1887

Abstract

Although classical type II secretion systems (T2SSs) are widely present in Gram-negative bacteria, atypical T2SSs can be found in some species. In in addition to the classical T2SS Xcp, it was reported that two genes, and , located outside the locus were organized in an operon () which encodes the orphan PaQa subunit. This subunit is able to associate with other components of the classical Xcp machinery to form a functional hybrid T2SS. In the present study, using a transcriptional fusion, we found that the operon was more efficiently expressed (i) on solid LB agar than in liquid LB medium, (ii) at 25 °C than at 37 °C and (iii) at an early stage of growth. These results suggested an adaptation of the hybrid system to particular environmental conditions. Transposon mutagenesis led to the finding that and genes are required for optimal expression of the orphan operon in the defined growth conditions used. Using an original culturing device designed to monitor secretion on solid medium, the ring-plate system, we found that T2SS-dependent secretion of exoproteins, namely the elastase LasB, was affected in a deletion mutant. Our findings led to the discovery of an interplay between FimV and the global regulator Vfr triggering the modulation of the level of Vfr and consequently the modulation of T2SS-dependent secretion on solid medium.

Funding
This study was supported by the:
  • CNRS
  • Agence Nationale de la Recherche Program ‘Jeune Chercheur’ (Award ANR-JC07-183230)
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2011-07-01
2024-04-23
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References

  1. Ahn K. S., Ha U., Jia J., Wu D., Jin S. ( 2004). The truA gene of Pseudomonas aeruginosa is required for the expression of type III secretory genes. Microbiology 150:539–547 [View Article][PubMed]
    [Google Scholar]
  2. Albus A. M., Pesci E. C., Runyen-Janecky L. J., West S. E., Iglewski B. H. ( 1997). Vfr controls quorum sensing in Pseudomonas aeruginosa. J Bacteriol 179:3928–3935[PubMed]
    [Google Scholar]
  3. Baysse C., Cullinane M., Dénervaud V., Burrowes E., Dow J. M., Morrissey J. P., Tam L., Trevors J. T., O'Gara F. ( 2005). Modulation of quorum sensing in Pseudomonas aeruginosa through alteration of membrane properties. Microbiology 151:2529–2542 [View Article][PubMed]
    [Google Scholar]
  4. Beatson S. A., Whitchurch C. B., Sargent J. L., Levesque R. C., Mattick J. S. ( 2002). Differential regulation of twitching motility and elastase production by Vfr in Pseudomonas aeruginosa. J Bacteriol 184:3605–3613 [View Article][PubMed]
    [Google Scholar]
  5. Bleves S., Gérard-Vincent M., Lazdunski A., Filloux A. ( 1999). Structure-function analysis of XcpP, a component involved in general secretory pathway-dependent protein secretion in Pseudomonas aeruginosa. J Bacteriol 181:4012–4019[PubMed]
    [Google Scholar]
  6. Bleves S., Viarre V., Salacha R., Michel G. P. F., Filloux A., Voulhoux R. ( 2010). Protein secretion systems in Pseudomonas aeruginosa: a wealth of pathogenic weapons. Int J Med Microbiol 300:534–543 [View Article][PubMed]
    [Google Scholar]
  7. Bodey G. P., Bolivar R., Fainstein V., Jadeja L. ( 1983). Infections caused by Pseudomonas aeruginosa. Rev Infect Dis 5:279–313 [View Article][PubMed]
    [Google Scholar]
  8. Bouley J., Condemine G., Shevchik V. E. ( 2001). The PDZ domain of OutC and the N-terminal region of OutD determine the secretion specificity of the type II out pathway of Erwinia chrysanthemi. J Mol Biol 308:205–219 [View Article][PubMed]
    [Google Scholar]
  9. Chapon-Hervé V., Akrim M., Latifi A., Williams P., Lazdunski A., Bally M. ( 1997). Regulation of the xcp secretion pathway by multiple quorum-sensing modulons in Pseudomonas aeruginosa. Mol Microbiol 24:1169–1178 [View Article][PubMed]
    [Google Scholar]
  10. de Groot A., Koster M., Gérard-Vincent M., Gerritse G., Lazdunski A., Tommassen J., Filloux A. ( 2001). Exchange of Xcp (Gsp) secretion machineries between Pseudomonas aeruginosa and Pseudomonas alcaligenes: species specificity unrelated to substrate recognition. J Bacteriol 183:959–967 [View Article][PubMed]
