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

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The FemAB-like factors Lif and Epr confer resistance to glycylglycine endopeptidases lysostaphin and Ale-1, respectively, by incorporating serine residues into the staphylococcal peptidoglycan interpeptide bridges specifically at positions 3 and 5. This required the presence of FemA and/or FemB, in contrast to earlier postulations.Glycylglycine endopeptidases are staphylolytic enzymes that cleave the pentaglycine interpeptide bridges of the staphylococcal peptidoglycan. Staphylococcus simulans biovar staphylolyticus, which produces lysostaphin, and Staphylococcus capitis, which produces Ale-1, protect themselves from their endopeptidases by the corresponding lysostaphin immunity factor Lif (1, 10) or the endopeptidase resistance factor Epr, respectively (9). Resistance is due to the integration of serine in place of glycine residues in the peptidoglycan pentaglycine interpeptide bridge (1, 9, 10). Pentaglycine interpeptide bridge formation in Staphylococcus aureus depends on at least three factors, FmhB (7), FemA (5,8), and FemB (4), which are needed for the addition of the first, the second and the third, and the fourth and the fifth glycines, respectively. Lif and Epr show up to 41% identity to FemA and FemB, suggesting that they may be catalyzing serine incorporation, and Lif was suggested to complement FemB (11). Here, we determined the positions of serine residues within the interpeptide bridge in a wild-type strain and different femAB mutants complemented with lif or epr and found a high specificity of Lif and Epr for serine incorporation at positions 3 and 5. Neither Lif nor Epr alone was able to extend the shortened cross bridges in the femAB mutants by the addition of serine residues, suggesting that both proteins depend on FemA and FemB for activity.Muropeptide profile of wild-type and femAB mutants expressing lif. Upon expression of lif from plasmid pCXlif (10), the amino acid fraction of the peptidoglycan in parent strain BB270 (NCTC 8325, mec) and in the corresponding femB mutant BB815 (mec, ⍀2006femB::Tn551) (4) showed an increased serine content, whereas that of the femA mutant UK17 (mec, ochre mutation in femA) (3) and the femAB null mutant AS145 (mec, ⌬femAB::tetK) (8) was not altered (11). The resulting muropeptide patterns of BB270/pCXlif and BB815/ pCXlif (11) showed additional peaks in the monomeric and dimeric fractions (Fig. 1a and b) when compared to the muropeptide profiles of the parent strains BB270 and BB815 (formerly UT43-2) determined earlier (5, 8). The main monomeric peaks of the two strains were collected and desalted as described (7), and their amino acid sequence was analyzed by automated Edman degradation (2). Since the major monomeric peak M4 could not be separated from the novel peak S1 (BB270/pCXlif) (Fig. 1a), the amino acid sequence of the isolated muropeptides revealed a mixture of the normal peptide with five glycine residues (M4; ϳ90%) and the peptide Gly-Gly-Ser-Gly-Ser (S1; ϳ10%). Furthermore, minor amounts of the sequence Gly-Gly-Ser-Gly-Gly could be detected. Consi...