Synthetic lac Operator Substitutions for Studying the Nitrate- and Nitrite-Responsive NarX-NarL and NarQ-NarP Two-Component Regulatory Systems of Escherichia coli K-12

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Abstract
The NarX and NarQ sensor-histidine kinases control phosphorylation of the NarL and NarP response regulators in response to the respiratory oxidants nitrate and nitrite. Target operon transcription is activated by the Fnr protein in response to anaerobiosis, and it is further activated and/or repressed by the phospho-NarL and phospho-NarP proteins, which bind to heptamer DNA sequences. The location and arrangement of heptamers vary widely among different target operon control regions. We have constructed a series of monocopy lac operon control region constructs in which the primary operator O1- lac has been replaced by 7-2-7 heptamer pairs from the nrfA , nirB , napF , and fdnG operon control regions. These constructs provide tools for dissecting various aspects of ligand interactions with sensor-kinases, sensor interactions with response regulators, and phospho-response regulator interactions with DNA targets. Expression of the lacZ gene from these constructs was repressed to various degrees by nitrate and nitrite. In response to nitrate, the nrfA and nirB operon 7-2-7 heptamer pairs at operator O1 each mediated greater than 100-fold repression of lacZ gene expression, whereas the napF operon 7-2-7 heptamer pair mediated approximately tenfold repression. Introduction of narL , narP , narX , and narQ null alleles in various combinations allowed the in vivo interactions between different sensor-regulator pairs to be evaluated and compared.

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