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.     

Discrimination between structurally related ligands nitrate and nitrite controls autokinase activity of the NarX transmembrane signal transducer of Escherichia coli K-12

Anaerobic respiratory gene expression in Escherichia coli is differentially controlled by nitrate and nitrite through dual interacting two-component regulatory systems. The NarX sensor is one of two membrane-spanning sensor kinases that control the phosphorylation state of two DNA-binding response regulators. We have studied NarX autophosphorylation in crude membrane preparations from cells that overexpress NarX protein. The low basal autophosphorylation rate was stimulated about sixfold and threefold by nitrate and nitrite respectively. This demonstrates that nitrate and nitrite differentially activate NarX autokinase activity. We also isolated single-residue substitutions in NarX that affect its ability to respond to or discriminate between nitrate and nitrite. Most of these substitutions affect residues within the conserved P-box sequence in the periplasmic domain. We characterized several of the mutants in vivo , by monitoring ligand-regulated gene expression, and in vitro , by monitoring ligand-responsive autophosphorylation. At least one change, K49I (Lys at position 49 changed to Ile), resulted in a protein with greatly impaired ability to discriminate between nitrate and nitrite. Other changes (H45E and R59K) resulted in proteins that responded normally to nitrate but were unable to respond to nitrite. These results implicate the P-box region in discrimination between subtly different small molecules.     

'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.     

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.     

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.     

Nitrate-dependent activation of the Dif signaling pathway of Myxococcus xanthus mediated by a NarX-DifA interspecies chimera

Myxococcus xanthus fibril exopolysaccharide (EPS), essential for the social gliding motility and development of this bacterium, is regulated by the Dif chemotaxis-like pathway. DifA, an MCP homolog, is proposed to mediate signal input to the Dif pathway. However, DifA lacks a prominent periplasmic domain, which in classical chemoreceptors is responsible for signal perception and for initiating transmembrane signaling. To investigate the signaling properties of DifA, we constructed a NarX-DifA (NafA) chimera from the sensory module of Escherichia coli NarX and the signaling module of M. xanthus DifA. We report here the first functional chimeric signal transducer constructed using genes from organisms in two different phylogenetic subdivisions. When expressed in M. xanthus, NafA restored fruiting body formation, EPS production, and S-motility to difA mutants in the presence of nitrate. Studies with various double mutants indicate that NafA requires the downstream Dif proteins to function. We propose that signal inputs to the Dif pathway and transmembrane signaling by DifA are essential for the regulation of EPS production in M. xanthus. Despite the apparent structural differences, DifA appears to share similar transmembrane signaling mechanisms with enteric sensor kinases and chemoreceptors.     

Chemotaxis mediated by NarX-FrzCD chimeras and nonadapting repellent responses in Myxococcus xanthus

Myxococcus xanthus requires gliding motility for swarming and fruiting body formation. It uses the Frz chemosensory pathway to regulate cell reversals. FrzCD is a cytoplasmic chemoreceptor required for sensing effectors for this pathway. NarX is a transmembrane sensor for nitrate from Escherichia coli. In this study, two NarX-FrzCD chimeras were constructed to investigate M. xanthus chemotaxis: NazD(F) contains the N-terminal sensory module of NarX fused to the C-terminal signalling domain of FrzCD; NazD(R) is similar except that it contains a G51R mutation in the NarX domain known to reverse the signalling output of a NarX-Tar chimera to nitrate. We report that while nitrate had no effect on the wild type, it decreased the reversal frequency of M. xanthus expressing NazD(F) and increased that of M. xanthus expressing NazD(R). These results show that directional motility in M. xanthus can be regulated independently of cellular metabolism and physiology. Surprisingly, the NazD(R) strain failed to adapt to nitrate in temporal assays as did the wild type to known repellents. The lack of temporal adaptation to negative stimuli appears to be a general feature in M. xanthus chemotaxis. Thus, the appearance of biased movements by M. xanthus in repellent gradients is likely due to the inhibition of net translocation by repellents.     

