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Antimicrobial Agents and Chemotherapy, May 2001, p. 1480-1486, Vol. 45, No. 5
0066-4804/01/$04.00+0   DOI: 10.1128/AAC.45.5.1480-1486.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Penicillin-Binding Protein 5 and Expression of Ampicillin Resistance in Enterococcus faecium

Louis B. Rice,1,2,* Lenore L. Carias,2 Rebecca Hutton-Thomas,1 Farid Sifaoui,3 Laurent Gutmann,3 and Susan D. Rudin2

Medical and Research Services, VA Medical Center,1 and Department of Medicine, Case Western Reserve University,2 Cleveland, Ohio, and L.R.M.A., Université Paris IV, 57270 Paris Cedex 06, Paris, France3

Received 6 September 2000/Returned for modification 2 November 2000/Accepted 30 January 2001

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 (ftsWEfm) alone; (ii) psr and pbp5; (iii) pbp5 alone; and (iv) ftsWEfm, 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 ftsWEfm 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 µg/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.

* Corresponding author. Mailing address: Medical Service 111(W), VA Medical Center, 10701 East Blvd., Cleveland, OH 44106. Phone: (216) 791-3800, ext. 4800. Fax: (216) 231-3289. E-mail: louis.rice{at}med.va.gov.

Antimicrobial Agents and Chemotherapy, May 2001, p. 1480-1486, Vol. 45, No. 5
0066-4804/01/$04.00+0   DOI: 10.1128/AAC.45.5.1480-1486.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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Copyright © 2001 by the American Society for Microbiology. All rights reserved.