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

Two-component regulatory proteins, histidine kinases and response regulator s, function in bacteria as sensing and adaptive factors in response to a wi de range of environmental stimuli. Conserved histidine and glycine regions of histidine kinase sensor proteins were used to design degenerate oligonuc leotide primers for amplification of DNA fragments from Mycobacterium tuber culosis. Two adjacent genes, trcR and trcS, which encode a response regulat or and a histidine kinase, respectively, have been identified. Full-length and truncated TrcR and TrcS proteins have been expressed in Escherichia col i. Difficulties in expressing recombinant full-length TrcS and a truncated N-terminal form of TrcS reveal that the transmembrane domains are toxic to E. coli. Overexpressed truncated C-terminal transmitter domains of TrcS hav e been autophosphorylated in vitro and have transphosphorylated both the fu ll-length recombinant TrcR protein and the N-terminal receiver/regulator do main of TrcR. In vitro autophosphorylation of TrcS requires the presence of Mn2+ or Ca2+ as a divalent cation cofactor and subsequent transphosphoryla tion of TrcR is evident in the presence of TrcS-phosphate and Ca2+. Transph osphorylation between these two proteins provides evidence that these M. tu berculosis genes encode functional two-component system regulatory proteins that are members of a signal transduction circuit. (C) 1999 Academic Press .