Bacteria rely on complex regulatory networks to coordinate growth, development, and environmental adaptation. In Caulobacter crescentus, cell cycle progression is tightly controlled by the essential CckA-ChpT-CtrA two-component signaling phosphorelay. CckA, a bifunctional sensor histidine kinase (SHK), regulates the phosphorylation state of CtrA, a master response regulator (RR) that directs the transcription of over 90 genes involved in cell cycle progression, cell division, and polar morphogenesis. The activity of CckA is influenced by intracellular signals such as cyclic-di-GMP and ADP, as well as environmental stress cues that enhance its phosphatase activity. Altogether, regulation of CckA kinase/phosphatase activity leads to the properly timed oscillation of CtrA inactivation and degradation during each cell cycle, as well as a block in cell division under stress. Despite the essential nature of CckA and CtrA, genetic studies have identified alternative pathways that can bypass the requirement for CckA function, highlighting the flexibility of the C. crescentus regulatory network. In this dissertation, I demonstrate that the bacterial enhancer binding protein (bEBP) NtrC, and its cognate SHK, NtrB, play critical but previously unrecognized roles in coordinating nitrogen metabolism with cell cycle progression and development in C. crescentus. NtrC is an unconventional bEBP that lacks the conserved GAFTGA motif required for σ54-RNA polymerase activation. I show that deletion of ntrC slows growth in complex medium and that ntrB and ntrC are essential when ammonium is the sole nitrogen source due to their requirement for glnA expression. Interestingly, spontaneous insertion of an IS3-family mobile genetic element frequently restored the growth defect of ntrC mutants by reactivating transcription of the glnBA operon, suggesting that IS3 transposition may play a role in evolutionary adaptation of C. crescentus to nutrient limitation. Genome-wide binding studies within this work identified numerous NtrC binding sites near genes involved in polysaccharide biosynthesis and cell cycle regulation, often overlapping with binding sites for the essential nucleoid-associated protein GapR and the cell cycle regulator MucR1. Loss of NtrC function resulted in elongated polar stalks and increased synthesis of cell envelope polysaccharides, implicating NtrC in the direct regulation of cell morphogenesis and development. Furthermore, genetic suppression of a temperature-sensitive cckA mutant revealed that mutant forms of NtrC can bypass the essential CckA-ChpT-CtrA phosphorelay through two mechanisms: 1) increased levels of the alarmone ppGpp due to intracellular glutamine limitation, which sustain CtrA protein levels, and 2) activation of transcription at select σ54-dependent promoters despite the absence of the GAFTGA motif. My results presented in this dissertation provide evidence that NtrC can function as a central integrator of nitrogen status and cell cycle progression in Caulobacter, linking nutrient availability with core developmental processes. My discovery of ntrC mutants that rescue cckA loss-of-function highlights the remarkable plasticity of bacterial regulatory networks and underscores the complex interplay between nitrogen metabolism, nucleotide signaling, and cell cycle control. This work establishes NtrC as a key regulator of cell cycle progression and developmental plasticity in C. crescentus, revealing new insights into the adaptive potential of bacterial signaling pathways.
Read
