Adaptation Through Mutation: Arginine Auxotrophy in Staphylococcus aureus

Staphylococcus aureus is a versatile pathogen capable of infecting all organ systems of the human host. Although S. aureus encodes biosynthetic pathways to synthesize arginine using proline or glutamate as a substrate, it remains a functional auxotroph when grown in a chemically defined medium lacking arginine (CDM-R). In these studies, we demonstrate that S. aureus clinical isolates and the laboratory strain JE2 select for mutations to grow in CDM-R. These mutations either enhance the intracellular ornithine pool or increase the catabolic ornithine carbamoyltransferase transcription, arcB1. Unexpectedly, we found that ArcB1 catalyzes an anabolic reaction by converting ornithine into citrulline, a key precursor for arginine biosynthesis, functioning outside its traditional catabolic scope. Furthermore, we identified that AhrC represses arginine biosynthesis via proline even in the absence of glucose and arginine. Mutations in ahrC or within regulatory regions upstream of arcA1B1D1C1 alleviated this repression, leading to increased transcription of arcB1 and arginine biosynthetic genes (argGH), thus supporting growth in CDM-R. Since all mutant strains utilized the alternative proline pathway for arginine biosynthesis, we investigated the functionality of the glutamate pathway encoded by the argDCJB operon. Our findings show that arginine biosynthesis from glutamate can be restored through ectopic argDCJB expression or compensatory mutations in spoVG and sarA. Transcriptional studies showed a significant increase in argDCJB expression in the JE2 spoVG sarA mutant in the absence of arginine, however, an additional ahrC mutation is needed to relieve repression when arginine is present. Building on these findings, we utilized mouse models of systemic infection to identify the niches where arginine biosynthesis is selected. We found that on day 5 post-infection, arginine auxotrophs predominated in all organs, including kidneys. However, by day 20, arginine prototrophs emerged in the kidneys, correlating with mature renal abscess formation. Whole-genome sequencing identified mutations in ahrC, upstream of arcA1B1D1C1, and pyrB. Clonality analysis revealed both clonal and non-clonal patterns, suggesting independent seeding followed by localized adaptation. Collectively, these findings demonstrate how S. aureus employs adaptive evolution to shift from arginine auxotrophy to prototrophy, enabling its persistence in nutrient-limited niches.