Skip to main content
PMC home page
Antimicrobial Agents and Chemotherapy logoLink to Antimicrobial Agents and Chemotherapy
. 1994 Sep;38(9):1899–1903. doi: 10.1128/aac.38.9.1899

Contribution of VanY D,D-carboxypeptidase to glycopeptide resistance in Enterococcus faecalis by hydrolysis of peptidoglycan precursors.

M Arthur 1, F Depardieu 1, H A Snaith 1, P E Reynolds 1, P Courvalin 1
PMCID: PMC284659  PMID: 7810996

Abstract

The vanR, vanS, vanH, vanA, and vanX genes of enterococcal transposon Tn1546 were introduced into the chromosome of Enterococcus faecalis JH2-2. Complementation of this portion of the van gene cluster by a plasmid encoding VanY D,D-carboxypeptidase led to a fourfold increase in the vancomycin MIC (from 16 to 64 micrograms/ml). Multicopy plasmids pAT80 (vanR vanS vanH vanA vanX) and pAT382 (vanR vanS vanH vanA vanX vanY) conferred similar levels of vancomycin resistance to JH2-2. The addition of D-alanine (100 mM) to the culture medium restored the vancomycin susceptibility of E. faecalis JH2-2/pAT80. The pentapeptide UDP-MurNAc-L-Ala-gamma-D-Glu-L-Lys-D-Ala-D-Ala partially replaced pentadepsipeptide UDP-MurNAc-L-Ala-gamma-D-Glu-L-Lys-D-Ala-D-Lac when the strain was grown in the presence of D-alanine. In contrast, resistance mediated by pAT382 was almost unaffected by the addition of the amino acid. Expression of the vanY gene of pAT382 resulted in the formation of the tetrapeptide UDP-MurNAc-L-Ala-gamma-D-Glu-L-Lys-D-Ala, indicating that a portion of the cytoplasmic precursors had been hydrolyzed. These results show that VanY contributes to glycopeptide resistance in conditions in which pentapeptide is present in the cytoplasm above a threshold concentration. However, the contribution of the enzyme to high-level resistance mediated by Tn1546 appears to be moderate, probably because hydrolysis of D-alanyl-D-alanine by VanX efficiently prevents synthesis of the pentapeptide.

