STRUCTURE OF THE LOW-AFFINITY PENICILLIN-BINDING-PROTEIN-5 PBP5FM IN WILD-TYPE AND HIGHLY PENICILLIN-RESISTANT STRAINS OF ENTEROCOCCUS-FAECIUM

Among its penicillin-binding proteins (PBPs), enterococcus faecium pos sesses a low-affinity PBP5, PBP5fm, which is the main target involved in beta-lactam resistance. A 7.7-kb EcoRI chromosomal fragment of E. f aecium D63r containing the pbp5fm gene was cloned and sequenced. Two o pen reading frames (ORFs) were found, A 2,037-bp ORF encoded tile dedu ced 73.8-kDa PBP5fm, the amino acid sequences of which were, respectiv ely, 99.8, 78.5, and 62% homologous to those of the low-affinity plasm id-encoded PBP3r of Enterococcus hirae S185r and the chromosome-encode d 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 appeare d to he 285 bp shorter than and 74% homologous with the regulatory gen e pst of E. hirae ATCC 9790, Different clinical isolates of E. faecium , for which a wide range of benzylpenicillin MICs were observed, showe d that the increases in MICs were related to two mechanisms. For some strains of intermediate resistance (MICs of 16 to 64 mu g/ml), the inc reased level of resistance could he explained by the presence of large r quantities of PBP5fm which had an affinity for benzylpenicillin (sec ond-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 resi stant strains, EFM-1 (MIC, 90 mu g/ml) and H80721 (MIG, 512 mu g/ml), the resistance was related to different amino acid substitutions yield ing very-low-affinity PBP5fm variants (k(+2)/k less than or equal to 1 .5 M(-1) s(-1)) which were synthesized in small quantities. More speci fically, it appeared, with a three-dimensional model of the C-terminal domain of PBP5fm, that the substitutions of Met-485, located in the t hird position after the conserved SDN triad, by Thr in EFM-1 and by Al a in H80721 were the most likely cause of the decreasing affinity of P BP5fm observed in these strains.