The inactivation of FemB by insertion of TnS51 in the central part of thefemB open reading frame was shown to increase susceptibility of methicillin-resistant Staphylococcus aureus strains toward 13-lactam antibiotics to the same extent as did inactivation offemA. Strains carrying the methicillin resistance determinant (mec) and expressing PBP 2' were affected to the same extent as were strains selected for in vitro resistance, which did not express PBP 2'. BothfemA andfemB, which form an operon, are involved in a yet unknown manner in the glycine interpeptide bridge formation of the S. aureus peptidoglycan. FemB inactivation was shown to reduce the glycine content of peptidoglycan by approximately 40%o, depending on the S. aureus strain. The reduction of the interpeptide bridge glycine content led to significant reduction in peptidoglycan cross-linking, as measured by gel permeation high-pressure liquid chromatography of muramidase-digested cell walls. Maximum peptide chain length was reduced by approximately 40%. It is shown that the complete pentaglycine interpeptide bridge is important for the sensitivity against 13-lactam antibiotics and for the undisturbed activity of the staphylococcal cell wall-synthesizing and hydrolyzing enzymes, as was also apparent from electron microscopic examinations, which revealed aberrant placement of cross walls and retarded cell separation, leading to a pseudomulticellular phenotype of the cells for bothfemA and femB mutants.Methicillin resistance in staphylococci is an intrinsic resistance of the cells to virtually all 1-lactam antibiotics, including cephalosporins and carbapenems, and does not involve drug destruction (12). The genetic determinant of methicillin resistance (mec) carries the structural gene mecA, coding for an additional low-affinity penicillin-binding protein, PBP 2' or 2a (25,37,40). PBP 2' is thought to be the only functional PBP in cell wall synthesis in the presence of otherwise inhibitory concentrations of methicillin (10,13,21,29,34) and is a prerequisite for methicillin resistance.mec-mediated methicillin resistance in clinical isolates of Staphylococcus aureus is typically heterogeneous in phenotypic expression despite genetic homogeneity (reviewed by Matthews and Stewart [30] 1-lactams (27).The product of femA is a 48-kDa protein involved in the pentaglycine cross-bridge formation of the S. aureus peptidoglycan (27). This conclusion was subsequently sustained by mass spectrometric analysis of related mutants (11). Whereas the inactivation offemA correlates with a 40 to 60% reduction in the interpeptide glycine content of S. aureus peptidoglycan, with a reduction in cross-linking and cell wall turnover, and with increased susceptibility to 1-lactams, no influence on the synthesis of PBP 2' was observed. Downstream and adjacent to femA lies a second factor, femB. femB mutants described earlier (4) apparently still retained part of their activity, presumably because they were insertionally inactivated at their outmost carboxy terminus. We show here t...

Tn551 inactivation offemA4, a factor involved in methicillin resistance of Staphylococcus aureus, caused the production of peptidoglycan in which the fraction of monoglycyl-and serine-containing muropeptides was increased at the expense of pentaglycyl muropeptides.femA mutants have a specific block in the biosynthesis of pentaglycine cross bridges after the addition of the first glycine residue.

To analyze if chemical cell wall alterations contribute to penicillin-induced bacteriolysis, changes in the amount, stability, and chemical composition of staphylococcal cell walls were investigated. All analyses were performed before onset of bacteriolysis i.e. during the first 60 min following addition of different penicillin G doses. Only a slight reduction of the amount of cell wall material incorporated after penicillin addition at the optimal lytic concentration was observed as compared to control cells. However, the presence of higher penicillin G concentrations reduced the incorporation of wall material progressively without bacteriolysis. Losses of wall material during isolation of dodecylsulfate insoluble cell walls were monitored to assess the stability of the wall material following penicillin addition. Wall material grown at the lytic penicillin concentration was least stable but about 30% of the newly incorporated wall material withstood even the harsh conditions of mechanical breakage and dodecylsulfate treatment. Dodecylsulfate insoluble cell walls were used for chemical analyses. While peptidoglycan chain length was unaffected in the presence of penicillin, other wall parameters were considerably altered: peptide cross-linking was reduced in the wall material synthesized after addition of penicillin; reductions from approx. 85% in controls to about 60% were similar for lytic and also for very high penicillin concentrations leading to nonlytic death. O-acetylation was also reduced after treatment with penicillin; this effect paralleled the occurrence of subsequent bacteriolysis at different drug concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)

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