Department of Biological Sciences, Faculty of Science, National University of Singapore, 10 Kent Ridge Crescent, Singapore1192601
Author for correspondence: K. Y. Leung. Tel: +65 8747835. Fax: +65 7792486. e-mail: dbslky{at}nus.edu.sg
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ABSTRACT |
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Abbreviations: CAS, chrome azurol S; EPC, epithelioma papillosum of carp; ROI, reactive oxygen intermediates
The GenBank accession numbers for the sequences determined in this work are AF324338AF324342 (Table 3).
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INTRODUCTION |
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A more systematic approach to the study of Ed. tarda pathogenesis is needed to identify and characterize the virulence factors and their mechanisms in mediating diseases in fish and other animals. To understand the mode of pathogenesis, it is imperative to study the virulence factors that predispose a stressed population of fish to the disease. Among these, flagella-mediated motility is believed to be crucial in the early stages of pathogenicity, which involve stable attachment and mucosal colonization (Otteman & Miller, 1997 ). The persistence of bacterial infection depends on the pathogens ability to acquire nutrients such as iron from the host (Guerinot, 1994
; Finlay & Falkow, 1997
; Payne, 1993
). Most pathogens secrete high-affinity iron chelators (siderophores) and transport siderophoreFe3+ complexes into the cell interior (Mietzner & Morse, 1994
; Ferguson et al., 1998
). Pathogens may also produce catalase against phagosomal reactive oxygen intermediates (ROI) such as H2O2 (Miller & Britigan, 1997
) and other factors to evade non-specific defence systems of the host like the bactericidal effect of serum (Yano, 1996
).
Transposon mutagenesis can be used to examine various aspects of bacterial virulence factors and their genetic determinants (Lodge et al., 1995 ; Berg & Berg, 1996
; Bina et al., 1997
). Attenuated mutants created by the insertion of transposons in genes associated with virulence can help to elucidate hostpathogen interactions and invasion pathways. Most virulence factors are either on the bacterial surface or secreted such that they can interact with host components (Finlay, 1996
). In this study we therefore chose a transposon, TnphoA, to identify potential virulence genes involved in the production of secretory or membrane proteins. We isolated 440 PhoA+ fusion mutants of Ed. tarda PPD130/91 employing TnphoA mutagenesis. Disruption of genes involved in iron transport, serum survival, motility and catalase could attenuate the pathogen, impairing its ability to cause infection in fish. Our results suggest the possible correlation of these factors with the virulence of the pathogen; the information elucidated will enrich the existing knowledge on Ed. tarda pathogenesis.
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METHODS |
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Cell culture and media.
Epithelioma papillosum of carp, Cyprinus carpio (EPC) cells (Wolf & Mann, 1980 ) were grown in minimal essential medium (MEM) with Hanks balanced salts solution (HBSS) (Sigma), 10 mM HEPES (pH 7·3), 2 mM glutamine, 0·23% NaHCO3 and 10% (v/v) heat-inactivated fetal bovine serum at 25 °C in a 5% (v/v) CO2 atmosphere. Cells were grown in 75 ml flasks and split at least once a week by trypsin/EDTA treatment with dilution at 1:10 in fresh media. All tissue culture reagents were obtained from Gibco-BRL.
Transposon mutagenesis.
Ed. tarda (recipient; Colr) and E. coli (donor; Ampr Neor) cultures were grown statically in TSB at 25 °C and 37 °C, respectively. Conjugative transfer of the suicide plasmid pJM703.1::TnphoA was performed by plate mating. Briefly, the bacterial cell ratio of E. coli to Ed. tarda PPD130/91 was adjusted to 4:1, with a total of about 107 c.f.u. of both donor and recipient during mating. After 24 h of mating at 25 °C on TSA plates, the cells were harvested and resuspended in 3 ml phosphate-buffered saline (PBS) (123·2 mM NaCl, 10·4 mM Na2HPO4 and 3·2 mM KH2PO4, pH 7·2). Appropriate dilutions were plated on TSA supplemented with neomycin, colistin and 5-bromo-3-chloro-3-indolyl phosphate (XP) (Sigma) (40 µg ml-1) (TSANCX) to select for transconjugants of Ed. tarda. Blue PhoA+ fusion clones were purified by streaking on TSANCX plates.
Detection of mutants defective in motility, siderophore production and catalase activity.
