PHOSPHORYLATION AND DEPHOSPHORYLATION OF THE NARQ, NARX, AND NARL PROTEINS OF THE NITRATE-DEPENDENT 2-COMPONENT REGULATORY SYSTEM OF ESCHERICHIA-COLI

The NarX, NarQ, and NarL proteins make up a nitrate-responsive regulat ory system responsible for control of the anaerobic respiratory pathwa y genes in Escherichia coli, including nitrate reductase (narGHJI), di methyl sulfoxide/trimethylamine-N-oxide reductase (dmsABC), and fumara te reductase (frdABCD) operons among others. The two membrane-bound pr oteins NarX and NarQ can independently sense the presence of nitrate a nd transfer this signal to the DNA-binding regulatory protein NarL, wh ich controls gene expression by transcriptional activation or repressi on. To establish the role of protein phosphorylation in this profess a nd to determine whether the NarX and NarQ proteins differ in their int eraction with NarL, the cytoplasmic domains of NarX and NarQ were over produced and purified. Both proteins were autophosphorylated in the pr esence of [gamma-P-32]ATP and MgCl2 but not with [alpha(32)P]ATP. Wher eas these autophosphorylation reactions were unaffected by the presenc e of nitrate, molybdate, GTP, or AMP, ADP was an inhibitor. The phosph orylated forms of 'NarX and 'NarQ were stable for hours at room temper ature. Each protein transferred its phosphoryl group to purified NarL protein, although 'NarQ-phosphate catalyzed the transfer reaction at a n apparently much faster rate than did 'NarX-phosphate, In addition, N arL was autophosphorylated with acetyl phosphate but not with ATP as a substrate. NarL-phosphate remained phosphorylated for at least 3 h. H owever, addition of 'NarX resulted in rapid dephosphorylation of NarL- phosphate. In contrast, 'NarQ exhibited a much slower phosphatase acti vity with NarL-phosphate. These studies establish that the cytoplasmic domains of the two nitrate sensors 'NarX and 'NarQ differ in their ab ility to interact with NarL.