Transcription analysis of the dnaA gene and oriC region of the chromosome of Mycobacterium smegmatis and Mycobacterium bovis BCG, and its regulation by the DnaA protein

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Abstract
The regions flanking the Mycobacterium dnaA gene have extensive sequence conservation, and comprise various DnaA boxes. Comparative analysis of the dnaA promoter and oriC region from several mycobacterial species revealed that the localization, spacing and orientation of the DnaA boxes are conserved. Detailed transcriptional analysis in M. smegmatis and M. bovis BCG shows that the dnaN gene of both species and the dnaA gene of M. bovis BCG are transcribed from two promoters, whereas the dnaA gene of M. smegmatis is transcribed from a single promoter. RT-PCR with total RNA showed that dnaA and dnaN were expressed in both species at all growth stages. Analysis of the promoter activity using dnaA–gfp fusion plasmids and DnaA expression plasmids indicates that the dnaA gene is autoregulated, although the degree of transcriptional autorepression was moderate. Transcription was also detected in the vicinity of oriC of M. bovis BCG, but not of M. smegmatis. These results suggest that a more complex transcriptional mechanism may be involved in the slow-growing mycobacteria, which regulates the expression of dnaA and initiation of chromosomal DNA replication.