Skip to main content
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1997 Dec;179(23):7468–7475. doi: 10.1128/jb.179.23.7468-7475.1997

Cloning and genetic analysis of the UV resistance determinant (uvr) encoded on the Enterococcus faecalis pheromone-responsive conjugative plasmid pAD1.

Y Ozawa 1, K Tanimoto 1, S Fujimoto 1, H Tomita 1, Y Ike 1
PMCID: PMC179699  PMID: 9393713

Abstract

The conjugative pheromone-responsive plasmid pAD1 (59.6 kb) of Enterococcus faecalis encodes a UV resistance determinant (uvr) in addition to the hemolysin-bacteriocin determinant. pAD1 enhances the UV resistance of wild-type E. faecalis FA2-2 and E. faecalis UV202, which is a UV-sensitive derivative of E. faecalis JH2-2. A 2.972-kb fragment cloned from between 27.7 and 30.6 kb of the pAD1 map conferred UV resistance function on UV202. Sequence analysis showed that the cloned fragment contained three open reading frames designated uvrA, uvrB, and uvrC. The uvrA gene is located on the pAD1 map between 28.1 and 29.4 kb. uvrB is located between 30.1 and 30.3 kb, and uvrC is located between 30.4 and 30.6 kb on the pAD1 map. The uvrA, uvrB, and uvrC genes encode sequences of 442, 60, and 74 amino acids, respectively. The deduced amino acid sequence of the uvrA-encoded protein showed 20% homology of the identical residues with the E. coli UmuC protein. Tn917 insertion mutagenesis and deletion mutant analysis of the cloned fragment showed that uvrA conferred UV resistance. A palindromic sequence, 5'-GAACNGTTC-3', which is identical to the consensus sequence found within the putative promoter region of the Bacillus subtilis DNA damage-inducible genes, was located within the promoter region of uvrA. Two uvrA transcripts of different lengths (i.e., 1.54 and 2.14 kb) which terminate at different points downstream of uvrA were detected in UV202 carrying the deletion mutant containing uvrA. The longer transcript, 2.14 kb, was not detected in UV202 carrying the deletion mutant containing both uvrA and uvrB, which suggests that uvrB encodes a terminator for the uvrA transcript. The uvrA transcript was not detected in any significant quantity in UV202 carrying the cloned fragment containing uvrA, uvrB, and uvrC; on the other hand, the 1.54-kb uvrA transcript was detected in the strain exposed to mitomycin C, which suggests that the UvrC protein functions as a regulator of uvrA.

Full Text

The Full Text of this article is available as a PDF (1.1 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bagg A., Kenyon C. J., Walker G. C. Inducibility of a gene product required for UV and chemical mutagenesis in Escherichia coli. Proc Natl Acad Sci U S A. 1981 Sep;78(9):5749–5753. doi: 10.1073/pnas.78.9.5749. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Booth M. C., Bogie C. P., Sahl H. G., Siezen R. J., Hatter K. L., Gilmore M. S. Structural analysis and proteolytic activation of Enterococcus faecalis cytolysin, a novel lantibiotic. Mol Microbiol. 1996 Sep;21(6):1175–1184. doi: 10.1046/j.1365-2958.1996.831449.x. [DOI] [PubMed] [Google Scholar]
  3. Burckhardt S. E., Woodgate R., Scheuermann R. H., Echols H. UmuD mutagenesis protein of Escherichia coli: overproduction, purification, and cleavage by RecA. Proc Natl Acad Sci U S A. 1988 Mar;85(6):1811–1815. doi: 10.1073/pnas.85.6.1811. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Cheo D. L., Bayles K. W., Yasbin R. E. Cloning and characterization of DNA damage-inducible promoter regions from Bacillus subtilis. J Bacteriol. 1991 Mar;173(5):1696–1703. doi: 10.1128/jb.173.5.1696-1703.