Identification of a Novel Gene Associated with High-Level β-Lactam Resistance in Heterogeneous Vancomycin-Intermediate Staphylococcus aureus Strain Mu3 and Methicillin-Resistant S. aureus Strain N315.

β-Lactam resistance levels vary among methicillin-resistant (MRSA) clinical isolates, mediated by chromosomal mutations and exogenous resistance gene However, MRSA resistance mechanisms are incompletely understood. A P440L mutation in the RNA polymerase β' subunit (RpoC) in slow-vancomycin-intermediate (sVISA) strain V6-5 is associated with conversion of heterogeneous VISA (hVISA) to sVISA. In this study, we found a V6-5-derivative strain (L4) with significantly decreased MICs to oxacillin (OX) and vancomycin. Whole-genome sequencing revealed that L4 has nonsense mutations in two genes, , encoding (p)ppGpp synthetase, an alarmone of the stringent response, and a gene of unknown function. deletion in the hVISA strain Mu3 did not affect OX MIC. However, introducing nonsense mutation of the unknown gene into Mu3 decreased OX MIC, whereas wild-type gene recovered high-level resistance. Thus, mutation of this unknown gene () decreased β-lactam resistance in Mu3 and L4. Presence of in a multicopy plasmid restored high-level resistance in strain L4 but not in the mutant Mu3 strain, indicating a genetic interaction between and depending on the L4 genetic background. While mupirocin (a stringent response inducer) can increase the β-lactam resistance of MRSA, mupirocin supplementation in an deletion mutant of N315 did not elevate resistance. expression in N315 was induced by mupirocin, and the relative amount of transcript in Mu3 was higher than in N315 induced by the stringent response. Our findings indicate that plays an essential role in high-level β-lactam resistance in MRSA via the stringent response.