A novel σB regulatory module in staphylococcus aureus: Unraveling the multifaceted roles of RsbU domains in stress response mechanisms.

Background: The σ^B factor in Staphylococcus aureus governs the environmental stress response and a wide spectrum of biological functions. σ^B activity is regulated by protein-protein interactions among RsbU, RsbV, RsbW, and σ^B. While the C-terminal PP2C phosphatase domain of RsbU is well-characterized, the function of its N-terminal domain remains unclear.

Methods: To analyze the molecular weight distributions of Rsb proteins and RsbV phosphorylation states, S. aureus cell lysates were subjected to gel filtration and Phos-tag gel electrophoresis. Protein associations were investigated through coelution experiments, immunoprecipitation, and a bacterial two-hybrid assay.

Results: Gel filtration revealed a shift in RsbV phosphorylation states following stress, with unphosphorylated monomeric RsbV predominating before stress and phosphorylated RsbV increasing afterward. This shift corresponded with a decrease in RsbV's ability to sequester RsbW. Under unstressed conditions, RsbU exhibited unexpectedly high phosphatase activity; however, unphosphorylated RsbV remained inactive in sequestering RsbW. Coelution and immunoprecipitation experiments demonstrated potential associations among RsbU, RsbW, and σ^B. The bacterial two-hybrid assay showed direct interactions between full-length RsbU and RsbV, while RsbU interacted with RsbW only in the presence of both RsbV and σ^B. Further experiments identified the N-terminal domain of RsbU as mediating interactions with RsbW.

Conclusion: These findings reveal a novel σ^B regulatory module in S. aureus that integrates interactions among the N- and C-terminal domains of RsbU and other Rsb proteins. This module differs from σ^B regulatory mechanisms described in other bacteria, advancing our understanding of stress response regulation in S. aureus.