A phosphoglucomutase-like gene essential for the optimal expression of methicillin resistance in Staphylococcus aureus: molecular cloning and DNA sequencing.
We describe here the cloning and sequencing of a new auxiliary gene identified by Tn551 insertional mutagenesis of the highly and homogeneously methicillin-resistant Staphylococcus aureus strain COL. The insertionally inactivated mutant RUSA315 had intact mecA and normal amounts of PBP2A, but drastically reduced antibiotic resistance (drop in methicillin MIC from 1600 to 1.5 micrograms ml-1), a unique heterogeneous phenotype, and a compositional change in the cell wall characterized by the complete disappearance of the unsubstituted disaccharide pentapeptide from the peptidoglycan. Cloning in E. coli followed by sequencing located the Tn551 insert omega 720 in an open reading frame of 451 codons, provisionally called femR315, defining a polypeptide with a deduced amino acid sequence that showed over 26% sequence identity and 57% overall sequence similarity with the phosphoglucomutase (PGM) gene of E. coli. The Tn551 insertion site of a previously described mutant 12F (femD) also lies in the same gene as femR315. The wild-type form of femR315 subcloned in a shuttle vector fully restored expression of high level (parental) methicillin resistance in mutant RUSA315. The exact biochemical function of femR315 is not known. However, enzymes similar to PGM catalyze the isomerization of hexose and hexosamine phosphates leading to the formation of glucosamine-1-P, which is an obligate precursor in the biosynthesis of UDP-N-acetylglucosamine (UDP-NAGA). We propose that the suppression of methicillin resistance in RUSA315 is related to some functional or quantitative abnormality of UDP-NAGA metabolism.
Wu
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Laboratory of Microbiology, Rockefeller University, New York, New York 10021-6399, USA.
de Lencastre
H
H
Sali
A
A
Tomasz
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A
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D003470:Culture Media; D004269:DNA, Bacterial / Q000032:analysis; D018274:Electroporation; D005798:Genes, Bacterial / Q000235:genetics*; D005816:Genetic Complementation Test; D016106:Methicillin Resistance / Q000235:genetics* / Q000502:physiology; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D010733:Phosphoglucomutase / Q000235:genetics* / Q000378:metabolism; D013211:Staphylococcus aureus / Q000201:enzymology / Q000235:genetics*
D003470:Culture Media; D004269:DNA, Bacterial; D010733:Phosphoglucomutase