1887

Abstract

is a Gram-negative plant-pathogenic bacterium that rots potato stems and tubers. Microarray analysis was used to identify genes that were differentially expressed when host extracts were added to the growth medium. Potato extracts downregulated the expression of ribosomal genes and genes related to uptake and metabolism of nutrients, and upregulated genes needed for nitrate or phosphonate use. Some of the observed changes in gene expression in host-extract-induced cultures are similar to those during attachment of the bacterium to host tissues. Other responses indicated defence against toxic metabolites in the extract. Tuber extract induced a large gene cluster having homology to type VI secretion genes shown to be virulence determinants in many, but not all, animal and human pathogens. Two of the genes in the type VI cluster were found to be expressed during infection in potato tubers and stems, and mutants with knockouts of the corresponding genes had increased virulence on potato. One of the type VI secretion mutants was further characterized and found to grow to higher cell density in culture in the presence of host extract and to produce slightly more extracellular tissue-macerating enzymes than the wild-type strain. Analysis of secreted proteins showed that this type VI mutant was affected in the production of haemolysin-coregulated proteins (Hcps), which have been suggested to be secreted by the type VI pathway in other bacteria. The results suggest that the type VI secretion system of is needed for secretion of Hcps but not for virulence on its host plant, potato.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.2008/017582-0
2008-08-01
2019-10-18
Loading full text...

Full text loading...

/deliver/fulltext/micro/154/8/2387.html?itemId=/content/journal/micro/10.1099/mic.0.2008/017582-0&mimeType=html&fmt=ahah

