies were obtained when the donor and recipient were mixed in different ratios for conjugation and no colonies were obtained on LB agar (gentamicin25) after plating of electro-transformed Y. ruckeri RS1154/RS7 cells. Thus, I did not get isogenic mutants for any of the five genes using the conjugation and electro- transformation protocols described. Some of the possible reasons for not getting isogenic mutants could be: (1) low frequency of transformation/conjugation/homologous recombination; (2) the flanking gene sequences were short (ranged between 220 bp - 875 bp on either side for different genes) for optimum homologous recombination; and (3) the gentamicin resistance gene used for mutating WT genes might some how be interfering in homologous recombination. As a positive control, I was able to transfer pUCGm plasmid DNA into Y. ruckeri RS1154/RS7 using the electro-transformation protocol described. The maximum transformation efficiency for pUCGm DNA was approximately 200 colonies, obtained with DNA concentrations between 50 ng to 250 ng. The transformation efficiency was lower for other pUCGm DNA concentrations. This was an indirect control for electro- transformation experiments, but these values can not be extrapolated for pCVD422 DNA as transformation efficiency may differ for plasmids. The transformation efficiency could not be calculated for pCVD422, as this plasmid can not replicate in Y. ruckeri cells. I made three modifications in the protocol for making isogenic mutants. First, I increased the length of flanking sequences to nearly 1 kb; second, I inserted the pKOBEG-sacB plasmid into Y. ruckeri RSI 154 cells; and third, I used a kanamycin 213 resistance gene for mutating WT gene. Derbise et al. (2003) reported that the frequency of generating isogenic mutants in Y. pseudotuberculosis was increased significantly when WT Yersinia cells were simultaneously expressing lambda phage redyPa operon on a plasmid. The modified approach was effective in generating an isogenic AznuACB mutant of Y. ruckeri as described in section 2.21 (Materials and Methods). 214 APPENDIX 3 STRATEGY FOR CONSTRUCTION OF AN aroA ISOGENIC MUTANT A. The 1180 bp sequence of the aroA was amplified by PCR. B and C. The PCR product was digested with Bst\J\ enzyme and a 430 bp product was gel purified, digested with Smal and cloned into pUCGM vector at the Smal site. D. To mutate the aroA, an 850 bp gentamicin resistance gene was cloned at an EcoRW site of aroA cloned into the pUCGM vector. E and F. The positive clone in pUCGM vector was digested with Xbal enzyme and a 1280 bp fragment containing aroA interrupted with gentamicin gene, was gel purified and cloned into Xbal site of pCVD422 vector. The positive clone in pCVD422 vector was used for conjugation or electro-transformation of WT Y. ruckeri RSI 154 in order to mutate the aroA by homologous recombination. 215 aroAF aroAR -? —? 1180bp Bstm Bstm (23 bp) (453 bp) EcoRV(331bp) B EcoRV Smdl aroA I Smal ^_ I *S Xbal iiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiniiiiiiiiiiimTtir Xbal aroA Gm aroA jlini mm Xbal Xbal D E aroA Gm aroA aroA Gm aroA R^J pCVD422 vector 1 W Levenaser ^^^m*^^^ 216 APPENDIX 4 STRATEGY FOR CONSTRUCTION OF AN ISOGENIC znuA MUTANT A. The 952 bp sequence of znuA was amplified by PCR. B and C. The PCR product was digested with BsaAI enzyme and a 716 bp product was gel purified and cloned into pUCGM vector at the Smal site. D. To mutate znuA, an 850 bp gentamicin resistance gene was cloned at the BsaBI site of the znuA cloned into the pUCGM vector. E and F. The positive clone in the pUCGM vector was digested with Xbal enzyme and a 1570 bp fragment, containing znuA interrupted with gentamicin gene, was gel purified and cloned into Xbal site of pCVD422 vector. The positive clone in the pCVD422 vector was used for conjugation or electro-transformation of WT Y. ruckeri RSI 154 in order to mutate znuA by homologous recombination. 