1887

Microbiology Society logo

Volume 152, Issue 3

The TraA relaxase autoregulates the putative type IV secretion-like system encoded by the broad-host-range plasmid pIP501 Free

Abstract

The conjugative multiple antibiotic resistance plasmid pIP501 can be transferred and stably maintained in a variety of Gram-positive genera, including multicellular , as well as in Gram-negative . The 15 putative pIP501 transfer () genes are organized in an operon-like structure terminating in a strong transcriptional terminator. This paper reports co-transcription of the pIP501 genes in exponentially growing JH2-2 cells, as shown by RT-PCR. The genes are expressed throughout the life cycle of , and the expression level is independent of the growth phase. Electrophoretic mobility shift assays indicated that the TraA relaxase, the first gene of the operon, binds to the promoter P, which partially overlaps with the origin of transfer (). DNase I footprinting experiments further delimited the TraA binding region and defined the nucleotides bound by TraA. -Galactosidase assays with P fusions proved P promoter activity, which was strongly repressed when TraA was supplied . Thus, it is concluded that the pIP501 operon is negatively autoregulated at the transcriptional level by the conjugative DNA relaxase TraA.

  • Received:
  • Accepted:
  • Revised:
  • Published Online:
Keyword(s): EMSA, electrophoretic mobility shift assay , G+, Gram positive , GAP-DH, glyceraldehyde-3-phosphate dehydrogenase , GST, glutathione S-transferase and G−, Gram negative
SGM
Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.28468-0
2006-03-01
2024-04-16
Loading full text...

Full text loading...

/deliver/fulltext/micro/152/3/637.html?itemId=/content/journal/micro/10.1099/mic.0.28468-0&mimeType=html&fmt=ahah

