Normally functioning murF is essential for the optimal expression of methicillin resistance in Staphylococcus aureus.
A carboxy-terminal fragment of murF was used to construct and insert a suicide plasmid into the chromosomal copy of the gene in the highly and homogeneously methicillin-resistant Staphylococcus aureus (MRSA) strain COL by Campbell type integration. The plasmid insertion generated a mutant in which the MIC value for oxacillin was reduced from 400 microg/ml of the parental strain to 0.75 microg/ml in 90% of the cells of the mutant cultures that were heterogeneous: they contained subpopulations of bacteria with a frequency of 10(-3) that were capable of expressing resistance at nearly the parental level. The impact of the murF mutation on antibiotic resistance was selective for beta-lactam antibiotics: there was no change in the susceptibility of the mutant to D-cycloserine, fosfomycin, beta-D-chloro-alanine, moenomycin, bacitracin, or vancomycin. Analysis of the mutant peptidoglycan showed decrease in the percentage of oligomeric components in rough proportion to the accumulation of several abnormal muropeptide components, which were identified as structural variants of the disaccharide tripeptide monomer. An abnormal cell wall precursor identified as UDP MurNac tripeptide was also detected in the cytoplasmic pool of the mutant strain. A normal proportion of oligomers and a greatly reduced representation of the disaccharide tripeptide were demonstrated in the cell wall of the murF mutant's subpopulation that has retained the parental level of resistance. Northern analysis demonstrated a drastic reduction in the transcription rate of mecA in mutant F9 whereas mecA transcription increased in the subpopulation of bacteria that retained high-level resistance.
Sobral
R G
RG
Molecular Genetics Laboratory, Instituto de Tecnologia Química e Biológica da Universidade Nova de Lisboa, 2780 Oeiras, Portugal.
Ludovice
A M
AM
Gardete
S
S
Tabei
K
K
De Lencastre
H
H
Tomasz
A
A
D000595:Amino Acid Sequence; D001483:Base Sequence; D015152:Blotting, Northern; D002473:Cell Wall / Q000737:chemistry; D002876:Chromosomes, Bacterial / Q000737:chemistry / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics / Q000502:physiology; D013058:Mass Spectrometry; D016106:Methicillin Resistance / Q000235:genetics*; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000235:genetics*; D016254:Mutagenesis, Insertional; D009154:Mutation / Q000235:genetics; D010457:Peptidoglycan / Q000737:chemistry / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D013211:Staphylococcus aureus / Q000187:drug effects* / Q000235:genetics*; D014158:Transcription, Genetic; D014530:Uridine Diphosphate / Q000737:chemistry
D004269:DNA, Bacterial; D009124:Muscle Proteins; D010457:Peptidoglycan; D014530:Uridine Diphosphate