Among its penicillin-binding proteins (PBPs), Enterococcus faecium possesses a low-affinity PBP5, PBP5fm, which is the main target involved in ␤-lactam resistance. A 7.7-kb EcoRI chromosomal fragment of E. faecium D63r containing the pbp5fm gene was cloned and sequenced. Two open reading frames (ORFs) were found. A 2,037-bp ORF encoded the deduced 73.8-kDa PBP5fm, the amino acid sequences of which were, respectively, 99.8, 78.5, and 62% homologous to those of the low-affinity plasmid-encoded PBP3r of Enterococcus hirae S185r and the chromosome-encoded PBP5 of E. hirae R40 and Enterococcus faecalis 56R. A second 597-bp ORF, designated psrfm, was found 2.3 kb upstream of pbp5fm. It appeared to be 285 bp shorter than and 74% homologous with the regulatory gene psr of E. hirae ATCC 9790. Different clinical isolates of E. faecium, for which a wide range of benzylpenicillin MICs were observed, showed that the increases in MICs were related to two mechanisms. For some strains of intermediate resistance (MICs of 16 to 64 g/ml), the increased level of resistance could be explained by the presence of larger quantities of PBP5fm which had an affinity for benzylpenicillin (second-order rate constant of protein acylation [k ؉2 /K] values of 17 to 25 M ؊1 s ؊1) that remained unchanged. For the two most highly resistant strains, EFM-1 (MIC, 90 g/ml) and H80721 (MIC, 512 g/ml), the resistance was related to different amino acid substitutions yielding very-low-affinity PBP5fm) which were synthesized in small quantities. More specifically, it appeared, with a three-dimensional model of the C-terminal domain of PBP5fm, that the substitutions of Met-485, located in the third position after the conserved SDN triad, by Thr in EFM-1 and by Ala in H80721 were the most likely cause of the decreasing affinity of PBP5fm observed in these strains.The genus Enterococcus, closely related to the genus Streptococcus, is involved in different clinical infections (37). Apart from their physiological properties, enterococci also differ from streptococci in that they generally are naturally 10-to 1,000-fold less susceptible to penicillins than streptococci (37). It was demonstrated that the natural low susceptibility of enterococci to penicillin is linked to the presence of at least one high-molecular-mass penicillin-binding protein (PBP) which has a low affinity for ␤-lactams (1, 17, 50). Enterococcus faecium appears to be the enterococcal species most resistant to ␤-lactam antibiotics, for which there are a wide range of benzylpenicillin MICs (0.5 to Ն 64 g/ml) for clinical isolates (22,30,50). Recently it became obvious that a new population of clinical E. faecium isolates for which the MICs of benzylpenicillin were very high (256 to 512 g/ml) had emerged in different countries (15,22,23,30).Among laboratory mutants and clinical isolates of E. faecium, two mechanisms have been shown to be involved in the high-level resistance to benzylpenicillin. These strains produced either an increased quantity of the essential low-affinity PBP5fm (15, 17) o...

SummaryA region of 7.8 kb of the plasmid pMB2 from Enterococcus faecalis S-48 carrying the information necessary for production and immunity of the peptide antibiotic AS-48 has been cloned and sequenced. It contains the as-48A structural gene plus five open reading frames (as-48B, as-48C, as-48C1, as-48D and as-48D1 ). Besides As-48D, all the predicted gene products are basic hydrophobic proteins with potential membrane-spanning domains (MSDs). None of them shows any homology with protein sequences stored in databanks, except for As-48D, which shows similarity to the C-terminal domain of ABC transporters and contains a highly conserved ATP-binding site. The gene products of as-48B, as-48C, as-48C1 and as-48D are thought to be involved in AS-48 production and secretion. The only gene able to provide resistance to AS-48 by itself is as-48D1. Immunity also seems to be enhanced at least by the products of as-48B, as-48C1 and as-48D genes. Transcription analysis using probes derived from the different ORFs revealed two large (3.5 and 2.7 kb) mRNAs, suggesting that the different genes are organized in two constitutive operons.

