ABSTRACTMethicillin-resistantStaphylococcus aureus(MRSA) infections are an increasing concern due to their intrinsic resistance to most standard-of-care β-lactam antibiotics. Recent studies of clinical isolates have documented a novel phenotype, termed NaHCO3responsiveness, in which a substantial proportion of MRSA strains exhibit enhanced susceptibility to β-lactams such as cefazolin and oxacillin in the presence of NaHCO3. A bicarbonate transporter, MpsAB (membranepotential-generatingsystem), was recently found inS. aureus, where it plays a role in concentrating NaHCO3for anaplerotic pathways. Here, we investigated the role of MpsAB in mediating the NaHCO3responsiveness phenotype. Radiolabeled NaH14CO3uptake profiling revealed significantly higher accumulation in NaHCO3-responsive vs nonresponsive MRSA strains when grown in ambient air. In contrast, under 5% CO2conditions, NaHCO3-responsive (but not nonresponsive) strains exhibited repressed uptake. Oxacillin MICs were measured in four prototype strains and theirmpsABCdeletion mutants in the presence of NaHCO3supplementation under 5% CO2conditions. NaHCO3-mediated reductions in oxacillin MICs were observed in the responsive parental strains but not inmpsABCdeletion mutants. No significant impact on oxacillin MICs was observed in the nonresponsive strains under the same conditions. Transcriptional and translational studies were carried out using both quantitative reverse transcription-PCR (qRT-PCR) andmpsA-green fluorescent protein (GFP) fusion constructs; these investigations showed thatmpsAexpression and translation were significantly upregulated during mid-exponential-phase growth in oxacillin-NaHCO3-supplemented medium in responsive versus nonresponsive strains. Taken together, these data show that the NaHCO3transporter MpsABC is a key contributor to the NaHCO3–β-lactam responsiveness phenotype in MRSA.IMPORTANCEMRSA infections are increasingly difficult to treat, due in part to their resistance to most β-lactam antibiotics. A novel and relatively common phenotype, termed NaHCO3responsiveness, has been identified in which MRSA strains show increased susceptibilityin vitroandin vivoto β-lactams in the presence of NaHCO3. A recently describedS. aureusNaHCO3transporter, MpsAB, is involved in intracellular NaHCO3concentration for anaplerotic pathways. We investigated the role of MpsAB in mediating the NaHCO3responsiveness phenotype in four prototype MRSA strains (two responsive and two nonresponsive). We demonstrated that MpsABC is an important contributor to the NaHCO3–β-lactam responsiveness phenotype. Our study adds to the growing body of well-defined characteristics of this novel phenotype, which could potentially translate to alternative targets for MRSA treatment using β-lactams.