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J. Biol. Chem., Vol. 277, Issue 39, 35801-35807, September 27, 2002

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Balance between Two Transpeptidation Mechanisms Determines the Expression of -Lactam Resistance in Enterococcus faecium^*

Jean-Luc Mainardi^§, Véronique Morel, Martine Fourgeaud, Julie Cremniter, Didier Blanot^¶, Raymond Legrand, Claude Fréhel^**, Michel Arthur, Jean van Heijenoort^¶, and Laurent Gutmann

From the  INSERM EMI-U 0004 Laboratoire de Recherche Moléculaire sur les Antibiotiques, UFR Broussais-Hôtel Dieu, Université Paris VI, 75270 Paris, France, the ^¶ Enveloppes Bactériennes et Antibiotiques, UMR 8619, CNRS, 91405 Orsay, France, the  Physics Department, Hoechst Marion Roussel, 93235 Romainville, France, and ^** INSERM U-411, Faculté de Médecine Necker Enfants Malades, 75730 Paris cedex 15, France

The D,D-transpeptidase activity of high molecular weight penicillin-binding proteins (PBPs) is essential to maintain cell wall integrity as it catalyzes the final cross-linking step of bacterial peptidoglycan synthesis. We investigated a novel -lactam resistance mechanism involving by-pass of the essential PBPs by L,D-transpeptidation in Enterococcus faecium. Determination of the peptidoglycan structure by reverse phase high performance liquid chromatography coupled to mass spectrometry revealed that stepwise selection for ampicillin resistance led to the gradual replacement of the usual cross-links generated by the PBPs (D-Ala₄  D-Asx-Lys₃) by cross-links resulting from L,D-transpeptidation (L-Lys₃  D-Asx-Lys₃). This was associated with no modification of the level of production of the PBPs or of their affinity for -lactams, indicating that altered PBP activity was not required for ampicillin resistance. A -lactam-insensitive L,D-transpeptidase was detected in membrane preparations of the parental susceptible strain. Acquisition of resistance was not because of variation of this activity. Instead, selection led to production of a -lactam-insensitive D,D-carboxypeptidase that cleaved the C-terminal D-Ala residue of pentapeptide stems in vitro and caused massive accumulation of cytoplasmic precursors containing a tetrapeptide stem in vivo. The parallel dramatic increase in the proportion of L-Lys₃  D-Asx-Lys₃ cross-links showed that the enzyme was activating the resistance pathway by generating the substrate for the L,D-transpeptidase.

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