217 ZnuAFor ZnuA/?ev ~? ? J- 952 bp BsaAl BsaBI BsaAI (223 bp) (431 bp) (939 bp) BsaBI ± B 211bp*-^ 511 bp BsaBI Xbal Xbal znuA Gm znuA E D Xbal Xbal znuA Gm znuA E El znuA Gm znuA Xbal Xbal AmpR Levenase 218 APPENDIX 5 STRATEGY FOR CONSTRUCTION OF AN ISOGENIC B3-2 MUTANT A. The 1171 bp B3-2 sequence was amplified by PCR. B and C. The PCR product was digested with BstUI enzyme and a 785 bp product was gel purified and cloned into the pUCGM vector at Smal site. D. To mutate the B3-2 gene, an 850 bp gentamicin resistance gene was cloned at an MscI site of the B3-2 gene cloned into pUCGM vector. E and F. The positive clone in pUCGM vector was digested with Xbal enzyme and a 1635 bp fragment containing B3-2 gene interrupted with gentamicin gene was gel purified and cloned into the Xbal site of pCVD422 vector. The positive clone in pCVD422 vector was used for conjugation or electro-transformation of WT Y. ruckeri RS1154 in order to mutate B3-2 gene by homologous recombination. 219 B3-2Rev A B3-2For —J If- 1177bp Bstm Mscl BstUl (301 bp) (524 bp) (1084 bp) Mscl B JL 223 bp*-> 562 bp Mscl Xbal Xbal B3-2 Gm B3-2 D Xbal Xbal B3-2 Gm B3-2 E urn inn Amp Levenasi 220 APPENDIX 6 STRATEGY FOR CONSTRUCTION OF AN ISOGENIC A6-11 (ptrA) MUTANT A. The 1331 bp sequence of ptrA was amplified by PCR. B and C. The PCR product was digested with Hindi enzyme and an 1168 bp product was gel purified and cloned into the pUCGM vector at Smal site. D. To mutate the ptrA, an 850 bp gentamicin resistance gene was cloned at the Kprii site of the ptrA cloned into pUCGM vector. E and F. The positive clone in pUCGM vector was digested with Xbal enzyme and a 2018 bp fragment containing ptrA gene interrupted with gentamicin gene was gel purified and cloned into the Xbal site of pCVD422 vector. The positive clone in pCVD422 vector was used for conjugation or electro-transformation of WT Y. ruckeri RSI 154 in order to mutate ptrA by homologous recombination. 221 A6-11 For A6-ll/?ev J t + + 1177 bp Hindi Kpnl Hindi B Kpnl -• 875 bp -«-+-293 bp«- Kpnl ptrA jiiiiiiiiiiiiiiiiiiiiiiiin iiiiiimmiLimiiiiL Xbal y ^Xbal t pUCG m vector \ ptrA Gm ptrA D Xbal Xbal ptrA Gm ptrA E ptrA Gm ptrA Xbal Xbal Amp Levenasi 222 APPENDIX 7 STRATEGY FOR CONSTRUCTION OF AN ISOGENIC uvrY MUTANT A. A 629 bp sequence of the uvrY was amplified by PCR. B and C. The PCR product was digested with BsiUI and Sspl enzymes and a 536 bp product was gel purified and cloned into the pUCGM vector at Smal site. D. To mutate the uvrY, an 850 bp gentamicin resistance gene was cloned at the Kpnl site of the uvrY cloned into pUCGM vector. E and F. The positive clone in pUCGM vector was digested with Xbal enzyme and a 1386 bp fragment, containing the uvrY interrupted with gentamicin gene, was gel purified and cloned into the Xbal site of pCVD422 vector. The positive clone in pCVD422 vector was used for conjugation or electro-transformation of WT Y. ruckeri RSI 154 in order to mutate uvrY by homologous recombination. 223 ¥2-AFor F2-4/?ev ""? -J 629bp BstUl Kpnl Sspl (41 bp) (301 bp) (576 bp) Kpnl 260 bp 273 bp B Xbal Xbal uvrY Gm uvrY Xbal Xbal D uvrY Gm uvrY E uvrY Gm uvrY Xbal-^ itiiiiiiiiii •iiiiimimi ^ Xbal pCVD422 vector AmpK Levenasi^ 224