In this study, we showed that the DnaA mutant protein in C. crescentus retains its ability to promote the initiation of chromosomal replication in vivo, and is even hyper-active as an initiator compared to the wild-type DnaA protein, as indicated by the severe over-replication phenotype of cells that over-express DnaA . In addition, we showed that the DnaA protein cannot replace DnaA , suggesting that the inactivation of the initiator DnaA is an essential process in C. crescentus. In contrast, we observed that the activity of DnaA as a transcription factor that stimulates the transcription of four genes is not higher than that of DnaA , indicating that the AAA+ domain of DnaA may not inactivate DnaA as a transcriptional regulator of these genes in C. crescentus. Below, we discuss the role of the AAA+ domain of DnaA in the regulation of both activities of DnaA in the control of the C. crescentus cell cycle. The R357A substitution in the AAA+ motif of the C. crescentus DnaA protein is equivalent to the previously characterized R334A mutation in the E. coli DnaA protein that inhibits RIDA and the intrinsic ATPase activity of DnaA in vivo and in vitro . It is thus likely that the R357 residue in the AAA+ domain of the C. crescentus DnaA protein participates in the hydrolysis of an ATP bound to DnaA, to inactivate DnaA immediately following the initiation of chromosome replication . Consistent with this model, the C. crescentus DnaA protein would be bound to ATP at all times of the cell cycle, as it is the case for the E. coli DnaA protein. Then, DnaA-ATP would initiate chromosome replication whenever and wherever active CtrA is absent, leading to C. crescentus cells that have undergone additional chromosome replication initiations like we observed . The C. crescentus HdaA protein may be a functional homolog of the E. coli Hda protein, stimulating the ATPase activity of the AAA+ domain of DnaA when bound to the replisome. In agreement with this model, we observed that the phenotype of HdaA-depleted cells resembles that of DnaA over-expressing cells , and that the HdaA protein dynamically co-localizes with the replisome . Our results suggest that the inactivation of the initiator DnaA by the hydrolysis of the ATP bound to DnaA by its AAA+ domain is an essential process in C. crescentus, as we were not able to replace the wild-type dnaA Abmole LY2157299 allele by the mutant dnaA allele on the C. crescentus chromosome . This could then explain why the HdaA protein is also essential for normal cell cycle progression in C. crescentus . When DnaA was expressed together with DnaA in strains such as JC367 or JC324, DnaA was probably competing with DnaA when CPI-613 Dehydrogenase inhibitor binding the Cori prior to the initiation of chromosomal replication, thereby maintaining cells alive by the inactivation of the wild-type subset of the multiple DnaA molecules bound to the Cori after the initiation of chromosomal replication.