Mutational analysis of a conserved signal-transducing element: the HAMP linker of the Escherichia coli nitrate sensor NarX

The HAMP linker, a predicted structural element observed in sensor proteins from all domains of life, is proposed to transmit signals between extracellular sensory input domains and cytoplasmic output domains. HAMP (histidine kinase, adenylyl cyclase, methyl-accepting chemotaxis protein, and phosphatase) linkers are located just inside the cytoplasmic membrane and are projected to form two short amphipathic alpha-helices (AS-1 and AS-2) joined by an unstructured connector. The presumed helices are comprised of hydrophobic residues in heptad repeats, with only three positions exhibiting strong conservation. We generated missense mutations at these three positions and throughout the HAMP linker in the Escherichia coli nitrate sensor kinase NarX and screened the resulting mutants for defective responses to nitrate. Most missense mutations in this region resulted in a constitutive phenotype mimicking the ligand-bound state, and only one residue (a conserved Glu before AS-2) was essential for HAMP linker function. We also scanned the narX HAMP linker with an overlapping set of seven-residue deletions. Deletions in AS-1 and the connector resulted in constitutive phenotypes. Two deletions in AS-2 resulted in a novel reversed response phenotype in which the response to ligand was the opposite of that seen for the narX(+) strain. These observations are consistent with the proposed HAMP linker structure, show that the HAMP linker plays an active role in transmembrane signal transduction, and indicate that the two amphipathic alpha-helices have different roles in signal transduction.     

Identification of a chemoreceptor zinc-binding domain common to cytoplasmic bacterial chemoreceptors

We report the identification and characterization of a previously unidentified protein domain found in bacterial chemoreceptors and other bacterial signal transduction proteins. This domain contains a motif of three noncontiguous histidines and one cysteine, arranged as Hxx[WFYL]x(21-28)Cx[LFMVI]Gx[WFLVI]x(18-27)HxxxH(boldface type indicates residues that are nearly 100% conserved). This domain was first identified in the soluble Helicobacter pylori chemoreceptor TlpD. Using inductively coupled plasma mass spectrometry on heterologously and natively expressed TlpD, we determined that this domain binds zinc with a subfemtomolar dissociation constant. We thus named the domain CZB, for chemoreceptor zinc binding. Further analysis showed that many bacterial signaling proteins contain the CZB domain, most commonly proteins that participate in chemotaxis but also those that participate in c-di-GMP signaling and nitrate/nitrite sensing, among others. Proteins bearing the CZB domain are found in several bacterial phyla. The variety of signaling proteins using the CZB domain suggests that it plays a critical role in several signal transduction pathways.     

Signal transduction in bacterial chemotaxis

Motile bacteria respond to environmental cues to move to more favorable locations. The components of the chemotaxis signal transduction systems that mediate these responses are highly conserved among prokaryotes including both eubacterial and archael species. The best-studied system is that found in Escherichia coli. Attractant and repellant chemicals are sensed through their interactions with transmembrane chemoreceptor proteins that are localized in multimeric assemblies at one or both cell poles together with a histidine protein kinase, CheA, an SH3-like adaptor protein, CheW, and a phosphoprotein phosphatase, CheZ. These multimeric protein assemblies act to control the level of phosphorylation of a response regulator, CheY, which dictates flagellar motion. Bacterial chemotaxis is one of the most-understood signal transduction systems, and many biochemical and structural details of this system have been elucidated. This is an exciting field of study because the depth of knowledge now allows the detailed molecular mechanisms of transmembrane signaling and signal processing to be investigated.     

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.     

Stimulus perception in bacterial signal-transducing histidine kinases.

Two-component signal-transducing systems are ubiquitously distributed communication interfaces in bacteria. They consist of a histidine kinase that senses a specific environmental stimulus and a cognate response regulator that mediates the cellular response, mostly through differential expression of target genes. Histidine kinases are typically transmembrane proteins harboring at least two domains: an input (or sensor) domain and a cytoplasmic transmitter (or kinase) domain. They can be identified and classified by virtue of their conserved cytoplasmic kinase domains. In contrast, the sensor domains are highly variable, reflecting the plethora of different signals and modes of sensing. In order to gain insight into the mechanisms of stimulus perception by bacterial histidine kinases, we here survey sensor domain architecture and topology within the bacterial membrane, functional aspects related to this topology, and sequence and phylogenetic conservation. Based on these criteria, three groups of histidine kinases can be differentiated. (i) Periplasmic-sensing histidine kinases detect their stimuli (often small solutes) through an extracellular input domain. (ii) Histidine kinases with sensing mechanisms linked to the transmembrane regions detect stimuli (usually membrane-associated stimuli, such as ionic strength, osmolarity, turgor, or functional state of the cell envelope) via their membrane-spanning segments and sometimes via additional short extracellular loops. (iii) Cytoplasmic-sensing histidine kinases (either membrane anchored or soluble) detect cellular or diffusible signals reporting the metabolic or developmental state of the cell. This review provides an overview of mechanisms of stimulus perception for members of all three groups of bacterial signal-transducing histidine kinases.     

