Confident protein-DNA interaction (PDI) data could significantly improve our understanding of transcriptional regulation network in both prokaryotes and eukaryotes. New methods need to be established for validating and mining the protein-DNA interaction data produced by bioinformatic tools and large-scale screening assays. In this study, we integrated bacterial one-hybrid technique (B1H) with classical chromatin immunoprecipitation (ChIP) assay to develop an innovative B1H-ChIP method, which has the advantages of being high-throughput, low cost, and easy-to-perform. Using this method, we validated two pairs of previously reported PDIs and further successfully discovered five novel target genes for Mce2R and four novel regulators of the gene dnaA in the human pathogen Mycobacterium tuberculosis. New PDI data suggest that Mce2R may play novel roles in the regulation of multi-drug resistance, cell wall synthesis, and intracellular growth of M. tuberculosis, and there exists a probable selective regulation of dnaA under different host conditions. Our findings provide important new information for understanding unique regulatory mechanisms in the pathogen. The B1H-ChIP approach has wide applications both in validating and discovering PDIs and in unraveling transcriptional regulatory network in prokaryotes and eukaryotes.