Bars, 1 μm. (C) qRT-PCR assays for the gene expression of M. smegmatis. The experiment was carried out as described in the “”Materials and Methods”". 16S rRNA gene, rrs, was used as control. All target

genes were amplified using specific primers. Different gene expressions were normalized to the levels of 16S rRNA gene transcripts, and the folds of expression change were calculated. Representative data are shown. When relative gene expression was measured via qRT-PCR as shown in Fig. 5C, the mtrA gene was only 0.38-fold that of the wild-type strain, indicating that the expression of the mtrA gene in recombinant M. smegmatis was greatly inhibited. The expression of the dnaA gene in the recombinant strain basically remained constant when compared with that in the LY294002 chemical structure wild-type strain. This was consistent with the fact that no conserved sequence motif existed within the regulatory region of this gene in M. smegmatis. Another approximately

26 potential target genes were randomly chosen to measure the expression change in the recombinant M. smegmatis strain (Fig. 5C). The expression levels of these genes clearly changed; iniA and mtrB NVP-AUY922 cell line gene expression increased 2.5-fold expression (Fig. 5C), while mraZ (Msmeg_4236) and rpfB (Msmeg_5439) gene expression decreased by about 0.2-fold (Fig. 5C). Therefore, the inhibition of the mtrA gene resulted in corresponding expression changes in many predicted target genes in M. smegmatis. The expression level of the mtrA gene consequently affected the drug resistance and cell morphology of M. smegmatis. Discussion MtrAB has been reported to regulate the expression of the M. tuberculosis replication

initiator gene, dnaA [12]. However, potential binding sites for MtrA have not been clearly characterized. In addition, there are many potential target genes that also appear to be regulated by MtrA. In the current study, we identified a 7 bp conserved sequence motif for the recognition of MtrA within the dnaA promoter. About 420 potential target genes regulated by MtrAB were predicted from the M. tuberculosis and M. smegmatis genomes Edoxaban upon searching their promoter databases. Many predicted target genes showed significant expression changes when the mtrA homologue of M. smegmatis was partially inhibited. The recombinant M. smegmatis cells increased in length and became sensitive to the anti-TB drugs isoniazid and streptomycin. The transcription of dnaA starts essentially at P1 dnaA , which is conserved in all mycobacterial species [18]. The analysis of the sequence in the upstream region of dnaA revealed a second promoter, P2 dnaA, in M. tuberculosis [18]. In previous in vivo experiments, MtrA bound with the regulatory region of the dnaA gene [12]. In the current study, two binding motifs for MtrA were located immediately downstream from the two promoters (Fig. 2C). Therefore, MtrA can apparently interfere with the promoter activity and thus regulate the expression of the replication initiator gene.