Full text

PDF
1901

Selected References

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

  1. Allen N. E., Hobbs J. N., Jr, Richardson J. M., Riggin R. M. Biosynthesis of modified peptidoglycan precursors by vancomycin-resistant Enterococcus faecium. FEMS Microbiol Lett. 1992 Nov 1;77(1-3):109–115. doi: 10.1016/0378-1097(92)90140-j. [DOI] [PubMed] [Google Scholar]
  2. Arthur M., Courvalin P. Genetics and mechanisms of glycopeptide resistance in enterococci. Antimicrob Agents Chemother. 1993 Aug;37(8):1563–1571. doi: 10.1128/aac.37.8.1563. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Arthur M., Molinas C., Bugg T. D., Wright G. D., Walsh C. T., Courvalin P. Evidence for in vivo incorporation of D-lactate into peptidoglycan precursors of vancomycin-resistant enterococci. Antimicrob Agents Chemother. 1992 Apr;36(4):867–869. doi: 10.1128/aac.36.4.867. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Arthur M., Molinas C., Courvalin P. Sequence of the vanY gene required for production of a vancomycin-inducible D,D-carboxypeptidase in Enterococcus faecium BM4147. Gene. 1992 Oct 12;120(1):111–114. doi: 10.1016/0378-1119(92)90017-j. [DOI] [PubMed] [Google Scholar]
  5. Arthur M., Molinas C., Courvalin P. The VanS-VanR two-component regulatory system controls synthesis of depsipeptide peptidoglycan precursors in Enterococcus faecium BM4147. J Bacteriol. 1992 Apr;174(8):2582–2591. doi: 10.1128/jb.174.8.2582-2591.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Arthur M., Molinas C., Depardieu F., Courvalin P. Characterization of Tn1546, a Tn3-related transposon conferring glycopeptide resistance by synthesis of depsipeptide peptidoglycan precursors in Enterococcus faecium BM4147. J Bacteriol. 1993 Jan;175(1):117–127. doi: 10.1128/jb.175.1.117-127.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Arthur M., Molinas C., Dutka-Malen S., Courvalin P. Structural relationship between the vancomycin resistance protein VanH and 2-hydroxycarboxylic acid dehydrogenases. Gene. 1991 Jul 15;103(1):133–134. doi: 10.1016/0378-1119(91)90405-z. [DOI] [PubMed] [Google Scholar]
  8. Bugg T. D., Dutka-Malen S., Arthur M., Courvalin P., Walsh C. T. Identification of vancomycin resistance protein VanA as a D-alanine:D-alanine ligase of altered substrate specificity. Biochemistry. 1991 Feb 26;30(8):2017–2021. doi: 10.1021/bi00222a002. [DOI] [PubMed] [Google Scholar]
  9. Bugg T. D., Wright G. D., Dutka-Malen S., Arthur M., Courvalin P., Walsh C. T. Molecular basis for vancomycin resistance in Enterococcus faecium BM4147: biosynthesis of a depsipeptide peptidoglycan precursor by vancomycin resistance proteins VanH and VanA. Biochemistry. 1991 Oct 29;30(43):10408–10415. doi: 10.1021/bi00107a007. [DOI] [PubMed] [Google Scholar]
  10. Cruz-Rodz A. L., Gilmore M. S. High efficiency introduction of plasmid DNA into glycine treated Enterococcus faecalis by electroporation. Mol Gen Genet. 1990 Oct;224(1):152–154. doi: 10.1007/BF00259462. [DOI] [PubMed] [Google Scholar]
  11. Dutka-Malen S., Molinas C., Arthur M., Courvalin P. The VANA glycopeptide resistance protein is related to D-alanyl-D-alanine ligase cell wall biosynthesis enzymes. Mol Gen Genet. 1990 Dec;224(3):364–372. doi: 10.1007/BF00262430. [DOI] [PubMed] [Google Scholar]
  12. Ferretti J. J., Gilmore K. S., Courvalin P. Nucleotide sequence analysis of the gene specifying the bifunctional 6'-aminoglycoside acetyltransferase 2"-aminoglycoside phosphotransferase enzyme in Streptococcus faecalis and identification and cloning of gene regions specifying the two activities. J Bacteriol. 1986 Aug;167(2):631–638. doi: 10.1128/jb.167.2.631-638.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Gutmann L., Billot-Klein D., al-Obeid S., Klare I., Francoual S., Collatz E., van Heijenoort J. Inducible carboxypeptidase activity in vancomycin-resistant enterococci. Antimicrob Agents Chemother. 1992 Jan;36(1):77–80. doi: 10.1128/aac.36.1.77. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Handwerger S., Pucci M. J., Volk K. J., Liu J., Lee M. S. The cytoplasmic peptidoglycan precursor of vancomycin-resistant Enterococcus faecalis terminates in lactate. J Bacteriol. 1992 Sep;174(18):5982–5984. doi: 10.1128/jb.174.18.5982-5984.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Jacob A. E., Hobbs S. J. Conjugal transfer of plasmid-borne multiple antibiotic resistance in Streptococcus faecalis var. zymogenes. J Bacteriol. 1974 Feb;117(2):360–372. doi: 10.1128/jb.117.2.360-372.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Le Bouguénec C., de Cespédès G., Horaud T. Presence of chromosomal elements resembling the composite structure Tn3701 in streptococci. J Bacteriol. 1990 Feb;172(2):727–734. doi: 10.1128/jb.172.2.727-734.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Messer J., Reynolds P. E. Modified peptidoglycan precursors produced by glycopeptide-resistant enterococci. FEMS Microbiol Lett. 1992 Jul 1;73(1-2):195–200. doi: 10.1016/0378-1097(92)90608-q. [DOI] [PubMed] [Google Scholar]
  18. Nieto M., Perkins H. R. Modifications of the acyl-D-alanyl-D-alanine terminus affecting complex-formation with vancomycin. Biochem J. 1971 Aug;123(5):789–803. doi: 10.1042/bj1230789. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Norrander J., Kempe T., Messing J. Construction of improved M13 vectors using oligodeoxynucleotide-directed mutagenesis. Gene. 1983 Dec;26(1):101–106. doi: 10.1016/0378-1119(83)90040-9. [DOI] [PubMed] [Google Scholar]
  20. Reynolds P. E. Structure, biochemistry and mechanism of action of glycopeptide antibiotics. Eur J Clin Microbiol Infect Dis. 1989 Nov;8(11):943–950. doi: 10.1007/BF01967563. [DOI] [PubMed] [Google Scholar]
  21. STROMINGER J. L. Microbial uridine-5'-pyrophosphate N-acetylamino sugar compounds. I. Biology of the penicillin-induced accumulation. J Biol Chem. 1957 Jan;224(1):509–523. [PubMed] [Google Scholar]
  22. Trieu-Cuot P., Carlier C., Poyart-Salmeron C., Courvalin P. An integrative vector exploiting the transposition properties of Tn1545 for insertional mutagenesis and cloning of genes from gram-positive bacteria. Gene. 1991 Sep 30;106(1):21–27. doi: 10.1016/0378-1119(91)90561-o. [DOI] [PubMed] [Google Scholar]
  23. Vieira J., Messing J. The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene. 1982 Oct;19(3):259–268. doi: 10.1016/0378-1119(82)90015-4. [DOI] [PubMed] [Google Scholar]
  24. Wright G. D., Molinas C., Arthur M., Courvalin P., Walsh C. T. Characterization of vanY, a DD-carboxypeptidase from vancomycin-resistant Enterococcus faecium BM4147. Antimicrob Agents Chemother. 1992 Jul;36(7):1514–1518. doi: 10.1128/aac.36.7.1514. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Zarlenga L. J., Gilmore M. S., Sahm D. F. Effects of amino acids on expression of enterococcal vancomycin resistance. Antimicrob Agents Chemother. 1992 Apr;36(4):902–905. doi: 10.1128/aac.36.4.902. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. al-Obeid S., Collatz E., Gutmann L. Mechanism of resistance to vancomycin in Enterococcus faecium D366 and Enterococcus faecalis A256. Antimicrob Agents Chemother. 1990 Feb;34(2):252–256. doi: 10.1128/aac.34.2.252. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Antimicrobial Agents and Chemotherapy are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES