Table 3.

P-box substitutions in E. coli NarQ.

Plasmid	Substitutions	P-box sequence	Phenotype
pBADEcQ	E. coli NarQ P-box	DAEAINIAGSLRMQSYRL	Nitrate and nitrite sensing
pRNW18	R54K	DAEAINIAGSLKMQSYRL	Ligand insensitive
pRNW19	N48E I49E	DAEAIEEAGSLRMQSYRL	Ligand insensitive
pRNW20	N48E I49E R54K	DAEAIEEAGSLKMQSYRL	Ligand insensitive
pRNW29	D43A E45S A46V N48E I49E	AASVIEEAGSLRMQSYRL	Ligand insensitive
pRNW30	D43A E45S A46V	AASVINIAGSLRMQSYRL	Ligand insensitive
pRNW31	D43A E45S A46V R54K	AASVINIAGSLKMQSYRL	Ligand insensitive
pRNW32	D43A E45S A46V N48E I49E R54K	AASVIEEAGSLKMQSYRL	Ligand insensitive
pRNW33	D43A E45S A46V S52N S57A	AASVINIAGNLRMQAYRL	Ligand insensitive
pRNW34	D43A E45S A46V N48E I49E S52N R54K S57A	AASVIEEAGNLKMQAYRL	Ligand insensitive

E. coli strains JCB391 (narXL narQ) or JCB12 (narXL narQ frdA::lacZ) transformed with pBADecQ expressing wild-type or mutated NarQ proteins were grown anaerobically in LB in the absence or presence of 20 mM NaNO₃ and the β-galactosidase activities determined. Mutations in the P box are shown in bold type. Wild-type NarQ was able to phosphorylate NarP in the presence of nitrate, activating expression of nirB and repressing frdA. Mutated NarQ proteins were inactive.