Antibiotic-resistant mutants of Pseudomonas aeruginosa were generated using chloramphenicol and ciprofloxacin as selective agents. These mutants displayed a multidrug phenotype and overexpressed an outer membrane protein of 50 kDa, which was shown by Western blot analysis to correspond to OprN. A cosmid clone harbouring the oprN gene was isolated by partial complementation of a mutant deficient in OprM, the outer membrane component of the mexAB-oprM efflux operon. Antibiotic-accumulation studies indicated that OprN was part of an energy-dependent antibiotic-efflux system. Sequencing of a 6180bp fragment from the complementing cosmid revealed the presence of three open reading frames (ORFs), which exhibited amino acid similarity to the components of the mexAB-oprM and mexCD-oprJ efflux operons of P. aeruginosa. The ORFs were designated MexE, MexF and OprN. Mutation of the mexE gene eliminated the multidrug-resistance phenotype in an OprN-overexpressing strain, but did not affect the susceptibility profile of the wild-type strain. Expression of the mexEF-oprN operon was shown to be positively regulated by a protein encoded on a 1.5 kb DNA fragment located upstream of mexE and belonging to the LysR family of transcriptional activators. The presence of a plasmid containing this DNA fragment was sufficient to confer a multidrug phenotype onto the wild-type strain but not onto the mexE mutant. Evidence is provided to show that the mexEF-oprN operon may be involved in the excretion of intermediates for the biosynthesis of pyocyanin, a typical secondary metabolite of P. aeruginosa.

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...

No abstract

The pbp4 gene of a Staphylococcus aureus strain selected stepwise in vitro for growth on increasing concentrations of penicillin and of its susceptible parent strain showed overall identity except in the promoter region. In the mutant a deletion upstream of the pbp4 structural gene removed 90 nucleotides (nt) that were framed by a 12 nt inverted repeat. This deletion occurred in step 4 of the in vitro selection procedure and was paralleled by a significant increase in the penicillin-binding protein 4 (PBP4) production. The in vitro step selected mutant showed a remarkable increase in the cross-linking of the peptidoglycan compared to its parent. This was linked to morphological changes in the appearance of the cells, which were surrounded by a very thick and fuzzy cell wall.

The Staphylococcus aureus mutant strain PVI selected in vitro for methicillin resistance overexpressed penicillin-binding protein (PBP) 4. In the wild-type parent strain the pbp4 gene was separated by 419 nucleotides from a divergently transcribed abcA locus coding for an ATP-binding cassette transporter. The mutant PVI was shown to have a deletion in the pbp4-abcA promoter region that affected pbp4 transcription but not expression of abcA. Introduction of the pbp4 gene plus the mutant promoter region into different genetic backgrounds revealed that PBP 4 overproduction was sufficient to increase in vitro-acquired methicillin resistance independently of other chromosomal genes. The role of the AbcA transporter in methicillin resistance remained unknown.