1887

Abstract

is a very significant fish pathogen that secretes two biochemically characterized extracellular proteolytic enzymes, Fpp1 and Fpp2. The genes encoding these enzymes are organized as an tandem in the genome of strain THC02/90. Analysis of the corresponding encoded proteins showed that they belong to two different protease families. For gene function analysis, new genetic tools were developed in by constructing stable isogenic and mutants via single-crossover homologous recombination. RT-PCR analysis of wild-type and mutant strains suggested that both genes are transcribed as a single mRNA from the promoter located upstream of the gene. Phenotypic characterization of the mutant showed lack of caseinolytic activity and higher colony spreading compared with the wild-type strain. Both characteristics were recovered in the complemented strain. One objective of this work was to assess the contribution to virulence of these proteolytic enzymes. LD experiments using the wild-type strain and mutants showed no significant differences in virulence in a rainbow trout challenge model, suggesting instead a possible nutritional role. The gene disruption procedure developed in this work, together with the knowledge of the complete genome sequence of , open new perspectives for the study of gene function in this bacterium.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.046938-0
2011-04-01
2024-04-19
Loading full text...

Full text loading...

/deliver/fulltext/micro/157/4/1196.html?itemId=/content/journal/micro/10.1099/mic.0.046938-0&mimeType=html&fmt=ahah

