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Journal of Bacteriology, December 2003, p. 7184-7192, Vol. 185, No. 24
0021-9193/03/$08.00+0 DOI: 10.1128/JB.185.24.7184-7192.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.
INSERM E0004-LRMA, UFR Broussais-Hôtel Dieu, Université Paris VI, 75270 Paris,1 Département Régulations, Développement et Diversité Moléculaire, Museum National d'Histoire Naturel, USM0502-CNRS UMR8041, 75005 Paris,France,4 University of Massachusetts Dartmouth, North Dartmouth, Massachusetts 02747,2 Department of Emergency Medicine, Somerville Hospital, Harvard Medical School, Somerville, Massachusetts 021433
Received 30 May 2003/ Accepted 17 September 2003
Enterococcus
faecalis produces a specific penicillin-binding protein (PBP5)
that mediates high-level resistance to the cephalosporin class of
ß-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 ß-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-Ala4
L-Ala-L-Ala-Lys3
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 ß-lactam resistance in
enterococci.
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