Antibiotic resistance as a stress response: Complete sequencing of a largenumber of chromosomal loci in Staphylococcus aureus strain COL that impacton the expression of resistance to methicillin

Tn551 inactivation has identified several determinants - fem or auxiliary g enes - that, in addition to the mecA gene, are also critical for the expres sion of high-level and homogeneous resistance to methicillin. Genetic and/o r biochemical analysis has shown that of the nearly dozen aux mutations des cribed so far most are in genes involved in cell wall synthesis (murE, pbp2 , glmM, glnR, femA/B, llm, etc.) or in complex regulatory functions (sigmaB ), suggesting that optimal expression of resistance may involve the coopera tive functioning of a number of genes in cell wall metabolism as well as st ress response. The exact mechanism of these functions is not known. In an a ttempt to explore this unusual aspect of methicillin resistance more fully, a Tn551 transposon library, constructed in the background of the highly an d homogeneously methicillin-resistant Staphylococcus aureus strain COL, was screened for all independent insertional mutants in which the level of met hicillin resistance of the parental strain (MIC, 1,600 mu g/ml) was reduced by at least 15-fold and up to 500-fold. We now describe the sequencing of 21 Tn551-inactivated genes and their vicinities in 23 new auxiliary mutants that have been studied before. Using the inverted polymerase chain reactio n (IPCR), we amplified fragments corresponding to the right and left juncti on of the Tn551 insertions, which were then sequenced by primer walking. Th e two largest groups of these new auxiliary genes encoded either proteins o f unknown functions (6 genes) or showed homology with genes encoding protei ns involved with putative sensory/regulatory activities (7 genes: protein k inases, ABC transporters, and a catabolite control protein). Sequencing ups tream and downstream allowed the identification of a number of additional o pen reading frames, some of which may also include functions relevant for t he expression of antibiotic resistance.