FIG 5.

The DosT sensor kinase is completely nonfunctional in the M. tuberculosis Beijing lineage. (A) A schematic representation of the DosT protein showing the relative position of each of its conserved functional domains (http://www.rcsb.org/pdb/protein/P9WGK1) is shown. The approximate location of the frameshift mutation (G775) identified within dosT of Beijing strains that is predicted to result in the introduction of a premature termination codon (STOP) is indicated, as is the autophosphorylation site at residue His392 (17). (B and C) qRT-PCR analysis of dosR (B) and dosT (C) expression in the H37Rv wild type, the H37Rv dosST KO mutant, and the H37Rv dosST KO mutant complemented with the dosT gene from either H37Rv (wt) or HN878 (mut). Results are shown as relative quantities (R.Q.), using sigA as the normalizing gene. The error bars represent standard deviations. Gray bars indicate samples prepared from cultures treated with the NO donor diethylenetriamine-nitric oxide (DETA-NO) for 2 h prior to RNA extraction. (D) Western immunoblotting using anti-FLAG M2 peroxidase to probe reduced protein extracts prepared from M. smegmatis transformants harboring N- or C-terminal FLAG epitope-tagged mutant (mut; Beijing) or wild-type (wt; non-Beijing) DosT. The negative control (neg.) corresponds to protein extracts from M. smegmatis carrying wild-type dosT (i.e., with no FLAG epitope). The arrow indicates the position of the full-length DosT protein. Molecular mass markers are indicated along the right.