Mrp - A new auxiliary gene essential for optimal expression of methicillinresistance in Staphylococcus aureus

Screening of a library of Tn551 insertional mutants selected for reduction in the methicillin resistance level of the parental Staphylococcus aureus s train COL resulted in the isolation of mutant RUSA266 in which the minimal inhibitory concentration (MIC) of the parent was reduced from 1,600 to 1.5 mu g/mL. Cloning and sequencing of the vicinity of the insertion site Omega 726 identified an open reading frame (orf1365) encoding a very large polyp eptide of more than 1,365 amino acids. A unique feature of the deduced amin o acid sequence was the presence of multiple tandem repeats of 75 amino aci ds in the polypeptide, reminiscent of the structure of high-molecular-weigh t cell-surface proteins EF* and Emb identified in some streptococcal strain s. Mutant RUSA266 with the inactivated gene, which we shall provisionally r efer to as mrp (for multiple repeat polypeptide), produced a peptidoglycan with altered muropeptide composition, and both the reduced antibiotic resis tance and the altered cell wall composition were co-transduced in back-cros ses into the parental strain COL, Additional sequencing upstream of mrp has revealed that this gene was part of a five-gene cluster occupying a 9.2-kh region of the staphylococcal chromosome and was composed of glmM (directly upstream of mrp), two open reading frames orf310 and orf269 coding for two hypothetical proteins, and the gene encoding the staphylococcal arginase ( arg), Transcriptional analysis demonstrated that the five genes in the clus ter were transcribed together.