Advertisement

Molecular and General Genetics MGG

, Volume 224, Issue 3, pp 364–372 | Cite as

The VANA glycopeptide resistance protein is related to d-alanyl-d-alanine ligase cell wall biosynthesis enzymes

  • Sylvie Dutka-Malen
  • Catherine Mohnas
  • Michel Arthur
  • Patrice Courvalin
Article

Summary

Inducible resistance to the glycopeptide antibiotics vancomycin and teicoplanin is mediated by plasmid pIP816 in Enterococcus faecium strain BM4147. Vancomycin induced the synthesis of a ca. 40 kDa membrane-associated protein designated VANA. The resistance protein was partially purified and its N-terminal sequence was determined. A 1761 by DNA restriction fragment of pIP816 was cloned into Escherichia coli and sequenced. When expressed in E. coli, this fragment encoded a ca. 40 kDa protein that comigrated with VANA from enterococcal membrane fractions. The ATG translation initiation codon for VANA specified the methionine present at the N-terminus of the protein indicating the absence of signal peptide processing. The amino acid sequence deduced from the sequence of the vanA gene consisted of 343 amino acids giving a protein with a calculated Mr of 37400. VANA was structurally related to the d-alanyl-d-alanine (d-ala-d-ala) ligases of Salmonella typhimurium (36% amino acid identity) and of E. coli (28%). The vanA gene was able to transcomplement an E. coli mutant with thermosensitive d-ala-d-ala ligase activity. Thus, the inducible resistance protein VANA was structurally and functionally related to cytoplasmic enzymes that synthesize the target of glycopeptide antibiotics. Based on these observations we discuss the possibility that resistance is due to modification of the glycopeptide target.

