1887

Microbiology Society logo

Volume 151, Issue 11

Fluorescence assays for F-pili and their application Free

Abstract

Conjugative pili are extracellular filaments elaborated by Gram-negative bacteria expressing certain type IV secretion systems. They are required at the earliest stages of conjugal DNA transfer to establish specific and secure cell–cell contacts. Conjugative pili also serve as adsorption organelles for both RNA and DNA bacteriophages. Beyond these facts, the structure, formation and function of these filaments are poorly understood. This paper describes a rapid, quantitative assay for F-pili encoded by the F plasmid type IV secretion system. The assay is based on the specific lateral adsorption of icosahedral RNA bacteriophage R17 by F-pili. Bacteriophage particles conjugated with a fluorescent dye, Alexa 488, and bound to F-pili defined filaments visible by immunofluorescence microscopy. F-pili attached to F cells and free F-pili were both visible by this method. For quantification, cell-bound bacteriophage were separated from free bacteriophage particles by sedimentation and released by suspending cell pellets in 0·1 % SDS. Fluorescence in cell-free supernatant fractions was measured by fluorometry. The authors present a characterization of this assay and its application to F-pilus formation by cells carrying mutations in the gene for the F-pilus subunit F-pilin. Each mutation introduced a cysteine, which F-pilin normally lacks, at a different position in its primary structure. Cysteine residues in the N-terminal domain I abolished filament formation as measured by fluorescent R17 binding. This was confirmed by measurements of DNA donor activity and filamentous DNA bacteriophage infection. With one exception (G53C), cysteines elsewhere in the F-pilin primary structure did not abolish filament formation, although some mutations differentially affected F-pilus functions.

  • Received:
  • Accepted:
  • Revised:
  • Published Online:
Keyword(s): FIU, fluorescence intensity unit(s) , R17, bacteriophage R17 conjugated with Alexa 488 and t.f.u., transductant-forming units
SGM
Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.28159-0
2005-11-01
2024-04-27
Loading full text...

Full text loading...

/deliver/fulltext/micro/151/11/3541.html?itemId=/content/journal/micro/10.1099/mic.0.28159-0&mimeType=html&fmt=ahah

