Recombinant Reporter Assay Using Transcriptional Machinery of Mycobacterium tuberculosis

Abstract

Development of anin vivogene reporter assay to assess interactions among the components of the transcription machinery inMycobacterium tuberculosisremains a challenge to scientists due to the tediousness of generation of mutant strains of the extremely slow-growing bacterium. We have developed a recombinant mCherry reporter assay that enables us to monitor the interactions ofMycobacterium tuberculosistranscriptional regulators with its promotersin vivoinEscherichia coli. The assay involves a three-plasmid expression system inE. coliwherein two plasmids are responsible forM. tuberculosisRNA polymerase (RNAP) production and the third plasmid harbors the mCherry reporter gene expression cassette under the control of either a σ factor or a transcriptional regulator-dependent promoter. We observed that the endogenousE. coliRNAP and σ factor do not interfere with the assay. By using the reporter assay, we found that the functional interaction ofM. tuberculosiscyclic AMP receptor protein (CRP) occurs with its own RNA polymerase, not with theE. colipolymerase. Performing the recombinant reporter assay inE. coliis much faster than if performed inM. tuberculosisand avoids the hazard of handling the pathogenic bacterium. The approach could be expanded to develop reporter assays for other pathogenic and slow-growing bacterial systems.