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Journal of Bacteriology, June 2001, p. 3436-3446, Vol. 183, No. 11
0021-9193/01/$04.00+0   DOI: 10.1128/JB.183.11.3436-3446.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Regulation of Expression of the vanD Glycopeptide Resistance Gene Cluster from Enterococcus faecium BM4339

Barbara Casadewall,1 Peter E. Reynolds,2 and Patrice Courvalin1,*

Unité des Agents Antibactériens, Institut Pasteur, 75724 Paris Cedex 15, France,1 and Department of Biochemistry, University of Cambridge, Cambridge, United Kingdom CB2 1QW2

Received 17 November 2000/Accepted 13 March 2001

A new open reading frame, encoding a putative integrase-like protein, was detected downstream from the six genes of the vanD glycopeptide resistance cluster in Enterococcus faecium BM4339 (B. Casadewall and P. Courvalin, J. Bacteriol. 181:3644-3648, 1999). In this cluster, genes coding for the VanRD-VanSD two-component regulatory system were cotranscribed from the PRD promoter, whereas transcription of the vanYD, vanHD, vanD, vanXD, and intD genes was initiated from the PYD promoter located between vanSD and vanYD (the D subscript indicates that the gene is part of the vanD operon). The VanRD-VanSD regulatory system is likely to activate transcription of the resistance genes from the promoter PYD. Glycopeptide-susceptible derivatives of BM4339 were obtained by trans complementation of the frameshift mutation in the ddl gene, restoring functional D-alanine:D-alanine ligase activity in this strain. The glycopeptide-susceptible transformant BM4409, producing only D-alanyl-D-alanine-terminating peptidoglycan precursors, did not express the resistance genes encoding the VanYD D,D-carboxypeptidase, the VanHD dehydrogenase, the VanD ligase, the VanXD D,D-dipeptidase, and also the IntD integrase, although the regulatory region of the vanD cluster was still transcribed. In BM4409, the absence of VanRD-VanSD, apparently dependent, transcription from promoter PYD correlated with the lack of D-alanyl-D-lactate-terminating precursors. The vanXD gene was transcribed in BM4339, but detectable amounts of VanXD D,D-dipeptidase were not synthesized. However, the gene directed synthesis of an active enzyme when cloned on a multicopy plasmid in Escherichia coli, suggesting that the enzyme was unstable in BM4339 or that it had very low activity that was detectable only under conditions of high gene dosage. This activity is not required for glycopeptide resistance in BM4339, since this strain cannot synthesize D-alanyl-D-alanine.

* Corresponding author. Mailing address: Unité des Agents Antibactériens, Institut Pasteur, 28 rue du Docteur Roux, 75724 Paris Cedex 15, France. Phone: 33 1 45 68 83 20. Fax: 33 1 45 68 83 19. E-mail: pcourval{at}pasteur.fr.

Journal of Bacteriology, June 2001, p. 3436-3446, Vol. 183, No. 11
0021-9193/01/$04.00+0   DOI: 10.1128/JB.183.11.3436-3446.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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Copyright © 2001 by the American Society for Microbiology. All rights reserved.