The structural gene of the enterococcal peptide antibiotic AS-48 (as-48) has been identified and cloned by using two degenerate 17-mer DNA oligonucleotides on the basis of the amino acid sequences of two peptides obtained by digestion of the antibiotic with Glu-C endoproteinase. That as-48 gene codes for a 105-amino-acid prepeptide, giving rise to a 70-amino-acid mature protein. Comparative analysis demonstrated that the 16-amino-acid sequence of one of the AS48 Glu-C peptides, designated V8-5, was composed of a 12-aminIo-acid sequence corresponding to the C-terminal end sequence (from isoleucine +59 to tryptophan +70 [I+'9 to W+70J) of the prepeptide and terminated in four residues forming the N terminus (M+1 to E+4) of a putative AS-48 propeptide. These data, combined with the characteristics of the gene sequence, strongly suggested that the antibiotic peptide was a 70-residue cyclic molecule. We propose that the AS-48 translated primary product is very likely submitted to a posttranslational modification during secretion (i) by an atypical or a typical signal peptidase that cleaves of a 35-residue or shorter signal peptide, respectively, from the prepeptide molecule and (ii) by the linkage of the methionine residue (M+1) to the C-terminal tryptophan residue (W+70) to obtain the cyclic peptide (a tall-head linkage).For many years, it has been known that enterococci produce inhibitory substances such as bacteriocins and hemolysins that are encoded by large widely disseminated and transmissible plasmids (4,9,13,20,22). These plasmids usually are able to transfer to recipient cells at a relatively high frequency in broth (4,13,24,25). The best-characterized bacteriocin produced by enterococci is the bacteriocin-hemolysin encoded by the sex pheromone plasmid pADi (11,14). The cytolytic system of pADi revealed two protein components, designated A (activator) and L (lytic), able to complement each other extracellularly (11,14) and to contribute to bacterial virulence in an animal model (15).AS-48 is a plasmid-encoded peptide antibiotic produced by Enterococcus faecalis S-48 with broad antimicrobial activities against gram-positive and gram-negative bacteria (6). Some pathogenic bacteria, including Staphylococcus, Enterococcus, and Salmonella species, are highly sensitive to AS-48 (7). Insertion of the peptide into the cytoplasmic membrane of target cells or artificial membranes renders the membrane permeable to small molecules (ions, amino acids), causing the release of cytoplasmic material and the lysis of sensitive cells (8).Because of its strongly cationic properties and biological activity, the AS-48 peptide antibiotic could be related to the group of bacteriocins and peptide antibiotics, mainly produced by lactic acid bacteria, which appear to function by permeabilizing the cytoplasmic membrane. They include lactococcins A, B, and G (12, 27, 39); lactocin S (26); and the group of lantibiotics, e.g., nisin (2, 10), epidermin (34)

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Abstract. Penicillin-binding proteins (PBPs) are membrane proteins involved in the final stages of peptidoglycan synthesis and represent the targets of b-lactam antibiotics. Enterococci are naturally resistant to these antibiotics because they produce a PBP, named PBP5fm in Enterococcus faecium, with low-level affinity for b-lactams. We report here the crystal structure of the acyl-enzyme complex of PBP5fm with benzylpenicillin at a resolution of 2.4 Å. A characteristic of the active site, which distinguishes PBP5fm from other PBPs of known structure, is the topology of the loop 451 -465 defining the left CMLS, Cell. Mol. Life Sci. 59 (2002) 1223 -1232 1420-682X/02/071223-10 $ 1.50 + 0.20/0 © Birkhäuser Verlag, Basel, 2002 CMLS Cellular and Molecular Life Sciencesedge of the cavity. The residue Arg464, involved in a salt bridge with the residue Asp481, confers a greater rigidity to the PBP5fm active site. In addition, the presence of the Val465 residue, which points into the active site, reducing its accessibility, could account for the low affinity of PBP5fm for b-lactam. This loop is common to PBPs of low affinity, such as PBP2a from Staphylococcus aureus and PBP3 from Bacillus subtilis. Moreover, the insertion of a serine after residue 466 in the most resistant strains underlines even more the determining role of this loop in the recognition of the substrates.