Swarming motility was assayed according to Janda et al. (1991b ). Fresh bacterial culture was spotted on 0·4% motility agar. Mutants were considered non-motile if diffused growth around the colony was not observed. Modified M9 minimal medium (Collins & Thune, 1996
) supplemented with chrome azurol S (CAS) and 1·5% agar was used for checking siderophore production according to Neilands (1994
). An orange halo around the bacterial colony indicated siderophore production. The results of both these tests were scored after 48 h incubation at 25 °C. To assay catalase activity, a drop of 3% H2O2 solution was added to fresh bacterial colonies on TSA plates. Brisk effervescence was associated with the breakdown of H2O2 by endogenous catalase (Hertel et al., 1998
). Mutants that produced less catalase generated significantly less effervescence than the wild-type.
H2O2 inhibition zone test.
Ed. tarda strains were grown overnight at 25 °C in TSB, and harvested by centrifugation. After washing, the cells were resuspended in fresh TSB at OD540 0·5, and 2 ml of the cell suspension was added into 18 ml top-agar medium, containing TSB and 1% agar at 50 °C. The top agar was immediately plated onto a Petri dish. After solidification, sterile Whatman 3MM disks (0·6 cm diameter) containing 10 µl 2 mM, 20 mM or 200 mM H2O2 were placed on the surface. Zones of inhibition were visualized after incubation overnight at 25 °C (Xu & Pan, 2000 ).
Survival assay in blue gourami serum.
Blood was obtained from naive blue gourami, Trichogaster trichopterus (Pallas), and serum was separated from the clot by centrifugation at 4 °C. Bacteria grown in TSB at 25 °C were collected by centrifugation and washed three times in PBS. The bacterial suspension was then mixed with fresh gourami serum to give a final serum concentration of 50% and the bacterial count was adjusted to approximately 5x107 c.f.u. ml-1. Tubes containing bacteria and serum were incubated at 25 °C and 0·1 ml samples were removed after 1 h for serial dilutions and plate counts on TSA. Serum survival ability was calculated by dividing the number of viable bacteria after the serum treatment by the number of viable bacteria before treatment (Wang et al., 1998 ). A value >1 was scored as serum-resistant, while a value <1 indicated serum sensitivity. The data were obtained from three independent experiments.
Superoxide anion assay.
Phagocytes were isolated from the head kidney of naive blue gourami and purified according to Secombes (1990 ). Briefly, head kidney tissue was macerated and filtered through a stainless-steel mesh to produce a cell suspension in L-15 medium (Sigma). Following separation on a 3451% (v/v) Percoll (Sigma) gradient, the cells lying in the 3451% interface were harvested, washed and resuspended in L-15 with 5% (v/v) heat-inactivated fetal calf serum (Sigma). The cells were counted and approximately 0·5x106 cells were seeded into each well of a 96-well tissue culture plate (Falcon) followed by incubation for 2 h at 25 °C in a 5% (v/v) CO2 atmosphere. The wells seeded with phagocytes were then inoculated with bacteria at a 1:1 cell ratio and incubated for 30 min at 25 °C. Then 100 µl of 1 mg ml-1 nitro blue tetrazolium (Sigma) was added into each well and the cells were further incubated at 25 °C for 30 min in a 5% (v/v) CO2 atmosphere. The reaction was arrested with 100% methanol followed by a single wash with 70% methanol. After drying the plate for 1 min, 120 µl 2 M potassium hydroxide and 140 µl dimethyl sulfoxide (Sigma) were added. The A630 was measured using a microplate reader (BIO-TEK Instruments); the values presented, a measure of ROI production, are means±SEM of quadruplicate wells from one of the three independent experiments.
Adhesion and internalization assays.
These assays were performed as described previously with minor modifications (Tan et al., 1998 ; Wang & Leung, 2000
). Briefly, monolayers of EPC cells were grown for 72 h in 24-well tissue culture plates to 100% confluence. The cells were then washed with MEM and incubated at 25 °C for 30 min before inoculation of bacteria. The inoculated plates were centrifuged for 5 min at 800 g at 4 °C and then incubated for a further 30 min at 25 °C. To measure the number of bacteria adhering to the monolayers, the plates were washed six times with HBSS, the EPC cells lysed with 1% (v/v) Triton X-100 in PBS, and then bacterial numbers quantified by plate counting (Elsinghorst, 1994
). To measure internalization, monolayers were washed twice with HBSS, then incubated for an additional 2 h in tissue culture medium containing 100 µg gentamicin ml-1 (Sigma) to kill extracellular bacteria. After incubation, the monolayers were washed twice with HBSS, the EPC cells lysed with 1% Triton X-100 in PBS and bacterial numbers quantified by plate counting. The adhesion and internalization rates were calculated from the mean of at least two wells in quadruplicate experiments.
DNA manipulations and Southern analysis.
