CHARACTERIZATION OF THE GENES AND PROTEINS OF A 2-COMPONENT SYSTEM FROM THE HYPERTHERMOPHILIC BACTERIUM THERMOTOGA-MARITIMA

As a step towards studying representative members of the two-component family of signal transduction proteins, we have cloned genes encoding a histidine protein kinase and a response regulator from the hyperthe rmophilic bacterium Thermotoga maritima, The genes have been designate d hpkA and drrA, respectively. The deduced HpkA sequence contains all five characteristic histidine protein kinase motifs with the same rela tive order and spacing found in the mesophilic bacterial proteins, A h ydropathy profile indicates that HpkA possesses only one membrane-span ning segment located at the extreme N terminus, The N-terminal region of DrrA exhibits all of the characteristics of the conserved domains o f mesophilic bacterial response regulators, and the C-terminal region shows high similarity to the OmpR-PhoB subfamily of DNA-binding protei ns, Recombinant T. maritima proteins, truncated HpkA lacking the putat ive membrane-spanning N-terminal amino acids and DrrA, were expressed in Escherichia coli, Partial purification of T. maritima proteins was achieved by heat denaturation of E. coli host proteins, In an in vitro assay, truncated HpkA protein was autophosphorylated in the presence of ATP, Thus, the N-terminal hydrophobic region is not required for ki nase activity, Phosphotransfer between truncated HpkA and DrrA was dem onstrated in vitro with the partially purified proteins, The phosphory lation reactions were strongly temperature dependent, The results indi cate that the recombinant T. maritima two-component proteins overexpre ssed in E. coli are stable as well as enzymatically active at elevated temperatures.