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chow J. W., Thal L. A., Perri M. B., Vazquez J. A., Donabedian S. M., Clewell D. B., Zervos M. J. Plasmid-associated hemolysin and aggregation substance production contribute to virulence in experimental enterococcal endocarditis. Antimicrob Agents Chemother. 1993 Nov;37(11):2474–2477. doi: 10.1128/aac.37.11.2474. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chung J. W., Bensing B. A., Dunny G. M. Genetic analysis of a region of the Enterococcus faecalis plasmid pCF10 involved in positive regulation of conjugative transfer functions. J Bacteriol. 1995 Apr;177(8):2107–2117. doi: 10.1128/jb.177.8.2107-2117.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Clewell D. B. Bacterial sex pheromone-induced plasmid transfer. Cell. 1993 Apr 9;73(1):9–12. doi: 10.1016/0092-8674(93)90153-h. [DOI] [PubMed] [Google Scholar]
  8. Clewell D. B., Tomich P. K., Gawron-Burke M. C., Franke A. E., Yagi Y., An F. Y. Mapping of Streptococcus faecalis plasmids pAD1 and pAD2 and studies relating to transposition of Tn917. J Bacteriol. 1982 Dec;152(3):1220–1230. doi: 10.1128/jb.152.3.1220-1230.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Colmar I., Horaud T. Enterococcus faecalis hemolysin-bacteriocin plasmids belong to the same incompatibility group. Appl Environ Microbiol. 1987 Mar;53(3):567–570. doi: 10.1128/aem.53.3.567-570.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Dunny G. M., Brown B. L., Clewell D. B. Induced cell aggregation and mating in Streptococcus faecalis: evidence for a bacterial sex pheromone. Proc Natl Acad Sci U S A. 1978 Jul;75(7):3479–3483. doi: 10.1073/pnas.75.7.3479. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Elledge S. J., Walker G. C. The muc genes of pKM101 are induced by DNA damage. J Bacteriol. 1983 Sep;155(3):1306–1315. doi: 10.1128/jb.155.3.1306-1315.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Fujimoto S., Hashimoto H., Ike Y. Low cost device for electrotransformation and its application to the highly efficient transformation of Escherichia coli and Enterococcus faecalis. Plasmid. 1991 Sep;26(2):131–135. doi: 10.1016/0147-619x(91)90053-y. [DOI] [PubMed] [Google Scholar]
  13. Fujimoto S., Tomita H., Wakamatsu E., Tanimoto K., Ike Y. Physical mapping of the conjugative bacteriocin plasmid pPD1 of Enterococcus faecalis and identification of the determinant related to the pheromone response. J Bacteriol. 1995 Oct;177(19):5574–5581. doi: 10.1128/jb.177.19.5574-5581.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Galli D., Friesenegger A., Wirth R. Transcriptional control of sex-pheromone-inducible genes on plasmid pAD1 of Enterococcus faecalis and sequence analysis of a third structural gene for (pPD1-encoded) aggregation substance. Mol Microbiol. 1992 May;6(10):1297–1308. doi: 10.1111/j.1365-2958.1992.tb00851.x. [DOI] [PubMed] [Google Scholar]
  15. Gilmore M. S., Segarra R. A., Booth M. C. An HlyB-type function is required for expression of the Enterococcus faecalis hemolysin/bacteriocin. Infect Immun. 1990 Dec;58(12):3914–3923. doi: 10.1128/iai.58.12.3914-3923.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Gilmore M. S., Segarra R. A., Booth M. C., Bogie C. P., Hall L. R., Clewell D. B. Genetic structure of the Enterococcus faecalis plasmid pAD1-encoded cytolytic toxin system and its relationship to lantibiotic determinants. J Bacteriol. 1994 Dec;176(23):7335–7344. doi: 10.1128/jb.176.23.7335-7344.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Ike Y., Clewell D. B. Evidence that the hemolysin/bacteriocin phenotype of Enterococcus faecalis subsp. zymogenes can be determined by plasmids in different incompatibility groups as well as by the chromosome. J Bacteriol. 1992 Dec;174(24):8172–8177. doi: 10.1128/jb.174.24.8172-8177.