ELEMENTS OF SIGNAL-TRANSDUCTION IN MYCOBACTERIUM-TUBERCULOSIS - IN-VITRO PHOSPHORYLATION IN-VIVO EXPRESSION OF THE RESPONSE REGULATOR MTRA

A putative two-component system, mtrA-mtrB, was isolated from M. tuber culosis H37Rv by using phoB from Pseudomonas aeruginosa as a hybridiza tion probe, The predicted gene product of mtrA displayed high similari ty with typical response regulators, including AfsQ1, PhoB, PhoP, and OmpR. The predicted gene product of mtrB displayed similarities with t he histidine protein kinases AfsQ2, PhoR, and EnvZ and other members o f this class of proteins. Expression analysis in the T7 system showed that mtrA encoded a polypeptide with an apparent molecular mass of 30 kDa. MtrA was overproduced, purified, and demonstrated to participate in typical phosphotransfer reactions using heterologous histidine prot ein kinase, CheA, as a phosphoryl group donor, Mycobacterium bovis BCG , harboring an mtrA-gfp (green fluorescent protein cDNA) transcription al fusion, was used to monitor mtrA expression iri infected J774 monol ayers. Flow cytometric and fluorescence microscopic analyses indicated that the mtrA promoter was activated upon entry and incubation in J77 4 macrophages. In contrast, the hsp60-gp fusion displayed no change in expression under the growth conditions tested. These results suggest a potential role for mtrA in adaptation of the M. tuberculosis complex organisms to environmental changes which may include intracellular co nditions.