Abstract
Opportunistic pathogens must coordinate virulence-factor production with changing conditions encountered during colonization and infection. Group B Streptococcus (GBS), a common human opportunistic pathogen, uses the CovRS two-component system to regulate virulence, but how the CovS sensor kinase is tuned to environmental conditions remains unclear. Here, we identify CflP, a noncatalytic membrane protein of the CPBP family, as an accessory regulator of CovS. CflP associates with CovS and promotes CovS-dependent phosphorylation of the response regulator CovR, thereby repressing virulence-associated genes, including the β-hemolysin/cytolysin operon. Loss of CflP also enhances cytotoxicity toward human immune cells. CflP acts independently of Abx1, a previously identified homologous CPBP protein that promotes CovS phosphatase activity. CflP abundance is regulated post-transcriptionally by Mg
2+
availability, linking an environmental cue to CovRS signaling. Comparative genomic analysis revealed distinct patterns of natural variation in these CovS regulators: cflP is predominantly maintained as an intact gene, although specific amino acid substitutions can compromise CflP abundance, whereas abx1 is frequently disrupted by recurrent frameshifts. Together, these findings reveal an accessory regulatory layer that tunes CovS activity in response to environmental conditions and whose natural variation may contribute to strain-dependent differences in CovRS regulation. More broadly, our findings illustrate how noncatalytic membrane proteins can modulate bacterial signaling to balance virulence-factor production with adaptation to changing host environments.