References

  1. Achtman M., Skurray R. 1977; A redefinition of the mating phenomenon in bacteria. In Microbial Interactions pp 234–279 Edited by Reissig J. London: Chapman & Hall;
     [Google Scholar]
  2. Achtman M., Willetts N., Clark J. 1972; Beginning a genetic analysis of conjugational transfer determined by the F factor in Escherichia coli by isolation and characterization of transfer-deficient mutants. J Bacteriol 106:529–538
     [Google Scholar]
  3. Anthony K., Sherburne C., Sherburne R., Frost L. 1994; The role of the pilus in recipient cell recognition during bacterial conjugation mediated by F-like plasmids. Mol Microbiol 13:939–953 [CrossRef]
     [Google Scholar]
  4. Biebricher C., Duker E.-M. 1984; Light-microscopic visualization of F and type 1 pili. J Gen Microbiol 130:941–949
     [Google Scholar]
  5. Casadaban M. 1976; Regulation of the regulatory gene for the arabinose pathway, araC . J Mol Biol 104:557–566 [CrossRef]
     [Google Scholar]
  6. Curtiss R., Caro L., Allison D., Stallions D. 1969; Early stages of conjugation in Escherichia coli . J Bacteriol 100:1091–1104
     [Google Scholar]
  7. Durrenberger M., Villiger W., Bachi Th. 1991; Conjugational junctions: morphology of specific contacts in conjugating Escherichia coli bacteria. J Struct Biol 107:146–156 [CrossRef]
     [Google Scholar]
  8. Eisenbrandt R., Kalkum M., Lai E., Lurz R., Kado C., Lanka E. 1999; Conjugative pili of IncP plasmids, and the Ti plasmid T pilus are composed of cyclic subunits. J Biol Chem 274:22548–22555 [CrossRef]
     [Google Scholar]
  9. Firth N., Ippen-Ihler K., Skurray R. others 1996; Structure and function of the F factor and mechanism of conjugation. In Escherichia coli and Salmonella, Cellular and Molecular Biology pp 2377–2401 Edited by Neidhardt F. C. Washington, DC: American Society for Microbiology;
     [Google Scholar]
  10. Folkhard W., Leonard K., Malsey S., Marvin D., Dubochet J., Engel A., Achtman M., Helmuth R. 1979; X-ray diffraction and electron microscope studies on the structure of F pili. J Mol Biol 130:145–160 [CrossRef]
     [Google Scholar]
  11. Freitag C., Eisenstein B. 1983; Genetic mapping and transcriptional orientation of the fimD gene. J Bacteriol 156:1052–1058
     [Google Scholar]
  12. Frost L., Paranchych W. 1988; DNA sequence analysis of point mutations in traA , the F-pilin gene, reveal two domains involved in F-specific phage attachment. Mol Gen Genet 213:134–139 [CrossRef]
     [Google Scholar]
  13. Frost L., Paranchych W., Willetts N. 1984; DNA sequence of the F traALE region that includes the gene for F-pilin. J Bacteriol 160:395–401
     [Google Scholar]
  14. Frost L., Finlay B., Opgenorth A., Paranchych W., Lee J. 1985; Characterization and sequence analysis of pilin from F-like plasmids. J Bacteriol 164:1238–1247
     [Google Scholar]
  15. Fullner K., Lara J., Nester E. 1996; Pilus assembly by Agrobacterium T DNA genes. Science 273:1107–1109 [CrossRef]
     [Google Scholar]
  16. Gilmour M., Lawley T., Rooker M., Newnham P., Taylor D. 2001; Cellular location and temperature-dependent assembly of IncH1 plasmid R27-encoded TrhC-associated conjugative transport protein complexes. Mol Microbiol 42:705–715
     [Google Scholar]
  17. Grahn A., Haase J., Bamford D., Lanka E. 2000; Components of the RP4 conjugative transfer apparatus form an envelope structure bridging inner and outer membranes of donor cells, implications for related macromolecular transport systems. J Bacteriol 182:1564–1574 [CrossRef]
     [Google Scholar]
  18. Grossman T., Silverman P. 1989; Structure and function of conjugative pili, inducible synthesis of functional F-pili by Escherichia coli K12 containing a lac-tra operon fusion. J Bacteriol 171:650–656
     [Google Scholar]
  19. Grossman T., Frost L., Silverman P. 1990; Structure and function of conjugative pili: monoclonal antibodies as probes for structural variants of F-pili. J Bacteriol 172:1174–1179
     [Google Scholar]
  20. Guzman L.-M., Belin D., Carson M., Beckwith J. 1995; Tight regulation, modulation, and high-level expression by vectors containing the arabinose pBAD promoter. J Bacteriol 177:4121–4130
     [Google Scholar]
  21. Harris R. L., Silverman P. M. 2004; Tra proteins characteristic of F-like Type IV secretion systems constitute an interaction group by yeast two-hybrid analysis. J Bacteriol 186:5480–5485 [CrossRef]
     [Google Scholar]
  22. Harris R., Sholl A., Conrad M., Dresser M., Silverman P. 1999; Interaction between the F plasmid TraA (F-pilin) and TraQ proteins. Mol Microbiol 34:780–791 [CrossRef]
     [Google Scholar]
  23. Harris R., Hombs V., Silverman P. 2001; Evidence that F-plasmid proteins TraV, TraK, and TraB assemble into an envelope-spanning structure in Escherichia coli . Mol Microbiol 42:757–766
     [Google Scholar]
  24. Ippen-Ihler K., Maneewannakul S. 1991; Conjugation among enteric bacteria: mating systems dependent on expression of pili. In Microbial Cell-Cell Interactions pp 35–69 Edited by Dworkin M. Washington, DC: American Society for Microbiology;
     [Google Scholar]
