A tripartite RpoS-CRP-cAMP-Cra circuit orchestrates metabolic reprogramming during stress adaptation.

The alternative sigma factor RpoS (σS) governs stress adaptation in many bacterial clades, yet how it coordinates stress responses with metabolic demands remains poorly understood. Thus, by integrating RNA-seq, ChIP-seq, and physiological and molecular assays, we redefined the RpoS regulon in Escherichia coli, expanding it to 667 genes and demonstrating that metabolic genes unexpectedly predominate over stress-response targets. RpoS directly drives a massive metabolic reprogramming event: it activates glycolytic and sugar transport genes while simultaneously repressing the trichloroacetic acid cycle and oxidative phosphorylation. This metabolic pivot is finely tuned by a novel RpoS-CRP-cAMP-Cra tripartite circuit to balance energy efficiency with survival during stationary phase. Furthermore, we provide a mechanistic framework for RpoS-mediated bidirectional regulation rooted in σS-factor competition, where transcriptional efficiency in the absence of RpoS dictates the regulatory mode: highly efficient promoters are repressed by RpoS displacement, whereas weak promoters are activated. This study advances our understanding of transcriptional precision during bacterial adaptation and offers an architectural blueprint for synthetic metabolic engineering.