Reduced methicillin resistance in a new Staphylococcus aureus transposon mutant that incorporates muramyl dipeptides into the cell wall peptidoglycan.

Screening of a new Tn551 library constructed in the background of a highly methicillin-resistant Staphylococcus aureus strain identified a new insertion site located on the SmaI B-fragment of the chromosome that reduced the minimal inhibitory concentration of the parent (1600 micrograms/ml) to 25-50 micrograms/ml in the mutant and caused heterogeneous expression of resistance and abnormality in peptidoglycan composition (absence of the unsubstituted pentapeptide and incorporation of alanylglutamate- and alanylisoglutamate-containing muropeptides). There was an accumulation of large amounts of the UDP-linked muramyl dipeptide in the cytoplasmic wall precursor pool of the mutant. Reduced (heterogeneous) antibiotic resistance and all the biochemical abnormalities were reproduced in genetic backcrosses by transduction with phage 80 alpha. Mutant RUSA235 appears to be impaired in the biosynthesis of the staphylococcal cell wall precursor muropeptide before the lysine addition step. We propose to provisionally call the gene inactivated in this mutant femF.

The peptidoglycan composition of a Staphylococcus aureus mutant selected for reduced methicillin resistance.

The peptidoglycan of a Tn551 mutant of Staphylococcus aureus (RUSA208) selected for reduced methicillin resistance was analyzed by reversed-phase high pressure liquid chromatography and mass spectrometry. RUSA208 is a member of a cluster of Tn551 mutants located on fragment A of SmaI digests but is distinct from the femA and femB class of mutants. The peptidoglycan of RUSA208 contained normal parental muropeptides but in diminished amounts only. The major muropeptides of RUSA208 were new components eluting with somewhat longer retention times from the column. Amino acid analysis of these new muropeptides showed identical compositions to the corresponding peaks in the parental strain, but mass spectrometry revealed increased molecular weights by the following mass units: 1 (in monomers), 1 or 2 (in dimers), and 2 or 3 (in trimers). These observations suggest that in RUSA208 the mutational block may be in the amidation of the stem peptide glutamate residues, resulting in the replacement of isoglutamine with free glutamic acid in one or more of the cell wall stem peptides.