Effects of (p)ppGpp on the Progression of the Cell Cycle of Caulobacter crescentus

Bacteria must control the progression of their cell cycle in response to nutrient availability. This regulation can be mediated by guanosine tetra-or pentaphosphate [(p)ppGpp], which are synthesized by enzymes of the RelA/SpoT homologue (Rsh) family, particularly under starvation conditions. Here, we study the effects of (p) ppGpp on the cell cycle of Caulobacter crescentus, an oligotrophic bacterium with a dimorphic life cycle. C. crescentus divides asymmetrically, producing a motile swarmer cell that cannot replicate its chromosome and a sessile stalked cell that is replication competent. The swarmer cell rapidly differentiates into a stalked cell in appropriate conditions. An artificial increase in the levels of (p) ppGpp in nonstarved C. crescentus cells was achieved by expressing a truncated relA gene from Escherichia coli, encoding a constitutively active (p) ppGpp synthetase. By combining single-cell microscopy, flow cytometry approaches, and swarming assays, we show that an increase in the intracellular concentration of (p) ppGpp is sufficient to slow down the swarmer-to-stalked cell differentiation process and to delay the initiation of chromosome replication. We also present evidence that the intracellular levels of two master regulators of the cell cycle of C. crescentus, DnaA and CtrA, are modulated in response to (p) ppGpp accumulation, even in the absence of actual starvation. CtrA proteolysis and DnaA synthesis seem indirectly inhibited by (p) ppGpp accumulation. By extending the life span of the motile nonreproductive swarmer cell and thus promoting dispersal and foraging functions over multiplication under starvation conditions, (p) ppGpp may play a central role in the ecological adaptation of C. crescentus to nutritional stresses.