References

  1. Bae T, Dunny G. M. 2001; Dominant-negative mutants of prgX : evidence for a role for PrgX dimerization in negative regulation of pheromone-inducible conjugation. Mol Microbiol 39:1307–1320 [CrossRef]
     [Google Scholar]
  2. Bae T, Kozlowicz B. K, Dunny G. M. 2002; Two targets in pCF10 DNA for PrgX binding: their role in production of Qa and prgX mRNA and in regulation of pheromone-inducible conjugation. J Mol Biol 315:995–1007 [CrossRef]
     [Google Scholar]
  3. Bae T, Kozlowicz B. K, Dunny G. M. 2004; Characterization of cis -acting prgQ mutants: evidence for two distinct repression mechanisms by Qa RNA and PrgX protein in pheromone-inducible enterococcal plasmid pCF10. Mol Microbiol 51:271–281
     [Google Scholar]
  4. Cascales E, Christie P. J. 2003; The versatile bacterial type IV secretion systems. Nat Rev Microbiol 1:137–149 [CrossRef]
     [Google Scholar]
  5. Chandler J. R, Dunny G. M. 2004; Enterococcal peptide sex pheromones: synthesis and control of biological activity. Peptides 25:1377–1388 [CrossRef]
     [Google Scholar]
  6. Chang A. C. Y, Cohen S. N. 1978; Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. J Bacteriol 134:1141–1156
     [Google Scholar]
  7. Christie P. J. 2004; Type IV secretion: the Agrobacterium VirB/D4 and related conjugation systems. Biochim Biophys Acta 1694219–234 [CrossRef]
     [Google Scholar]
  8. Christie P. J, Cascales E. 2005; Structural and dynamic properties of bacterial type IV secretion systems. Mol Membr Biol 22:51–61 [CrossRef]
     [Google Scholar]
  9. Christie P. J, Atmakuri K, Krishnamoorthy V, Jakubowski S, Cascales E. 2005; Biogenesis, architecture, and function of bacterial type IV secretion systems. Annu Rev Microbiol (in press)
    [Google Scholar]
  10. Clewell D. B, Dunny G. M. 2002; Conjugation and genetic exchange in Enterococci. In The Enterococci: Pathogenesis, Molecular Biology, and Antibiotic Resistance, 1st edn. pp  265–300 Edited by Gilmore M. S., Clewell D. B., Courvalin P., Dunny G. M., Murray B. E., Rice L. B. Washington, DC: American Society for Microbiology;
     [Google Scholar]
  11. de Antonio C, Farías M. E, de Lacoba M. G, Espinosa M. 2004; Features of the plasmid pMV158-encoded MobM, a protein involved in its mobilization. J Mol Biol 335:733–743 [CrossRef]
     [Google Scholar]
  12. Farías M. E, Grohmann E, Espinosa M. 1999; Expression of the mobM gene of the streptococcal plasmid pMV158 in Lactococcus lactis subsp. lactis . FEMS Microbiol Lett 176:403–410 [CrossRef]
     [Google Scholar]
  13. Frost L. S, Manchak J. 1998; F[sup]−[/sup] phenocopies: characterization of expression of the F transfer region in stationary phase. Microbiology 144:2579–2587 [CrossRef]
     [Google Scholar]
  14. Grohmann E. 2005; Cell–cell channels in conjugating bacteria. In Cell–Cell Channels Edited by Baluska F., Volkmann D., Barlow P. W. Georgetown, TX: Landes Bioscience (in press)
    [Google Scholar]
  15. Grohmann E, Guzmán L. M, Espinosa M. 1999; Mobilisation of the streptococcal plasmid pMV158: interactions of MobM protein with its cognate oriT DNA region. Mol Gen Genet 261:707–715 [CrossRef]
     [Google Scholar]
  16. Grohmann E, Muth G, Espinosa M. 2003; Conjugative plasmid transfer in Gram-positive bacteria. Microbiol Mol Biol Rev 67:277–301 [CrossRef]
     [Google Scholar]
  17. Hayes W. 1964 The Genetics of Bacteria and their Viruses New York: Wiley;
     [Google Scholar]
  18. Horii T, Nagasawa H, Nakayama J. 2002; Functional analysis of TraA, the sex pheromone receptor encoded by pPD1, in a promoter region essential for the mating response in Enterococcus faecalis . J Bacteriol 184:6343–6350 [CrossRef]
     [Google Scholar]
  19. Horodniceanu T, Bouanchaud D. H, Bieth G, Chabbert Y. A. 1976; R plasmids in Streptococcus agalactiae (group B). Antimicrob Agents Chemother 10:795–801 [CrossRef]
     [Google Scholar]
  20. Jacob A. E, Hobbs S. J. 1974; Conjugal transfer of plasmid-borne multiple antibiotic resistance in Streptococcus faecalis var. zymogenes . J Bacteriol 117:360–372
     [Google Scholar]
  21. Kopeć J, Bergmann A, Fritz G, Grohmann E, Keller W. 2005; TraA and its N-terminal relaxase domain of the Gram-positive plasmid pIP501 show specific oriT binding and behave as dimers in solution. Biochem J 387:401–409 [CrossRef]
     [Google Scholar]
  22. Kurenbach B, Grothe D, Farías M. E, Szewzyk U, Grohmann E. 2002; The tra region of the conjugative plasmid pIP501 is organized in an operon with the first gene encoding the relaxase. J Bacteriol 184:1801–1805 [CrossRef]
     [Google Scholar]
  23. Kurenbach B, Bohn C, Prabhu J, Abudukerim M, Szewzyk U, Grohmann E. 2003; Intergeneric transfer of the Enterococcus faecalis plasmid pIP501 to Escherichia coli and Streptomyces lividans and sequence analysis of its tra region. Plasmid 50:86–93 [CrossRef]
     [Google Scholar]
  24. Leblanc B, Moss T. 1994; Dnase I footprinting. In DNA–Protein Interactions, Principles and Protocols (Methods in Molecular Biology vol. 30). pp  1–10 Edited by Kneale G. G. Tatowa, NJ: Humana Press;
  25. Llosa M, de la Cruz F. 2005; Bacterial conjugation: a potential tool for genomic engineering. Res Microbiol 156:1–6 [CrossRef]
     [Google Scholar]
  26. Miller J. H. 1972 Experiments in Molecular Genetics, 1st edn.. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
     [Google Scholar]
  27. Ronald S. L, Kropinski A. M, Farinha M. A. 1990; Construction of broad-host-range vectors for the selection of divergent promoters. Gene 90:145–148 [CrossRef]
     [Google Scholar]
  28. 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]
  29. Schulein R, Guye P, Rhomberg T. A, Schmid M. C, Schroder G, Vergunst A. C, Carena I, Dehio C. 2005; A bipartite signal mediates the transfer of type IV secretion substrates of Bartonella henselae into human cells. Proc Natl Acad Sci U S A 102:856–861 [CrossRef]
     [Google Scholar]
  30. Szpirer C. Y, Faelen M, Couturier M. 2001; Mobilization function of the pBHR1 plasmid, a derivative of the broad-host-range plasmid pBBR1. J Bacteriol 183:2101–2110 [CrossRef]
     [Google Scholar]
  31. van der Lelie D, Wösten H. A, Bron S, Oskam L, Venema G. 1990; Conjugal mobilization of streptococcal plasmid pMV158 between strains of Lactococcus lacti ssubsp. lactis . J Bacteriol 172:47–52
     [Google Scholar]
  32. Wang A, Macrina F. L. 1995a; Streptococcal plasmid pIP501 has a functional oriT site. J Bacteriol 177:4199–4206
     [Google Scholar]
  33. Wang A, Macrina F. L. 1995b; Characterization of six linked open reading frames necessary for pIP501-mediated conjugation. Plasmid 34:206–210 [CrossRef]
     [Google Scholar]
  34. Zuker M. 2003; MFold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res 31:3406–3415 [CrossRef]
     [Google Scholar]
  35. Zuniga M, Pardo I, Ferrer S. 2003; Conjugative plasmid pIP501 undergoes specific deletions after transfer from Lactococcus lactis to Oenococcus oeni . Arch Microbiol 180:367–373 [CrossRef]
     [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.28468-0
Loading
The TraA relaxase autoregulates the putative type IV secretion-like system encoded by the broad-host-range Streptococcus agalactiae plasmid pIP501
Microbiology 152, 637 (2006); https://doi.org/10.1099/mic.0.28468-0
/content/journal/micro/10.1099/mic.0.28468-0
/content/journal/micro/10.1099/mic.0.28468-0
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