System-level reconstruction of RpoD, RpoS, and RpoN regulatory networks in Pseudomonas putida KT2440

Background Pseudomonas putida KT2440 is a widely recognized industrial chassis with broad metabolic versatility. Its large repertoire of σ-factors enables coordinated transcriptional response to diverse environmental and physiological conditions, yet the regulatory landscapes of its major σ-factors remain poorly defined. Here, we provide a comprehensive genome-wide characterization of the RpoD and RpoS regulons, and systematically compare RpoN binding across nitrogen conditions. By integrating ChIP-seq, RNA-seq, and independent component analysis, we establish a systems-level framework for resolving σ-factor regulation and its condition-dependent coordination. Results RpoN occupied a largely conserved set of genomic targets across nitrogen sources, with binding intensity broadly increased under amino acid substrates relative to ammonium. An intragenic RpoN site within rlmD , upstream of the stringent-response gene relA , recapitulated a regulatory architecture previously identified in Escherichia coli and Salmonella Typhimurium, suggesting a conserved link between RpoN and the stringent response. Decomposition of transcriptomes using i-modulons uncovered cross-σ regulatory relationships including activation of RpoS-associated modules following rpoN deletion and activation of previously uncharacterized modules associated with genomic islands and an alternative Type VI secretion system under a nitrogen-limiting condition. During stationary phase, RpoS regulated TCA-cycle activity, while increased RpoD occupancy at promoters derepressed in the rpoS mutant provided evidence of σ-factor competition. Conclusions Together, these results delineate the functional architecture of major σ-factors regulation in P. putida and provide a foundational reference for future studies that leverage σ-factor for metabolic engineering.