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...

ABSTRACT

The nitrate reductase operon (narGHJI) of Escherichia cohl encodes an anaerobic respiratory enzyme. Previous work has identified two cis-acting sites in the nar operon control region: a proximal site required for anaerobic induction mediated by the activator Fnr and a remote upstream site required for nitrate induction mediated by the activator NarL [Li, S. & DeMoss, J. A. (1988) J. Biol. Chem. 263, 13700-137051. Our search for nar regulatory mutants yielded one strain with a mutation in himD, the structural gene for one of the subunits of integration host factor (IHF). Strains carrying null alleles of the IHF structural genes, himD and himA, had severe defects in nitrate induction of the nar operon but were normal for nitrate induction of the coordinately regulated fdn operon. Anaerobic expression of both operons was normal in him mutants. Gel-mobility-shift and DNase I protection experiments revealed a single I1F binding site in the nar operon control region, located midway between the upstream activation site and the promoter. We conclude that an IHFmediated DNA bend is essential for efficient nitrate induction of the a70-dependent nar operon promoter. This requirement of IHF for transcriptional activation had been noted for several o-'-dependent promoters.Analysis of prokaryotic promoters has revealed two classes of sites important for transcriptional activation: proximal sites, which are located within 65 nucleotides upstream ofthe transcription initiation site, and remote sites, which are located further upstream (1). Several examples of remote activation sites have been found in conjunction with -54_ dependent promoters. Well-documented examples include promoters that are activated by NtrC in Salmonella typhimurium and Escherichia coli (glnA) and NifA in Klebsiella pneumoniae (nifI) (2,3). The NtrC protein, bound to its DNA sites "140 and "110 base pairs upstream of the transcription initiation site (4), interacts with o-54-containing RNA polymerase at the promoter via a DNA loop (5,6) to catalyze open complex formation (2). Formation of an analogous loop in the nifH regulatory region requires an accessory protein, integration host factor (IHF; ref. 7). IHF, a heterodimer encoded by the himA and himD (hip) genes, is a sequence-specific DNA-binding protein involved in bending DNA (8). IHF mediates the formation of specific bends that bring the upstream-bound regulatory protein into proper proximity to RNA polymerase and enhances transcriptional activation by NifA at the nifH promoter (7) and by NtrC at the glnH promoter (9).Operons for E. coli formate dehydrogenase-N (fdnGHI) and nitrate reductase (narGHJI), enzymes involved in anaerobic respiration, are subject to dual positive regulation (10, 11). Anaerobic expression of these operons requires the activator protein Fnr, which interacts with proximal sites -42 base pairs upstream of the transcription initiation site in each operon (12, 13). Available evidence suggests that the nar operon promoter requires oa70 (12), and it is established that it d...

In wild-type Azorhizobium caulinodans ORS571, nicotinate served both as anabolic substrate for NAD+ production and as catabolic substrate for use as the N source. Catabolic enzyme activities were greatest from cultures grown with nicotinate as the N source and least when cultures were grown with ammonium as the N source. Vector insertion mutants unable to catabolize nicotinate (nic::Vi mutants) still required micromolar quantities of this compound for growth. Therefore, A. caulinodans wild type is NAD+ auxotrophic. As the first two intermediates in A. caulinodans nicotinate catabolism, two cyclic compounds, 6-hydroxynicotinate and 1,4,5,6-tetrahydro-6-oxonicotinate, were identified. These compounds were purified from the growth medium of strain 61009 (a nic::Vi mutant) by high-performance liquid chromatography; their identities were subsequently confirmed by UV absorbance, nuclear magnetic resonance, and mass spectra. The conversion of 1 mol of nicotinate to 6-hydroxynicotinate consumed 0.5 mol of 02. From 180 isotopic incorporation experiments, water was the hydroxyl-equivalent source. A nicotinate hydroxylase activity proved to be cell wall-membrane associated; this activity served as direct electron donor (not indirect via NADP+) to 02 via membrane electron transport. These catabolic reactions have not previously been witnessed together in the same organism. A. caulinodans nicotinate catabolism seems coupled to N2 fixation, although the explicit mechanism of this coupling remains to be determined.

No abstract