Phosphorylation and dephosphorylation catalyzed in vitro by purified components of the nitrate sensing system, NarX and NarL

Abstract

The regulation of specific gene expression by nitrate in Escherichia coli is mediated by the NarX/NarQ-NarL system. Based on sequence homologies with a family of two-component regulatory systems in bacteria, NarL has been identified as a putative response regulator while NarX and NarQ were proposed to be alternative membrane-associated sensors that activate NarL in the presence of nitrate. To investigate the interaction of NarX and NarL in vitro, both proteins were purified from overproducing strains. Purified NarX was rapidly labeled when incubated with [gamma-32P] ATP but not with [alpha-32P]ATP in a reaction that required Mg2+ but was unaffected by nitrate. Incubation of the labeled NarX with purified NarL resulted in the transient phosphorylation of NarL. Both the phosphorylation and dephosphorylation of NarL required Mg2+, and neither reaction was affected by the presence of nitrate. NarL-phosphate, stabilized by the addition of EDTA, ran as a monomer on gel filtration. Dephosphorylation of the isolated NarL-phosphate required the addition of both Mg2+ and the NarX protein. The relative stabilities of the phosphorylated forms of the two proteins at different pH values were consistent with the proposal that, in analogy to other related two-component regulatory systems, NarX and NarL were phosphorylated on specific histidine and aspartate residues, respectively.