Functional dissection of the transmitter module of the histidine kinase NtrB in Escherichia coli.
Signal transduction by two-component systems involves phosphorylation and thereby activation of the response regulator by the cognate histidine kinase. Bifunctional histidine kinases have two opposing activities: depending on the environmental stimuli they either promote phosphorylation or stimulate the rapid dephosphorylation of the response regulator. To determine the mechanism of this switch, we analyzed the domain organization of the bifunctional histidine kinase NtrB. Based on sequence alignments with other histidine kinases and a deletion analysis, we defined three separate subdomains of the transmitter module, the H domain (amino acids 123-221), the N domain (amino acids 221-269), and the G domain (amino acids 269-349). The transmitter module, when separately expressed, exhibited a constitutive positive phenotype. In contrast, in the absence of the G domain, the H domain exhibits a constitutive negative phenotype. This negative regulatory activity of the H domain is inhibited by the G domain. The G domain could be physically uncoupled; when coexpressed with the H-N fragment, the constitutive positive phenotype of the transmitter was restored. We demonstrate, in vitro, that the constitutive negative phenotype of the fragments lacking the G domain is caused by stimulation of dephosphorylation of the response regulator NtrC-P. Based on our analysis, we suggest that the function of the sensor domain is to control the interaction of the H and G domains. If these subdomains interact, NtrB acts as a positive regulator; if they cannot interact, NtrB acts as a negative regulator.
Kramer
G
G
Department of Biology, University of Konstanz, D-78464 Konstanz, Germany.
Weiss
V
V
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology*; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics; D000071677:Histidine Kinase; D010446:Peptide Fragments / Q000378:metabolism; D010749:Phosphoprotein Phosphatases / Q000737:chemistry / Q000235:genetics; D010766:Phosphorylation; D011494:Protein Kinases / Q000737:chemistry* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D016415:Sequence Alignment; D015398:Signal Transduction
D001426:Bacterial Proteins; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D011494:Protein Kinases; D000071677:Histidine Kinase; D010749:Phosphoprotein Phosphatases