Cloning and characterization of a gene affecting the methicillin resistance level and the autolysis rate in Staphylococcus aureus

Abstract

Tn918 mutagenesis of a high-level methicillin-resistant Staphylococcus aureus (methicillin MIC, 800 micrograms/ml) led to the isolation of a low-resistance mutant. The Tn918 insert was transferred back to the parent to produce strain SRM563 (methicillin MIC, 12.5 micrograms/ml), which showed heterogeneous resistance. Twenty-two clinical isolates of methicillin-resistant S. aureus were transformed with DNA of SRM563. In the transformants of most strains, instances of reduced resistance were observed with concomitant increases of autolysis rate induced by Triton X-100 and were generally more profound in high-resistance strains. Two transformants exhibited a decrease of the autolysis rate and little reduction of resistance. In the transformant of methicillin-susceptible strain RN2677, an increase of the autolysis rate and little reduction of resistance were observed. The production of low-affinity penicillin-binding protein (PBP2') did not significantly decrease in the mutants. Insertion of Tn918 occurred within the 3'-terminal region of a novel gene designated llm, which was cloned and sequenced. RNA blot analysis demonstrated that the gene was transcribed. The encoded protein was composed of 351 amino acid residues with a molecular weight of 38,512 and was hydrophobic, suggesting its location on the membrane. The gene was detected by PCR in all S. aureus strains tested but not in the other 26 staphylococcal species. Comparison of the 3'-terminal sequences of the gene among several S. aureus strains showed that, whereas nucleotide substitutions occurred at the third position in seven of eight 3'-terminal codons, only C-terminal amino acid variation of glutamate or aspartate was observed.