References

  1. Abe N., Kadowaki T., Okamoto K., Nakayama K., Ohishi M., Yamamoto K. 1998; Biochemical and functional properties of lysine-specific cysteine proteinase (Lys-gingipain) as a virulence factor of Porphyromonas gingivalis in periodontal disease. J Biochem 123:305–312
    [Google Scholar]
  2. Agarwal S., Hunnicutt D. W., McBride M. J. 1997; Cloning and characterization of the Flavobacterium johnsoniae ( Cytophaga johnsonae ) gliding motility gene, gldA . Proc Natl Acad Sci U S A 94:12139–12144
    [Google Scholar]
  3. Álvarez B., Guijarro J. A. 2007; Recovery of Flavobacterium psychrophilum viable cells using a charcoal-based solid medium. Lett Appl Microbiol 44:569–572
    [Google Scholar]
  4. Álvarez B., Secades P., McBride M. J., Guijarro J. A. 2004; Development of genetic techniques for the psychrotrophic fish pathogen Flavobacterium psychrophilum . Appl Environ Microbiol 70:581–587
    [Google Scholar]
  5. Álvarez, B., Álvarez, J., Menéndez A., Guijarro J. A. 2008; A mutant in one of two exbD loci of a TonB system in Flavobacterium psychrophilum shows attenuated virulence and confers protection against cold water disease. Microbiology 154:1144–1151
    [Google Scholar]
  6. Arai H., Morita Y., Izumi S., Katagiri T., Kimura H. 2007; Molecular typing by pulsed-field gel electrophoresis of Flavobacterium psychrophilum isolates derived from Japanese fish. J Fish Dis 30:345–355
    [Google Scholar]
  7. Bertolini J. M., Wakabayashi H., Watral V. G., Whipple M. J., Rohovec J. S. 1994; Electrophoretic detection of proteases from selected strains of Flexibacter psychrophilum and assessment of their variability. J Aquat Anim Health 6:224–233
    [Google Scholar]
  8. Bhogal P. S., Slakeski N., Reynolds E. C. 1997; A cell-associated protein complex of Porphyromonas gingivalis W50 composed of Arg- and Lys-specific cysteine proteinases and adhesins. Microbiology 143:2485–2495
    [Google Scholar]
  9. Bjornsdottir B., Fridjonsson O. H., Magnusdottir S., Andresdottir V., Hreggvidsson G. O., Gudmundsdottir B. K. 2009; Characterisation of an extracellular vibriolysin of the fish pathogen Moritella viscosa . Vet Microbiol 136:326–334
    [Google Scholar]
  10. Cepeda C., Santos Y. 2000; Rapid and low-level toxic PCR-based method for routine identification of Flavobacterium psychrophilum . Int Microbiol 3:235–238
    [Google Scholar]
  11. Cepeda C., , García-Márquez S., Santos Y. 2004; Improved growth of Flavobacterium psychrophilum using a new culture medium. Aquaculture 238:75–82
    [Google Scholar]
  12. Chakroun C., Grimont F., Urdaci M. C., Bernardet J.-F. 1998; Fingerprinting of Flavobacterium psychrophilum isolates by ribotyping and plasmid profiling. Dis Aquat Organ 33:167–177
    [Google Scholar]
  13. Chen S., Bagdasarian M., Kaufman M. G., Walker E. D. 2007; Characterization of strong promoters from an environmental Flavobacterium hibernum strain by using a green fluorescent protein-based reporter system. Appl Environ Microbiol 73:1089–1100
    [Google Scholar]
  14. Clemmer K. M., Rather P. N. 2008; The Lon protease regulates swarming motility and virulence gene expression in Proteus mirabilis . J Med Microbiol 57:931–937
    [Google Scholar]
  15. Curtis M. A., Aduse-Opoku J., Rangarajan M. 2001; Cysteine proteases of Porphyromonas gingivalis . Crit Rev Oral Biol Med 12:192–216
    [Google Scholar]
  16. del Cerro A., Mendoza M. C., Guijarro J. A. 2002; Usefulness of a TaqMan-based polymerase chain reaction assay for the detection of the fish pathogen Flavobacterium psychrophilum . J Appl Microbiol 93:149–156
    [Google Scholar]
  17. Duchaud E., Boussaha M., Loux V., Bernardet J. F., Michel C., Kerouault B., Mondot S., Nicolas P., Bossy R. other authors 2007; Complete genome sequence of the fish pathogen Flavobacterium psychrophilum . Nat Biotechnol 25:763–769
    [Google Scholar]
  18. Fernández L., López J. R., Secades P., Menéndez A., Márquez I., Guijarro J. A. 2003; In vitro and in vivo studies of the Yrp1 protease from Yersinia ruckeri and its role in protective immunity against enteric red mouth disease of salmonids. Appl Environ Microbiol 69:7328–7335
    [Google Scholar]
  19. Fujiwara-Nagata E., Eguchi M. 2009; Development and evaluation of a loop-mediated isothermal amplification assay for rapid and simple detection of Flavobacterium psychrophilum . J Fish Dis 32:873–881
    [Google Scholar]
  20. Gardel C. L., Mekalanos J. J. 1996; Alterations in Vibrio cholerae motility phenotypes correlate with changes in virulence factor expression. Infect Immun 64:2246–2255
    [Google Scholar]
  21. Gupta M., Rao K. K. 2009; Epr plays a key role in DegU-mediated swarming motility of Bacillus subtilis . FEMS Microbiol Lett 295:187–194
    [Google Scholar]
  22. Izumi S., Aranishi F., Wakabayashi H. 2003; Genotyping of Flavobacterium psychrophilum using PCR-RFLP analysis. Dis Aquat Organ 56:207–214
    [Google Scholar]