    [Google Scholar]
  11. Figurski D. H., Helinski D. R. ( 1979). Replication of an origin-containing derivative of plasmid RK2 dependent on a plasmid function provided in trans. Proc Natl Acad Sci U S A 76:1648–1652 [View Article][PubMed]
    [Google Scholar]
  12. Filloux A. ( 2004). The underlying mechanisms of type II protein secretion. Biochim Biophys Acta 1694:163–179 [View Article][PubMed]
    [Google Scholar]
  13. Fox A., Haas D., Reimmann C., Heeb S., Filloux A., Voulhoux R. ( 2008). Emergence of secretion-defective sublines of Pseudomonas aeruginosa PAO1 resulting from spontaneous mutations in the vfr global regulatory gene. Appl Environ Microbiol 74:1902–1908 [View Article][PubMed]
    [Google Scholar]
  14. Fuchs E. L., Brutinel E. D., Jones A. K., Fulcher N. B., Urbanowski M. L., Yahr T. L., Wolfgang M. C. ( 2010). The Pseudomonas aeruginosa Vfr regulator controls global virulence factor expression through cyclic AMP-dependent and -independent mechanisms. J Bacteriol 192:3553–3564 [View Article][PubMed]
    [Google Scholar]
  15. Fulcher N. B., Holliday P. M., Klem E., Cann M. J., Wolfgang M. C. ( 2010). The Pseudomonas aeruginosa Chp chemosensory system regulates intracellular cAMP levels by modulating adenylate cyclase activity. Mol Microbiol 76:889–904 [View Article][PubMed]
    [Google Scholar]
  16. Fürste J. P., Pansegrau W., Frank R., Blöcker H., Scholz P., Bagdasarian M., Lanka E. ( 1986). Molecular cloning of the plasmid RP4 primase region in a multi-host-range tacP expression vector. Gene 48:119–131 [View Article][PubMed]
    [Google Scholar]
  17. Gambello M. J., Iglewski B. H. ( 1991). Cloning and characterization of the Pseudomonas aeruginosa lasR gene, a transcriptional activator of elastase expression. J Bacteriol 173:3000–3009[PubMed]
    [Google Scholar]
  18. Gambello M. J., Kaye S., Iglewski B. H. ( 1993). LasR of Pseudomonas aeruginosa is a transcriptional activator of the alkaline protease gene (apr) and an enhancer of exotoxin A expression. Infect Immun 61:1180–1184[PubMed]
    [Google Scholar]
  19. Heeb S., Blumer C., Haas D. ( 2002). Regulatory RNA as mediator in GacA/RsmA-dependent global control of exoproduct formation in Pseudomonas fluorescens CHA0. J Bacteriol 184:1046–1056 [View Article][PubMed]
    [Google Scholar]
  20. Herrero M., de Lorenzo V., Timmis K. N. ( 1990). Transposon vectors containing non-antibiotic resistance selection markers for cloning and stable chromosomal insertion of foreign genes in Gram-negative bacteria. J Bacteriol 172:6557–6567[PubMed]
    [Google Scholar]
  21. Hoang T. T., Kutchma A. J., Becher A., Schweizer H. P. ( 2000). Integration-proficient plasmids for Pseudomonas aeruginosa: site-specific integration and use for engineering of reporter and expression strains. Plasmid 43:59–72 [View Article][PubMed]
    [Google Scholar]
  22. Kanack K. J., Runyen-Janecky L. J., Ferrell E. P., Suh S. J., West S. E. ( 2006). Characterization of DNA-binding specificity and analysis of binding sites of the Pseudomonas aeruginosa global regulator, Vfr, a homologue of the Escherichia coli cAMP receptor protein. Microbiology 152:3485–3496 [View Article][PubMed]
    [Google Scholar]
  23. Kaniga K., Delor I., Cornelis G. R. ( 1991). A wide-host-range suicide vector for improving reverse genetics in gram-negative bacteria: inactivation of the blaA gene of Yersinia enterocolitica. Gene 109:137–141 [View Article][PubMed]
    [Google Scholar]
  24. Kulasekara H. D., Ventre I., Kulasekara B. R., Lazdunski A., Filloux A., Lory S. ( 2005). A novel two-component system controls the expression of Pseudomonas aeruginosa fimbrial cup genes. Mol Microbiol 55:368–380 [View Article][PubMed]
    [Google Scholar]
  25. Laemmli U. K. ( 1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685 [View Article][PubMed]
    [Google Scholar]
  26. Levin A. M., de Vries R. P., Wösten H. A. ( 2007). Localization of protein secretion in fungal colonies using a novel culturing technique; the ring-plate system. J Microbiol Methods 69:399–401 [View Article][PubMed]
    [Google Scholar]
