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Volume 148, Issue 7

Isolation and analysis of a protease gene with an ABC transport system in the fish pathogen : insertional mutagenesis and involvement in virulence

aThe GenBank accession numbers for the sequences reported in this paper are AJ318052 () and AJ421517 ( and ).

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Abstract

is a Gram-negative pathogen that causes enteric redmouth disease in salmonids. A gene from encoding an extracellular protease termed ( rotease ) was cloned from a 3AI library constructed in pUC19 and analysed in gelatin-supplemented medium. The nucleotide sequence of the gene indicated an ORF encoding a protein of 477 aa. On the basis of the high degree of homology in the amino acid sequence as well as its conservative motifs, this protein was included within the serralysin metalloendopeptidase subfamily (EC 3.4.24.12). The N-terminal sequence showed a 14 aa propeptide followed by a 10 aa sequence identical to the one deduced previously from the 47 kDa purified protease. Additional results demonstrated that the gene encodes the 47 kDa protein. In contrast to other species, the protease is secreted by a type I Gram-negative bacterial ABC exporter protein secretion system composed of three genes termed , and , and a protease inhibitor . The development of genetic methods for this species has allowed the exploration of the organization and the putative role of the Yrp1 genetic locus. Thus, site-directed insertion mutations into the and the genes were constructed by the integration of the mobilizable suicide vector pIVET8 containing internal portions of both coding sequences. Complementation studies of those mutants with different loci indicated that they are organized as a single operon. The mutant strains lacked protease activity as well as the Yrp1 protein and, although physiologically similar to the parental strain when growing on nutrient broth medium, they were attenuated in virulence when bacteria were injected intraperitoneally into rainbow trout (). This is the first report of defined mutations in to show the implication of a factor such as an extracellular protease in pathogenesis.

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Keyword(s): enteric redmouth disease , pathogenicity and type I secretion system
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2002-07-01
2024-04-19
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References

