Unraveling chromatin dynamics and regulatory networks in Epsilonproteobacteria: development of the 4f-SAMMY-seq in Helicobacter pylori and characterization of the CosR regulon in Campylobacter jejuni

Helicobacter pylori and Campylobacter jejuni are phylogenetically related Gram-negative pathogens responsible for widespread gastric and enteric diseases. Their persistence and increasing antibiotic resistance pose a major public health challenge. These bacteria display remarkable resilience to environmental stressors, including nutrient limitation, acidic pH, and sub-inhibitory antibiotic exposure. Understanding how they regulate gene expression and reorganize their genomes under stress is therefore crucial for identifying novel antimicrobial targets. This work focused on the coccoid form of Helicobacter pylori, a morphological state associated with the viable but non-culturable condition, which is increasingly recognized as a key contributor to persistence and transmission. To investigate genome organization in this state, the 4f-SAMMY-seq method was successfully adapted and optimized for H. pylori, enabling analysis of nucleoid architecture under native conditions. This approach, based on sequential chromatin fractionation according to DNA accessibility, revealed reproducible patterns of genomic compaction across growth phases. Distinct chromatin rearrangements were observed during the transition to coccoid and biofilm states, indicating that nucleoid remodeling accompanies dormancy and may support transcriptional reprogramming linked to persistence. In parallel, a genome-wide ChIP-seq analysis was performed to define the regulon of CosR, an essential OmpR-type transcriptional regulator in Campylobacter jejuni and the functional homolog of HP1043 in Helicobacter pylori. Using a FLAG-tagged strain, 168 high-confidence CosR-binding sites were identified, predominantly in promoter regions of genes involved in translation, energy metabolism, and stress response. Electrophoretic mobility shift assays confirmed specific binding to selected promoters and demonstrated CosR autoregulation. Moreover, oxidative stress altered the CosR DNA-binding profile, suggesting redox-dependent modulation of its activity. Overall, these findings provide new insights into the interplay between chromosomal organization and essential regulatory networks in Epsilonproteobacteria. This study represents the adaptation of the SAMMY technique to prokaryotic cells and the definition of the CosR regulon under native conditions, highlighting CosR and HP1043 as targets.