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Mrp--a new auxiliary gene essential for optimal expression of methicillin resistance in Staphylococcus aureus
1Laboratory of Microbiology, Rockefeller University, New York, NY, USA.
Abstract:
Screening of a library of Tn551 insertional mutants selected for reduction in the methicillin resistance level of the parental Staphylococcus aureus strain 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 micrograms/mL. Cloning and sequencing of the vicinity of the insertion site omega 726 identified an open reading frame (orf1365) encoding a very large polypeptide of more than 1,365 amino acids. A unique feature of the deduced amino acid sequence was the presence of multiple tandem repeats of 75 amino acids in the polypeptide, reminiscent of the structure of high-molecular-weight cell-surface proteins EF* and Emb identified in some streptococcal strains. Mutant RUSA266 with the inactivated gene, which we shall provisionally refer to as mrp (for multiple repeat polypeptide), produced a peptidoglycan with altered muropeptide composition, and both the reduced antibiotic resistance and the altered cell wall composition were co-transduced in back-crosses 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-kb 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 cluster were transcribed together.
Insights
Researchers identified a novel gene, mrp, in Staphylococcus aureus that significantly reduces methicillin resistance when inactivated. This finding offers new insights into antibiotic resistance mechanisms and potential therapeutic targets.
Area of Science:
- Microbiology
- Genetics
- Molecular Biology
Background:
- Methicillin resistance in Staphylococcus aureus (MRSA) is a major public health concern.
- Understanding the genetic basis of antibiotic resistance is crucial for developing new treatment strategies.
Purpose of the Study:
- To identify genes involved in regulating methicillin resistance levels in Staphylococcus aureus.
- To characterize a novel gene, mrp, and its role in antibiotic resistance and cell wall composition.
Main Methods:
- Screening of Tn551 insertional mutants of Staphylococcus aureus COL.
- Cloning, sequencing, and characterization of the insertion site and surrounding genes.
- Analysis of peptidoglycan composition and antibiotic resistance levels.
- Co-transduction experiments to confirm gene function.
- Transcriptional analysis of the identified gene cluster.
Main Results:
- Isolation of mutant RUSA266 with a significant reduction in methicillin resistance (MIC from 1,600 to 1.5 µg/mL).
- Identification of a novel gene, mrp (orf1365), encoding a large polypeptide with multiple tandem repeats.
- Inactivation of mrp resulted in altered peptidoglycan composition and reduced antibiotic resistance.
- The mrp gene is part of a co-transcribed five-gene cluster including glmM, orf310, orf269, and arg.
Conclusions:
- The mrp gene plays a significant role in maintaining high-level methicillin resistance in Staphylococcus aureus.
- Alterations in mrp expression impact both antibiotic resistance and cell wall synthesis.
- The identified five-gene cluster is likely involved in a coordinated cellular process affecting resistance and cell wall structure.