Bacterial genomic DNA was extracted according to the manuals of the QIAGEN Genomic DNA Purification Kit and the BIO 101 Genomic DNA Kit. Plasmid DNA was extracted using QIAGEN columns. Restriction endonuclease digestion was accomplished by standard methods (Sambrook et al., 1989 ). Southern blot analysis was carried out to characterize the transposon mutants of Ed. tarda PPD130/91 using the BluGene Non-Radioactive Nucleic Acid Detection System (Gibco-BRL). Transfer of DNA to nylon membrane (GeneScreen, NEN Research Products) and hybridization conditions were in accordance with standard methods (Sambrook et al., 1989
). Genomic DNA from Ed. tarda 130/91 and its mutants was digested with EcoRV, hybridized with HindIII-digested 14-dATP biotinylated (BioNick Labelling System, Gibco-BRL) pJM703.1 plasmid probe, and then visualized with streptavidinalkaline phosphatase conjugate (BluGene Nonradioactive Nucleic Acid Detection System, Gibco-BRL).
Characterization of transposon insertion sites and DNA sequencing.
Genomic DNA of the mutants was digested with EcoRV, which did not cut the transposon. Adaptors from the PCR-Select Bacterial Genome Subtraction Kit (Clontech) were then ligated to the Ed. tarda DNA fragments, by overnight incubation at 14 °C. PCR was performed using pfu Turbo polymerase (Stratagene) with the following cycle parameters: 30 cycles of 1·0 min at 94 °C, 30 s at 58 °C, 30 s at 72 °C and a final extension of 10 min at 72 °C. Primer 1 (Clontech) specific to the adaptor and forward (5'-GCACGATGAAGAGCAGAAGT-3') and reverse (5'-GGCATAATTACGTGCGGCAGT-3') primers specific to TnphoA, synthesized by Gibco-BRL, were used for amplification. The PCR product was cloned into pT-Adv (Clontech) or pGEM-T Easy vector (Ampr) (Promega), transformed into E. coli Top 10F' competent cells (Clontech) and spread on LB agar plates containing ampicillin, 40 µl 100 mM IPTG and 40 µl X-Gal (40 mg ml-1) for bluewhite screening of cloned DNA. Alternatively, fragments of mutant genomic DNA flanking the transposon obtained by complete digestion with BamHI were cloned into pBluescript SK+ (Ampr) vector cut with the same enzyme and transformed into E. coli TOP10F' cells as described above. Transformants bearing TnphoA and flanking Ed. tarda chromosomal sequences were selected by their ability to grow on LB agar containing ampicillin and neomycin.
DNA sequencing was carried out on an Applied Biosystems PRISM 377 automated DNA sequencer by the dye termination method. The ABI PRISM BigDye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems) was used. The sequences were edited by using the manufacturers software. Sequence assembly and further editing were done with DNASIS DNA analysis software (Hitachi Software). TBLASTN, TBLASTX and FASTA sequence homology analyses were performed by using the National Centre for Biotechnology Information BLAST network service.
LD50 determinations.
Naïve blue gourami weighing approximately 14 g each were purchased from commercial fish farms and acclimatized for 3 weeks. Three groups of 10 fish were injected intramuscularly or intraperitoneally with 0·1 ml PBS-washed bacterial cells adjusted to the required concentrations. A control group of fish each received 0·1 ml PBS. Fish were monitored for mortalities for 14 d and LD50 values were calculated by the method of Reed & Muench (1938 ).
Statistical analysis.
Data obtained from adhesion and internalization, serum survival and superoxide anion production assays are expressed as means±SEM. The data were analysed using one-way ANOVA and a Duncan multiple range test (SAS software, SAS Institute). Values of P<0·05 were considered statistically significant.
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RESULTS |
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Screening of Ed. tarda PPD130/91 mutants for four virulence factors
The putative virulence factors expressed by Ed. tarda PPD130/91 include siderophore and catalase production, motility and serum resistance. The 440 PhoA+ transposon mutants derived from this strain were screened to isolate mutants deficient in one or more of these phenotypes; eight such mutants were identified, and further grouped into four classes (Table 1).
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The five mutants belonging to classes IIa (210) and b (40, 172, 179 and 230) were sensitive to serum. Two of these (210 and 230) were also partially motile (Table 1). The class III mutant (34) was unable to swarm and showed completely defective motility when compared to the wild-type. It also produced less catalase. The class IV mutant (402) was deficient only in catalase production. Brisk effervescence was associated with the breakdown of H2O2 by the wild-type while catalase-deficient mutants (34 and 402) generated significantly less effervescence.
Further characterization of the TnphoA mutants
All eight of the above mutants were Amps, suggesting that there was no suicide plasmid retention and integration.