  25. Krall L., Wiedemann U., Unsin G., Weiss S., Domke N., Baron C. 2002; Detergent extraction identifies different VirB protein subassemblies of the type IV secretion machinery in the membranes of Agrobacterium tumefaciens . Proc Natl Acad Sci U S A 99:11405–11410 [CrossRef]
     [Google Scholar]
  26. Kumar R., Xie Y.-H., Das A. 2000; Subcellular localization of the Agrobacterium tumefaciens T-DNA transport pore proteins: VirB8 is essential for the assembly of the transport pore. Mol Microbiol 36:608–617
     [Google Scholar]
  27. Lai E.-M., Kado C. 1998; Processed VirB2 is the major subunit of the promiscuous pilus of Agrobacterium tumefaciens . J Bacteriol 180:2711–2717
     [Google Scholar]
  28. Lawley T., Gordon G., Wright A., Taylor D. 2002; Bacterial conjugative transfer: visualization of successful mating pairs and plasmid establishment in live Escherichia coli . Mol Microbiol 44:947–956 [CrossRef]
     [Google Scholar]
  29. Majdalani N., Ippen-Ihler K. 1996; Membrane insertion of the F-pilin subunit is Sec independent but requires leader peptidase B and the proton motive force. J Bacteriol 178:3742–3747
     [Google Scholar]
  30. Majdalani N., Moore D., Maneewannakul S., Ippen-Ihler K. 1996; Role of the propilin leader peptide in the maturation of F-pilin. J Bacteriol 178:3748–3754
     [Google Scholar]
  31. Manchak J., Anthony K., Frost L. 2002; Mutational analysis of F-pilin reveals domains for pilus assembly, phage infection, and DNA transfer. Mol Microbiol 43:195–205 [CrossRef]
     [Google Scholar]
  32. Maneewannakul K., Maneewannakul S., Ippen-Ihler K. 1993; Synthesis of F-pilin. J Bacteriol 175:1384–1391
     [Google Scholar]
  33. Maneewannakul K., Maneewannakul S., Ippen-Ihler K. 1995; Characterization of traX, the F plasmid locus required for acetylation of F-pilin subunits. J Bacteriol 177:2957–2964
     [Google Scholar]
  34. Manning P., Achtman M. 1979; Cell-to-cell interactions in conjugating Escherichia coli : the involvement of the cell envelope. In Bacterial Outer Membranes pp 409–448 Edited by Inouye M. New York: Wiley Interscience;
     [Google Scholar]
  35. Moore D., Sowa B., Ippen-Ihler K. 1981a; Location of an F-pilin pool in the inner membrane. J Bacteriol 146:251–259
     [Google Scholar]
  36. Moore D., Sowa B., Ippen-Ihler K. 1981b; The effect of tra mutations on the F-pilin polypeptide. Mol Gen Genet 184:260–264
     [Google Scholar]
  37. Moore D., Hamilton C., Maneewannakul K., Mintz Y., Frost L., Ippen-Ihler K. 1993; The Escherichia coli K-12 F plasmid gene traX is required for acetylation of F-pilin. J Bacteriol 175:1375–1383
     [Google Scholar]
  38. Otto G., Wu M., Clarke M., Lu H., Anderson O., Hilbi H., Shuman H., Kessin R. 2004; Autophagy is dispensable for intracellular replication of Legionella pneumophila in Dictyostelium discoideum . Mol Microbiol 51:63–72
     [Google Scholar]
  39. Paiva W., Grossman T., Silverman P. 1992; Characterization of F-pilin as an inner membrane component of Escherichia coli K12. J Biol Chem 267:26191–26197
     [Google Scholar]
  40. Panicker M., Minkley E. Jr 1985; DNA transfer occurs during a cell surface contact stage of F sex factor-mediated bacterial conjugation. J Bacteriol 162:584–590
     [Google Scholar]
  41. Paranchych W., Frost L. 1988; The physiology and biochemistry of pili. Adv Microb Physiol 29:53–114
     [Google Scholar]
  42. Parmley S., Smith G. 1988; Antibody-selectable filamentous fd phage vectors: affinity purification of target genes. Gene 73:305–318 [CrossRef]
     [Google Scholar]
  43. Samuels A., Lanka E., Davies J. 2000; Conjugative junctions in RP-4-mediated mating of Escherichia coli . J Bacteriol 182:2709–2715 [CrossRef]
     [Google Scholar]
  44. Silverman P. 1997; Towards a structural biology of bacterial conjugation. Mol Microbiol 23:423–429 [CrossRef]
     [Google Scholar]
  45. Sowa B., Moore D., Ippen-Ihler K. 1983; Physiology of F-pilin synthesis and utilization. J Bacteriol 153:962–968
     [Google Scholar]
  46. Thorstenson Y., Kuldau G., Zambryski P. 1993; Subcellular localization of seven VirB proteins of Agrobacterium tumefaciens : implications for the formation of a T-DNA transport structure. J Bacteriol 175:5233–5241
     [Google Scholar]
  47. Valentine R., Silverman P., Ippen K., Mobach H. 1969; The F-pilus of Escherichia coli . Adv Microb Physiol 3:1–52
     [Google Scholar]
  48. Vogel H., Bonner D. 1956; Acetylornithinase of Escherichia coli : partial purification and some properties. J Biol Chem 218:97–106
     [Google Scholar]
  49. Weber K., Konigsberg W. 1975; Proteins of RNA phages. In RNA Phages pp 51–84 Edited by Zinder N. D. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
     [Google Scholar]
  50. Yamamoto K., Alberts B., Benziger R., Lawhorne L., Treiber G. 1972; Rapid bacteriophage sedimentation in the presence of polyethylene glycol and its application to large-scale virus purification. Virology 40:734–744
     [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.28159-0
Loading
Fluorescence assays for F-pili and their application
Microbiology 151, 3541 (2005); https://doi.org/10.1099/mic.0.28159-0
/content/journal/micro/10.1099/mic.0.28159-0
/content/journal/micro/10.1099/mic.0.28159-0
Loading

Data & Media loading...

Most cited Most Cited RSS feed

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