1887

Abstract

The subcellular localization of the major type II secretion system of , the Xcp system, was studied microscopically using a biarsenical ligand that becomes fluorescent upon binding to a tetracysteine motif (Lumio tag), which was fused to several Xcp components. Fusion of the Lumio tag to the C termini of the XcpR and XcpS proteins did not affect the functionality of these proteins. Fluorescence microscopy showed that they were predominantly localized to the poles of cells, when produced at levels comparable to chromosomally encoded XcpR and XcpS. In most labelled cells, the proteins were found at one of the poles, although bipolar localization was also observed. When produced in the absence of other Xcp components, labelled XcpS was still found to locate at the poles, whereas XcpR was evenly distributed in the cell. These data suggest that XcpS, but not XcpR, contains information required for polar localization. The polar location of the Xcp machinery was further confirmed by the visualization of protease secretion with an intramolecularly quenched casein conjugate.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.2008/018069-0
2008-10-01
2019-10-13
Loading full text...

Full text loading...

/deliver/fulltext/micro/154/10/3025.html?itemId=/content/journal/micro/10.1099/mic.0.2008/018069-0&mimeType=html&fmt=ahah

References

  1. Adams, S. R., Campbell, R. E., Gross, L. A., Martin, B. R., Walkup, G. K., Yao, Y., Llopis, J. & Tsien, R. Y. ( 2002; ). New biarsenical ligands and tetracysteine motifs for protein labeling in vitro and in vivo: synthesis and biological applications. J Am Chem Soc 124, 6063–6076.[CrossRef]
    [Google Scholar]
  2. Arts, J., de Groot, A., Ball, G., Durand, E., El Khattabi, M., Filloux, A., Tommassen, J. & Koster, M. ( 2007; ). Interaction domains in the Pseudomonas aeruginosa type II secretory apparatus component XcpS (GspF). Microbiology 153, 1582–1592.[CrossRef]
    [Google Scholar]
  3. Ball, G., Chapon-Hervé, V., Bleves, S., Michel, G. & Bally, M. ( 1999; ). Assembly of XcpR in the cytoplasmic membrane is required for extracellular protein secretion in Pseudomonas aeruginosa. J Bacteriol 181, 382–388.
    [Google Scholar]
  4. Ball, G., Durand, E., Lazdunski, A. & Filloux, A. ( 2002; ). A novel type II secretion system in Pseudomonas aeruginosa. Mol Microbiol 43, 475–485.[CrossRef]
    [Google Scholar]
  5. Bally, M., Filloux, A., Akrim, M., Ball, G., Lazdunski, A. & Tommassen, J. ( 1992; ). Protein secretion in Pseudomonas aeruginosa: characterization of seven xcp genes and processing of secretory apparatus components by prepilin peptidase. Mol Microbiol 6, 1121–1131.[CrossRef]
    [Google Scholar]
  6. Bitter, W., Koster, M., Latijnhouwers, M., de Cock, H. & Tommassen, J. ( 1998; ). Formation of oligomeric rings by XcpQ and PilQ, which are involved in protein transport across the outer membrane of Pseudomonas aeruginosa. Mol Microbiol 27, 209–219.[CrossRef]
    [Google Scholar]
  7. Bleves, S., Lazdunski, A. & Filloux, A. ( 1996; ). Membrane topology of three Xcp proteins involved in exoprotein transport by Pseudomonas aeruginosa. J Bacteriol 178, 4297–4300.
    [Google Scholar]
  8. Bleves, S., Voulhoux, R., Michel, G., Lazdunski, A., Tommassen, J. & Filloux, A. ( 1998; ). The secretion apparatus of Pseudomonas aeruginosa: identification of a fifth pseudopilin, XcpX (GspK family). Mol Microbiol 27, 31–40.[CrossRef]