  27. Liberati N. T., Urbach J. M., Miyata S., Lee D. G., Drenkard E., Wu G., Villanueva J., Wei T., Ausubel F. M. ( 2006). An ordered, nonredundant library of Pseudomonas aeruginosa strain PA14 transposon insertion mutants. Proc Natl Acad Sci U S A 103:2833–2838 [View Article][PubMed]
    [Google Scholar]
  28. Lindeberg M., Salmond G. P., Collmer A. ( 1996). Complementation of deletion mutations in a cloned functional cluster of Erwinia chrysanthemi out genes with Erwinia carotovora out homologues reveals OutC and OutD as candidate gatekeepers of species-specific secretion of proteins via the type II pathway. Mol Microbiol 20:175–190 [View Article][PubMed]
    [Google Scholar]
  29. Martínez A., Ostrovsky P., Nunn D. N. ( 1998). Identification of an additional member of the secretin superfamily of proteins in Pseudomonas aeruginosa that is able to function in type II protein secretion. Mol Microbiol 28:1235–1246 [View Article][PubMed]
    [Google Scholar]
  30. Michel G. P., Voulhoux R. ( 2009). The type II secretory system (T2SS) in Gram negative bacteria: a molecular nanomachine for secretion of Sec and Tat-dependent extracellular proteins. Bacterial Secreted Proteins vol. 167–92 Wooldridge K. Norfolk: Caister Academic Press;
    [Google Scholar]
  31. Michel G. P., Durand E., Filloux A. ( 2007). XphA/XqhA, a novel GspCD subunit for type II secretion in Pseudomonas aeruginosa. J Bacteriol 189:3776–3783 [View Article][PubMed]
    [Google Scholar]
  32. Miller J. H. ( 1972). Experiments in Molecular Genetics Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  33. Morales V. M., Bäckman A., Bagdasarian M. ( 1991). A series of wide-host-range low-copy-number vectors that allow direct screening for recombinants. Gene 97:39–47 [View Article][PubMed]
    [Google Scholar]
  34. Passador L., Cook J. M., Gambello M. J., Rust L., Iglewski B. H. ( 1993). Expression of Pseudomonas aeruginosa virulence genes requires cell-to-cell communication. Science 260:1127–1130 [View Article][PubMed]
    [Google Scholar]
  35. 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]
  36. Schuster M., Lostroh C. P., Ogi T., Greenberg E. P. ( 2003). Identification, timing, and signal specificity of Pseudomonas aeruginosa quorum-controlled genes: a transcriptome analysis. J Bacteriol 185:2066–2079 [View Article][PubMed]
    [Google Scholar]
  37. Semmler A. B., Whitchurch C. B., Leech A. J., Mattick J. S. ( 2000). Identification of a novel gene, fimV, involved in twitching motility in Pseudomonas aeruginosa. Microbiology 146:1321–1332[PubMed]
    [Google Scholar]
  38. Suh S. J., Runyen-Janecky L. J., Maleniak T. C., Hager P., MacGregor C. H., Zielinski-Mozny N. A., Phibbs P. V. Jr, West S. E. ( 2002). Effect of vfr mutation on global gene expression and catabolite repression control of Pseudomonas aeruginosa. Microbiology 148:1561–1569[PubMed]
    [Google Scholar]
  39. Swain M., Ross N. W. ( 1995). A silver stain protocol for proteins yielding high resolution and transparent background in sodium dodecyl sulfate-polyacrylamide gels. Electrophoresis 16:948–951 [View Article][PubMed]
    [Google Scholar]
  40. Termine E., Michel G. P. ( 2009). Transcriptome and secretome analyses of the adaptive response of Pseudomonas aeruginosa to suboptimal growth temperature. Int Microbiol 12:7–12[PubMed]
    [Google Scholar]
  41. Wagner V. E., Bushnell D., Passador L., Brooks A. I., Iglewski B. H. ( 2003). Microarray analysis of Pseudomonas aeruginosa quorum-sensing regulons: effects of growth phase and environment. J Bacteriol 185:2080–2095 [View Article][PubMed]
    [Google Scholar]
  42. Wolfgang M. C., Lee V. T., Gilmore M. E., Lory S. ( 2003). Coordinate regulation of bacterial virulence genes by a novel adenylate cyclase-dependent signaling pathway. Dev Cell 4:253–263 [View Article][PubMed]
    [Google Scholar]
  43. Wretlind B., Pavlovskis O. R. ( 1984). Genetic mapping and characterization of Pseudomonas aeruginosa mutants defective in the formation of extracellular proteins. J Bacteriol 158:801–808[PubMed]
    [Google Scholar]
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