In Staphylococcus aureus , MbcS is a refunctionalized acyl-CoA synthetase that confers a fitness advantage during intra-species competition

ABSTRACT Staphylococcus aureus is one of the most frequently co-isolated pathogens in polymicrobial infections, where interspecies interactions contribute to enhanced virulence, persistence, and antimicrobial tolerance. Nutrient availability plays a central role in these interactions as microorganisms compete for resources to sustain essential cellular processes. For instance, branched-chain amino acids (BCAAs) are critical for protein synthesis, and valine synthesis pathway precursors are essential for energy production. In S. aureus , BCAAs are also the precursors for branched-chain fatty acids (BCFAs), the dominant fatty acids in the S. aureus membrane. We previously identified a second pathway that uses branched-chain carboxylic acids (BCCAs) and the high-affinity acyl-CoA synthetase MbcS to catalyze the formation of primers for BCFA synthesis. However, the physiological role of this pathway and the conditions triggering its activation remain unclear. Here, we show that mbcS is restricted to S. aureus and closely related human-associated staphylococci. Phylogenetic analyses suggest that MbcS arose from a refunctionalization event and represents a non-orthologous replacement for the phosphotransbutyrylase (Ptb) and butyrate kinase (Buk) enzymes. Consistent with this model, Ptb and Buk from Staphylococcus pseudintermedius catalyze the formation of branched-chain acyl-CoAs from BCCAs, but only at high substrate concentrations. We further show that mbcS expression is upregulated in a CodY-deficient strain, implicating this pathway in BCAA-limited conditions. In support, we show that MbcS is required for optimal fitness during intra-species competition. Together, our findings support a model in which the MbcS-dependent pathway enables S. aureus to scavenge BCFA precursors under nutrient-limited conditions, providing a competitive advantage in polymicrobial environments. IMPORTANCE Staphylococcus aureus is a major contributor to polymicrobial infections, where competition for nutrients can influence bacterial physiology and survival. A deeper understanding of how S. aureus adapts to nutrient limitation is therefore essential to explain its success as a human pathogen. In S. aureus , the acyl-CoA synthetase MbcS supports branched-chain fatty acid (BCFA) synthesis from branched-chain amino acid (BCAA)-derived carboxylic acids and aldehydes, which are released into the environment as by-products of bacterial metabolism. Herein, we provide evidence that S. aureus acquired the acyl-CoA synthetase MbcS as an adaptive trait. This metabolic innovation allows this bacterium to maintain membrane homeostasis under nutrient limitation and compete against neighboring bacteria. Our findings highlight an adaptive strategy that may contribute to the persistence of S. aureus in polymicrobial infections.