    [Google Scholar]
  9. 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.
    [Google Scholar]
  10. Braun, P., de Groot, A., Bitter, W. & Tommassen, J. ( 1998; ). Secretion of elastinolytic enzymes and their propeptides by Pseudomonas aeruginosa. J Bacteriol 180, 3467–3469.
    [Google Scholar]
  11. Brok, R., Van Gelder, P., Winterhalter, M., Ziese, U., Koster, A. J., de Cock, H., Koster, M., Tommassen, J. & Bitter, W. ( 1999; ). The C-terminal domain of the Pseudomonas secretin XcpQ forms oligomeric rings with pore activity. J Mol Biol 294, 1169–1179.[CrossRef]
    [Google Scholar]
  12. Buddelmeijer, N., Francetic, O. & Pugsley, A. P. ( 2006; ). Green fluorescent chimeras indicate nonpolar localization of pullulanase secreton components PulL and PulM. J Bacteriol 188, 2928–2935.[CrossRef]
    [Google Scholar]
  13. Camberg, J. L. & Sandkvist, M. ( 2005; ). Molecular analysis of the Vibrio cholerae type II secretion ATPase EpsE. J Bacteriol 187, 249–256.[CrossRef]
    [Google Scholar]
  14. 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.[CrossRef]
    [Google Scholar]
  15. Chiang, P., Habash, M. & Burrows, L. L. ( 2005; ). Disparate subcellular localization patterns of Pseudomonas aeruginosa type IV pilus ATPases involved in twitching motility. J Bacteriol 187, 829–839.[CrossRef]
    [Google Scholar]
  16. De Kievit, T. R. & Iglewski, B. H. ( 1999; ). Quorum sensing, gene expression, and Pseudomonas biofilms. Methods Enzymol 310, 117–128.
    [Google Scholar]
  17. Duong, F., Soscia, C., Lazdunski, A. & Murgier, M. ( 1994; ). The Pseudomonas fluorescens lipase has a C-terminal secretion signal and is secreted by a three-component bacterial ABC-exporter system. Mol Microbiol 11, 1117–1126.[CrossRef]
    [Google Scholar]
  18. Enderle, P. J. & Farwell, M. A. ( 1998; ). Electroporation of freshly plated Escherichia coli and Pseudomonas aeruginosa cells. Biotechniques 25, 954–958.
    [Google Scholar]
  19. Filloux, A. ( 2004; ). The underlying mechanisms of type II protein secretion. Biochim Biophys Acta 1694, 163–179.[CrossRef]
    [Google Scholar]
  20. Filloux, A., Bally, M., Murgier, M., Wretlind, B. & Lazdunski, A. ( 1989; ). Cloning of xcp genes located at the 55 min region of the chromosome and involved in protein secretion in Pseudomonas aeruginosa. Mol Microbiol 3, 261–265.[CrossRef]
    [Google Scholar]
  21. Filloux, A., Michel, G. & Bally, M. ( 1998; ). GSP-dependent protein secretion in gram-negative bacteria: the Xcp system of Pseudomonas aeruginosa. FEMS Microbiol Rev 22, 177–198.[CrossRef]
    [Google Scholar]
  22. Gaietta, G., Deerinck, T. J., Adams, S. R., Bouwer, J., Tour, O., Laird, D. W., Sosinsky, G. E., Tsien, R. Y. & Ellisman, M. H. ( 2002; ). Multicolor and electron microscopic imaging of connexin trafficking. Science 296, 503–507.[CrossRef]
    [Google Scholar]
  23. Gérard-Vincent, M., Robert, V., Ball, G., Bleves, S., Michel, G. P., Lazdunski, A. & Filloux, A. ( 2002; ). Identification of XcpP domains that confer functionality and specificity to the Pseudomonas aeruginosa type II secretion apparatus. Mol Microbiol 44, 1651–1665.[CrossRef]
    [Google Scholar]
  24. Griffin, B. A., Adams, S. R. & Tsien, R. Y. ( 1998; ). Specific covalent labeling of recombinant protein molecules inside live cells. Science 281, 269–272.[CrossRef]