Structure of the Sensor Domain of Mycobacterium tuberculosis PknH Receptor Kinase Reveals a Conserved Binding Cleft

Since their discovery over 20 years ago, eukaryotic-like transmembrane receptor Ser/Thr protein kinases (STPKs) have been shown to play critical roles in the virulence, growth, persistence, and reactivation of many bacteria. Information regarding the signals transmitted by these proteins, however, remains scarce. To enhance understanding of the basis for STPK receptor signaling, we determined the 1.7-Å-resolution crystal structure of the extracellular sensor domain of the Mycobacterium tuberculosis receptor STPK, PknH (Rv1266c). The PknH sensor domain adopts an unanticipated fold containing two intramolecular disulfide bonds and a large hydrophobic and polar cleft. The residues lining the cleft and those surrounding the disulfide bonds are conserved. These results suggest that PknH binds a small-molecule ligand that signals by changing the location or quaternary structure of the kinase domain.     

Genetic and Biochemical Dissection of a HisKA Domain Identifies Residues Required Exclusively for Kinase and Phosphatase Activities

Two-component signal transduction systems, composed of histidine kinases (HK) and response regulators (RR), allow bacteria to respond to diverse environmental stimuli. The HK can control both phosphorylation and subsequent dephosphorylation of its cognate RR. The majority of HKs utilize the HisKA subfamily of dimerization and histidine phosphotransfer (DHp) domains, which contain the phospho-accepting histidine and directly contact the RR. Extensive genetics, biochemistry, and structural biology on several prototypical TCS systems including NtrB-NtrC and EnvZ-OmpR have provided a solid basis for understanding the function of HK–RR signaling. Recently, work on NarX, a HisKA_3 subfamily protein, indicated that two residues in the highly conserved region of the DHp domain are responsible for phosphatase activity. In this study we have carried out both genetic and biochemical analyses on Myxococcus xanthus CrdS, a member of the HisKA subfamily of bacterial HKs. CrdS is required for the regulation of spore formation in response to environmental stress. Following alanine-scanning mutagenesis of the α1 helix of the DHp domain of CrdS, we determined the role for each mutant protein for both kinase and phosphatase activity. Our results indicate that the conserved acidic residue (E372) immediately adjacent to the site of autophosphorylation (H371) is specifically required for kinase activity but not for phosphatase activity. Conversely, we found that the conserved Thr/Asn residue (N375) was required for phosphatase activity but not for kinase activity. We extended our biochemical analyses to two CrdS homologs from M. xanthus, HK1190 and HK4262, as well as Thermotoga maritima HK853. The results were similar for each HisKA family protein where the conserved acidic residue is required for kinase activity while the conserved Thr/Asn residue is required for phosphatase activity. These data are consistent with conserved mechanisms for kinase and phosphatase activities in the broadly occurring HisKA family of sensor kinases in bacteria.     

Sensor domain of the Mycobacterium tuberculosis receptor Ser/Thr protein kinase, PknD, forms a highly symmetric beta propeller

Diverse pathogenic bacteria produce transmembrane receptor Ser/Thr protein kinases (STPKs), but little is known about the signals mediated by these "eukaryotic-like" proteins. To explore the basis for signaling in the bacterial STPK receptor family, we determined the structure of the sensor domain of Mycobacterium tuberculosis PknD. In two crystal forms, the PknD sensor domain forms a rigid, six-bladed beta-propeller with a flexible tether to the transmembrane domain. The PknD sensor domain is the most symmetric beta-propeller structure described. All residues that vary most among the blade subdomains cluster in the large "cup" motif, analogous to the ligand-binding surface in many beta-propeller proteins. These results suggest that PknD binds a multivalent ligand that signals by changing the quaternary structure of the intracellular kinase domain.