In vitro evidence of two-component system phosphorylation between the Mycobacterium tuberculosis TrcR/TrcS proteins

Two-component regulatory proteins, histidine kinases and response regulators, function in bacteria as sensing and adaptive factors in response to a wide range of environmental stimuli. Conserved histidine and glycine regions of histidine kinase sensor proteins were used to design degenerate oligonucleotide primers for amplification of DNA fragments from Mycobacterium tuberculosis. Two adjacent genes, trcR and trcS, which encode a response regulator and a histidine kinase, respectively, have been identified. Full-length and truncated TrcR and TrcS proteins have been expressed in Escherichia coli. Difficulties in expressing recombinant full-length TrcS and a truncated N-terminal form of TrcS reveal that the transmembrane domains are toxic to E. coil. Overexpressed truncated C-terminal transmitter domains of TrcS have been autophosphorylated in vitro and have transphosphorylated both the full-length recombinant TrcR protein and the N-terminal receiver/regulator domain of TrcR. In vitro autophosphorylation of TrcS requires the presence of Mn²⁺ or Ca²⁺ as a divalent cation cofactor and subsequent transphosphorylation of TrcR is evident in the presence of TrcS-phosphate and Ca²⁺. Transphosphorylation between these two proteins provides evidence that these M. tuberculosis genes encode functional two-component system regulatory proteins that are members of a signal transduction circuit.