The H2O2 inhibition zone tests showed that the catalase-deficient mutants 34 and 402 were more vulnerable to killing by H2O2 than the wild-type. The inhibition zones for mutants 34 and 402 were 10·3±0·3 mm (n=3) and 14·3±0·3 mm (n=3), respectively, when they were incubated overnight with disks containing 200 mM H2O2. Smaller inhibition zones were observed for the wild-type and other transposon mutants (6·7±0·2 mm, n=3). All the mutants were analysed for their ability to induce production of reactive oxygen intermediates (ROI) in phagocytes. No significant differences were found between the catalase-deficient mutants and the wild-type (Table 2).
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Southern hybridization
The results of the Southern hybridization are shown in Fig. 1. All the mutants showed a single TnphoA insertion, except for mutants 210 (class IIa) and 402 (class IV), which had two distinct transposon insertions each. The size of the third band identified for mutant 402 was smaller than that of the transposon (7·7 kb), and therefore should not be due to a third transposon insertion. The hybridized fragments of all the classes were more than 8·0 kb in size. No band was found for the wild-type genomic DNA.
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Attenuation of virulence in fish
Both intramuscular and intraperitoneal routes of delivery were used to determine the LD50 values of the eight mutants. The dead fish had external signs of haemorrhage that were consistent with Ed. tarda infection. The LD50 calculations indicated that mutants 2A (class I) and 34 (class III) were attenuated compared to the wild-type (Table 2). There was a 1·1 (intraperitoneal route) to 1·5 (intramuscular route) log increase in the LD50 of mutant 34 when compared to the wild-type, while in the case of mutant 2A, the increase was about 1·0 log. The LD50 values of mutants belonging to classes IIa and b were slightly higher than that of mutant 402 (class IV) and the wild-type.
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DISCUSSION |
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Characterization of TnphoA mutants
The eight mutants isolated from the library were grouped into four classes. Class I consisted of the attenuated and siderophore-deficient mutant 2A. Classes IIa and b consisted of the slightly attenuated and serum-sensitive mutants 40, 172, 179, 210 and 230. Two of these (210 and 230) were also partially defective in motility. Class IIb mutants (40, 172, 179 and 230) were pleiotropic, exhibiting lower internalization rates and poor growth in phosphate-limiting medium. They had transposon insertions in the pst operon, homologous to that of Ent. cloacae and E. coli. Mutant 210 (class IIa) differed from class IIb mutants, in having the TnphoA insertion outside the pst operon. This mutant also showed a significantly higher rate of adhesion and internalization into EPC cells. The class III mutant was attenuated, non-motile and deficient in catalase production; it had the transposon insertion in a gene homologous to a catalase precursor gene (katB) of P. aeruginosa. The class IV mutant was deficient only in catalase production but it retained its virulence.
Siderophores and bacterial virulence
Mutant 2A (class I), deficient in siderophore production, exhibited lower virulence than the parent strain PPD130/91. The siderophore-mediated iron acquisition system is common to all species of Enterobacteriaceae (Dhaenens et al., 1999 ) including Ed. tarda (Kokubo et al., 1990
), and is necessary for bacterial survival in the host. Since most pathogens produce siderophores to secure iron from the host (Weinberg, 1998
), the lack of siderophore synthesis may reduce pathogenicity, as seen in mutant 2A and similar studies done on Neisseria gonorrhoeae (Yancey & Finkelstein, 1981
) and Vibrio anguillarum (Wertheimer et al., 1999
). Iron is important for the process of bacterial colonization and survival within the host (Mietzner & Morse, 1994
). However, iron metabolism may not be the only limiting factor for establishing septicaemia, as most Ed. tarda isolates, including avirulent strains, produce siderophores (Thune et al., 1993
).
Mutant 2A showed transposon interruption in a gene with significant homology to the gene encoding the arylsulfate sulfotransferase protein, AstA, of Ent. amnigenus (Baek et al., 1996 ). This enzyme catalyses the transfer of sulfate groups from phenylsulfate esters to phenolic compounds (Konishi-Imamura et al., 1995
) and may play an important role in the metabolism of phenolic compounds (Konishi-Imamura et al., 1994
). It is presently not clear how arylsulfate sulfotransferase affects the production of siderophores in Ed. tarda. Enterobactin is a catecholate (phenolate) siderophore produced by enteric bacteria (Neilands, 1981
). Interruption of the arylsulfate sulfotransferase gene as in mutant 2A may directly or indirectly affect the biosynthesis of siderophores such as enterobactin in Ed. tarda. Since siderophores are essential for intracellular survival of Mycobacterium tuberculosis (DeVoss et al., 2000
), it could be speculated that mutant 2A may have a reduced virulence in fish because it is deficient in siderophore production.
