1887

Abstract

This report describes a high-throughput assay to identify substances that reduce the frequency of conjugation in Gram-negative bacteria. Bacterial conjugation is largely responsible for the spread of multiple antibiotic resistances in human pathogens. Conjugation inhibitors may provide a means to control the spread of antibiotic resistance. An automated conjugation assay was developed that used plasmid R388 and a laboratory strain of as a model system, and bioluminescence as a reporter for conjugation activity. Frequencies of conjugation could be measured continuously in real time by the amount of light produced, and thus the effects of inhibitory compounds could be determined quantitatively. A control assay, run in parallel, allowed elimination of compounds affecting cell growth, plasmid stability or gene expression. The automated conjugation assay was used to screen a database of more than 12 000 microbial extracts known to contain a wide variety of bioactive compounds (the NatChem library). The initial hit rate was 1·4 %. From these, 48 extracts containing active compounds and representing a variety of organisms and extraction conditions were subjected to fractionation (24 fractions per extract). The 52 most active fractions were subjected to a secondary analysis to determine the range of plasmid inhibition. Plasmids R388, R1 and RP4 were used as representatives of a variety of plasmid transfer systems. Only one fraction (of complex composition) affected transfer of all three plasmids, while four other fractions were active against two of them. Two separate compounds were identified from these fractions: linoleic acid and dehydrocrepenynic acid. Downstream analysis showed that the chemical class of unsaturated fatty acids act as true inhibitors of conjugation.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.28216-0
2005-11-01
2019-10-18
Loading full text...

Full text loading...

/deliver/fulltext/micro/151/11/3517.html?itemId=/content/journal/micro/10.1099/mic.0.28216-0&mimeType=html&fmt=ahah

