Role in cell permeability of an essential two-component system in Staphylococcus aureus

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.

FIG. 1

Partial chromosomal map detailing the local organization around the yycFG locus. The location in the C-terminal portion of purA of the Tn917lac insertion used in the construction of the isogenic strains SAM1010 and SAM1011 is shown (Ω), along with the HpaI (H) and EcoRV (V) restriction sites used to create ermC insertions in either yycF or yycG. Predicted tRNA sequences are denoted by an inverted triangle. The relative locations of the original complementing clone (pMP373), the clone rescued from SAM970 (pMP749), and the two inactivation constructs are shown below the sequence. The ORFs that have clear orthologues in B. subtilis are labeled; the last ORF exhibits only limited sequence similarity to yycH from B. subtilis (data not shown).

FIG. 2

Structural alignment of RRs with a high degree of similarity to YycF. The conserved residues necessary for proper phosphotransfer (56) in the regulatory domain are boxed and highlighted with an inverted triangle. The location of the amino acid substitution (E63K) in the variant form of the RR is highlighted by a star. Abbreviations: Bsu, B. subtilis; Sau, S. aureus; Efa, E. faecalis; Spn, S. pneumoniae; Spy, Streptococcus pyogenes. The S. pneumoniae (type 4) unpublished genomic sequence data used to identify the YycF orthologue were provided by The Institute for Genomic Research (25a). The E. faecalis sequence (strain V583) was obtained by designing PCR primers based on the unpublished genomic sequence data provided by The Institute for Genomic Research (25a) and then resolving frameshifts by sequencing PCR-derived amplicons from strain ATCC 29212. The S. pyogenes sequence (strain M1 GAS) was provided by the University of Oklahoma (44a).

FIG. 3

Comparison of the growth of isogenic strains SAM1010 and SAM1011 at 41°C. (a) The SAM1010 mutant could not survive above 40°C in the presence of oxygen in a variety of liquid media, including TSB. (b) The rate of growth of the yycF1 mutant was only partially restored under anoxic conditions at a nonpermissive temperature in the same liquid media. OD, optical density; ts, temperature sensitive; wt, wild type.

FIG. 4

Resolution frequencies for chromosomal duplications. The ability to resolve a tandem duplication of the yycFG locus with an intervening tetK marker (hatched box) was compared for strains either bearing a complete duplication (left) or bearing an ermC insertion (shaded box) within the second copy of yycG (right). Not shown is the parallel insertion in yycF.