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Journal of Bacteriology, June 2009, p. 3649-3656, Vol. 191, No. 11
0021-9193/09/$08.00+0     doi:10.1128/JB.01834-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Role of Class A Penicillin-Binding Proteins in the Expression of β-Lactam Resistance in Enterococcus faecium{triangledown}

Louis B. Rice,1,2,4* Lenore L. Carias,4 Susan Rudin,4 Rebecca Hutton,2 Steven Marshall,2 Medhat Hassan,3 Nathalie Josseaume,5,6,7 Lionel Dubost,8,9 Arul Marie,8,9 and Michel Arthur5,6,7

Medical,1 Research,2 Pathology and Laboratory Medicine Services, Louis Stokes Cleveland VA Medical Center,3 Department of Medicine, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106,4 INSERM, U872, LRMA-Equipe 12, Paris, F-75006, France,5 Centre de Recherche des Cordeliers, Université Pierre et Marie Curie, UMR S 872, Paris F-75006, France,6 Université Paris Descartes, UMR S 872, Paris F-75006, France,7 Muséum National d'Histoire Naturelle, USM0502, Plateforme de Spectrométrie de Masse et de Protéomique du Muséum, Département Recherche Développement et Diversité Moléculaire, Paris F-75005, France,8 CNRS, UMR8041, Paris F-75005, France9

Received 30 December 2008/ Accepted 17 March 2009

Peptidoglycan is polymerized by monofunctional D,D-transpeptidases belonging to class B penicillin-binding proteins (PBPs) and monofunctional glycosyltransferases and by bifunctional enzymes that combine both activities (class A PBPs). Three genes encoding putative class A PBPs (pbpF, pbpZ, and ponA) were deleted from the chromosome of Enterococcus faecium D344R in all possible combinations in order to identify the glycosyltransferases that cooperate with low-affinity class B Pbp5 for synthesis of peptidoglycan in the presence of β-lactam antibiotics. The viability of the triple mutant indicated that glycan strands can be polymerized independently from class A PBPs by an unknown glycosyltranferase. The susceptibility of the {Delta}pbpF {Delta}ponA mutant and triple mutants to extended spectrum cephalosporins (ceftriaxone and cefepime) identified either PbpF or PonA as essential partners of Pbp5 for peptidoglycan polymerization in the presence of the drugs. Mass spectrometry analysis of peptidoglycan structure showed that loss of PonA and PbpF activity led to a minor decrease in the extent of peptidoglycan cross-linking by the remaining PBPs without any detectable compensatory increase in the participation of the L,D-transpeptidase in peptidoglycan synthesis. Optical density measurements and electron microscopy analyses showed that the {Delta}pbpF {Delta}ponA mutant underwent increased stationary-phase autolysis compared to the parental strain. Unexpectedly, deletion of the class A pbp genes revealed dissociation between the expression of resistance to cephalosporins and penicillins, although the production of Pbp5 was required for resistance to both classes of drugs. Thus, susceptibility of Pbp5-mediated peptidoglycan cross-linking to different β-lactam antibiotics differed as a function of its partner glycosyltransferase.

* Corresponding author. Mailing address: Medical Service 111(W), Louis Stokes Cleveland VA Medical Center, 10701 East Blvd., Cleveland, OH 44106. Phone: (216) 791-3800. Fax: (216) 231-3289. E-mail: louis.rice{at}va.gov

{triangledown} Published ahead of print on 20 March 2009.

Journal of Bacteriology, June 2009, p. 3649-3656, Vol. 191, No. 11
0021-9193/09/$08.00+0     doi:10.1128/JB.01834-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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