References

  1. Bell, K. S., Sebaihia, M., Pritchard, L., Holden, M. T., Hyman, L. J., Holeva, M. C., Thomson, N. R., Bentley, S. D., Churcher, L. J. & other authors ( 2004; ). Genome sequence of the enterobacterial phytopathogen Erwinia carotovora subsp. atroseptica and characterization of virulence factors. Proc Natl Acad Sci USA 101, 11105–11110.[CrossRef]
    [Google Scholar]
  2. Bingle, L. E. H., Bailey, C. M. & Pallen, M. J. ( 2008; ). Type VI secretion: a beginner's guide. Curr Opin Microbiol 11, 3–8.[CrossRef]
    [Google Scholar]
  3. Bladergroen, M. R., Badelt, K. & Spaink, H. P. ( 2003; ). Infection-blocking genes of a symbiotic Rhizobium leguminosarum strain that are involved in temperature-dependent protein secretion. Mol Plant Microbe Interact 16, 53–64.[CrossRef]
    [Google Scholar]
  4. Caldo, R. A., Nettleton, D. & Wise, R. P. ( 2004; ). Interaction-dependent gene expression in Mla-specified response to barley powdery mildew. Plant Cell 16, 2514–2528.[CrossRef]
    [Google Scholar]
  5. Datsenko, K. A. & Wanner, B. L. ( 2000; ). One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci U S A 97, 6640–6645.[CrossRef]
    [Google Scholar]
  6. de Bruin, O. M., Ludu, J. S. & Nano, F. E. ( 2007; ). The Francisella pathogenicity island protein IglA localizes to the bacterial cytoplasm and is needed for intracellular growth. BMC Microbiol 7, 1 [CrossRef]
    [Google Scholar]
  7. Filloux, A., Hachani, A. & Bleves, S. ( 2008; ). The bacterial type VI secretion machine: yet another player for protein transport across membranes. Microbiology 154, 1570–1583.[CrossRef]
    [Google Scholar]
  8. Fox, E. M. & Mendz, G. L. ( 2006; ). Phosphonate degradation in microorganisms. Enzyme Microb Technol 40, 145–150.[CrossRef]
    [Google Scholar]
  9. Hinton, J. C., Perombelon, M. C. & Salmond, G. P. ( 1985; ). Efficient transformation of Erwinia carotovora subsp. carotovora and E. carotovora subsp. atroseptica. J Bacteriol 161, 786–788.
    [Google Scholar]
  10. Huynh, T. V., Dahlbeck, D. & Staskawicz, B. J. ( 1989; ). Bacterial blight of soybean: regulation of a pathogen gene determining host cultivar specificity. Science 245, 1374–1377.[CrossRef]
    [Google Scholar]
  11. Kachlany, S. C., Planet, P. J., DeSalle, R., Fine, D. H., Figurski, D. H. & Kaplan, J. B. ( 2001; ). flp-1, the first representative of a new pilin gene subfamily, is required for non-specific adherence of Actinobacillus actinomycetemcomitans. Mol Microbiol 40, 542–554.[CrossRef]
    [Google Scholar]
  12. Mattinen, L., Nissinen, R., Riipi, T., Kalkkinen, N. & Pirhonen, M. ( 2007; ). Host-extract-induced changes in the secretome of plant pathogenic bacterium Pectobacterium atrosepticum. Proteomics 7, 3527–3537.[CrossRef]
    [Google Scholar]
  13. McCarter-Zorner, N. J., Harrison, M. D., Franc, G. D., Quinn, C. E., Sells, I. A. & Graham, D. C. ( 1985; ). Soft rot Erwinia bacteria in the rhizosphere of weeds and crop plants in Colorado, United States and Scotland. J Appl Bacteriol 59, 357–368.[CrossRef]
    [Google Scholar]
  14. Mougous, J. D., Cuff, M. E., Raunser, S., Shen, A., Zhou, M., Gifford, C. A., Goodman, A. L., Joachimiak, G., Ordoñez, C. L. & other authors ( 2006; ). A virulence locus of Pseudomonas aeruginosa encodes a protein secretion apparatus. Science 312, 1526–1530.[CrossRef]
    [Google Scholar]
  15. O'Connell, K. L. & Stults, J. T. ( 1997; ). Identification of mouse liver proteins on two-dimensional electrophoresis gels by matrix-assisted laser desorption/ionization mass spectrometry of in situ enzymatic digests. Electrophoresis 18, 349–359.[CrossRef]
    [Google Scholar]
  16. O'Toole, G. A. & Kolter, R. ( 1998; ). The initiation of biofilm formation in Pseudomonas fluorescens WCS365 proceeds via multiple, convergent signaling pathways: a genetic analysis. Mol Microbiol 28, 449–461.[CrossRef]
    [Google Scholar]
  17. Parsons, D. A. & Heffron, F. ( 2005; ). sciS an icmF homolog in Salmonella enterica serovar typhimurium, limits intracellular replication and decreases virulence. Infect Immun 73, 4338–4345.[CrossRef]
    [Google Scholar]
  18. Pérombelon, M. C. M. ( 2002; ). Potato diseases caused by soft rot erwinias: an overview of pathogenesis. Plant Pathol 51, 1–12.[CrossRef]
    [Google Scholar]
  19. Pirhonen, M., Saarilahti, H. T., Karlsson, M. & Palva, E. T. ( 1991; ). Identification of pathogenicity determinants of Erwinia carotovora subsp. carotovora by transposon mutagenesis. Mol Plant Microbe Interact 4, 276–283.[CrossRef]
    [Google Scholar]
  20. Planet, P. J., Kachlany, S. C., Fine, D. H., DeSalle, R. & Figurski, D. H. ( 2003; ). The widespread colonization island of Actinobacillus actinomycetemcomitans. Nat Genet 34, 193–198.[CrossRef]
    [Google Scholar]
  21. Preston, G. M., Studholme, D. J. & Caldelari, I. ( 2005; ). Profiling the secretomes of plant pathogenic Proteobacteria. FEMS Microbiol Rev 29, 331–360.
    [Google Scholar]
