Bacterial Strains and Recombinant Plasmid Construction

Cloning and protein overexpression were carried out in E. coli DH10β and BL21 Arctic Express^TM (Agilent Technologies, United States) (Ferrer et al., 2003), respectively. The strains were grown in LB medium with 100 μg/ml ampicillin or 50 μg/ml gentamycin as required. The primers used for PCR and cloning are listed in the Supplementary Table. Recombinant plasmids used for protein overexpression have been reported previously (Nambi et al., 2010; Agrawal et al., 2015). Recombinant plasmids for expressing chimeric MtrB-GFP protein, the nucleotide region from the 851 to 1754 bp region of mtrB, capable of coding for the 300 aa long cytosolic catalytic domain of the MtrB protein, was PCR amplified from H37Rv genomic DNA using specific primers and cloned in pProEx-Htc vector at NcoI and BamHI restriction sites. The gfp gene was cloned between BamHI and XhoI sites. Similarly, for MtrA-RFP chimeric protein, the mtrA gene was cloned in NcoI and BamHI restriction sites, and the rfp gene was cloned between BamHI and XhoI sites in the pPRoEx-Htb expression vector. For in cellulo and in vivo analysis, the gene coding for wild-type MtrA protein was cloned in pMV261 vector between PstI and HindIII sites, and the recombinant plasmid was introduced in M. tuberculosis H37Ra for further analysis. Various lysine mutations were introduced by a quick-change, site-directed mutagenesis technique directly on pPROEx or pMV261 plasmids. All the recombinant constructs were verified by DNA sequencing.