Role of the periplasmic domain of the Escherichia coli NarX sensor-transmitter protein in nitrate-dependent signal transduction and gene regulation

The narX, narQ and narL genes of Escherichia coli encode a nitrate-responsive two-component regulatory system that controls the expression of many anaerobic electron-transport- and fermentation-related genes. When nitrate is present, the NarX and NarQ sensor-transmitter proteins function to activate the response-regulator protein, NarL, which in turn binds to its DNA-recognition sites to modulate gene expression. The sensor-transmitter proteins are anchored in the cytoplasmic membrane by two transmembrane domains that are separated by a periplasmic region of ≈115 amino acids. In this study we report the isolation and characterization of narX* (star) mutants that constitutively activate nitrate reductase (narGHJI) gene expression and repress fumarate reductase (frdABCD) gene expression when no nitrate is provided for the cell. An additional narX mutant was identified that has lost its ability to respond to environmental signals. Each narX defect was caused by a single amino acid substitution within a conserved 17 amino acid sequence, called the ‘P-box’, in the periplasmic exposed region of the NarX protein. As a result, DNA binding is then ‘locked-on’ or ‘locked-off’ to give the observed pattern of gene expression. Diploid analysis of these narX mutants showed that a NarX P-box mutant which confered a ‘locked-on’ phenotype was trans dominant over wild-type NarX. Both were also trans dominant over the NarX P-box mutant which conferred a ‘locked-off’ phenotype. Certain narX P-box mutations, when combined with a narX‘linker’ region mutation, were recessive to the NarX linker mutation. Finally, a truncated form of the NarX protein that lacked the periplasmic and membrane regions also showed a ‘locked-on’ phenotype in vivo. Thus, the periplasmic and membrane domains are essential for signal transduction to NarL. From these findings, we propose that nitrate is detected in the periplasmic space of the cell, and that a signal-transduction event through the cytoplasmic membrane into the interior of the cell modulates the NarX-dependent phosphorylation/dephosphorylation of NarL.     

Signal-Dependent Phosphorylation of the Membrane-Bound NarX Two-Component Sensor-Transmitter Protein of Escherichia coli: Nitrate Elicits a Superior Anion Ligand Response Compared to Nitrite

The Nar two-component regulatory system, consisting of the dual sensor-transmitters NarX and NarQ and the dual response regulators NarL and NarP, controls the expression of various anaerobic respiratory pathway genes and fermentation pathway genes. Although both NarX and NarQ are known to detect the two environmental signals nitrate and nitrite, little is known regarding the sensitivity and selectivity of ligand for detection or activation of the sensor-transmitters. In this study, we have developed a sensitive anion-specific in vitro assay for NarX autophosphorylation by using Escherichia coli membranes highly enriched in the full-length NarX protein. In this ATP- and magnesium-dependent reaction, nitrate elicited a greater signal output (i.e., NarX autophosphorylation) than did nitrite. Nitrate stimulation occurred at concentrations as low as 5 microM, and the half-maximal level of NarX autophosphorylation occurred at approximately 35 microM nitrate. In contrast, nitrite-dependent stimulation was detected only at 500 microM, while 3.5 mM nitrite was needed to achieve half-maximal NarX autophosphorylation. Maximal nitrate- and nitrite-stimulated levels of NarX phosphorylation were five and two times, respectively, over the basal level of NarX autophosphorylation. The presence of Triton X-100 eliminated the nitrate-stimulated kinase activity and lowered the basal level of activity, suggesting that the membrane environment plays a crucial role in nitrate detection and/or regulation of kinase activity. These results provide in vitro evidence for the differential detection of dual signaling ligands by the NarX sensor-transmitter protein, which modulates the cytoplasmic NarX autokinase activity and phosphotransfer to NarL, the cognate response regulator.     

'Locked-on' and 'locked-off' signal transduction mutations in the periplasmic domain of the Escherichia coli NarQ and NarX sensors affect nitrate- and nitrite-dependent regulation by NarL and NarP

