The regulation of DnaA in «Caulobacter crescentus»

All growing cells must ensure that their genetic material is faithfully replicated and divided equally to the newly formed daughter cells. Once chromosome replication has begun the cell must assure that it does not start replication until the next cell cycle. In nearly all bacteria chromosome replication is initiated by the highly conserved DnaA protein. In Escherichia coli the activity of the initiator protein DnaA is down regulated by the regulatory inactivation of DnaA (RIDA) system. Shortly after the initiation of replication, the intrinsic ATPase activity of DnaA is stimulated by Hda, a protein with homology to the ATPase domain of DnaA. Therefore, Hda converts DnaA from its active ATP bound form to its inactive ADP bound form. The current models of chromosome replication have been developed using the model bacteria E. coli. However, DnaA proteins form other bacteria differ from DnaA in E. coli in their structures and biochemical properties. A regulatory model based solely on E. coli does not take into account the diversity found among bacteria. Another model is provided by the free-living Gram-negative bacterium Caulobacter crescentus which is found in nutrient poor aqueous environments such as fresh water lakes. Genomic analysis predicts C. crescentus encodes a protein (HdaA) with homology to Hda of E. coli. C. crescentus also makes use of novel regulatory mechanisms not found in E. coli to regulate DnaA. Unlike DnaA of E. coli the DnaA protein of C. crescentus is unstable in growing cells, rapidly degraded during stationary phase and upon nutrient starvation. I have created a strain of C. crescentus with hdaA expressed under the control of a xylose dependent promoter. I demonstrated that C. crescentus depends upon a RIDA mechanism to prevent multiple initiations of chromosome replication in the same cell cycle, as blocking hdaA expression causes an increased frequency of chromosome replication as well as a blockage of cell division. I have also uncovered an unexpected role for HdaA in the stability control of DnaA. Removing HdaA from C. crescentus stabilizes the DnaA protein in growing cells and prevents complete DnaA protein removal from stationary phase cells. My experiments have identified a new role for HdaA in DnaA proteolysis that presumably works in exponentially growing cells to aid the RIDA mechanism that restricts chromosome replication to once per cell cycle. I also demonstrate that HdaA participates in the programmed transition from exponentially growing cells to the stationary phase. C. crescentus also contains a second gene (hdaB) predicted to encode a protein with homology to the DNA binding domain of DnaA. The hdaB gene is not essential for the normal growth of C. crescentus. The fact that homologs of HdaB are found in the genomes of other alphaproteobacteria suggests that, hdaB has an evolutionary conserved role in these bacteria. Form these studies I propose that HdaA not only alters DnaA activity but also DnaA protein stability. This hypothesis further suggests that C. crescentus employs both nucleotide binding/hydrolysis, as seen in E. coli and a novel proteolytic mechanism to regulate DnaA. However, no previous studies have directly addressed the nucleotide binding and ATP hydrolysis of C. crescentus DnaA. To clarify the link between ATPase activity and protein stability I created four independent single amino acid mutations in two conserved positions of C. crescentus DnaA, DnaAR300 and DnaAR357. I showed that mutations in either position reduce DnaA ATPase activity in vitro. Further in vivo studies showed that mutations C. crescentus DnaAR357 increased chromosome replication and increased DnaA stability. These results indicate that the stability of DnaA protein is linked with its ATPase activity state. Combined with the established E. coli RIDA mechanism, my results imply a similar feedback mechanism that also ties DnaA proteolysis with C. crescentus cell cycle progression.

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