Catalase and bacterial virulence
The production of ROI such as superoxide and H2O2 is crucial for the optimal microbicidal activities of phagocytes in host defence (Miller & Britigan, 1997 ). In response, bacteria have developed complex strategies including cell-surface structures such as lipopolysaccharide and peptidoglycan, and production of enzymes such as catalase, to overcome the hostile environments. Our catalase-deficient mutants (34 and 402) were more vulnerable to killing by H2O2 than the wild-type, suggesting that catalase might be one of the virulence factors in Ed. tarda. The ability of the eight mutants to induce ROI production by phagocytes was also determined. There was no direct correlation between catalase deficiency and increased induction of ROI production in the mutants. The TnphoA library can be used further to screen for mutants sensitive to phagocyte-mediated killing. The resulting mutants and corresponding genes will be valuable for understanding the intimate relationship between phagocytes and intracellular pathogens.
Motility and bacterial virulence
Ed. tarda PPD130/91 exhibited pronounced motility when assayed on soft agar. Mutant 34 (class III) was deficient in motility and was also attenuated in fish. Its motility deficiency may have hindered its spread and colonization in the host. However, this mutant was not impaired in its ability to adhere to, and be internalized into, EPC cells. Other studies showed that non-motile mutants of Aeromonas hydrophila and V. anguillarum were unable to interact with fish cells (Merino et al., 1997 ; Ormonde et al., 2000
) and exhibited reduced pathogenicity in fish (Norqvist & Wolf-Watz, 1993
; OToole et al., 1996
). On the other hand, a partially motile mutant 210 (class IIa) obtained in this study, was found to be hyperadhesive and hyperinvasive, suggesting that motility might influence bacterial interaction with fish cells.
The reduction in catalase production and virulence in the non-motile mutant (34) might be due to a mutation of the catalase precursor gene homologous to the katB gene of P. aeruginosa (Brown et al., 1995 ). Since bacterial colonization of host tissue is affected by motility (Comolli et al., 1999
) and also catalase activity (Visick & Ruby, 1998
), mutant 34 may be attenuated due to its deficiency of both motility and catalase production. However, it cannot be concluded which of the two phenotypes are important for bacterial virulence since the catalase-deficient, but motile and non-attenuated mutant (402, class IV), showed double insertions of TnphoA. Further studies are needed to understand the pleiotropic nature and polar effects of the transposon mutations. Our results on bacterial motility and virulence need to be investigated further by green fluorescent protein (GFP) tagging using an immersion challenge model in blue gourami, to determine whether mutants defective in motility can invade and colonize fish.
Serum-sensitive mutants
Mutants 40, 172, 179 and 230 (class IIb), having TnphoA insertion in the pstS, pstA and pstB genes of the pst operon, were identified as being deficient in several phenotypes: serum resistance, motility, internalization into EPC cells and reduced growth in phosphate-limiting medium. Serum resistance of E. coli depends on the pst operon (Daigle et al., 1995 ) and perhaps also in Ed. tarda as seen in this study. The partial motility of mutant 230 may perhaps be due to its mutation in a gene homologous to the pstB gene of Ent. cloacae, which regulates phosphate transport, since both bacterial chemotaxis (Kusaka et al., 1997
) and motility (Rashid & Kornberg, 2000
) require phosphorus. However, this defect in motility did not abrogate virulence in fish. Mutants in class IIb had a lower internalization rate, compared to the wild-type (Table 2
). Although the precise mechanism of bacterial internalization remains unknown, it may be regulated by the pst operon whose mutation can also retard growth in phosphate-limiting medium.
Under phosphate-limiting conditions, phosphate assimilation is aided by the pst operon. Since mutation in this operon resulted in considerable changes in several phenotypes, perhaps this operon and phosphorus uptake may be important in the regulation of bacterial growth and metabolism (Wanner, 1987 ; Muda et al., 1992
). The regulation of Ed. tarda pathogenesis by the inorganic phosphate level is not known but this nutrient has been implicated in bacterial virulence (Demain, 1994
), and virulence factors such as haemolysin with phospholipase C (PLC) activity are induced in low-phosphate environments (Gray et al., 1982
). Our future experiments will focus on virulence factors that are specifically expressed under phosphate-limiting conditions.
