(p)ppGpp modulates cell size and the initiation of DNA replication in Caulobacter crescentus in response to a block in lipid biosynthesis.

Stress conditions, such as a block in fatty acid synthesis, signal bacterial cells to exit the cell cycle. Caulobacter crescentus FabH is a cell-cycle-regulated ß-ketoacyl-acyl carrier protein synthase that initiates lipid biosynthesis and is essential for growth in rich media. To explore how C. crescentus responds to a block in lipid biosynthesis, we created a FabH-depletion strain. We found that FabH depletion blocks lipid biosynthesis in rich media and causes a cell cycle arrest that requires the alarmone (p)ppGpp for adaptation. Notably, basal levels of (p)ppGpp coordinate both a reduction in cell volume and a block in the over-initiation of DNA replication in response to FabH depletion. The gene ctrA encodes a master transcription factor that directly regulates 95 cell-cycle-controlled genes while also functioning to inhibit the initiation of DNA replication. Here, we demonstrate that ctrA transcription is (p)ppGpp-dependent during fatty acid starvation. CtrA fails to accumulate when FabH is depleted in the absence of (p)ppGpp due to a substantial reduction in ctrA transcription. The (p)ppGpp-dependent maintenance of ctrA transcription during fatty acid starvation initiated from only one of the two ctrA promoters. In the absence of (p)ppGpp, the majority of FabH-depleted cells enter a viable but non-culturable state, with multiple chromosomes, and are unable to recover from the miscoordination of cell cycle events. Thus, basal levels of (p)ppGpp facilitate C. crescentus' re-entry into the cell cycle after termination of fatty acid starvation.

The Caulobacter crescentus ctrA P1 promoter is essential for the coordination of cell cycle events that prevent the overinitiation of DNA replication.

The master regulator CtrA oscillates during the Caulobacter cell cycle due to temporally regulated proteolysis and transcription. It is proteolysed during the G1-S transition and reaccumulates in predivisional cells as a result of transcription from two sequentially activated promoters, P1 and P2. CtrA reinforces its own synthesis by directly mediating the activation of P2 concurrently with repression of P1. To explore the role of P1 in cell cycle control, we engineered a mutation into the native ctrA locus that prevents transcription from P1 but not P2. As expected, the ctrA P1 mutant exhibits striking growth, morphological and DNA replication defects. Unexpectedly, we found CtrA and its antagonist SciP, but not DnaA, GcrA or CcrM accumulation to be dramatically reduced in the ctrA P1 mutant. SciP levels closely paralleled CtrA accumulation, suggesting that CtrA acts as a rheostat to modulate SciP abundance. Furthermore, the reappearance of CtrA and CcrM in predivisional cells was delayed in the P1 mutant by 0.125 cell cycle unit in synchronized cultures. High levels of ccrM transcription despite low levels of CtrA and increased transcription of ctrA P2 in the ctrA P1 mutant are two examples of robustness in the cell cycle. Thus, Caulobacter can adjust regulatory pathways to partially compensate for reduced and delayed CtrA accumulation in the ctrA P1 mutant.