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Ike Y., Clewell D. B. Genetic analysis of the pAD1 pheromone response in Streptococcus faecalis, using transposon Tn917 as an insertional mutagen. J Bacteriol. 1984 Jun;158(3):777–783. doi: 10.1128/jb.158.3.777-783.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Ike Y., Clewell D. B., Segarra R. A., Gilmore M. S. Genetic analysis of the pAD1 hemolysin/bacteriocin determinant in Enterococcus faecalis: Tn917 insertional mutagenesis and cloning. J Bacteriol. 1990 Jan;172(1):155–163. doi: 10.1128/jb.172.1.155-163.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Ike Y., Flannagan S. E., Clewell D. B. Hyperhemolytic phenomena associated with insertions of Tn916 into the hemolysin determinant of Enterococcus faecalis plasmid pAD1. J Bacteriol. 1992 Mar;174(6):1801–1809. doi: 10.1128/jb.174.6.1801-1809.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Ike Y., Hashimoto H., Clewell D. B. Hemolysin of Streptococcus faecalis subspecies zymogenes contributes to virulence in mice. Infect Immun. 1984 Aug;45(2):528–530. doi: 10.1128/iai.45.2.528-530.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Ike Y., Hashimoto H., Clewell D. B. High incidence of hemolysin production by Enterococcus (Streptococcus) faecalis strains associated with human parenteral infections. J Clin Microbiol. 1987 Aug;25(8):1524–1528. doi: 10.1128/jcm.25.8.1524-1528.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Jett B. D., Jensen H. G., Nordquist R. E., Gilmore M. S. Contribution of the pAD1-encoded cytolysin to the severity of experimental Enterococcus faecalis endophthalmitis. Infect Immun. 1992 Jun;60(6):2445–2452. doi: 10.1128/iai.60.6.2445-2452.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Kao S. M., Olmsted S. B., Viksnins A. S., Gallo J. C., Dunny G. M. Molecular and genetic analysis of a region of plasmid pCF10 containing positive control genes and structural genes encoding surface proteins involved in pheromone-inducible conjugation in Enterococcus faecalis. J Bacteriol. 1991 Dec;173(23):7650–7664. doi: 10.1128/jb.173.23.7650-7664.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Leonard B. A., Podbielski A., Hedberg P. J., Dunny G. M. Enterococcus faecalis pheromone binding protein, PrgZ, recruits a chromosomal oligopeptide permease system to import sex pheromone cCF10 for induction of conjugation. Proc Natl Acad Sci U S A. 1996 Jan 9;93(1):260–264. doi: 10.1073/pnas.93.1.260. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Love P. E., Yasbin R. E. Genetic characterization of the inducible SOS-like system of Bacillus subtilis. J Bacteriol. 1984 Dec;160(3):910–920. doi: 10.1128/jb.160.3.910-920.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Love P. E., Yasbin R. E. Induction of the Bacillus subtilis SOS-like response by Escherichia coli RecA protein. Proc Natl Acad Sci U S A. 1986 Jul;83(14):5204–5208. doi: 10.1073/pnas.83.14.5204. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Morris T. W., Reed K. E., Cronan J. E., Jr Lipoic acid metabolism in Escherichia coli: the lplA and lipB genes define redundant pathways for ligation of lipoyl groups to apoprotein. J Bacteriol. 1995 Jan;177(1):1–10. doi: 10.1128/jb.177.1.1-10.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Murli S., Walker G. C. SOS mutagenesis. Curr Opin Genet Dev. 1993 Oct;3(5):719–725. doi: 10.1016/s0959-437x(05)80089-9. [DOI] [PubMed] [Google Scholar]
