Phosphorylation and dephosphorylation of the NarQ, NarX, and NarL proteins of the nitrate-dependent two-component regulatory system of Escherichia coli

Abstract

The NarX, NarQ, and NarL proteins make up a nitrate-responsive regulatory system responsible for control of the anaerobic respiratory pathway genes in Escherichia coli, including nitrate reductase (narGIJI), dimethyl sulfoxide/trimethylamine-N-oxide reductase (dnsABC), and fumarate reductase (frdABCD) operons among others. The two membrane-bound proteins NarX and NarQ can independently sense the presence of nitrate and transfer this signal to the DNA-binding regulatory protein NarL, which controls gene expression by transcriptional activation or repression. To establish the role of protein phosphorylation in this process and to determine whether the NarX and NarQ proteins differ in their interaction with NarL, the cytoplasmic domains of NarX and NarQ were overproduced and purified. Both proteins were autophosphorylated in the presence of [_y-32P]ATP and MgCl2 but not with [a-32P]ATP. Whereas these autophosphorylation reactions were unaffected by the presence of nitrate, molybdate, GTP, or AMP, ADP was an inhibitor. The phosphorylated forms of 'NarX and 'NarQ were stable for hours at room temperature. Each protein transferred its phosphoryl group to purified NarL protein, although 'NarQ-phosphate catalyzed the transfer reaction at an apparently much faster rate than did 'NarX-phosphate. In addition, NarL was autophosphorylated with acetyl phosphate but not with ATP as a substrate. NarL-phosphate remained phosphorylated for at least 3 h. However, addition of 'NarX resulted in rapid dephosphorylation of NarL-phosphate. In contrast, 'NarQ exhibited a much slower phosphatase activity with NarL-phosphate. These studies establish that the cytoplasmic domains of the two nitrate sensors 'NarX and 'NarQ differ in their ability to interact with NarL.

Nitrate repression of the Escherichia coli pfl operon is mediated by the dual sensors NarQ and NarX and the dual regulators NarL and NarP

“…Therefore, NarX and NarQ are equally effective in sensing the presence of nitrate and relaying this signal to the regulator components of the pathway, a finding which has been extensively studied for other nitrate-responsive operons (21,28,33). Either of the response regulators, NarL or NarP, was sufficient to confer maximal nitrate and nitrite repression in rich medium, while the NarL protein appeared to be marginally more effective when cells were grown in minimal medium.…”

“…3, lane 3). It has been demonstrated that incubation of the NarL protein with acetyl phosphate results in the transfer of the phosphate group to the protein (28).…”

“…For example, nitrate induction of the narGHJI operon (21) and the narK gene (12) is controlled exclusively by NarL, while induction of the fdnGHI operon is controlled principally by NarL, but NarP also effects a degree of control (observed with a narL mutant) (21). Expression of the dmsABC and the frdABCD operons is repressed in the presence of nitrate, and this repression is dependent on the NarL protein (8,10,21,28). A more complex scenario is presented by the regulation of two more recently identified genes, termed aeg-46.5 and aeg-93 (4).…”

“…A complex system of proteins that is involved in sensing nitrate in the environment and transmitting its presence to the level of gene expression has been discovered (33). The proteins include two sensor-regulator pairs termed NarX-NarL and NarQ-NarP of which NarX and NarQ are membrane-associated histidine kinases and NarL and NarP are transcription regulators (3,7,14,20,21,28,35,39). The NarQ and NarX proteins are not only capable of sensing nitrate but they are also responsive to nitrite.…”

Purification and Characterization of the Selenate Reductase from Thauera selenatis

“…Gel Electrophoresis-Protein samples were prepared in 2.5% SDS, 5% ␤-mercaptoethanol, and 0.005% bromphenol blue as described (17). After heating for 5 min at 90°C, 1 l of the sample was applied to a 12.5% homogenous polyacrylamide SDS gel (Phastsystem, Pharmacia).…”