The divergent cts1 type VI secretion system locus of Citrobacter rodentium is controlled by multiple promoters that are silenced by H-NS, with CpxR contributing to their activation upon derepression.

The type VI secretion system (T6SS) is a molecular nanomachine that provides Gram-negative bacteria with competitive advantages during host colonization and interbacterial interactions. Citrobacter rodentium, a mouse-specific attaching and effacing pathogen, employs a T6SS encoded by the cts1 locus to outcompete the gut microbiota and establish infection. However, under standard laboratory growth conditions, cts1 expression remains silent and the regulatory mechanisms governing its activation are unknown. Here, we investigated the regulation of cts1, which is organized into two divergent gene clusters, cts1L and cts1R. Transcriptional fusions of the cts1L-cts1R intergenic region in both orientations were inactive in wild-type C. rodentium, but became active in the absence of the global regulator H-NS. Systematic deletion and site-directed mutagenesis of this region identified four promoter regions, one driving cts1L transcription and three directing cts1R expression (P1, P2 and P3). Additional promoters were identified within the cts1R cluster. All cts1 promoters were differentially repressed by H-NS. Moreover, expression of the response regulator CpxR from a low-copy plasmid enhanced promoter activity in the Δhns background. Electrophoretic mobility shift assays showed that H-NS binds the cts1 regulatory region directly, whereas phosphorylated CpxR bound the internal cts1R promoters but not the intergenic P1-P3 regions, suggesting that CpxR activates the latter indirectly. Together, these findings demonstrate that cts1 expression is controlled by a complex hierarchical regulatory network in which H-NS acts as the primary repressor and CpxR contributes to activation only once H-NS-mediated repression is relieved, in a context-dependent manner. This multifactorial regulation likely involves additional, yet-unidentified factors that fine-tune cts1 expression in response to environmental cues.