The Escherichia coli NarX, NarQ, NarL and NarP proteins comprise a two-component regulatory system that controls the expression of many anaerobic electron-transport and fermentation-related genes in response to nitrate and nitrite. Either of the two sensor-transmitter proteins, NarX and NarQ, can activate the response-regulator proteins, NarL and NarP, which in turn are able to bind at their respective DNA regulatory sites to modulate gene expression. NarX contains a conserved 17 amino acid sequence, designated the ‘P-box’ element, that is essential for nitrate sensing. In this study we characterize narQ mutants that also confer altered nitrate control of NarL-dependent nitrate reductase (narGHJI ) and fumarate reductase (frdABCD) gene expression. While some narQ mutations cause the constitutive activation or repression of reporter-gene expression even when the cells are grown in the absence of the nitrate signal (i.e. a ‘locked-on’ phenotype), other mutations abolish nitrate-dependent control (i.e. a ‘locked-off’ phenotype). Interestingly the narQ (A42→T) and narQ (R50→Q) mutations along with the analogous narX18 (A46→T) and narX902 (R54→E) mutations also confer a ‘locked-on’ or a ‘locked-off’ phenotype in response to nitrite, the second environmental signal detected by NarQ and NarX. Furthermore, these narQ and narX mutations also affect NarP-dependent gene regulation of nitrite reductase (nrfABCDEFG) and aeg-46.5 gene expression in response to nitrite. We therefore propose that the NarQ sensor-transmitter protein also detects nitrate and nitrite in the periplasmic space via its periplasmic domain. A signal transduction model, which we previously proposed for NarX, is now extended to NarQ, in which a nitrate- or nitrite-detection event in the periplasmic region of the cell is followed by a signal transduction event through the inner membrane to the cytoplasmic domain of NarQ and NarX proteins to modulate their protein kinase/phosphatase activities.     

Asymmetric cross‐regulation between the nitrate‐responsive NarX–NarL and NarQ–NarP two‐component regulatory systems from Escherichia coli K‐12

The NarX–NarL and NarQ–NarP sensor–response regulator pairs control Escherichia coli gene expression in response to nitrate and nitrite. Previous analysis suggests that the Nar two‐component systems form a cross‐regulation network in vivo. Here we report on the kinetics of phosphoryl transfer between different sensor–regulator combinations in vitro. NarX exhibited a noticeable kinetic preference for NarL over NarP, whereas NarQ exhibited a relatively slight kinetic preference for NarL. These findings were substantiated in reactions containing one sensor and both response regulators, or with two sensors and a single response regulator. We isolated 21 NarX mutants with missense substitutions in the cytoplasmic central and transmitter modules. These confer phenotypes that reflect defects in phospho‐NarL dephosphorylation. Five of these mutants, all with substitutions in the transmitter DHp domain, also exhibited NarP‐blind phenotypes. Phosphoryl transfer assays in vitro confirmed that these NarX mutants have defects in catalysing NarP phosphorylation. By contrast, the corresponding NarQ mutants conferred phenotypes indicating comparable interactions with both NarP and NarL. Our overall results reveal asymmetry in the Nar cross‐regulation network, such that NarQ interacts similarly with both response regulators, whereas NarX interacts preferentially with NarL.     

Mutationally altered signal output in the Nart (NarX-Tar) hybrid chemoreceptor

Signal-transducing proteins that span the cytoplasmic membrane transmit information about the environment to the interior of the cell. In bacteria, these signal transducers include sensor kinases, which typically control gene expression via response regulators, and methyl-accepting chemoreceptor proteins, which control flagellar rotation via the CheA kinase and CheY response regulator. We previously reported that a chimeric protein (Nart) that joins the ligand-binding, transmembrane, and linker regions of the NarX sensor kinase to the signaling and adaptation domains of the Tar chemoreceptor elicits a repellent response to nitrate and nitrite. As with NarX, nitrate evokes a stronger response than nitrite. Here we show that mutations targeting a highly conserved sequence (the P box) in the periplasmic domain alter chemoreception by Nart and signaling by NarX similarly. In particular, the G51R substitution converts Nart from a repellent receptor into an attractant receptor for nitrate. Our results underscore the conclusion that the fundamental mechanism of transmembrane signaling is conserved between homodimeric sensor kinases and chemoreceptors. They also highlight the plasticity of the coupling between ligand binding and signal output in these systems.     

Probing conservation of HAMP linker structure and signal transduction mechanism through analysis of hybrid sensor kinases

The HAMP linker, a predicted structural element observed in many sensor kinases and methyl-accepting chemotaxis proteins, transmits signals between sensory input modules and output modules. HAMP linkers are located immediately inside the cytoplasmic membrane and are predicted to form two short amphipathic alpha-helices (AS-1 and AS-2) joined by an unstructured connector. HAMP linkers are found in the Escherichia coli nitrate- and nitrite-responsive sensor kinases NarX and NarQ (which respond to ligand by increasing kinase activity) and the sensor kinase CpxA (which responds to ligand by decreasing kinase activity). We constructed a series of hybrids with fusion points throughout the HAMP linker, in which the sensory modules of NarX or NarQ are fused to the transmitter modules of NarX, NarQ, or CpxA. A hybrid of the NarX sensor module and the CpxA HAMP linker and transmitter module (NarX-CpxA-1) responded to nitrate by decreasing kinase activity, whereas a hybrid in which the HAMP linker of NarX was replaced by that of CpxA (NarX-CpxA-NarX-1) responded to nitrate by increasing kinase activity. However, sequence variations between HAMP linkers do not allow free exchange of HAMP linkers or their components. Certain deletions in the NarX HAMP linker resulted in characteristic abnormal responses to ligand; similar deletions in the NarQ and NarX-CpxA-1 HAMP linkers resulted in responses to ligand generally similar to those seen in NarX. We conclude that the structure and action of the HAMP linker are conserved and that the HAMP linker transmits a signal to the output domain that ligand is bound.     