    [Google Scholar]
  25. Holloway, B. W. ( 1955; ). Genetic recombination in Pseudomonas aeruginosa. J Gen Microbiol 13, 572–581.[CrossRef]
    [Google Scholar]
  26. Judd, P. K., Kumar, R. B. & Das, A. ( 2005; ). Spatial location and requirements for the assembly of the Agrobacterium tumefaciens type IV secretion apparatus. Proc Natl Acad Sci U S A 102, 11498–11503.[CrossRef]
    [Google Scholar]
  27. Koster, M., Bitter, W. & Tommassen, J. ( 2000; ). Protein secretion mechanisms in Gram-negative bacteria. Int J Med Microbiol 290, 325–331.[CrossRef]
    [Google Scholar]
  28. Laemmli, U. K. ( 1970; ). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685.[CrossRef]
    [Google Scholar]
  29. Mattick, J. S. ( 2002; ). Type IV pili and twitching motility. Annu Rev Microbiol 56, 289–314.[CrossRef]
    [Google Scholar]
  30. Nunn, D. N. & Lory, S. ( 1991; ). Product of the Pseudomonas aeruginosa gene pilD is a prepilin leader peptidase. Proc Natl Acad Sci U S A 88, 3281–3285.[CrossRef]
    [Google Scholar]
  31. Nunn, D. N. & Lory, S. ( 1992; ). Components of the protein-excretion apparatus of Pseudomonas aeruginosa are processed by the type IV prepilin peptidase. Proc Natl Acad Sci U S A 89, 47–51.[CrossRef]
    [Google Scholar]
  32. Peabody, C. R., Chung, Y. J., Yen, M. R., Vidal-Ingigliardi, D., Pugsley, A. P. & Saier, M. H. ( 2003; ). Type II protein secretion and its relationship to bacterial type IV pili and archaeal flagella. Microbiology 149, 3051–3072.[CrossRef]
    [Google Scholar]
  33. Py, B., Loiseau, L. & Barras, F. ( 2001; ). An inner membrane platform in the type II secretion machinery of Gram-negative bacteria. EMBO Rep 2, 244–248.[CrossRef]
    [Google Scholar]
  34. Robert, V., Filloux, A. & Michel, G. P. ( 2005a; ). Role of XcpP in the functionality of the Pseudomonas aeruginosa secreton. Res Microbiol 156, 880–886.[CrossRef]
    [Google Scholar]
  35. Robert, V., Filloux, A. & Michel, G. P. ( 2005b; ). Subcomplexes from the Xcp secretion system of Pseudomonas aeruginosa. FEMS Microbiol Lett 252, 43–50.[CrossRef]
    [Google Scholar]
  36. Robien, M. A., Krumm, B. E., Sandkvist, M. & Hol, W. G. ( 2003; ). Crystal structure of the extracellular protein secretion NTPase EpsE of Vibrio cholerae. J Mol Biol 333, 657–674.[CrossRef]
    [Google Scholar]
  37. Sambrook, J., Fritsch, E. F. & Maniatis, T. ( 1989; ). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.
  38. Scott, M. E., Dossani, Z. Y. & Sandkvist, M. ( 2001; ). Directed polar secretion of protease from single cells of Vibrio cholerae via the type II secretion pathway. Proc Natl Acad Sci U S A 98, 13978–13983.[CrossRef]
    [Google Scholar]
  39. Tommassen, J., van Tol, H. & Lugtenberg, B. ( 1983; ). The ultimate localization of an outer membrane protein of Escherichia coli K-12 is not determined by the signal sequence. EMBO J 2, 1275–1279.
    [Google Scholar]
  40. Weiss, R. L. ( 1971; ). The structure and occurrence of pili (fimbriae) on Pseudomonas aeruginosa. J Gen Microbiol 67, 135–143.[CrossRef]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.2008/018069-0
Loading
/content/journal/micro/10.1099/mic.0.2008/018069-0
Loading

Data & Media loading...

vol. , part 10, pp. 3025 - 3032

Oligonucleotides used [ PDF] (39 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