The global regulator CodY links nutrient availability to the regulation of virulence factor gene expression in Staphylococcus aureus, including many genes whose products affect biofilm formation. Antithetical phenotypes of both biofilm deficiency and accumulation have been reported for codY-null mutants; thus, the role of CodY in biofilm development remains unclear. codY mutant cells of a strain producing a robust biofilm elaborate proaggregation surface-associated features not present on codY mutant cells that do not produce a robust biofilm. Biochemical analysis of the clinical isolate SA564, which aggregates when deficient for CodY, revealed that these features are sensitive to nuclease treatment and are resistant to protease exposure. Genetic analyses revealed that disrupting lgt (the diacylglycerol transferase gene) in codY mutant cells severely weakened aggregation, indicating a role for lipoproteins in the attachment of the biofilm matrix to the cell surface. An additional and critical role of IcaB in producing functional poly-N-acetylglucosamine (PIA) polysaccharide in extracellular DNA (eDNA)-dependent biofilm formation was shown. Moreover, overproducing PIA is sufficient to promote aggregation in a DNA-dependent manner regardless of source of nucleic acids. Taken together, our results point to PIA synthesis as the primary determinant of biofilm formation when CodY activity is reduced and suggest a modified electrostatic net model for matrix attachment whereby PIA associates with eDNA, which interacts with the cell surface via covalently attached membrane lipoproteins. This work counters the prevailing view that polysaccharide- and eDNA/protein-based biofilms are mutually exclusive. Rather, we demonstrate that eDNA and PIA can work synergistically to form a biofilm. IMPORTANCE Staphylococcus aureus remains a global health concern and exemplifies the ability of an opportunistic pathogen to adapt and persist within multiple environments, including host tissue. Not only does biofilm contribute to persistence and immune evasion in the host environment, it also may aid in the transition to invasive disease. Thus, understanding how biofilms form is critical for developing strategies for dispersing biofilms and improving biofilm disease-related outcomes. Using biochemical, genetic, and cell biology approaches, we reveal a synergistic interaction between PIA and eDNA that promotes cell aggregation and biofilm formation in a CodY-dependent manner in S. aureus. We also reveal that envelope-associated lipoproteins mediate attachment of the biofilm matrix to the cell surface.

Previous studies describing mechanisms for K+ uptake in S. aureus revealed that the Ktr-mediated K+ transport system was required for normal growth under alkaline conditions but not under neutral or acidic conditions. This work focuses on the effect of K+ uptake on S. aureus metabolism, including intracellular pH and carbon flux, and is the first to utilize a pH-dependent green fluorescent protein (GFP) to measure S. aureus cytoplasmic pH. These studies highlight the role of K+ uptake in supporting proton efflux under alkaline conditions and uncover a critical role for K+ uptake in establishing efficient carbon utilization.

In , the global transcriptional regulator CodY modulates the expression of hundreds of genes in response to the availability of GTP and the branched-chain amino acids isoleucine, leucine, and valine (ILV). CodY DNA-binding activity is high when GTP and ILV are abundant. When GTP and ILV are limited, CodY's affinity for DNA drops, altering expression of CodY regulated targets. In this work, we investigated the impact of guanine nucleotides on physiology and CodY activity by constructing a null mutant (Δ). biosynthesis of guanine monophosphate is abolished due to the mutation; thus, the mutant cells require exogenous guanosine for growth. We also found that CodY activity was reduced when we knocked out , activating the Agr two-component system and increasing secreted protease activity. Notably, in a rich, complex medium, we detected an increase in alternative sigma factor B activity in the Δ mutant, which results in a 5-fold increase in production of the antioxidant pigment staphyloxanthin. Under biologically relevant flow conditions, Δ cells failed to form robust biofilms when limited for guanine or guanosine. RNA-seq analysis of transcriptome during growth in guanosine-limited chemostats revealed substantial CodY-dependent and -independent alteration of gene expression profiles. Importantly, these changes increase production of proteases and δ-toxin, suggesting that exhibits a more invasive lifestyle when limited for guanosine. Further, gene-products upregulated under GN limitation, including those necessary for lipoic acid biosynthesis and sugar transport, may prove to be useful drug targets for treating Gram-positive infections. infections impose a serious economic burden on healthcare facilities and patients because of the emergence of strains resistant to last-line antibiotics. Understanding the physiological processes governing fitness and virulence of in response to environmental cues is critical for developing efficient diagnostics and treatments. purine biosynthesis is essential for both fitness and virulence in , since inhibiting production cripples's ability to cause infection. Here, we corroborate these findings and show that blocking guanine nucleotide synthesis severely affects fitness by altering metabolic and virulence gene expression. Characterizing pathways and gene products upregulated in response to guanine limitation can aid in the development of novel adjuvant strategies to combat infections.

Staphylococcus aureus is a leading cause of biofilm-associated infections, including infective endocarditis, worldwide. A greater understanding of metabolic forces driving biofilm formation in S. aureus is essential for the identification of novel therapeutic targets and for the development of new strategies to combat this medically important pathogen.

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