A NarX-Tar chimera mediates repellent chemotaxis to nitrate and nitrite

Membrane receptors communicate between the external world and the cell interior. In bacteria, these receptors include the transmembrane sensor kinases, which control gene expression via their cognate response regulators, and chemoreceptors, which control the direction of flagellar rotation via the CheA kinase and CheY response regulator. Here, we show that a chimeric protein that joins the ligand-binding, transmembrane and linker domains of the NarX sensor kinase to the signalling and adaptation domains of the Tar chemoreceptor of Escherichia coli mediates repellent responses to nitrate and nitrite. Nitrate induces a stronger response than nitrite and is effective at lower concentrations, mirroring the relative sensitivity to these ligands exhibited by NarX itself. We conclude that the NarX-Tar hybrid functions as a bona fide chemoreceptor whose activity can be predicted from its component parts. This observation implies that ligand-dependent activation of a sensor kinase and repellent-initiated activation of receptor-coupled CheA kinase involve a similar transmembrane signal.     

Nitrate- and nitrite-sensing protein NarX of Escherichia coli K-12: mutational analysis of the amino-terminal tail and first transmembrane segment.

Nitrate and nitrite control of anaerobic respiratory gene expression is mediated by dual two-component regulatory systems. The sensors NarX and NarQ each communicate nitrate and nitrite availability to the response regulators NarL and NarP. In the presence of nitrate, the NarX protein acts as a positive regulator ("kinase") of both NarL and NarP activity. In the presence of nitrite, the NarX protein acts primarily as a negative regulator ("phosphatase") of NarL activity but remains a positive regulator of NarP activity. In other topologically similar sensory proteins, such as the methyl-accepting chemotaxis proteins, the transmembrane regions are important for signal transduction. We therefore used localized mutagenesis of the amino-terminal coding region to isolate mutations in narX that confer an altered signaling phenotype. Five of the mutations studied alter residues in the amino-terminal cytoplasmic tail, and five alter residues in the first transmembrane segment. Based on patterns of target operon expression in various regulatory mutant strain backgrounds, most of the mutant NarX proteins appear to have alterations in negative control function. One mutant, with a change of residue Leu-11 to Pro in the cytoplasmic tail, exhibits strikingly altered patterns of NarL- and NarP-dependent gene expression. We conclude that the amino terminus of the NarX protein is important for the differential response to nitrate and nitrite.     

Posttranslational regulation of nitrate assimilation in the cyanobacterium Synechocystis sp. strain PCC 6803

Posttranslational regulation of nitrate assimilation was studied in the cyanobacterium Synechocystis sp. strain PCC 6803. The ABC-type nitrate and nitrite bispecific transporter encoded by the nrtABCD genes was completely inhibited by ammonium as in Synechococcus elongatus strain PCC 7942. Nitrate reductase was insensitive to ammonium, while it is inhibited in the Synechococcus strain. Nitrite reductase was also insensitive to ammonium. The inhibition of nitrate and nitrite transport required the PII protein (glnB gene product) and the C-terminal domain of NrtC, one of the two ATP-binding subunits of the transporter, as in the Synechococcus strain. Mutants expressing the PII derivatives in which Ala or Glu is substituted for the conserved Ser49, which has been shown to be the phosphorylation site in the Synechococcus strain, showed ammonium-promoted inhibition of nitrate uptake like that of the wild-type strain. The S49A and S49E substitutions in GlnB did not affect the regulation of the nitrate and nitrite transporter in Synechococcus either. These results indicated that the presence or absence of negative electric charge at the 49th position does not affect the activity of the PII protein to regulate the cyanobacterial ABC-type nitrate and nitrite transporter according to the cellular nitrogen status. This finding suggested that the permanent inhibition of nitrate assimilation by an S49A derivative of PII, as was previously reported for Synechococcus elongatus strain PCC 7942, is likely to have resulted from inhibition of nitrate reductase rather than the nitrate and nitrite transporter.     