  22. Pukatzki, S., Ma, A. T., Sturtevant, D., Krastins, B., Sarracino, D., Nelson, W. C., Heidelberg, J. F. & Mekalanos, J. J. ( 2006; ). Identification of a conserved bacterial protein secretion system in Vibrio cholerae using the Dictyostelium host model system. Proc Natl Acad Sci U S A 103, 1528–1533.[CrossRef]
    [Google Scholar]
  23. Pukatzki, S., Ma, A. T., Revel, A. T., Sturtevant, D. & Mekalanos, J. J. ( 2007; ). Type VI secretion system translocates a phage tail spike-like protein into target cells where it cross-links actin. Proc Natl Acad Sci U S A 104, 15508–15513.[CrossRef]
    [Google Scholar]
  24. Rao, P. S., Yamada, Y., Tan, Y. P. & Leung, K. Y. ( 2004; ). Use of proteomics to identify novel virulence determinants that are required for Edwardsiella tarda pathogenesis. Mol Microbiol 53, 573–586.[CrossRef]
    [Google Scholar]
  25. Saeed, A. I., Sharov, V., White, J., Li, J., Liang, W., Bhagabati, N., Braisted, J., Klapa, M., Currier, T. & other authors ( 2003; ). TM4: a free, open-source system for microarray data management and analysis. Biotechniques 34, 374–378.
    [Google Scholar]
  26. Sambrook, J., Fritsch, E. F. & Maniatis, T. ( 1989; ). Molecular Cloning: a Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
  27. Savli, H., Karadenizli, A., Kolayli, F., Gundes, S., Ozbek, U. & Vahaboglu, H. ( 2003; ). Expression stability of six housekeeping genes: a proposal for resistance gene quantification studies of Pseudomonas aeruginosa by real-time quantitative RT-PCR. J Med Microbiol 52, 403–408.[CrossRef]
    [Google Scholar]
  28. Schell, M. A., Ulrich, R. L., Ribot, W. J., Brueggemann, E. E., Hines, H. B., Chen, D., Lipscomb, L., Kim, H. S., Mrázek, J. & other authors ( 2007; ). Type VI secretion is a major virulence determinant in Burkholderia mallei. Mol Microbiol 64, 1466–1485.[CrossRef]
    [Google Scholar]
  29. Shalom, G., Shaw, J. G. & Thomas, M. S. ( 2007; ). In vivo expression technology identifies a type VI secretion system locus in Burkholderia pseudomallei that is induced upon invasion of macrophages. Microbiology 153, 2689–2699.[CrossRef]
    [Google Scholar]
  30. Shevchenko, A., Wilm, M., Vorm, O. & Mann, M. ( 1996; ). Mass spectrometric sequencing of proteins from silver-stained polyacrylamide gels. Anal Chem 68, 850–858.[CrossRef]
    [Google Scholar]
  31. Singh, B. K. & Walker, A. ( 2006; ). Microbial degradation of organophosphorus compounds. FEMS Microbiol Rev 30, 428–471.[CrossRef]
    [Google Scholar]
  32. Smyth, G. K. ( 2005; ). Limma: linear models for microarray data. In Bioinformatics and Computational Biology Solutions Using R and Bioconductor, pp. 397–420. Edited by R. Gentleman, V. Carey, S. Dudoit, R. Irizarry & W. Huber. New York: Springer.
  33. Southey-Pillig, C. J., Davies, D. G. & Sauer, K. ( 2005; ). Characterization of temporal protein production in Pseudomonas aeruginosa biofilms. J Bacteriol 187, 8114–8126.[CrossRef]
    [Google Scholar]
  34. Summers, W. C. ( 1970; ). A simple method for extraction of RNA of E. coli utilizing DEPC. Anal Biochem 33, 459–463.
    [Google Scholar]
  35. Takle, G. W., Toth, I. A. & Brurberg, M. B. ( 2007; ). Evaluation of reference genes for real-time RT-PCR expression studies in the plant pathogen Pectobacterium atrosepticum. BMC Plant Biol 7, 50 [CrossRef]
    [Google Scholar]
  36. Whitehead, N. A., Byers, J. T., Commander, P., Corbett, M. J., Coulthurst, S. J., Everson, L., Harris, A. K. P., Pemberton, C. L., Simpson, N. J. L. & other authors ( 2002; ). The regulation of virulence in phytopathogenic Erwinia species: quorum sensing, antibiotics and ecological considerations. Antonie Van Leeuwenhoek 81, 223–231.[CrossRef]
    [Google Scholar]
  37. Williams, S. G., Varcoe, L. T., Attridge, S. R. & Manning, P. A. ( 1996; ). Vibrio cholerae Hcp, a secreted protein coregulated with HlyA. Infect Immun 64, 283–289.
    [Google Scholar]
  38. Wu, H. Y., Chung, P. C., Shih, H. W., Wen, S. R. & Lai, E. M. ( 2008; ). Secretome analysis uncovers an Hcp-family protein secreted via a type VI secretion system in Agrobacterium tumefaciens. J Bacteriol 190, 2841–2850.[CrossRef]
    [Google Scholar]
  39. Zheng, J. & Leung, K. Y. ( 2007; ). Dissection of a type VI secretion system in Edwardsiella tarda. Mol Microbiol 66, 1192–1206.[CrossRef]
    [Google Scholar]
  40. Zusman, T., Feldman, M., Halperin, E. & Segal, G. ( 2004; ). Characterization of the icmH and icmF genes required for Legionella pneumophila intracellular growth, genes that are present in many bacteria associated with eukaryotic cells. Infect Immun 72, 3398–3409.[CrossRef]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.2008/017582-0
Loading
/content/journal/micro/10.1099/mic.0.2008/017582-0
Loading

Data & Media loading...

Supplements

vol. , part 8, pp. 2387 - 2396

Microarray data [ Excel file] (1274 kb)



EXCEL

vol. , part 8, pp. 2387 - 2396

Because of a file conversion error at the typesetters, an incorrect version of Fig. 2 was published (the labels in the text box are wrongly spaced and/or have hyphens missing). The correct version of the figure is here(PDF, 63 kb).



PDF
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