Inhibition of chromosome replication in Mycobacterium smegmatis: effect of the rpmH–dnaA promoter region

Abstract

In a previous study a functional mycobacterial origin of replication, oriC, was isolated on a plasmid. However, it was found that origin function was inhibited by the presence of the adjacent dnaA gene or its regulatory region, so that plasmids containing both of these regions next to the origin did not yield transformants. This inhibition could be due either to overexpression of dnaA on a plasmid being toxic, the transcription of dnaA into the downstream origin topologically inhibiting its function, or to the DnaA boxes upstream of dnaA somehow interacting with the DnaA boxes in the origin to prevent its function. To distinguish between these possibilities, plasmids were constructed lacking different parts of the dnaA gene : the promoter, the DnaA boxes, or both. Additionally, the putative dnaA promoter region was replaced by mycobacterial sequences that exhibit weaker or null promoter activity. The results indicate that the rpmH-dnaA promoter region, but not the DnaA boxes, is the principal cause of the incompatibility observed and suggest that this region could be playing a role in the inhibition of chromosome replication.

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

“…The resulting fragments were cloned into the BamHI site of pFPV27 generating plasmids pGFP85, pGFP11, pGFP61, pGFP8, pGFPS5, pGFPB7, pGFPS12 and pGFPB11. The pGFP87 and pGFP71 plasmids were derived from pGFP85 and have been previously described (Salazar, 2000). The pGFP22 and pGFP16 plasmids were obtained by subcloning from pGFP11.…”

“…Deletion of the three DnaA boxes (compare pGFP85 with pGFP87, and pGFP30 with pGFP22-4) resulted in an increase in the fluorescence emission ( Fig. 1 and Salazar, 2000).…”