Conclusions
In conclusion, TnphoA transposon mutagenesis was used to construct a library of fusion mutants from which several genes were identified that might play a role in Ed. tarda virulence. Attenuation of one or more of these virulence factors can be used to develop live vaccines to combat edwardsiellosis. More work needs to be done, especially in terms of characterizing and screening our remaining library of fusion mutants to identify more virulence genes. The information generated from this study will contribute to a better understanding of how Ed. tarda causes diseases in fish and other animals.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Amemura, M., Makino, K., Shinagawa, H., Kobayashi, A. & Nakata, A.(1985). Nucleotide sequence of the genes involved in phosphate transport and regulation of the phosphate regulon in Escherichia coli. J Mol Biol 184, 241-250.[Medline]
Baek, M. C., Kim, S. K., Kim, D. H., Kim, B. K. & Choi, E. C.(1996). Cloning and sequencing of the Klebsiella K-36 astA gene, encoding an arylsulfate sulphotransferase. Microbiol Immunol 40, 531-537.[Medline]
Berg, C. M. & Berg, D. E. (1996). Transposable element tools for microbial genetics. In Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology. pp. 25882612. Edited by F. C. Neihardt and others. Washington, DC: American Society for Microbiology.
Bina, J. E., Nano, F. & Hancock, R. E. W.(1997). Utilization of alkaline phosphatase fusions to identify secreted proteins, including potential efflux proteins and virulence factors from Helicobacter pylori. FEMS Microbiol Lett 148, 63-68.[Medline]
Brown, S. M., Howell, M. L., Vasil, M. L., Anderson, A. J. & Hassett, D. J.(1995). Cloning and characterisation of the katB gene of Pseudomonas aeruginosa encoding a hydrogen peroxide-inducible catalase: purification of katB, cellular localization, and demonstration that it is essential for optimal resistance to hydrogen peroxide. J Bacteriol 177, 6536-6544.[Abstract]
Chen, J. D., Lai, S. Y. & Huang, S. L.(1996). Molecular cloning, characterisation and sequencing of the hemolysin gene from Edwardsiella tarda. Arch Microbiol 165, 9-17.[Medline]
Collins, L. A. & Thune, R. L.(1996). Development of a defined minimal medium for the growth of Edwardsiella ictaluri. Appl Environ Microbiol 62, 848-852.[Abstract]
Comolli, J. C., Hauser, A. R., Waite, L., Whitchurch, C. B., Mattick, J. S. & Engel, J. N.(1999). Pseudomonas aeruginosa gene products PilT and PilU are required for cytotoxicity in vitro and virulence in a mouse model of acute pneumonia. Infect Immun 67, 3625-3630.
Daigle, F., Fairbrother, J. M. & Harel, J.(1995). Identification of a mutation in the pstphoU operon that reduces pathogenicity of an Escherichia coli strain causing septicemia in pigs. Infect Immun 63, 4924-4927.[Abstract]
Demain, A. L.(1994). Introduction: phosphate and survival of bacteria. In Phosphate in Microorganisms: Cellular and Molecular Biology , pp. 119. Edited by A. Torriani-Gorini, E. Yagil & S. Silver. Washington, DC:American Society for Microbiology.
DeVoss, J. J., Rutter, K., Schroeder, B. G., Su, H., Zhu, Y. & Barry, C. E.III(2000). The salicylate-derived mycobactin siderophores of Mycobacterium tuberculosis are essential for growth in macrophages. Proc Natl Acad Sci USA 97, 1252-1257.
Dhaenens, L., Szczebara, F., Van Nieuwenhuyse, S. & Husson, M. O.(1999). Comparison of iron uptake in different Helicobacter species. Res Microbiol 150, 475-481.[Medline]
Elsinghorst, E. A.(1994). Measurement of invasion by gentamicin resistance. Methods Enzymol 236, 405-420.[Medline]
Ferguson, A. D., Hofmann, E., Coulton, J. W., Diedrichs, K. & Welte, W.(1998). Siderophore-mediated iron transport: crystal structure of FhuA with bound polysaccharide. Science 282, 2215-2220.
Finlay, B. B.(1996). Bacterial diseases in diverse hosts. Cell 96, 315-318.
Finlay, B. B. & Falkow, S.(1997). Common themes in microbial pathogenicity revisited. Microbiol Mol Biol Rev 61, 136-169.[Abstract]
Gray, G. L., Berka, R. M. & Vasil, M. L.(1982). Phospholipase C regulatory mutation of Pseudomonas aeruginosa that results in constitutive synthesis of several phosphate-repressible proteins. J Bacteriol 150, 1221-1226.[Medline]
Guerinot, M. L.(1994). Microbial iron transport. Annu Rev Microbiol 48, 743-772.[Medline]
Hertel, C., Schmidt, G., Fischer, M., Oellers, K. & Hammes, W. P.(1998). Oxygen-dependent regulation of the expression of the catalase gene katA of Lactobacillus sakei LTH677. Appl Environ Microbiol 64, 1359-1365.
