Co-regulation of HexC fine-tunes HexA-dependent transcription and acid stress response in Pectobacterium carotovorum

Environmental fluctuations and host-derived stresses demand precise transcriptional regulation in bacterial pathogens to ensure survival and pathogenic success. In Pectobacterium sp., causal agents of soft rot disease, the global regulator HexA is known to repress genes involved in plant cell wall-degrading enzyme (PCWDE) production, quorum sensing (QS), and virulence. However, its broader regulatory scope and physiological significance remain poorly understood. Here, we examined HexA function in P. carotovorum PCC27 through integrated ChIP-seq and RNA-seq analyses, revealing that HexA controls a diverse regulon, including transcriptional regulators, hypothetical proteins, and post-transcriptional regulators such as RsmA and RsmB. Among the newly identified targets, HexC, previously annotated as a hypothetical protein, was identified as a co-regulator that physically interacts with HexA and increases its DNA-binding affinity. Biochemical and genetic analyses demonstrated that HexC reinforces HexA-mediated transcriptional repression. The hexA/hexC double mutant produced higher levels of QS signals than the hexA single mutant but did not exhibit further increase in virulence. Phenotypic assays showed that the hexA/hexC mutant exhibited reduced fitness under acidic conditions yet outcompeted the wild-type at neutral pH in LB medium. However, in planta, the hexA, hexC, and hexA/hexC mutants consistently displayed reduced competitiveness compared to the wild-type in both single and mixed infections. These findings establish HexC as a co-repressor that fine-tunes HexA activity, providing mechanistic insights into how transcriptional regulation evolves to balance virulence and environmental fitness.