C. crescentus is an aquatic bacterium that divides asymmetrically into a stalked cell (which is a sessile, proliferative cell) and a swarmer cell (which is motile and does not replicate). Swarmer cells can differentiate into stalked cells in a process that was previously shown to be dependent on c-di-GMP. Furthermore, c-di-GMP levels are known to oscillate during the bacterial cell cycle, with levels being low in swarmer cells and peaking in stalked cells, but whether such oscillations contribute to cell cycle progression was unknown.
The authors performed a genetic screen for synthetic lethal mutants in a C. crescentus strain that lacks all diguanylate cyclases and is therefore unable to synthesize c-di-GMP. They identified the response regulator DivK, suggesting that DivK and c-di-GMP co-regulate cell cycle progression. DivK is known to regulate the activity of CckA, a bifunctional protein with both kinase and phosphatase activities that regulates the activity of the response regulator CtrA via a phosphorelay; CtrA is phosphorylated in swarmer cells and prevents chromosome replication by binding to the origin of replication, whereas in stalked cells CtrA is dephosphorylated, which promotes replication initiation. Thus, the authors analysed the effects of c-di-GMP on the CckA–CtrA phosphorelay and found that c-di-GMP inhibits CckA kinase activity and stimulates its phosphatase activity. Furthermore, the authors showed that c-di-GMP binds to the catalytic ATP-binding domain of CckA, and a single mutation in CckA (Y514D) strongly diminished c-di-GMP binding. Moreover, the mutant CckA was unable to switch from the kinase state to the phosphatase state, demonstrating that c-di-GMP binding to CckA is required to induce a change in its activity.
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