Characterization of the genes and proteins of a two-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 hyperthermophilic bacterium Thermotoga maritima. The genes have been designated HpkA and drrA, respectively. The deduced HpkA sequence contains all five characteristic histidine protein kinase motifs with the same relative order and spacing found in the mesophilic bacterial proteins. A hydropathy profile indicates that HpkA possesses only one membrane-spanning segment located at the extreme N terminus. The N-terminal region of DrrA exhibits all of the characteristics of the conserved domains of mesophilic bacterial response regulators, and the C-terminal region shows high similarity to the OmpR-PhoB subfamily of DNA-binding proteins. Recombinant T. maritima proteins, truncated HpkA lacking the putative 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 kinase activity. Phosphotransfer between truncated HpkA and DrrA was demonstrated in vitro with the partially purified proteins. The phosphorylation reactions were strongly temperature dependent. The results indicate that the recombinant T. maritima two-component proteins overexpressed in E. coli are stable as well as enzymatically active at elevated temperatures.
Lee
P J
PJ
Center for Advanced Biotechnology and Medicine and Department of Molecular Biology and Biochemistry, Rutgers University, Piscataway, New Jersey 08854, USA.
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D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D005798:Genes, Bacterial; D006089:Gram-Negative Anaerobic Bacteria / Q000235:genetics*; D000071677:Histidine Kinase; D006358:Hot Temperature; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011494:Protein Kinases / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction / Q000235:genetics
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; C101895:DrrA protein, Bacteria; D011994:Recombinant Proteins; D011494:Protein Kinases; D000071677:Histidine Kinase