Disrupting NtrC function reveals unexpected robustness in a central cell cycle regulatory network.

Cell cycle progression relies on coordinated signaling systems that integrate diverse cellular and environmental cues. In and related Alphaproteobacteria, the essential sensor histidine kinase CckA initiates a phosphorelay signaling cascade that controls both the transcriptional activity and proteolytic stability of the master cell cycle regulator, CtrA. To uncover regulatory connections between this essential signaling pathway and other cellular processes, we selected for mutations that bypassed the loss of CckA function. Among the identified mutations were structurally distinct loss-of-function alleles of the conserved nitrogen assimilation regulator , which differentially suppressed the viability defect of a temperature-sensitive (ts) mutant. Complete loss of NtrC function reduces intracellular glutamine, elevates levels of the alarmone ppGpp, and sustains CtrA protein levels, partially rescuing (ts) viability. In contrast, loss-of-function NtrC mutants that retain intact AAA+ ATPase and phosphoreceiver domains but lack a DNA-binding domain fully rescue (ts) viability and display unexpected transcriptional properties. Specifically, strains expressing these NtrC alleles activate a subset of σ-dependent flagellar genes, even though NtrC does not contain the conserved GAFTGA motif required for NtrC-related enhancer-binding proteins to interact with σ-RNA polymerase. We propose that bypass of CckA function by NtrC DNA-binding mutants involves a combination of metabolic changes induced by glutamine limitation and an emergent NtrC activity that modulates transcription from non-native sites on the chromosome. This study reveals unexpected genetic routes to bypass an essential signaling process, highlighting the mutational robustness of a conserved cell cycle control system.IMPORTANCEThe study of essential genes offers insight into the core biological processes required for life. Yet, gene essentiality is often conditional, shaped by both environmental factors and genomic context. Here, we show that mutations in , a conserved regulator of nitrogen assimilation, enable bypass of an essential signaling process catalyzed by the CckA cell cycle kinase. These mutations coordinately alter metabolism, protein levels, and gene expression in ways that sustain cell growth and division even when the essential regulatory activity of CckA is severely impaired. Our results highlight the capacity of bacterial cells to maintain essential functions through evolutionary reconfiguration of metabolic and regulatory networks.