The femAB operon is involved in the formation of the characteristic pentaglycine side chain of the staphylococcal peptidoglycan. Allele replacement of the femAB operon with the tetracycline resistance determinant tetK in a methicillin-resistant Staphylococcus aureus strain resulted in impaired growth, methicillin hypersusceptibility, and lysostaphin resistance. The usual pentaglycine cross-bridges were replaced by monoglycine bridges exclusively, and cross-linking of the peptidoglycan strands was drastically reduced. Complementation of the femAB null mutant by either femA or femAB resulted in the extension of the cross-bridges to a triglycine or a pentaglycine, respectively. This finding suggests that FemA is responsible for the formation of glycines 2 and 3, and FemB is responsible for formation of glycines 4 and 5, of the pentaglycine side chain of the peptidoglycan precursor. Moreover, it can be deduced that addition of the first glycine must occur by a femABindependent mechanism.The chromosomal femAB operon belongs to the Staphylococcus aureus housekeeping genes and is found in all S. aureus strains (15, 18), and femAB alleles similar in organization and sequence were identified in Staphylococcus epidermidis and Staphylococcus haemolyticus (1). The operon arose by gene duplication and codes for two similar cytoplasmic proteins that are produced mainly during the exponential phase (7, 16). FemA and FemB are involved in a yet unknown way in the formation of the pentaglycine side chain that is attached to the L-lysine of the peptidoglycan stem peptide (7,14,21). This long and flexible pentaglycine peptide allows the high cross-linking between the peptide moiety of the single peptidoglycan strands observed in S. aureus cell walls. While FemB-less cells are able to form only triglycine cross-bridges, the cell wall composition of leaky femAB mutants suggests that FemA might be responsible for the addition of the second and third glycine residues (9, 14). A femAB null mutant would be expected to attach only a single glycine to the stem peptide, and it was questionable if such a mutant would be still viable.The consequences of either femB inactivation or lowering of femAB transcription are pleiotropic. Besides the reduction of the glycine content of the cell walls, overall peptidoglycan cross-linking and cell wall turnover are reduced, there is an aberrant cell septum formation, and cell separation is retarded (13,14,21). Methicillin resistance in S. aureus is abolished, and ␤-lactam susceptibility in susceptible strains is increased. The production of the low-affinity penicillin-binding protein PBP2Ј in methicillin-resistant (Mc r ) strains, the prerequisite for methicillin resistance, is thereby not affected (6), suggesting that PBP2Ј is unable to function properly in femAB mutants. This makes FemA and/or FemB suitable targets for inhibitors which could restore the efficacy of ␤-lactam antibiotics against Mc r strains. The construction of a femAB null mutant is the first step in unravelling the biological function...

The factor catalyzing the first step in the synthesis of the characteristic pentaglycine interpeptide in Staphylococcus aureus peptidoglycan was found to be encoded by the essential gene fmhB. We have analyzed murein composition and structure synthesized when fmhB expression is reduced. The endogenous fmhB promoter was substituted with the xylose regulon from Staphylococcus xylosus, which allowed glucose-controlled repression of fmhB transcription. Repression of fmhB reduced growth and triggered a drastic accumulation of uncrosslinked, unmodified muropeptide monomer precursors at the expense of the oligomeric fraction, leading to a substantial decrease in overall peptidoglycan crosslinking. The composition of the predominant muropep

(")-Epicatechin gallate (ECg), a component of green tea, sensitizes meticillin-resistant Staphylococcus aureus (MRSA) to b-lactam antibiotics, promotes staphylococcal cell aggregation and increases cell-wall thickness. The potentiation of b-lactam activity against MRSA by ECg was not due to decreased bacterial penicillin-binding protein (PBP) 2a expression or ECg binding to peptidoglycan. A 5-10 % reduction in peptidoglycan cross-linking was observed. Reduced cross-linking was insufficient to compromise the integrity of the cell wall and no evidence of PBP2a activity was detected in the muropeptide composition of ECg-grown cells. ECg increased the quantity of autolysins associated with the cell wall, even though the cells were less susceptible to Triton X-100-induced autolysis than cells grown in the absence of ECg. ECg promoted increased lysostaphin resistance that was not due to alteration of the pentaglycine cross-bridge configuration or inhibition of lysostaphin activity. Rather, decreased lysostaphin susceptibility was associated with structural changes to wall teichoic acid (WTA), an acid-labile component of peptidoglycan. ECg also promoted lipoteichoic acid (LTA) release from the cytoplasmic membrane. It is proposed that ECg reduces b-lactam resistance in MRSA either by binding to PBPs at sites distinct from the penicillin-binding site or by intercalation into the cytoplasmic membrane, displacing LTA from the phospholipid palisade. Thus, ECg-mediated alterations to the physical nature of the bilayer will elicit structural changes to WTA that result in modulation of the cell-surface properties necessary to maintain the b-lactam-resistant phenotype.

. Analysis of the resistance profile of the mutant revealed a loss of ␤-lactam resistance with a concomitant increase in resistance to glycopeptides. In both strains, cell wall thickness was 1.4-fold greater than that of control isolates. However, cross-linking of the cell wall was drastically lower in SA137/93A than in SA137/93G. The sensitivity of strain SA137/93G to ␤-lactams was due to loss of the ␤-lactamase plasmid and a deletion that comprises 32.5 kb of the methicillin resistance cassette SCCmec, as well as 65.4 kb of chromosomal DNA. A spontaneous mutant of SA137/93G with higher sensitivity to vancomycin displayed a cell wall profile similar, in some respects, to that of an fmhB mutant. Results described here and elsewhere show that the only feature common to all VISA strains is a thickened cell wall, which may play a central role in the vancomycin resistance mechanism.Staphylococcus aureus is one of the most common pathogens and is a trigger of community-acquired and nosocomial disease. The drugs of choice against methicillin-resistant S. aureus (MRSA) strains are the glycopeptide antibiotics vancomycin and teicoplanin. Since 1996, emergence of MRSA strains resistant to vancomycin has been reported in many countries (4-6, 13, 16, 21, 30, 36, 41, 45, 53). According to the NCCLS, strains for which the MIC of vancomycin is less than or equal to 4 g/ml are considered susceptible whereas those for which the MIC is greater than or equal to 32 g/ml are considered resistant. Strains for which the MICs are 8 to 16 g/ml are intermediately resistant. Detailed characterization of clinical isolates and in vitro-selected vancomycin-resistant mutants showed that the resistance phenotype of at least some vancomycin intermediately resistant S. aureus (VISA) strains is caused by activation of cell wall biosynthesis and a significant increase in free D-alanyl-D-alanine termini in the cell wall, which represent false target sites for vancomycin. Resistance was shown to go along with thickening of the cell wall, changes in the composition of the peptidoglycan, and the expression of penicillin-binding proteins (PBPs) (7,8,18,31,37,44,(46)(47)(48). Some authors have suggested that the genes involved in glycopeptide resistance are probably under the control of a comprehensive regulatory system (8, 33). However, the reported changes in cell wall biochemistry are not restricted to glycopeptide-resistant strains and the magnitude of the changes does not always correlate with the resistance level (7,39).In this study, we characterize two VISA isolates and one revertant strain. SA137/93A was identified by the screening of 457 clinical isolates of MRSA from the strain collection of the Reference Center for Staphylococci of the University of Bonn (4). A spontaneous ␤-lactam-susceptible mutant of SA137/93A with an increased vancomycin MIC was named SA137/93G. SA137/93G1 is a vancomycin-susceptible revertant of strain SA137/93G. We examined the three strains with regard to the previously reported characteristics of glycopeptide-resist...