Nitrate- and molybdenum-independent signal transduction mutations in narX that alter regulation of anaerobic respiratory genes in Escherichia coli.

Escherichia coli can respire anaerobically by reducing nitrate, trimethylamine-N-oxide, dimethyl sulfoxide, or fumarate. When nitrate is present, expression of the genes for fumarate (frdABCD), trimethylamine-N-oxide, and dimethyl sulfoxide (dmsABC) is repressed while expression of the nitrate reductase (narGHJI) gene is induced. This regulation requires molybdate and is mediated by the narX and narL gene products, which together form a two-component regulatory system. We provide evidence that NarX is a nitrate and molybdenum sensor which activates NarL when nitrate is available to cells. Mutants generated by hydroxylamine mutagenesis were repressed for frdA-lacZ expression even when cells were grown in the absence of nitrate. The mutations responsible for three of these nitrate independence (NarX*) phenotypes were localized to narX and further characterized in vivo for their ability to repress frdA-lacZ expression. Two of the mutants (the narX64 and narX71 mutants) had a greatly reduced requirement for molybdenum to function but still responded to nitrate. In contrast, a third mutant (the narX32 mutant) required molybdenum but did not exhibit full repression of frdA-lacZ expression even when nitrate was present. These narX* alleles also caused the induction of nitrate reductase gene expression and the repression of a dmsA-lacZ fusion in the absence of nitrate. Each narX* mutation was determined to lie in an 11-amino-acid region of the NarX polypeptide that follows a proposed transmembrane domain. We suggest that the conformation of the narX* gene products is altered such that even in the absence of nitrate each of these gene products more closely resembles the wild-type NarX protein when nitrate is present. These data establish a clear role for the narX gene product in gene regulation and strongly suggest its role in sensing nitrate and molybdenum.     

Nitrate regulation of anaerobic respiratory gene expression in narX deletion mutants of Escherichia coli K-12.

Previous studies have shown that narL+ is required for nitrate regulation of anaerobic respiratory enzyme synthesis, including formate dehydrogenase-N, nitrate reductase, and fumarate reductase. Insertions in the closely linked narX gene decrease, but do not abolish, nitrate regulation of anaerobic enzyme synthesis. Analysis of sequence similarities suggests that NarX and NarL comprise a two-component regulatory pair. We constructed lacZ operon and gene fusions to investigate the operon structure of narXL. We found evidence for a complex operon with at least two promoters; PXL-narX-PL-narL. We also investigated the role of NarX in nitrate regulation of anaerobic respiratory enzyme synthesis by constructing nonpolar loss of function narX alleles. These deletions were studied on narL+ lambda specialized transducing bacteriophage. The narX deletions had no effect on nitrate regulation in delta (narXL) strains. This finding suggest that the subtle effects of previously studied narX insertions are due to decreased expression of narL and that narX+ is not essential for normal nitrate regulation. The role of NarX in nitrate regulation remains to be determined.     

The effect of Glc6P uptake and its subsequent oxidation within pea root plastids on nitrite reduction and glutamate synthesis

In roots, nitrate assimilation is dependent upon a supply of reductant that is initially generated by oxidative metabolism including the pentose phosphate pathway (OPPP). The uptake of nitrite into the plastids and its subsequent reduction by nitrite reductase (NiR) and glutamate synthase (GOGAT) are potentially important control points that may affect nitrate assimilation. To support the operation of the OPPP there is a need for glucose 6-phosphate (Glc6P) to be imported into the plastids by the glucose phosphate translocator (GPT). Competitive inhibitors of Glc6P uptake had little impact on the rate of Glc6P-dependent nitrite reduction. Nitrite uptake into plastids, using (13)N labelled nitrite, was shown to be by passive diffusion. Flux through the OPPP during nitrite reduction and glutamate synthesis in purified plastids was followed by monitoring the release of (14)CO(2) from [1-(14)C]-Glc6P. The results suggest that the flux through the OPPP is maximal when NiR operates at maximal capacity and could not respond further to the increased demand for reductant caused by the concurrent operation of NiR and GOGAT. Simultaneous nitrite reduction and glutamate synthesis resulted in decreased rates of both enzymatic reactions. The enzyme activity of glucose 6-phosphate dehydrogenase (G6PDH), the enzyme supporting the first step of the OPPP, was induced by external nitrate supply. The maximum catalytic activity of G6PDH was determined to be more than sufficient to support the reductant requirements of both NiR and GOGAT. These data are discussed in terms of competition between NiR and GOGAT for the provision of reductant generated by the OPPP.