  23. Kadowaki T., Yoneda M., Okamoto K., Maeda K., Yamamoto K. 1994; Purification and characterization of a novel arginine-specific cysteine proteinase (argingipain) involved in the pathogenesis of periodontal disease from the culture supernatant of Porphyromonas gingivalis . J Biol Chem 269:21371–21378
    [Google Scholar]
  24. Karlsson E. N., Hachem M. A., Ramchuran S., Costa H., Holst O., Svenningsen S. F., Hreggvidsson G. O. 2004; The modular xylanase Xyn10A from Rhodothermus marinus is cell-attached, and its C-terminal domain has several putative homologues among cell-attached proteins within the phylum Bacteroidetes. FEMS Microbiol Lett 241:233–242
    [Google Scholar]
  25. Li L.-Y., Shoemaker N. B., Salyers A. A. 1995; Location and characteristics of the transfer region of a Bacteroides conjugative transposon and regulation of transfer genes. J Bacteriol 177:4992–4999
    [Google Scholar]
  26. Lorenzen E., Olesen N. J. 1997; Characterization of isolated Flavobacterium psychrophilum associated with coldwater disease or rainbow trout fry syndrome. II. Serological studies. Dis Aquat Organ 31:209–220
    [Google Scholar]
  27. Madsen L., Dalsgaard I. 1998; Characterization of Flavobacterium psychrophilum ; comparison of proteolytic activity and virulence of strains isolated from trout ( Oncorhynchus mykiss . In Methodology in Fish Disease Research pp 45–52 Edited by Barnes A. C., Davidson G. A., Hiney M. P., McIntosh D. Aberdeen, Scotland: Fisheries Research Service;
    [Google Scholar]
  28. Michel C., Antonio D., Hedrick R. P. 1999; Production of viable cultures of Flavobacterium psychrophilum : approach and control. Res Microbiol 150:351–358
    [Google Scholar]
  29. Møller J. D., Larsen J. L., Madsen L., Dalsgaard I. 2003; Involvement of a sialic acid-binding lectin with hemagglutination and hydrophobicity of Flavobacterium psychrophilum . Appl Environ Microbiol 69:5275–5280
    [Google Scholar]
  30. Nicolas P., Mondot S., Achaz G., Bouchenot C., Bernardet J.-F., Duchaud E. 2008; Population structure of the fish-pathogenic bacterium Flavobacterium psychrophilum . Appl Environ Microbiol 74:3702–3709
    [Google Scholar]
  31. Pathirana R. D., O'Brien-Simpson N. M., Veith P. D., Riley P. F., Reynolds E. C. 2006; Characterization of proteinase–adhesin complexes of Porphyromonas gingivalis . Microbiology 152:2381–2394
    [Google Scholar]
  32. Pérez-Pascual, D., Menéndez, A., Fernández, L., Méndez J., Reimundo P., Navais R., Guijarro J. A. 2009; Spreading versus biomass production by colonies of the fish pathogen Flavobacterium psychrophilum : role of the nutrient concentration. Int Microbiol 12:207–214
    [Google Scholar]
  33. Pike R. N., McGraw W., Potempa J., Travis J. 1994; Lysine- and arginine-specific proteinases from Porphyromonas gingivalis . Isolation, characterization, and evidence for the existence of complexes with hemagglutinins. J Biol Chem 269:406–411
    [Google Scholar]
  34. Rangarajan M., Smith S. J. M., Curtis M. A. 1997; Biochemical characterisation of the arginine-specific proteases of Porphyromonas gingivalis W50 suggests a common precursor. Biochem J 323:701–709
    [Google Scholar]
  35. Reed L. J., Muench H. 1938; A simple method of estimating fifty percent endpoints. Am J Hyg 27:493–497
    [Google Scholar]
  36. Secades P., , Álvarez 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
    [Google Scholar]
  37. Secades P., , Álvarez B., Guijarro J. A. 2003; Purification and properties of a new psychrophilic metalloprotease (Fpp2) in the fish pathogen Flavobacterium psychrophilum . FEMS Microbiol Lett 226:273–279
    [Google Scholar]
  38. 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
    [Google Scholar]
  39. Soule M., LaFrentz S., Cain K., LaPatra S., Call D. R. 2005; Polymorphisms in 16S rRNA genes of Flavobacterium psychrophilum correlate with elastin hydrolysis and tetracycline resistance. Dis Aquat Organ 65:209–216
    [Google Scholar]
  40. Szeto C. Y. Y., Leung G. S., Kwan H. S. 2007; Le.MAPK and its interacting partner, Le.DRMIP, in fruiting body development in Lentinula edodes . Gene 393:87–93
    [Google Scholar]
  41. Urdaci M. C., Chakroun C., Faure D., Bernardet J.-F. 1998; Development of a polymerase chain reaction assay for identification and detection of the fish pathogen Flavobacterium psychrophilum . Res Microbiol 149:519–530
    [Google Scholar]
  42. Yang H., Chen J., Yang G., Zhang X.-H., Li Y. 2007; Mutational analysis of the zinc metalloprotease EmpA of Vibrio anguillarum . FEMS Microbiol Lett 267:56–63
    [Google Scholar]
  43. Zhang W. W., Hu Y. H., Wang H. L., Sun L. 2009; Identification and characterization of a virulence-associated protease from a pathogenic Pseudomonas fluorescens strain. Vet Microbiol 139:183–188
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.046938-0
Loading
/content/journal/micro/10.1099/mic.0.046938-0
Loading

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error