Hirono, I., Tange, N. & Aoki, T.(1997). Iron-regulated hemolysin gene from Edwardsiella tarda. Mol Microbiol 24, 851-856.[Medline]
Janda, J. M., Abbott, S. L. & Oshiro, L. S.(1991a). Penetration and replication of Edwardsiella spp. in Hep-2 cells. Infect Immun 59, 154-161.[Medline]
Janda, J. M., Abbott, S. L., Kroske-Bystrom, S., Cheung, W. K. W., Powers, C., Kokka, R. P. & Tamura, K.(1991b). Pathogenic properties of Edwardsiella species. J Clin Microbiol 29, 1997-2001.[Medline]
Kokubo, T., Iida, T. & Wakabayashi, H.(1990). Production of siderophore by Edwardsiella tarda. Fish Pathol 25, 237-241.
Konishi-Imamura, L., Sato, M., Dohi, K., Kadota, S., Namba, T. & Kobashi, K.(1994). Enzymatic sulfation of glycosides and their corresponding aglycones by arylsulfate sulfotransferase from a human intestinal bacterium. Biol Pharm Bull 17, 1018-1022.[Medline]
Konishi-Imamura, L., Kim, D. H., Koizumi, M. & Kobashi, K.(1995). Regulation of arylsulfate sulfotransferase from a human intestinal bacterium by nucleotides and magnesium ion. J Enzyme Inhib 8, 233-241.[Medline]
Kusaka, K., Shibata, K., Kuroda, A., Kato, J. & Ohtake, H.(1997). Isolation and characterisation of Enterobacter cloacae mutants which are defective in chemotaxis toward inorganic phosphate. J Bacteriol 179, 6192-6195.[Abstract]
Kusuda, R. & Salati, F.(1993). Major bacterial diseases affecting mariculture in Japan. Annu Rev Fish Dis 3, 69-85.
Ling, S. H. M., Wang, X. H., Xie, L., Lim, T. M. & Leung, K. Y.(2000). Use of green fluorescent protein (GFP) to study the invasion pathways of Edwardsiella tarda in in vivo and in vitro fish models. Microbiology 146, 7-19.
Lodge, J., Douce, G. R., Amin, I., Bolton, A. J., Martin, G. D., Chatfield, S., Dougan, G., Brown, N. L. & Stephen, J.(1995). Biological and genetic characterisation of TnphoA mutants of Salmonella typhimurium TML in the context of gastroenteritis. Infect Immun 63, 762-769.[Abstract]
Manoil, C. & Beckwith, J.(1985). TnphoA: a transposon probe for protein export signals. Proc Natl Acad Sci USA 82, 8129-8133.[Abstract]
Mietzner, T. A. & Morse, S. A.(1994). The role of iron-binding proteins in the survival of pathogenic bacteria. Annu Rev Nutr 14, 471-493.[Medline]
Miller, R. A. & Britigan, B. E.(1997). Role of oxidants in microbial pathophysiology. Clin Microbiol Rev 10, 1-18.[Abstract]
Merino, S., Rubires, X., Aguilar, A. & Tomas, J. M.(1997). The role of flagella and motility in the adherence and invasion to fish cell lines by Aeromonas hydrophila serogroup O:34 strains. FEMS Microbiol Lett 151, 213-217.[Medline]
Muda, M., Rao, N. N. & Torriani, A.(1992). Role of PhoU in phosphate transport and alkaline phosphatase regulation. J Bacteriol 174, 8057-8064.[Abstract]
Neilands, J. B.(1981). Microbial iron compounds. Annu Rev Biochem 50, 715-731.[Medline]
Neilands, J. B.(1994). Identification and isolation of mutants defective in iron acquisition. Methods Enzymol 235, 352-356.[Medline]
Norqvist, A. & Wolf-Watz, H.(1993). Characterisation of a novel chromosomal virulence locus involved in expression of a major surface flagellar sheath antigen of the fish pathogen Vibrio anguillarum. Infect Immun 61, 2434-2444.[Abstract]
Ormonde, P., Hörstedt, P., OToole, R. & Milton, D. L.(2000). Role of motility in adherence to and invasion of a fish cell line by Vibrio anguillarum. J Bacteriol 182, 2326-2328.