  1. Bearden S. W., Perry R. D. 1999; The Yfe system of Yersinia pestis transports iron and manganese is required for full virulence of plague. Mol Microbiol 32:403–414 [CrossRef]
     [Google Scholar]
  2. Bertolini J. M., Wakabayashi H., Watral V. G., Whipple M. J., Rohovec J. S. 1994; Electrophoretic detection of proteases from selected strains of Flexibacter psychrophilus and assessment of their variability. J Aquat Anim Health 6:224–233 [CrossRef]
     [Google Scholar]
  3. Binet R., Létoffé S., Ghigo J., Delapelaire P., Wandersman C. 1997; Protein secretion by gram negative bacteria ABC export: a review. Gene 192:7–11 [CrossRef]
     [Google Scholar]
  4. Birnboim H. C., Doly J. 1979; A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res 7:1513–1523 [CrossRef]
     [Google Scholar]
  5. Braunagel S. C., Benedin M. S. 1990; The metalloprotease gene of Serratia marcescens strain SM6. Mol Gen Genet 222:446–451 [CrossRef]
     [Google Scholar]
  6. Busch R. A., Lingg A. J. 1974; Establishment of a symptomatic carrier state infection of enteric redmouth disease in rainbow trout ( Salmo gairdneri) . J Fish Res Board Can 32:2429–2432
     [Google Scholar]
  7. Carniel E. 1999; The Yersinia high-pathogenicity island. Int Microbiol 2:161–167
     [Google Scholar]
  8. Cascón A., Fregeneda J., Aller M., Yugueros J., Temprano A., Hernanz C., Sánchez M., Rodriguez-Aparicio L., Naharro G. 2000a; Cloning, characterization, and insertional inactivation of a major extracellular serine protease gene with elastolytic activity from Aeromonas hydrophila . J Fish Dis 23:1–11 [CrossRef]
     [Google Scholar]
  9. Cascón A., Yugueros J., Temprano A., Sánchez M., Hernanz C., Luengo J. M., Naharro G. 2000b; A major secreted elastase is essential for pathogenicity of Aeromonas hydrophila . Infect Immun 68:3233–3241 [CrossRef]
     [Google Scholar]
  10. Dahler G. S., Barras F., Keen N. T. 1990; Cloning of genes encoding extracellular metalloproteases from Erwinia chrysanthemi EC16. J Bacteriol 172:5803–5815
     [Google Scholar]
  11. Davies R. L. 1991; Yersinia ruckeri produces four iron-regulated outer membrane proteins but does not produce detectable siderophores. J Fish Dis 14:563–570 [CrossRef]
     [Google Scholar]
  12. Denkin S. M., Nelson D. R. 1999; Induction of protease activity in Vibrio anguillarum by gastrointestinal mucus. Appl Environ Microbiol 65:3555–3560
     [Google Scholar]
  13. Doug F., Lazdunski A., Cami B., Murgier M. 1992; Sequence of a cluster of genes controlling synthesis and secretion of alkaline protease in Pseudomonas aeruginosa : relationships to other secretory pathways. Gene 121:47–54 [CrossRef]
     [Google Scholar]
  14. Everlyn T. P. T. 1996; Infection and disease. In The Fish Immune System: Organism, Pathogen and Environment pp 339–362 Edited by Iwana G., Nakanishi T. San Diego: Academic Press;
     [Google Scholar]
  15. Furones M. D., Gilpin M. L., Alderman D. J., Munn C. B. 1990; Virulence of Yersinia ruckeri serotype 1 strains is associated with a heat sensitive factor (HSF) in cell extracts. FEMS Microbiol Lett 66:339–344 [CrossRef]
     [Google Scholar]
  16. Furones M. D., Gilpin M. L., Munn C. B. 1993; Culture media for the differentiation of isolates of Yersinia ruckeri based on detection of a virulence factor. J Appl Bacteriol 74:360–366 [CrossRef]
     [Google Scholar]
  17. Ghigo J. M., Wandersman C. 1992a; Cloning, nucleotide sequence and characterization of the gene encoding the Erwinia chrysanthemi B374 PrtA metalloprotease: a third metalloprotease secreted via a C-terminal secretion signal. Mol Gen Genet 236:135–144
     [Google Scholar]
  18. Ghigo J. M., Wandersman C. 1992b; A fourth metalloprotease gene in Erwinia chrysanthemi . Res Microbiol 143:857–867 [CrossRef]
     [Google Scholar]
  19. Gong S., Bearden S. W., Geoffroy V. A., Fetherston J. D., Perry R. D. 2001; Characterization of the Yersinia pestis Yfu ABC inorganic iron transport system. Infect Immun 67:2829–2837
     [Google Scholar]
  20. Griffin B. R. 1987; Columnaris disease: recent advances in research. Aquaculture 13:48–50
     [Google Scholar]
  21. Gunnlaugsdottir B., Gudmundsdottir B. K. 1997; Pathogenicity of atypical Aeromonas salmonicida in Atlantic salmon compared with protease production. J Appl Microbiol 83:543–541
     [Google Scholar]
  22. Huek C. 1998; Type III protein secretion systems in bacterial pathogens in plants and animals. Microbiol Mol Biol Rev 62:379–433
     [Google Scholar]
  23. Idei A., Kawai E., Akatsura H., Omori H. 1999; Cloning and characterization of the Pseudomonas fluorescens ATP-binding cassette exporter Has DEF, for the heme acquisition protein Has A. J Bacteriol 181:7545–7551
     [Google Scholar]
  24. Létoffé S., Delapelaire P., Wandersman C. 1990; Protease secretion by Erwinia chrysanthemi . EMBO J 9:1375–1382
     [Google Scholar]
