The contribution of penicillin-binding protein 5 (PBP 5) to intrinsic and acquired ␤-lactam resistance was investigated by constructing isogenic strains of Enterococcus faecium producing different PBP 5. The pbp5 genes from three E. faecium clinical isolates (BM4107, D344, and H80721) were cloned into the shuttle vector pAT392 and introduced into E. faecium D344S, a spontaneous derivative of E. faecium D344 highly susceptible to ampicillin due to deletion of pbp5 (MIC, 0.03 g/ml). Immunodetection of PBP5 indicated that cloning of the pbp5 genes into pAT392 resulted in moderate overproduction of PBP 5 in comparison to wild-type strains. This difference may be attributed to a difference in gene copy number. Expression of the pbp5 genes from BM4107 (MIC, 2 g/ml), D344 (MIC, 24 g/ml), and H80721 (MIC, 512 g/ml) in D344S conferred relatively low levels of resistance to ampicillin (MICs, 6, 12, and 20 g/ml, respectively). A methionine-to-alanine substitution was introduced at position 485 of the BM4107 PBP 5 by site-directed mutagenesis. In contrast to previous hypotheses based on comparison of nonisogenic strains, this substitution resulted in only a 2.5-fold increase in the ampicillin MIC. The reversed-phase high-performance liquid chromatography muropeptide profiles of D344 and D344S were similar, indicating that deletion of pbp5 was not associated with a detectable defect in cell wall synthesis. These results indicate that pbp5 is a nonessential gene responsible for intrinsic resistance to moderate levels of ampicillin and by itself cannot confer high-level resistance.Enterococcus faecium is intrinsically resistant to moderate levels of ampicillin by production of the low-affinity penicillinbinding protein 5 (PBP 5). Bacterial growth occurs at ␤-lactam concentrations sufficient to inactivate all the other PBPs, suggesting that PBP5 is the only transpeptidase required for peptidoglycan synthesis under such conditions (4,20). Acquired resistance to higher levels of ampicillin in clinical isolates of E. faecium has been associated with increased production of PBP 5 or decreased affinity for the ␤-lactam antibiotics (7,8,10,13,18,20,21). The latter mechanism was inferred from comparison of the pbp5 genes from clinical isolates in which amino acid substitutions at specific positions near or in the conserved motifs of the PBP module were associated with decreased interaction with ␤-lactams and expression of resistance (8,13,18,21). However, the role of the PBP 5 in the level of resistance was not rigorously established since isogenic strains were not constructed. In the present study, pbp5 genes from E. faecium clinical isolates expressing various levels of ampicillin resistance were cloned into a shuttle vector and introduced into E. faecium D344S, a spontaneous mutant in which the chromosomal pbp5 locus is deleted. Expression of the different pbp5 genes in this host resulted in similar low levels of ampicillin resistance, indicating that alterations of PBP 5 alone do not account for acquired high-level ␤-lactam resist...

We report a structural and transcriptional analysis of the pbp5 region of Enterococcus faecium C68. pbp5 exists within a larger operon that includes upstream open reading frames (ORFs) corresponding to previously reported psr (penicillin-binding protein synthesis repressor) and ftsW (whose product is a transmembrane protein that interacts with PBP3 in Escherichia coli septum formation) genes. Hybridization of mRNA from C68, CV133, and four ampicillin-resistant CV133 mutants revealed four distinct transcripts from this region, consisting of (i) E. faecium ftsW (ftsW Efm ) alone; (ii) psr and pbp5; (iii) pbp5 alone; and (iv) ftsW Efm , psr, and pbp5. Quantities of the different transcripts varied between strains and did not always correlate with quantities of PBP5 or levels of ampicillin resistance. Since the psr of C68 is presumably nonfunctional due to an insertion of an extra nucleotide in the codon for the 44th amino acid, the region extending from the ftsW Efm promoter through the pbp5 gene of C68 was cloned in E. coli to facilitate mutagenesis. The psr ORF was regenerated using site-directed mutagenesis and introduced into E. faecium D344-SRF on conjugative shuttle vector pTCV-lac (pCWR558 [psr ORF interrupted]; pCWR583 [psr ORF intact]). Ampicillin MICs for both D344-SRF-(pCWR558) and D344-SRF(pCWR583) were 64 g/ml. Quantities of pbp5 transcript and protein were similar in strains containing either construct regardless of whether they were grown in the presence or absence of ampicillin, arguing against a role for PSR as a repressor of pbp5 transcription. However, quantities of psr transcript were increased in D344-SRF(pCWR583) compared to D344-SRF(pCWR558), especially after growth in ampicillin; suggesting that PSR acts in some manner to activate its own transcription.Penicillin resistance in Enterococcus faecium is associated with production of low-affinity penicillin-binding protein PBP5. The presence of this penicillin-binding protein (PBP) in virtually all clinical E. faecium strains that have been investigated (including those susceptible to clinically achievable levels of penicillin [L. B. Rice, unpublished data]) suggests that it is intrinsic to this species, rather than an acquired gene. Supportive evidence for the role of PBP5 in penicillin resistance is derived from experiments indicating that PBP5-expressing cells replicate when incubated with penicillin at concentrations sufficient to saturate all of the other PBPs, as well as from studies demonstrating that E. faecium strains lacking PBP5 are highly susceptible to penicillin (10-12, 25, 26).Early studies on Enterococcus hirae 9790 (which until 1985 was considered to be a type strain for Enterococus faecalis [8,16]) reported that elevated levels of penicillin resistance (to ca. 64 g/ml) were associated with increased quantities of detectable PBP5. Increased PBP5 production in one resistant mutant (R40) was associated with deletion of the N-terminal portion and some upstream DNA of an open reading frame (ORF) located ca. 1 kb upstream of the pbp5...