title: Recombinant reporter assay using transcriptional machinery of Mycobacterium tuberculosis.
abstract: Development of an in vivo gene reporter assay to assess interactions among the components of the transcription machinery in Mycobacterium tuberculosis remains 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 of Mycobacterium tuberculosis transcriptional regulators with its promoters in vivo in Escherichia coli. The assay involves a three-plasmid expression system in E. coli wherein two plasmids are responsible for M. tuberculosis RNA 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 endogenous E. coli RNAP and σ factor do not interfere with the assay. By using the reporter assay, we found that the functional interaction of M. tuberculosis cyclic AMP receptor protein (CRP) occurs with its own RNA polymerase, not with the E. coli polymerase. Performing the recombinant reporter assay in E. coli is much faster than if performed in M. tuberculosis and 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.
authors.affiliation: Department of Chemistry, Bose Institute, Kolkata, India.
authors.forename: Rajdeep
authors.initials: R
authors.lastname: Banerjee
authors.affiliation: Department of Chemistry, Bose Institute, Kolkata, India.
authors.forename: Paulami
authors.initials: P
authors.lastname: Rudra
authors.affiliation: Department of Biochemistry, Bose Institute, Kolkata, India.
authors.forename: Abinit
authors.initials: A
authors.lastname: Saha
authors.affiliation: Department of Chemistry, Bose Institute, Kolkata, India jayanta@jcbose.ac.in.
authors.forename: Jayanta
authors.initials: J
authors.lastname: Mukhopadhyay
mesh_terms: D019677:Artificial Gene Fusion; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D017930:Genes, Reporter; D005827:Genetics, Microbial / Q000379:methods*; D008164:Luminescent Proteins / Q000032:analysis / Q000235:genetics; D008967:Molecular Biology / Q000379:methods*; D009169:Mycobacterium tuberculosis / Q000235:genetics*; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D011995:Recombination, Genetic; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic; D000097573:Red Fluorescent Protein
chemical_list: D001426:Bacterial Proteins; D008164:Luminescent Proteins; D011994:Recombinant Proteins; D014157:Transcription Factors; D000097573:Red Fluorescent Protein; C513697:CRP protein, Mycobacterium tuberculosis