Revisiting the regulation of the capsular polysaccharide biosynthesis gene cluster in Staphylococcus aureus

In Staphylococcus aureus , the capsular polysaccharide (CP) protects against phagocytosis, but also hinders adherence to endothelial cells and matrix proteins. Its biosynthesis is tightly controlled resulting in a heterogeneous phenotype within a population and CP being mainly detectable in non-growing cells. Capsular biosynthesis genes are encoded by a conserved capA-P operon whose expression is driven by an upstream promoter element (P cap ) in front of capA . The organization of P cap is poorly understood, as is the interplay of different regulators that influence the early-Off/late-Heterogeneous cap transcription pattern. Here, we demonstrate that P cap contains a main SigB-dependent promoter. The SigB consensus motif overlaps with a previously described inverted repeat that is crucial for cap expression. The essentiality of the inverted repeat is derived from this region acting as a SigB binding site rather than as an operator site for the proposed cap activators RbsR and MsaB. Furthermore, P cap contains an extensive upstream region harboring a weak SigA-dependent promoter and binding sites for the cap repressors SaeR, CodY and Rot. We show that heterogeneous CP synthesis is determined by the combination of SigB activity and repressor binding to the upstream region. The direct SigB dependency and the upstream repressors are also sufficient to explain the temporal gene expression pattern at the transcriptional level. However, CP synthesis remains growth phase-dependent even when capA transcription is rendered constitutive, suggesting additional post-transcriptional regulatory circuits. Thus, the interference of multiple repressors with SigB-dependent promoter activity as well as post-transcriptional mechanisms ensure the appropriate regulation of CP synthesis. Importance The majority of bacterial pathogens produce an array of polysaccharides on their surface which are important virulence factors and thus serve as attractive vaccine candidates. However, the synthesis and assembly of these structures is highly variable and tightly regulated at various levels. In the human pathogen Staphylococcus aureus , the synthesis of the capsular polysaccharide (CP) is dependent on a complex regulatory network which ensures that CP is produced only in a fraction of stationary phase cells. Here, we determined main regulators that drive the peculiar CP expression pattern. We found that the interplay of the transcriptional repressors Sae, CodY and Rot with the alternative Sigma factor B is responsible for early-Off/late-Heterogeneous expression at the transcriptional level. The data also implicates post-transcriptional mechanisms that may act to avoid conflict in precursor usage by machineries involved in either synthesis of CP or other glycopolymers in growing bacterial cells.