When we performed flow cytometry experiments without a rifampicin treatment, we observed a profile drastically shifted towards a higher amount of DNA content per cell, compared to what we observed with the control strain or with the strain that over-expresses the wild-type DnaA protein at similar levels . To confirm that these cells over-initiated DNA replication, we also measured the Cori/ter ratio by Q-PCR from genomic DNA extracted from these cells. Consistent with our single cell flow cytometry results, we observed that the Cori/ter ratio in cells expressing DnaA is about two fold higher than that of cells expressing DnaA as similar levels . All together, these results indicate that DnaA can initiate new rounds of chromosomal replication, but in an uncontrolled manner, suggesting that DnaA is hyper-active for its function as an initiator of chromosomal replication. It is very likely that DnaA remains bound to ATP, and thus active for PI3K inhibitor replication initiation, at all times of the cell cycle of C. crescentus, as it is the case for the DnaA protein in E coli. We previously demonstrated that the HdaA protein is essential for normal cell cycle progression in C. crescentus and that a DhdaA strain can not be constructed in the absence of an extra copy of the hdaA gene expressed in trans . These results suggested that the switch of DnaA activity promoted by HdaA after chromosome replication has initiated could be essential for the viability of C. crescentus. If this prediction is correct, it Torin 1 manufacturer should be very difficult, or impossible, to construct a strain carrying the dnaA allele as the sole copy of dnaA on the C. crescentus chromosome. We attempted to construct such a strain by two different and complementary approaches. The JC125 strain has no apparent phenotype and grows like the wild-type strain: it was used as a control for transduction efficiency. The V cassette confers resistance to spectinomycin and streptomycin. Both phage lysates were used for transduction assays using the NA1000 wild-type strain and the JC323 and JC324 strains containing a copy of the dnaA or the dnaA gene, respectively, under the control of the xylX promoter at the xylX locus. Upon selection on spectinomycin and streptomycin containing plates, we isolated numerous DCC1613::V transductants in the three different genetic backgrounds, regardless of the presence of the xylose inducer , demonstrating that transduction efficiency is not significantly affected by the expression of dnaA or dnaA . We also isolated numerous DdnaA::V mutant colonies from our transduction assay into JC323, but only on xylose-containing plates .