  30. Nakayama J., Yoshida K., Kobayashi H., Isogai A., Clewell D. B., Suzuki A. Cloning and characterization of a region of Enterococcus faecalis plasmid pPD1 encoding pheromone inhibitor (ipd), pheromone sensitivity (traC), and pheromone shutdown (traB) genes. J Bacteriol. 1995 Oct;177(19):5567–5573. doi: 10.1128/jb.177.19.5567-5573.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Nohmi T., Battista J. R., Dodson L. A., Walker G. C. RecA-mediated cleavage activates UmuD for mutagenesis: mechanistic relationship between transcriptional derepression and posttranslational activation. Proc Natl Acad Sci U S A. 1988 Mar;85(6):1816–1820. doi: 10.1073/pnas.85.6.1816. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Nohmi T., Hakura A., Nakai Y., Watanabe M., Murayama S. Y., Sofuni T. Salmonella typhimurium has two homologous but different umuDC operons: cloning of a new umuDC-like operon (samAB) present in a 60-megadalton cryptic plasmid of S. typhimurium. J Bacteriol. 1991 Feb;173(3):1051–1063. doi: 10.1128/jb.173.3.1051-1063.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Perry K. L., Elledge S. J., Mitchell B. B., Marsh L., Walker G. C. umuDC and mucAB operons whose products are required for UV light- and chemical-induced mutagenesis: UmuD, MucA, and LexA proteins share homology. Proc Natl Acad Sci U S A. 1985 Jul;82(13):4331–4335. doi: 10.1073/pnas.82.13.4331. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Perry K. L., Walker G. C. Identification of plasmid (pKM101)-coded proteins involved in mutagenesis and UV resistance. Nature. 1982 Nov 18;300(5889):278–281. doi: 10.1038/300278a0. [DOI] [PubMed] [Google Scholar]
  35. Pontius L. T., Clewell D. B. Conjugative transfer of Enterococcus faecalis plasmid pAD1: nucleotide sequence and transcriptional fusion analysis of a region involved in positive regulation. J Bacteriol. 1992 May;174(10):3152–3160. doi: 10.1128/jb.174.10.3152-3160.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Pontius L. T., Clewell D. B. Regulation of the pAD1-encoded sex pheromone response in Enterococcus faecalis: nucleotide sequence analysis of traA. J Bacteriol. 1992 Mar;174(6):1821–1827. doi: 10.1128/jb.174.6.1821-1827.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Ruhfel R. E., Manias D. A., Dunny G. M. Cloning and characterization of a region of the Enterococcus faecalis conjugative plasmid, pCF10, encoding a sex pheromone-binding function. J Bacteriol. 1993 Aug;175(16):5253–5259. doi: 10.1128/jb.175.16.5253-5259.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Sedgwick S. G., Ho C., Woodgate R. Mutagenic DNA repair in enterobacteria. J Bacteriol. 1991 Sep;173(18):5604–5611. doi: 10.1128/jb.173.18.5604-5611.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Segarra R. A., Booth M. C., Morales D. A., Huycke M. M., Gilmore M. S. Molecular characterization of the Enterococcus faecalis cytolysin activator. Infect Immun. 1991 Apr;59(4):1239–1246. doi: 10.1128/iai.59.4.1239-1246.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Shaw J. H., Clewell D. B. Complete nucleotide sequence of macrolide-lincosamide-streptogramin B-resistance transposon Tn917 in Streptococcus faecalis. J Bacteriol. 1985 Nov;164(2):782–796. doi: 10.1128/jb.164.2.782-796.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Shinagawa H., Iwasaki H., Kato T., Nakata A. RecA protein-dependent cleavage of UmuD protein and SOS mutagenesis. Proc Natl Acad Sci U S A. 1988 Mar;85(6):1806–1810. doi: 10.1073/pnas.85.6.1806. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Smith C. M., Eisenstadt E. Identification of a umuDC locus in Salmonella typhimurium LT2. J Bacteriol. 1989 Jul;171(7):3860–3865. doi: 10.1128/jb.171.7.3860-3865.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Tanimoto K., An F. Y., Clewell D. B. Characterization of the traC determinant of the Enterococcus faecalis hemolysin-bacteriocin plasmid pAD1: binding of sex pheromone. J Bacteriol. 1993 Aug;175(16):5260–5264. doi: 10.1128/jb.175.16.5260-5264.