Key words

Resistance Glycopeptide Vancomycin Enterococcus d-alanyl-d-alanine ligase 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Al-Obeid S, Collatz E, Gutmann L (1990) Mechanism of resistance to vancomycin in Enterococcus faecium D366 and Enterococcus faecalis A256. Antimicrob Agents Chemother 34:252–256Google Scholar
  2. Brisson-Noel A, Dutka-Malen S, Molinas C, Leclercq R, Courvalin P (1990) Cloning and heterospecific expression of the resistance determinant vanA encoding high-level resistance to glycopeptides in Enterococcus faecium BM4147. Antimicrob Agents Chemother 34:924–927Google Scholar
  3. Claverie J-M (1984) A common philosophy and FORTRAN 77 software package for implementing and searching sequence databases. Nucleic Acids Res 12:397–407Google Scholar
  4. Daub E, Zawadzke LE, Botstein D, Walsh CT (1988) Isolation, cloning, and sequencing of the Salmonella typhimurium ddlA gene with purification and characterization of its product, {sdd-alanine-d-alanine ligase (ADP forming)}. Biochemistry 27:3701–3708Google Scholar
  5. Edman P (1967) A protein sequanator. Eur J Biochem 1:80–81Google Scholar
  6. Fry DC, Kuby SA, Mildvan AS (1986) ATP-binding site of adenylate kinase: mechanistic implications of its homology with rasencoded p21, F1-ATPase and other nucleotide-binding proteins. Proc Natl Acad Sci USA 83:907–911Google Scholar
  7. Gale EF, Cundliffe E, Reynolds PE, Richmond MH, Waring MJ (1981) The molecular basis of antibiotic action, 2nd edition. Wiley-Interscience Publications, London, pp 144–174Google Scholar
  8. Hammes WP, Neuhaus FC (1974) On the mechanism of action of vancomycin: inhibition of peptidoglycan synthesis in Gaffkya homari. Antimicrob Agents Chemother 6:722–728Google Scholar
  9. Hanahan D (1983) Studies on transformation of Escherichia coli with plasmids. J Mol Biol 166:557–580PubMedGoogle Scholar
  10. Jeffs PW, Nisbet LJ (1988) Glycopeptide antibiotics: a comprehensive approach to discovery, isolation, and structure determination. In: Actor P, Daneo-Moore L, Higgins ML, Salton MRJ, Shockman GD (ed) Antibiotic inhibition of bacterial cell surface assembly and function. American Society for Microbiology, Washington DC, pp 509–530Google Scholar
  11. Kyte J, Doolitle RF (1982)A simple method for displaying the hydropathic character of a protein. J Mol Biol 157:105–132PubMedGoogle Scholar
  12. Laemmli Uk (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685PubMedGoogle Scholar
  13. Leclercq R, Derlot E, Duval J, Courvalin P (1988) Plasmid-mediated resistance to vancomycin and teicoplanin in Enterococcus faecium. N Engl J Med 319:157–161Google Scholar
  14. Lipman DJ, Pearson WR (1985) Rapid and sensitive protein similarity searches. Science 227:1435–1440Google Scholar
  15. Lugtenberg EJJ (1972) Studies on Escherichia coli enzymes involved in the synthesis of uridine diphosphate-N-acetyl-muramyl-pentapeptide. J Bacteriol 110:26–34PubMedGoogle Scholar
  16. Lugtenberg EJJ, van Schijndel-van Dam A (1973) Temperaturesensitive mutant of Escherichia coli K-12 with an impaired d-alanine-d-alanine ligase. J Bacteriol 113:96–104Google Scholar
  17. Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning, a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NYGoogle Scholar
  18. Martin P, Jullien E, Courvalin P (1988) Nucleotide sequence of Acinetobacter baumannii aphA-6 gene: evolutionary and functional implications of sequence homologies with nucleotidebinding proteins, kinases and others aminoglycoside-modifying enzymes. Mol Microbiol 2:615–625Google Scholar
  19. Messing J (1979) A multipurpose cloning system based on singlestranded bacteriophage M13 Recombinant DNA Tech Bull 1:43–44Google Scholar
  20. Moran CP, Lang N, Le Grice SF, Lee G, Stephens M, Sonenshein AL, Pero J, Losick R (1982) Nucleotide sequences that signal the initiation of transcription and translation in Bacillus suhtilis. Mol Gen Genet 186:339–346Google Scholar
  21. Neuhaus FC (1962) The enzymatic synthesis of d-alanyl-d-alanine. I. Purification and properties of d-alanyl-d-alanine synthetase. J Biol Chem 237:778–786Google Scholar
  22. Nicas TI, Wu CYE, Hobbs JN, Preston Jr DA, Allen NE (1989) Characterization of vancomycin resistance in Enterococcus faecium and Enterococcus faecalis. Antimicrob Agents Chemother 33:1121–1I24Google Scholar
  23. Norrander J, Kempe T, Messing J (1983) Construction of improved M13 vectors using oligodeoxynucleotide-directed mutagenesis. Gene 26:101–106Google Scholar
  24. Ogasawara N, Moriya S, von Meyenburg K, Hansen FG, Yoshikawa H (1985) Conservation of genes and their organization in the chromosomal origin region of Bacillus subtilis and Escherichia coli. EMBO J 4:3345–3350Google Scholar
  25. Perkins HR (1969) Specificity of combination between mucopeptide precursors and vancomycin or ristocetin. Biochem J 111:195–205Google Scholar
  26. Perkins HR, Nieto M (1970) The preparation of iodinated vancomycin and its distribution in bacteria treated with the antibiotic. Biochem J 116:83–92Google Scholar
  27. Rambach A, Hogness DS (1977) Translation of Drosophila melanogaster sequences in Escherichia coli. Proc Natl Acad Sci USA 74:5041–5045Google Scholar
  28. Reynolds PE (1961) Studies on the mode of action of vancomycin. Biochim Biophys Acta 52:403–405Google Scholar
  29. Reynolds PE (1989) Structure, biochemistry and mechanism of action of glycopeptide antibiotics. Eur J Microbiol Infect Dis 8:943–950Google Scholar
  30. Robinson AC, Kenan DJ, Sweeney J, Donachie WD (1986) Further evidence for overlapping transcriptional units in an Escherichia coli cell envelope-cell division gene cluster: DNA sequence and transcriptional organization of the ddl fts Q region. J Bacteriol 167:809–817Google Scholar
  31. Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467PubMedGoogle Scholar
  32. Shlaes DM, Al-Obeid S, Shlaes JH, Boisivon A, Williamson R (1989) Inducible transferable resistance to vancomycin in Enterococcus faecium D399. J Antimicrob Chemother 23:503–508Google Scholar
  33. Staden R (1980) A new computer method for the storage and manipulation of DNA gel reading data. Nucleic Acids Res 8:3673–3694Google Scholar
  34. Trieu-Cuot P, Carlier C, Martin P, Courvalin P (1987) Plasmid transfer by conjugation from Escherichia coli to Gram-positive bacteria. FEMS Microbiol Lett 48:289–294Google Scholar
  35. Walsh CT (1989) Enzymes in the D-alanine branch of bacterial cell wall peptidoglycan assembly. J Biol Chem 264:2393–2396Google Scholar
  36. Watson MEE (1984) Compilation of published signal sequences. Nucleic Acids Res 12:5145–5164Google Scholar
  37. Wilbur WJ, Lipman DJ (1983) Rapid similarity searches of nucleic acid and protein data banks. Proc Natl Acad Sci USA 80:726–730Google Scholar
  38. Williamson R, Al. Obeid S, Shlaes JH, Goldstein FW, Shlaes DM (1989) Inducible resistance to vancomycin in Enterococcus faecium D366. J Infect Dis 159:1095–1104Google Scholar

Copyright information

© Springer-Verlag 1990

Authors and Affiliations

  • Sylvie Dutka-Malen
    • 1
  • Catherine Mohnas
    • 1
  • Michel Arthur
    • 1
  • Patrice Courvalin
    • 1
  1. 1.Unité des Agents Antibactériens, Centre National de la Recherche Scientifique, Unité Associée 27IInstitut PasteurParis Cedex 15France

Personalised recommendations