REASSESSMENT OF THE NUMBER OF AUXILIARY GENES ESSENTIAL FOR EXPRESSION OF HIGH-LEVEL METHICILLIN RESISTANCE IN STAPHYLOCOCCUS-AUREUS

A new transposon library constructed in the background of the highly a nd homogeneously methicillin-resistant Staphylococcus aureus strain CO L yielded 70 independent insertional mutants with reduced levels of an tibiotic resistance. Restriction analysis with HindIII, EcoRV, EcoRI, and PstI and then Southern hybridization with probes for the transposo n and for the femA-femB gene demonstrated that 41 of the 70 Tn551 muta nts carried distinct and novel, as yet undescribed insertion sites, al l of which were outside of the mecA gene and were also outside the alr eady-characterized auxiliary genes femA, femB, femC, and femD. All pre viously described Tn551 mutations of this type were in genes located e ither on SmaI fragment A or SmaI fragment I. In contrast, inserts of t he new library were located in 7 of the 16 SmaI chromosomal fragments, fragments A, B, C, D, E, F, and I. In all of the mutants, expression of methicillin resistance became heterogeneous, and the MTC for the ma jority of cells was reduced (1.5 to 200 mu g ml(-1)) from the homogene ous methicillin MIC (1,600 mu g ml(-1)) of the parental cells. Althoug h identification of the exact number of genes inactivated through the new set of transposon inserts will require cloning and sequencing, a r ough estimate of this number from mapping data suggests a minimum of a t least 10 to 12 new genetic determinants, all of which are needed tog ether with femA, femB, femC, and femD for the optimal expression of me thicillin resistance.