OToole, R., Milton, D. M. & Wolf-Watz, H.(1996). Chemotactic motility is required for invasion of the host by the fish pathogen Vibrio anguillarum. Mol Microbiol 19, 625-637.[Medline]
Otteman, K. M. & Miller, J. F.(1997). Roles for motility in bacterialhost interactions. Mol Microbiol 24, 1109-1117.[Medline]
Payne, S. M.(1993). Iron acquisition in microbial pathogenesis. Trends Microbiol 1, 66-69.[Medline]
Plumb, J. A. (1993). Bacterial Diseases of Fish. Edited by N. R. Bromage, V. Inglis & R. J. Roberts. Oxford: Blackwell Scientific.
Rashid, M. H. & Kornberg, A.(2000). Inorganic polyphosphate is needed for swimming, swarming, and twitching motilities of Pseudomonas aeruginosa. Proc Natl Acad Sci USA 97, 4885-4890.
Rashid, M. M., Honda, K., Nakai, T. & Muroga, K.(1994). An ecological study on Edwardsiella tarda in flounder farms. Fish Pathol 29, 221-227.
Reed, L. J. & Muench, H.(1938). A simple way of estimating fifty percent end points. Am J Hyg 27, 493-497.
Sambrook, J. Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Secombes, C. J.(1990). Isolation of salmonid macrophages and analysis of their killing activity. In Techniques in Fish Immunology , pp. 137-154. Edited by J. S. Stolen, T. C. Fletcher, D. P. Anderson, B. S. Robertson & W. B. van Muiswinkel. Fair Haven, NJ:SOS.
Strauss, E. J., Ghori, N. & Falkow, S.(1997). An Edwardsiella tarda strain containing a mutation in a gene with homology to sglB and hpmB is defective for entry into epithelial cells in culture. Infect Immun 65, 3924-3932.[Abstract]
Suprapto, H., Hara, T., Nakai, T. & Muroga, K.(1996). Purification of a lethal toxin of Edwardsiella tarda. Fish Pathol 31, 203-207.
Tan, E., Low, K. W., Wong, W. S. F. & Leung, K. Y.(1998). Internalization of Aeromonas hydrophila by fish cells can be inhibited with a tyrosine kinase inhibitor. Microbiology 144, 299-307.[Abstract]
Thune, R. L., Stanley, L. A. & Cooper, R. K.(1993). Pathogenesis of gram-negative bacterial infections in warmwater fish. Annu Rev Fish Dis 3, 37-68.
Visick, K. L. & Ruby, E. G.(1998). The periplasmic Group III catalase of Vibrio fischeri is required for normal symbiotic competence and is induced both by oxidative stress and by approach to stationary phase. J Bacteriol 180, 2087-2092.
Wang, X. H. & Leung, K. Y.(2000). Biochemical characterization of different types of adherence of Vibrio species to fish epithelial cells. Microbiology 146, 989-998.
Wang, X. H., Oon, H. L., Ho, G. W. P., Wong, W. S. F., Lim, T. M. & Leung, K. Y.(1998). Internalization and cytotoxicity are important virulence mechanisms in vibriofish epithelial cell interactions. Microbiology 144, 2987-3002.[Abstract]
Wanner, B. L.(1987). Bacterial alkaline phosphatase gene regulation and the phosphate response in Escherichia coli. In Phosphate Metabolism and Cellular Regulation in Microorganisms , pp. 12-19. Edited by A. Torriani-Gorini, F. G. Rothman, S. Silver, A. Wright & E. Yagil. Washington, DC:American Society for Microbiology.
Weinberg, E. D.(1998). Patho-ecological implications of microbial acquisition of host iron. Rev Med Microbiol 9, 171-178.
Wertheimer, A. M., Verweij, W., Chen, Q., Crosa, L. M., Nagasawa, M., Tolmasky, M. E., Actis, L. A. & Crosa, J. H.(1999). Characterisation of the angR gene of Vibrio anguillarum: essential role in virulence. Infect Immun 67, 6496-6509.
Wolf, K. & Mann, J. A.(1980). Poikilotherm vertebrate cell lines and viruses, a current listing for fishes. In Vitro 16, 168-179.[Medline]
Xu, X. Q. & Pan, S. Q.(2000). An Agrobacterium catalase is a virulence factor involved in tumourigenesis. Mol Microbiol 35, 407-414.[Medline]
Yancey, R. J. & Finkelstein, R. A.(1981). Siderophore production by pathogenic Neisseria spp. Infect Immun 32, 600-608.[Medline]
Yano, T.(1996). The nonspecific immune system: humoral defense. In The Fish Immune System: Organism, Pathogen, and Environment , pp. 105-157. Edited by G. K. Iwama & T. Nakanishi. New York:Academic Press.
Received 28 July 2000;
revised 25 October 2000;
accepted 6 November 2000.