More than 60 years after the introduction of penicillin, the class of ␤-lactam antibiotics remains the most commonly used to treat severe infections despite the emergence of various resistance mechanisms, including drug detoxification by production of ␤-lactamases (1), decreased affinity of the target (2), and decreased permeability (3). The success story of ␤-lactams involves the development of several generations of ␤-lactams to defeat the resistance mechanisms and their association with inhibitors of ␤-lactamases (4, 5). All ␤-lactams have a common mechanism of action, as they share structural similarity with bacterial cell wall precursors. These drugs act as suicide substrates of the DD-transpeptidase catalytic domain of the penicillin-binding proteins (PBPs) 2 responsible for the last cross-linking step of cell wall assembly (Fig. 1A) (2, 6, 7).Among the DD-transpeptidases of Enterococcus faecium, the low affinity PBP5 is unessential for growth but responsible for intrinsic low level resistance to ampicillin (8, 9), the first-line drug used at high dosage to treat severe enterococcal infections. In clinical isolates, higher levels of resistance to ampicillin are commonly associated with multiple amino acid substitutions in the transpeptidase domain of PBP5 (10, 11). We have shown previously that even higher levels of resistance to ampicillin can be achieved by a novel mechanism in the absence of PBP5 (12 (Fig. 1B). In the presence of ampicillin, the novel L-Lys 3 3D-iAsn-L-Lys 3 cross-links were exclusively found in the peptidoglycan of E. faecium M512, indicating that the DD-transpeptidase activity of the PBPs did not participate in the formation of the cross-links. Selection of E. faecium M512 from E. faecium D344S in five consecutive steps on increasing concentrations of ampicillin led to the gradual activation of the LD-transpeptidation pathway because the proportion of L-Lys 3 3D-iAsn-L-Lys 3 was 3.1% for D344S and increased at each selection step (13). Activation of the resistance pathway was associated with the production of a DD-carboxypeptidase that generated precursors containing a tetrapeptide stem in the cytoplasm of E. faecium M512 (13). Based on these observations, we have speculated that precursors containing a tetrapeptide stem are essential for the formation of the L-Lys 3 3D-iAsn-L-Lys 3 cross-links. In the present work, we have identified in E. faecium an LD-transpeptidase as the key enzyme of this alternate transpeptidation pathway and have shown that its substrate does not bear structural similarity to ␤-lactams. The bypass mechanism could invalidate the ␤-lactams as the major class of antibiotics. EXPERIMENTAL PROCEDURES Purification of the LD-Transpeptidase and N-terminal Sequencing-The LD-transpeptidase from E. faecium (Ldt fm ) was partially purified in four chromatographic steps using a radioactive exchange assay (13) to detect active fractions. Briefly, E. faecium M512 (12) was grown to an A 650 of 0.7 in 24 liters of brain heart infusion broth (Difco), harvested by centrifugati...

Enterococcus faecalis produces a specific penicillin-binding protein (PBP5) that mediates high-level resistance to the cephalosporin class of ␤-lactam antibiotics. Deletion of a locus encoding a previously uncharacterized two-component regulatory system of E. faecalis (croRS) led to a 4,000-fold reduction in the MIC of the expanded-spectrum cephalosporin ceftriaxone. The cytoplasmic domain of the sensor kinase (CroS) was purified and shown to catalyze ATP-dependent autophosphorylation followed by transfer of the phosphate to the mated response regulator (CroR). The croR and croS genes were cotranscribed from a promoter (croRp) located in the rrnC-croR intergenic region. A putative seryl-tRNA synthetase gene (serS) located immediately downstream from croS did not appear to be a target of CroRS regulation or to play a role in ceftriaxone resistance. A plasmid-borne croRp-lacZ fusion was trans-activated by the CroRS system in response to the presence of ceftriaxone in the culture medium. The fusion was also induced by representatives of other classes of ␤-lactam antibiotics and by inhibitors of early and late steps of peptidoglycan synthesis. The croRS null mutant produced PBP5, and expression of an additional copy of pbp5 under the control of a heterologous promoter did not restore ceftriaxone resistance. Deletion of croRS was not associated with any defect in the synthesis of the nucleotide precursor UDP-MurNAc-pentapeptide or of the D-Ala 4 3L-Ala-L-Ala-Lys 3 peptidoglycan cross-bridge. Thus, the croRS mutant was susceptible to ceftriaxone despite the production of PBP5 and the synthesis of wild-type peptidoglycan precursors. These observations constitute the first description of regulatory genes essential for PBP5-mediated ␤-lactam resistance in enterococci.Enterococcus faecalis and E. faecium are opportunistic pathogens that are common causes of urinary tract infections, bacteremia, and endocarditis (20). Enterococcal infections are difficult to treat, as enterococci are intrinsically resistant to various antibiotics and can acquire, mainly by horizontal gene transfer, high-level resistance to virtually all antimicrobial agents. The complete genome sequence of E. faecalis strain V583 revealed an unusually high (25%) content of mobile elements and exogenously acquired DNA, including virulence factors and antibiotic resistance genes (22). The plasticity of the genome correlates with the facility of enterococci to acquire novel resistance mechanisms and to transfer the corresponding genes to other genera, as exemplified by the emergence of high-level glycopeptide resistance in E. faecalis and E. faecium in the late 1980s and the dissemination of the same gene cluster in Staphylococcus aureus 15 years later (8).Enterococci are resistant to the newer cephalosporins which have been developed to treat infections due to gram-negative bacteria producing ␤-lactamases. Treatment with cephalosporins is one of the risk factors for colonization and infection by multidrug-resistant enterococci. Cephalosporin resistance is me...

Peptidoglycan polymerization complexes contain multimodular penicillin-binding proteins (PBP) of classesA 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 ␤-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 ␤-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.