References

  1. Avila, P., Nuñez, B. & de la Cruz, F. ( 1996; ). Plasmid R6K contains two functional oriTs which can assemble simultaneously in relaxosomes in vivo. J Mol Biol 261, 135–143.[CrossRef]
    [Google Scholar]
  2. Black, P. N. & DiRusso, C. C. ( 2003; ). Transmembrane movement of exogenous long-chain fatty acids: proteins, enzymes, and vectorial esterification. Microbiol Mol Biol Rev 67, 454–472.[CrossRef]
    [Google Scholar]
  3. Bu'lock, J. D. & Gregory, H. ( 1959; ). The biosynthesis of polyacetylenes. 2. Origin of the carbon atoms. Biochem J 72, 322–325.
    [Google Scholar]
  4. Cabezón, E., Sastre, J. & de la Cruz, F. ( 1997; ). Genetic evidence of a coupling role for the TraG protein family in bacterial conjugation. Mol Gen Genet 254, 400–406.[CrossRef]
    [Google Scholar]
  5. Chandler, M. & Galas, D. ( 1983; ). Cointegrate formation mediated by Tn9. II. Activity of IS1 is modulated by external DNA sequences. J Mol Biol 170, 61–91.[CrossRef]
    [Google Scholar]
  6. Christie, P. J. & Vogel, J. P. ( 2000; ). Bacterial type IV secretion: conjugation systems adapted to deliver effector molecules to host cells. Trends Microbiol 8, 354–360.[CrossRef]
    [Google Scholar]
  7. Conter, A., Sturny, R., Gutierrez, C. & Cam, K. ( 2002; ). The RcsCB His-Asp phosphorelay system is essential to overcome chlorpromazine-induced stress in Escherichia coli. J Bacteriol 184, 2850–2853.[CrossRef]
    [Google Scholar]
  8. Datta, N., Hedges, R. W., Shaw, E. J., Sykes, R. B. & Richmond, R. H. ( 1971; ). Properties of an R factor from Pseudomonas aeruginosa. J Bacteriol 108, 1244–1249.
    [Google Scholar]
  9. de la Cruz, F. & Davies, J. ( 2000; ). Horizontal gene transfer and the origin of species: lessons from bacteria. Trends Microbiol 8, 128–133.[CrossRef]
    [Google Scholar]
  10. de Lorenzo, V. & Timmis, K. N. ( 1994; ). Analysis and construction of stable phenotypes in gram-negative bacteria with Tn5- and Tn10-derived minitransposons. Methods Enzymol 235, 386–405.
    [Google Scholar]
  11. de Lorenzo, V., Herrero, M., Jakubzik, U. & Timmis, K. N. ( 1990; ). Mini-Tn5 transposon derivatives for insertion mutagenesis, promoter probing, and chromosomal insertion of cloned DNA in gram-negative eubacteria. J Bacteriol 172, 6568–6572.
    [Google Scholar]
  12. Deuschle, U., Gentz, R. & Bujard, H. ( 1986; ). lac repressor blocks transcribing RNA polymerase and terminates transcription. Proc Natl Acad Sci U S A 83, 4134–4137.[CrossRef]
    [Google Scholar]
  13. Francia, M. V., Varsaki, A., Garcillan-Barcia, M. P., Latorre, A., Drainas, C. & de la Cruz, F. ( 2004; ). A classification scheme for mobilization regions of bacterial plasmids. FEMS Microbiol Rev 28, 79–100.[CrossRef]
    [Google Scholar]
  14. Gomis-Ruth, F. X., Moncalian, G., Perez-Luque, R., Gonzalez, A., Cabezon, E., de la Cruz, F. & Coll, M. ( 2001; ). The bacterial conjugation protein TrwB resembles ring helicases and F1-ATPase. Nature 409, 637–641.[CrossRef]
    [Google Scholar]
  15. Grant, S. G. N., Jesee, J., Bloom, F. R. & Hanahan, D. ( 1990; ). Differential plasmid rescue from transgenic mouse DNAs into Escherichia coli methylation-restriction mutants. Proc Natl Acad Sci U S A 87, 4645–4649.[CrossRef]
    [Google Scholar]
  16. Guasch, A., Lucas, M., Moncalian, G., Cabezas, M., Perez-Luque, R., Gomis-Ruth, F. X., de la Cruz, F. & Coll, M. ( 2003; ). Recognition and processing of the origin of transfer DNA by conjugative relaxase TrwC. Nat Struct Biol 10, 1002–1010.[CrossRef]
    [Google Scholar]
  17. Hansen, L. H., Sorensen, S. J. & Jensen, L. B. ( 1997; ). Chromosomal insertion of the entire Escherichia coli lactose operon, into two strains of Pseudomonas, using a modified mini-Tn5 delivery system. Gene 186, 167–173.[CrossRef]
    [Google Scholar]
  18. Hooper, D. C., Wolfson, J. S., Tung, C., Souza, K. S. & Swartz, M. N. ( 1989; ). Effects of inhibition of the B subunit of DNA gyrase on conjugation in Escherichia coli. J Bacteriol 171, 2235–2237.
    [Google Scholar]
  19. Koonin, E. V., Makarova, K. S. & Aravind, L. ( 2001; ). Horizontal gene transfer in prokaryotes: quantification and classification. Annu Rev Microbiol 55, 709–742.[CrossRef]
    [Google Scholar]
  20. Llosa, M., Bolland, S. & de la Cruz, F. ( 1991; ). Structural and functional analysis of the origin of conjugal transfer of the broad-host-range IncW plasmid R388 and comparison with the related IncN plasmid R46. Mol Gen Genet 226, 473–483.[CrossRef]
    [Google Scholar]
  21. Mandi, Y. & Molnar, J. ( 1981; ). Effect of chlorpromazine on conjugal plasmid transfer and sex pili. Acta Microbiol Acad Sci Hung 28, 205–210.
    [Google Scholar]
  22. Martínez, E. & de la Cruz, F. ( 1988; ). Transposon Tn21 encodes a recA independent site-specific integration system. Mol Gen Genet 211, 320–325.[CrossRef]
    [Google Scholar]
  23. Mazel, D. & Davies, J. ( 1999; ). Antibiotic resistance in microbes. Cell Mol Life Sci 56, 742–754.[CrossRef]
    [Google Scholar]
  24. Michel-Briand, Y. & Laporte, J. M. ( 1985; ). Inhibition of conjugal transfer of R plasmids by nitrofurans. J Gen Microbiol 131, 2281–2284.
    [Google Scholar]
  25. Ou, J. T. & Reim, R. ( 1976; ). Effect of 1,10-phenanthroline on bacterial conjugation in Escherichia coli K-12: inhibition of maturation from preliminary mates into effective mates. J Bacteriol 128, 363–371.
    [Google Scholar]
  26. Sandler, J. R., Tecklenburg, M. & Betz, J. L. ( 1980; ). Plasmid containing many tandem repeats of a synthetic lactose operator. Gene 8, 279–300.[CrossRef]
    [Google Scholar]
  27. Winans, S. C. & Walker, G. C. ( 1985; ). Conjugal transfer system of the IncN plasmid pKM101. J Bacteriol 161, 402–410.
    [Google Scholar]
  28. Winson, M. K., Swift, S., Fish, L., Throup, J. P., Jorgensen, F., Chhabra, S. R., Bycroft, B. W., Williams, P. & Stewart, G. S. ( 1998a; ). Construction and analysis of luxCDABE-based plasmid sensors for investigating N-acylhomoserine lactone-mediated quorum sensing. FEMS Microbiol Lett 163, 185–192.[CrossRef]
    [Google Scholar]
  29. Winson, M. K., Swift, S., Hill, P. J., Sims, C. M., Griesmayr, G., Bycroft, B. W., Williams, P. & Stewart, G. S. ( 1998b; ). Engineering the luxCDABE genes from Photorhabdus luminescens to provide a bioluminescent reporter for constitutive and promoter probe plasmids and mini-Tn5 constructs. FEMS Microbiol Lett 163, 193–202.[CrossRef]
    [Google Scholar]
  30. Yung, B. Y. & Kornberg, A. ( 1988; ). Membrane attachment activates DnaA protein, the initiation protein of chromosome replication in Escherichia coli. Proc Natl Acad Sci U S A 85, 7202–7205.[CrossRef]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.28216-0
Loading
/content/journal/micro/10.1099/mic.0.28216-0
Loading

Data & Media loading...

Supplements

Adobe PDF - SupplementaryData.pdf 

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