  25. Leung K. Y., Stevenson R. M. W. 1988; Tn 5 induced protease deficient strains of Aeromonas hydrophila with reduced virulence for fish. Infect Immun 56:2639–2644
     [Google Scholar]
  26. Liao C. H., McCallus D. E. 1998; Biochemical and genetic characterization of an extracellular protease from Pseudomonas fluorescens CY091. Appl Environ Microbiol 64:914–921
     [Google Scholar]
  27. Mahan M. J., Tobias J. W., Slauch J. M., Hanna P. C., Collier R. J., Mekalanos J. J. 1995; Antibiotic-based selection for bacterial genes that are specifically induced during infection of a host. Proc Natl Acad Sci USA 92:669–673 [CrossRef]
     [Google Scholar]
  28. Milton D. L., Norquist A., Wolf-Watz H. 1992; Cloning of a metalloprotease gene involved in the virulence mechanism of Vibrio anguillarum . J Bacteriol 174:7235–7244
     [Google Scholar]
  29. Nakahama K., Yoshimura K., Marumoto R., Kikuchi M., Lee I. S., Hase T., Matsubara H. 1986; Cloning and sequencing of Serratia protease gene. Nucleic Acids Res 14:5843–5855 [CrossRef]
     [Google Scholar]
  30. Norqvist A., Norrman B., Wolf-Watz H. 1990; Identification and characterization of a zinc metalloprotease associated with invasion by the fish pathogen Vibrio anguillarum . Infect Immun 58:3731–3736
     [Google Scholar]
  31. Rawlings N. D., Barrett A. J. 1995; Evolutionary families of metallopeptidases. Methods Enzymol 248:183–228
     [Google Scholar]
  32. Reed L. J., Muench H. 1938; A simple method of estimating fifty percent endpoints. Am J Hyg 27:493–497
     [Google Scholar]
  33. Romalde J. L., Toranzo A. E. 1993; Pathological activities of Yersinia ruckeri , the enteric redmouth (ERM) bacterium. FEMS Microbiol Lett 112:291–300 [CrossRef]
     [Google Scholar]
  34. Romalde J. L., Lemos M. L., Conchas R. F., Bandı́n I., Toranzo A. E. 1990; Adhesive properties and other virulence factors in Yersinia ruckeri . In Pathology in Marine Science pp 123–139 Edited by Cheng T. C., Perkins F. O. New York: Academic Press;
     [Google Scholar]
  35. Romalde J. L., Conchas R. F., Toranzo A. E. 1991; Evidence that Yersinia ruckeri possesses a high affinity iron uptake system. FEMS Microbiol Lett 80:121–126 [CrossRef]
     [Google Scholar]
  36. Rossi M. S., Fetherston J. D., Létoffé S., Carniel E., Perry R. D., Ghigo J. M. 2001; Identification and characterization of the hemophore-dependent heme acquisition system of Yersinia pestis . Infect Immun 69:6707–6717 [CrossRef]
     [Google Scholar]
  37. Sakai D. K. 1985; Loss of virulence in a protease-deficient mutant of Aeromonas salmonicida . Infect Immun 48:146–152
     [Google Scholar]
  38. Saken E., Rakin A., Heesemann J. 2000; Molecular characterization of a novel siderophore-independent iron transport system in Yersinia . Int J Med Microbiol 290:51–60 [CrossRef]
     [Google Scholar]
  39. Sambrook J., Fritsch E. F., Maniatis T. 1989 Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
     [Google Scholar]
  40. Secades P., Guijarro J. A. 1999; Purification and characterization of an extracellular protease from the fish pathogen Yersinia ruckeri and effect of culture conditions on production. Appl Environ Microbiol 65:3969–3975
     [Google Scholar]
  41. Secades P., Alvarez B., Guijarro J. A. 2001; Purification and characterization of a psychrophilic calcium induced, growth-phase-dependent metalloprotease from the fish pathogen Flavobacterium psychrophilum . Appl Environ Microbiol 67:2436–2444 [CrossRef]
     [Google Scholar]
  42. Simon R., Priefer V., Puhler A. 1983; A broad host range mobilization system for in vitro genetic engineering: transposon mutagenesis in Gram negative bacteria. Biotechnology 1:784–791 [CrossRef]
     [Google Scholar]
  43. Stevenson R. M. W. 1997; Immunization with bacterial antigens: yersiniosis. Dev Biol Stand 90:117–124
     [Google Scholar]
  44. Stover C. K., Pham X. Q., Erwin A. L. 23 other authors 2000; Complete genome sequence of Pseudomonas aeruginosa PAO1, an opportunistic pathogen. Nature 406:954–964 [CrossRef]
     [Google Scholar]
  45. Vieira J., Messing J. 1991; New pUC derived cloning vectors with different selectable markers and DNA replication origins. Gene 100:189–194 [CrossRef]
     [Google Scholar]
  46. Vipond R., Bricknell I. R., Durant E., Bowden T. J., Ellis A. E., Smith M., MacIntyre S. 1998; Defined deletion mutants demonstrate that the major secreted toxins are not essential for the virulence of Aeromonas salmonicida . Infect Immun 66:1990–1998
     [Google Scholar]
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Isolation and analysis of a protease gene with an ABC transport system in the fish pathogen Yersinia ruckeri: insertional mutagenesis and involvement in virulenceaaThe GenBank accession numbers for the sequences reported in this paper are AJ318052 (yrp1) and AJ421517 (yrpDEF and inh).
Microbiology 148, 2233 (2002); https://doi.org/10.1099/00221287-148-7-2233
/content/journal/micro/10.1099/00221287-148-7-2233
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