Affiliation:
1. Microcide Pharmaceuticals, Inc., Mountain View, California 94043
Abstract
ABSTRACT
A temperature-sensitive lethal mutant of
Staphylococcus aureus
was found to harbor a mutation in the uncharacterized two-component histidine kinase (HK)-response regulator (RR) pair encoded by
yycFG
; orthologues of
yycFG
could be identified in the genomes of
Bacillus subtilis
and other gram-positive bacteria. Sequence analysis of the mutant revealed a point mutation resulting in a nonconservative change (Glu to Lys) in the regulator domain of the RR at position 63. To confirm that this signal transduction system was essential, a disrupted copy of either the RR (
yycF
) or the HK (
yycG
) was constructed with a set of suicide vectors and used to generate tandem duplications in the chromosome. Resolution of the duplications, leaving an insertion in either the
yycF
or the
yycG
coding region, was achieved only in the presence of an additional wild-type copy of the two open reading frames. Phenotypic characterization of the conditional lethal mutant showed that at permissive growth conditions, the mutant was hypersusceptible to macrolide and lincosamide antibiotics, even in the presence of the
ermB
resistance determinant. Other mutant phenotypes, including hypersensitivity to unsaturated long-chain fatty acids and suppression of the conditional lethal phenotype by high sucrose and NaCl concentrations, suggest that the role of the two-component system includes the proper regulation of bacterial cell wall or membrane composition. The effects of this point mutation are strongly bactericidal at the nonpermissive temperature, indicating that this pathway provides an excellent target for the identification of novel antibiotics.
Publisher
American Society for Microbiology
Subject
Molecular Biology,Microbiology