Genome-wide definition of the CosR regulon and DNA-binding properties in Campylobacter jejuni

CosR is an essential OmpR-family transcriptional regulator of Campylobacter jejuni, but its direct regulon and DNA-binding properties in vivo remain poorly defined. Here, we used ChIP-seq with a functional chromosomal CosR::3xFLAG allele to define the genome-wide CosR-binding landscape. CosR binding was strongly enriched at promoters and accumulated around transcription start sites, consistent with a primary role in transcriptional control. Functional analysis of promoter-bound targets revealed significant enrichment for translation- and transcription-related functions, identifying CosR as a regulator of core cellular processes. Motif analysis of summit-centered ChIP-seq regions identified a CosR-associated bipartite sequence signature characterized by TTAA-like elements separated by an A/T-rich spacer. DNase I footprinting confirmed direct promoter binding at nucleotide resolution and revealed heterogeneous architectures, including single, multipartite, and bidirectional binding arrangements. A footprint-derived motif was significantly similar to the ChIP-derived motif, supporting a shared recognition signature across in vivo-enriched regions and in vitro-protected segments. Footprinting also validated binding at non-coding RNA promoters and at the cosR promoter, indicating autoregulation. Hydrogen peroxide treatment differentially remodeled CosR promoter occupancy in vivo, reducing binding at translation-associated promoters while increasing enrichment at other targets. Redox-dependent footprinting showed that oxidative conditions directly impaired CosR binding at selected promoters, consistent with previously reported C218-dependent redox modulation. Despite these opposite occupancy patterns, most tested transcripts decreased after oxidative stress, indicating that CosR redox responsiveness is integrated with broader stress-dependent regulatory inputs. Together, these data define CosR as a condition-responsive regulator linking promoter recognition, core physiology and oxidative-stress-associated transcriptional remodeling. IMPORTANCECampylobacter jejuni is a major foodborne pathogen that must adapt to changing oxygen levels and stress conditions during transmission and infection. CosR is essential for this bacterium, but previous studies could not clearly distinguish direct regulation from secondary effects caused by perturbing an essential protein. By mapping CosR binding across the genome and validating binding at nucleotide resolution, this study shows that CosR directly targets promoters associated with fundamental cellular functions, particularly translation and RNA metabolism, showing that the role of CosR extends well beyond oxidative-stress regulation. We also show that a brief oxidative challenge redistributes CosR occupancy among promoters in a target-dependent manner. These findings identify CosR as a condition-responsive regulator linking the gene-expression machinery and other core physiological functions to stress adaptation, and provide a framework for understanding how C. jejuni adjusts its physiology in changing environments.