Dual response regulators (NarL and NarP) interact with dual sensors (NarX and NarQ) to control nitrate- and nitrite-regulated gene expression in Escherichia coli K-12

Abstract

Two sensor proteins, NarX and NarQ, mediate nitrate regulation of anaerobic respiratory gene expression.Either of these sensors is sufficient to signal the presence of nitrate to the response regulator protein, NarL, a transcriptional activator and repressor. Two observations suggested the existence of a second response regulator that is also involved in nitrate regulation. First, narL null mutants retain residual nitrate induction offdnG operon expression; this residual induction is absent in narX narQ double-null strains. Second, nitrate induction of aeg-46.5 operon expression is substantially enhanced in narL null strains (M. H. Choe and W. S. Reznikoff, J. Bacteriol. 173:6139-6146, 1991). We found that this nitrate induction requires either the NarX or the NarQ protein, consistent with the existence of a second response regulator. We designate this second regulator NarP. We isolated insertion mutants that are defective in aeg-46.5 operon expression. These insertions are in the narP gene, which encodes a response regulator that is 44% identical to the NarL protein.Null alleles of narP abolished aeg-46.5 induction and also eliminated the residual NarL-independent nitrate induction offdnG operon expression. Both the NarX and NarQ proteins communicate with both the NarP and NarL proteins. We found that the primary signal for NarP-dependent aeg-46.5 operon induction is nitrite rather than nitrate. By contrast, nitrite is a relatively weak signal for NarL-dependent induction. In narX null strains, nitrite was an efficient signal for NarL-dependent induction, and this induction required the NarQ protein. We conclude that, in wild-type strains, the NarQ protein communicates the presence of nitrite to both the NarP and NarL proteins and that the NarX protein inhibits this communication with the NarL protein.Eschenchia coli is a facultative aerobe and can synthesize a variety of respiratory chains during anaerobic growth. Anaerobic induction of respiratory enzyme synthesis is mediated by the activator protein Fnr (reviewed in reference 35). The energetically most efficient anaerobic respiratory chain is formate-nitrate oxidoreductase, which consists of formate dehydrogenase-N (encoded by the fdnGHI operon), quinone, and nitrate reductase (encoded by the narGHJI operon) (reviewed in reference 38). During anaerobic growth, nitrate (NO3-) induces the synthesis of formate dehydrogenase-N and nitrate reductase through the action of the activator protein NarL (2, 37; reviewed in reference 39). The NarL protein also represses the synthesis of alternate respiratory enzymes such as fumarate reductase (encoded by the frdABCD operon) during anaerobic growth with nitrate (17,19,40 This shows that the NarL protein represses aeg-46. 5 expression even in the absence of nitrate. Regulation of the aeg-93 operon resembles that of the nrf operon, encoding formatedependent respiratory nitrite reductase (26). Additionally, the nrf genes map at 93 min (9). Thus, it is likely that the aeg-93 operon fusion resides in one of the nrf genes. Ex...

The construction and application of a lux ‐based nitrate biosensor

“…Furthermore, the existence of E. coli narL transposon mutants could potentially facilitate the cloning of these corresponding narL loci. This work also reinforces the previously described observations that a sensor-regulator coupling pathway is involved in regulating nitrate reductase expression in E. coli (Stewart and MacGregor 1982 ;Rabin and Stewart 1993).…”

“…Phoswashes in 2×SSC, 0·1% SDS were carried out at 68°C. The phorylated NarL then binds to the nitrate reductase promoter hybrids were detected using anti-dig-alkaline phosphatase upstream of the structural genes narGHJI and activates antibody conjugate as described in the manufacturer's nitrate reductase expression (Stewart 1993). Transformation instructions (Boehringer Mannheim).…”

“…Nitrate-induced NarX and NarQ sensor proteins for nitrate is not absolute incubation at 37°C). ž, Nitrate ; E, nitrite ; Ž, ammonium and it has been demonstrated that nitrite-induced narGHJI transcription does occur (Rabin and Stewart 1993). With this in mind, the response of the biosensor to nitrite was examined and was shown to be induced by nitrite to lower levels at high concentrations (Fig.…”

“…In mock fermentations O. proteus has been shown to catalyse the The reduction of nitrate by Escherichia coli, a paradigm for reduction of wort nitrate to nitrite via nitrate reductase (Kara the family Enterobacteriaceae, has been reviewed recently et al 1987). In a brewery fermentation, nitrate arises from (Stewart 1993 ;Bonnefoy and DeMoss 1994). In this organtwo main sources : water (liquor) and hops.…”

Differential regulation by the homologous response regulators NarL and NarP of Escherichia coli K‐12 depends on DNA binding site arrangement

“…This nitrate and nitrite regulation of anaerobic gene expression is mediated by a dual two-component regulatory system. Homologous membrane-bound sensor proteins (NarX and NarQ; Williams and Stewart, 1997) monitor the availability of nitrate and nitrite and control the phosphorylation state of homologous DNA-binding response regulators (NarL and NarP; Rabin and Stewart, 1993; reviewed by . The phosphorylated response regulators then bind to target operon control regions and regulate their expression.…”