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Journal of Bacteriology, March 2004, p. 1221-1228, Vol. 186, No. 5
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.5.1221-1228.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Role of Class A Penicillin-Binding Proteins in PBP5-Mediated ß-Lactam Resistance in Enterococcus faecalis

Ana Arbeloa,¹ Heidi Segal,^1, Jean-Emmanuel Hugonnet,¹ Nathalie Josseaume,¹ Lionnel Dubost,² Jean-Paul Brouard,² Laurent Gutmann,¹ Dominique Mengin-Lecreulx,³ and Michel Arthur^1*

INSERM E0004-LRMA, Université Paris VI, 75270 Paris,¹ Département Régulations, Développement et Diversité Moléculaire, Museum National d'Histoire Naturelle, USM0502-CNRS UMR8041, 75005 Paris,² Institut de Biochimie et de Biophysique Moléculaire et Cellulaire, UMR 8619 CNRS, Université Paris-Sud, 91405 Orsay, France³

Received 21 July 2003/ Accepted 21 November 2003

Peptidoglycan polymerization complexes contain multimodular penicillin-binding proteins (PBP) of classes A and B that associate a conserved C-terminal transpeptidase module to an N-terminal glycosyltransferase or morphogenesis module, respectively. In Enterococcus faecalis, class B PBP5 mediates intrinsic resistance to the cephalosporin class of ß-lactam antibiotics, such as ceftriaxone. To identify the glycosyltransferase partner(s) of PBP5, combinations of deletions were introduced in all three class A PBP genes of E. faecalis JH2-2 (ponA, pbpF, and pbpZ). Among mutants with single or double deletions, only JH2-2 ponA pbpF was susceptible to ceftriaxone. Ceftriaxone resistance was restored by heterologous expression of pbpF from Enterococcus faecium but not by mgt encoding the monofunctional glycosyltransferase of Staphylococcus aureus. Thus, PBP5 partners essential for peptidoglycan polymerization in the presence of ß-lactams formed a subset of the class A PBPs of E. faecalis, and heterospecific complementation was observed with an ortholog from E. faecium. Site-directed mutagenesis of pbpF confirmed that the catalytic serine residue of the transpeptidase module was not required for resistance. None of the three class A PBP genes was essential for viability, although deletion of the three genes led to an increase in the generation time and to a decrease in peptidoglycan cross-linking. As the E. faecalis chromosome does not contain any additional glycosyltransferase-related genes, these observations indicate that glycan chain polymerization in the triple mutant is performed by a novel type of glycosyltransferase. The latter enzyme was not inhibited by moenomycin, since deletion of the three class A PBP genes led to high-level resistance to this glycosyltransferase inhibitor.

Present address: Department of Medical Microbiology, Medical School, University of Cape Town, 7925 Cape Town, South Africa.

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