The NarX and NarQ sensor-transmitter proteins of Escherichia coli each require two conserved histidines for nitrate-dependent signal transduction to NarL

Abstract

The NarX, NarQ, and NarL proteins of Escherichia coli constitute a two-component regulatory system that controls the expression of a number of anaerobic respiratory pathway genes in response to nitrate. NarX and NarQ are sensor-transmitter proteins that can independently detect the presence of nitrate in the cell environment and transmit this signal to the response regulator, NarL. Upon activation, NarL binds DNA and modulates the expression of its target genes by the repression or activation of transcription. NarX and NarQ each contain a conserved histidine residue that corresponds to the site of autophosphorylation of other sensor-transmitter proteins. They also contain a second conserved histidine residue that is present in the NarX, NarQ, UhpB, DegS, and ComP subfamily of sensor-transmitter proteins. The second histidine is located near a universally conserved asparagine residue, the role of which in signal transduction is unknown. To investigate the role of these conserved amino acids in the NarX and NarQ proteins, we mutated the narX and narQ genes by site-directed mutagenesis. In vivo, each mutation severely impaired NarL-dependent activation or repression of reporter gene expression in response to nitrate. The in vivo data suggest that the environmental signal nitrate controls both the kinase and phosphatase activities of the two sensor-transmitter proteins. The altered NarX and NarQ proteins were purified and shown to be defective in their ability to autophosphorylate in the presence of [gamma-32P]ATP. The NarX and NarQ proteins with amino acid substitutions at the first conserved histidine position were also unable to dephosphorylate NarL-phosphate in vitro. In contrast, the proteins containing amino acid substitutions at the second conserved histidine or at the conserved asparagine residue retained NarL-phosphate dephosphorylation activity. The conserved histidine and asparagine residues are essential for NarX and NarQ function, and this suggests that other two-component sensor-transmitter proteins may function in a similar fashion.