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Tanimoto K., Clewell D. B. Regulation of the pAD1-encoded sex pheromone response in Enterococcus faecalis: expression of the positive regulator TraE1. J Bacteriol. 1993 Feb;175(4):1008–1018. doi: 10.1128/jb.175.4.1008-1018.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Tanimoto K., Ozawa Y., Tomita H., Fujimoto S., Ike Y. Cloning and characterization of the uvr (ultraviolet resistance) gene on conjugative plasmid pAD1 of Enterococcus faecalis. Dev Biol Stand. 1995;85:83–87. [PubMed] [Google Scholar]
  46. Tanimoto K., Tomita H., Ike Y. The traA gene of the Enterococcus faecalis conjugative plasmid pPD1 encodes a negative regulator for the pheromone response. Plasmid. 1996 Jul;36(1):55–61. doi: 10.1006/plas.1996.0032. [DOI] [PubMed] [Google Scholar]
  47. Thomas S. M., Sedgwick S. G. Cloning of Salmonella typhimurium DNA encoding mutagenic DNA repair. J Bacteriol. 1989 Nov;171(11):5776–5782. doi: 10.1128/jb.171.11.5776-5782.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Tomich P. K., An F. Y., Clewell D. B. Properties of erythromycin-inducible transposon Tn917 in Streptococcus faecalis. J Bacteriol. 1980 Mar;141(3):1366–1374. doi: 10.1128/jb.141.3.1366-1374.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Tomich P. K., An F. Y., Damle S. P., Clewell D. B. Plasmid-related transmissibility and multiple drug resistance in Streptococcus faecalis subsp. zymogenes strain DS16. Antimicrob Agents Chemother. 1979 Jun;15(6):828–830. doi: 10.1128/aac.15.6.828. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Walker G. C. Inducible DNA repair systems. Annu Rev Biochem. 1985;54:425–457. doi: 10.1146/annurev.bi.54.070185.002233. [DOI] [PubMed] [Google Scholar]
  51. Walker G. C. Mutagenesis and inducible responses to deoxyribonucleic acid damage in Escherichia coli. Microbiol Rev. 1984 Mar;48(1):60–93. doi: 10.1128/mr.48.1.60-93.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Weaver K. E., Clewell D. B. Construction of Enterococcus faecalis pAD1 miniplasmids: identification of a minimal pheromone response regulatory region and evaluation of a novel pheromone-dependent growth inhibition. Plasmid. 1989 Sep;22(2):106–119. doi: 10.1016/0147-619x(89)90020-6. [DOI] [PubMed] [Google Scholar]
  53. Weaver K. E., Clewell D. B. Regulation of the pAD1 sex pheromone response in Enterococcus faecalis: effects of host strain and traA, traB, and C region mutants on expression of an E region pheromone-inducible lacZ fusion. J Bacteriol. 1990 May;172(5):2633–2641. doi: 10.1128/jb.172.5.2633-2641.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Wirth R., An F. Y., Clewell D. B. Highly efficient protoplast transformation system for Streptococcus faecalis and a new Escherichia coli-S. faecalis shuttle vector. J Bacteriol. 1986 Mar;165(3):831–836. doi: 10.1128/jb.165.3.831-836.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Woodgate R., Sedgwick S. G. Mutagenesis induced by bacterial UmuDC proteins and their plasmid homologues. Mol Microbiol. 1992 Aug;6(16):2213–2218. doi: 10.1111/j.1365-2958.1992.tb01397.x. [DOI] [PubMed] [Google Scholar]
  56. Yagi Y., Clewell D. B. Recombination-deficient mutant of Streptococcus faecalis. J Bacteriol. 1980 Aug;143(2):966–970. doi: 10.1128/jb.143.2.966-970.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Yagi Y., Kessler R. E., Shaw J. H., Lopatin D. E., An F., Clewell D. B. Plasmid content of Streptococcus faecalis strain 39-5 and identification of a pheromone (cPD1)-induced surface antigen. J Gen Microbiol. 1983 Apr;129(4):1207–1215. doi: 10.1099/00221287-129-4-1207. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES