Two-component signal transduction mediates a wide range of phenotypes in microbes and plants. The sensor transmitter module controls the phosphorylation state of the cognate-responseregulator receiver domain. Whereas the two-component autokinase and phosphotransfer reactions are well-understood, the mechanism by which sensors accelerate the rate of phosphoresponse regulator dephosphorylation, termed "transmitter phosphatase activity," is unknown. We identified a conserved DxxxQ motif adjacent to the phospho-accepting His residue in the HisKA_3 subfamily of two-component sensors. We used site-specific mutagenesis to make substitutions for these conserved Gln and Asp residues in the nitrate-responsive NarX sensor and analyzed function both in vivo and in vitro. Results show that the Gln residue is critical for transmitter phosphatase activity, but is not essential for autokinase or phosphotransfer activities. The documented role of an amide moiety in phosphoryl group hydrolysis suggests an analogous catalytic function for this Gln residue in HisKA_3 members. Results also indicate that the Asp residue is important for both autokinase and transmitter phosphatase activities. Furthermore, we noted that sensors of the HisKA subfamily exhibit an analogous E/DxxT/N motif, the conserved Thr residue of which is critical for transmitter phosphatase activity of the EnvZ sensor. Thus, twocomponent sensors likely use similar mechanisms for receiver domain dephosphorylation.histidine kinase | DHp domain | DesK

SummaryThe 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 phosphoNarL 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.

NarX-NarL and NarQ-NarP are paralogous two-component regulatory systems that control Escherichia coli gene expression in response to the respiratory oxidants nitrate and nitrite. Nitrate stimulates the autophosphorylation rates of the NarX and NarQ sensors, which then phosphorylate the response regulators NarL and NarP to activate and repress target operon transcription. Here, we investigated both the autophosphorylation and dephosphorylation of soluble sensors in which the maltose binding protein (MBP) has replaced the amino-terminal transmembrane sensory domain. The apparent affinities (K m ) for ADP were similar for both proteins, about 2 M, whereas the affinity of MBP-NarQ for ATP was lower, about 23 M. At a saturating concentration of ATP, the rate constant of MBP-NarX autophosphorylation (about 0.5 ؋ 10 ؊4 s ؊1 ) was lower than that observed for MBP-NarQ (about 2.2 ؋ 10 ؊4 s ؊1 ). At a saturating concentration of ADP, the rate constant of dephosphorylation was higher than that of autophosphorylation, about 0.03 s ؊1 for MBP-NarX and about 0.01 s ؊1 for MBP-NarQ. For other studied sensors, the published affinities for ADP range from about 16 M (KinA) to about 40 M (NtrB). This suggests that only a small proportion of NarX and NarQ remain phosphorylated in the absence of nitrate, resulting in efficient response regulator dephosphorylation by the remaining unphosphorylated sensors.

Two transmembrane proteins were tentatively classified as NarK1 and NarK2 in the Pseudomonas genome project and hypothesized to play an important physiological role in nitrate/nitrite transport in Pseudomonas aeruginosa. The narK1 and narK2 genes are located in a cluster along with the structural genes for the nitrate reductase complex. Our studies indicate that the transcription of all these genes is initiated from a single promoter and that the gene complex narK1K2GHJI constitutes an operon. Utilizing an isogenic narK1 mutant, a narK2 mutant, and a narK1K2 double mutant, we explored their effect on growth under denitrifying conditions. While the ⌬narK1::Gm mutant was only slightly affected in its ability to grow under denitrification conditions, both the ⌬narK2::Gm and ⌬narK1K2::Gm mutants were found to be severely restricted in nitratedependent, anaerobic growth. All three strains demonstrated wild-type levels of nitrate reductase activity. Nitrate uptake by whole-cell suspensions demonstrated both the ⌬narK2::Gm and ⌬narK1K2::Gm mutants to have very low yet different nitrate uptake rates, while the ⌬narK1::Gm mutant exhibited wild-type levels of nitrate uptake. Finally, Escherichia coli narK rescued both the ⌬narK2::Gm and ⌬narK1K2::Gm mutants with respect to anaerobic respiratory growth. Our results indicate that only the NarK2 protein is required as a nitrate/nitrite transporter by Pseudomonas aeruginosa under denitrifying conditions. Denitrification involves four separate nitrogen oxide reductases and ultimately reduces nitrate to dinitrogen (37). Respiratory nitrate reductase, which is the first enzyme in this denitrification pathway, has its active site on the cytoplasmic side of the membrane (23). The enzyme substrate, nitrate, is an ion and cannot be taken up by the simple process of passive diffusion (18). Both of these factors require the bacterium to synthesize a transport protein(s) to carry nitrate into the cytoplasm, where the reduction of nitrate to nitrite takes place. It has been demonstrated for Pseudomonas aeruginosa, Pseudomonas stutzeri, and Escherichia coli (7,11,24) that the product of nitrate respiration, i.e., nitrite, is immediately excreted to the external environment, presumably protecting the organism from potential toxic effects. These toxic effects are due to the ability of this anion to bind to the heme groups in electron carriers, thereby inhibiting the flow of electrons (25). Genetic and physiological data suggest that nitrate transport in some bacteria occurs through two different uptake systems. Thus, for the process of nitrate assimilation, ABC transporters as well as secondary transporters are postulated to be used. On the other hand, anaerobically, for the purpose of nitrate respiration, it is postulated that bacteria rely solely on secondary transporters (18).Originally, John (14) demonstrated that membrane permeabilization of the cells significantly enhanced nitrate uptake, suggesting the need for a transport protein specific for nitrate. This was corroborated by sever...

SUMMARY Negative control in two-component signal transduction results from sensor transmitter phosphatase activity for phospho-receiver dephosphorylation. A hypothetical mechanism for this reaction involves a catalytic residue in the H-box active site region. However, a complete understanding of transmitter phosphatase regulation is hampered by the abundance of kinase-competent, phosphatase-defective missense substitutions (K+ P− phenotype) outside of the active site region. For the Escherichia coli NarX sensor, a model for the HisKA_3 sequence family, DHp domain K+ P− mutants defined two classes. Interaction mutants mapped to the active site-distal base of the DHp helix 1, whereas conformation mutants were affected in the X-box region of helix 2. Thus, different types of perturbations can influence transmitter phosphatase activity indirectly. By comparison, K+ P− substitutions in the HisKA sensors EnvZ and NtrB additionally map to a third region, at the active site-proximal top of the DHp helix 1, independently identified as important for DHp-CA domain interaction in this sensor class. Moreover, the NarX transmitter phosphatase activity was independent of nucleotides, in contrast to the activity for many HisKA family sensors. Therefore, distinctions involving both the DHp and CA domains suggest functional diversity in the regulation of HisKA and HisKA_3 transmitter phosphatase activities.

The requirement for the mobA gene in key assimilatory and respiratory nitrogen metabolism of Pseudomonas aeruginosa PAO1 was investigated by mutational analysis of PA3030 (mobA; MoCo guanylating enzyme), PA1779 (nasA; assimilatory nitrate reductase), and PA3875 (narG; respiratory nitrate reductase). The mobA mutant was deficient in both assimilatory and respiratory nitrate reductase activities, whereas xanthine dehydrogenase activity remained unaffected. Thus, P. aeruginosa requires both the molybdopterin (MPT) and molybdopterin guanine dinucleotide (MGD) forms of the molybdenum cofactor for a complete spectrum of nitrogen metabolism, and one form cannot substitute for the other. Regulation studies using a Phi(PA3030-lacZGm) reporter strain suggest that expression of mobA is not influenced by the type of nitrogen source or by anaerobiosis, whereas assimilatory nitrate reductase activity was detected only in the presence of nitrate.

In two-component signal transduction, a sensor protein transmitter module controls cognate receiver domain phosphorylation. Most receiver domain sequences contain a small residue (Gly or Ala) at position T ؉ 1 just distal to the essential Thr or Ser residue that forms part of the active site. However, some members of the NarL receiver subfamily have a large hydrophobic residue at position T ؉ 1. Our laboratory previously isolated a NarL mutant in which the T ؉ 1 residue Val-88 was replaced with an orthodox small Ala. This NarL V88A mutant confers a striking phenotype in which high-level target operon expression is both signal (nitrate) and sensor (NarX and NarQ) independent. This suggests that the NarL V88A protein is phosphorylated by cross talk from noncognate sources. Although cross talk was enhanced in ackA null strains that accumulate acetyl phosphate, it persisted in pta ackA double null strains that cannot synthesize this compound and was observed also in narL ؉ strains. This indicates that acetate metabolism has complex roles in mediating NarL cross talk. Contrariwise, cross talk was sharply diminished in an arcB barA double null strain, suggesting that the encoded sensors contribute substantially to NarL V88A cross talk. Separately, the V88A substitution altered the in vitro rates of NarL autodephosphorylation and transmitter-stimulated dephosphorylation and decreased affinity for the cognate sensor, NarX. Together, these experiments show that the residue at position T ؉ 1 can strongly influence two distinct aspects of receiver domain function, the autodephosphorylation rate and cross talk inhibition. IMPORTANCEMany bacterial species contain a dozen or more discrete sensor-response regulator two-component systems that convert a specific input into a distinct output pattern. Cross talk, the unwanted transfer of signals between circuits, occurs when a response regulator is phosphorylated inappropriately from a noncognate source. Cross talk is inhibited in part by the high interaction specificity between cognate sensor-response regulator pairs. This study shows that a relatively subtle missense change from Val to Ala nullifies cross talk inhibition, enabling at least two noncognate sensors to enforce an inappropriate output independently of the relevant input.T wo-component signal transduction determines numerous phenotypes in microorganisms. The sensor transmitter module, in response to the input signal, governs phosphorylation of the response regulator receiver domain to control the output (1). Although conserved in structure and sequence, receiver domains nevertheless catalyze autophosphorylation and autodephosphorylation over a broad range of rates (2) and interact specifically with their cognate transmitter modules (3).Receiver domains, approximately 120 residues, share a topology in which a central five-stranded parallel ␤-sheet is enveloped by five ␣-helices. The active site for phosphorylation and dephosphorylation includes five critical residues, as exemplified by the intensively studied C...

In this study, oxygen and nitrate regulation of transcription and subsequent protein expression of the unique narK1K2GHJI respiratory operon of Pseudomonas aeruginosa were investigated. Under the control of P LAC , P. aeruginosa was able to transcribe nar and subsequently express methyl viologen-linked nitrate reductase activity under aerobic conditions without nitrate. Modulation of P LAC through the LacI repressor enabled us to assess both transcriptional and posttranslational regulation by oxygen during physiological whole-cell nitrate reduction.Pseudomonas aeruginosa is a ubiquitous gram-negative bacterium capable of growth and/or survival anaerobically through arginine catabolism (36), pyruvate fermentation (6), or denitrification in the presence of nitrogen oxides (37). The latter process allows this organism to persist in soil as part of the global nitrogen cycle. Additionally, denitrification has been implicated in infections by this opportunistic pathogen in the airways of cystic fibrosis patients (9,18,35).During anaerobic growth of Escherichia coli, the Fnr protein is responsible for activation of the synthesis of anaerobic respiratory enzymes such as nitrate reductase (28,31). In addition, the presence of external nitrate induces the transcription of the nitrate reductase operon through the dual twocomponent regulatory systems of narX-narL (31, 32) and narQnarP (3,21,22). Parallel studies of P. aeruginosa have resulted in the characterization of a unique nar operon (24, 27) regulated by the proteins Anr and Dnr (40) as well as narX and narL (24). However, a narQ homologue has not been identified (30,34).Posttranslationally, oxygen also has the capacity to inhibit denitrification immediately at the level of nitrate uptake and nitrite excretion (10,11,33) as well as through the diversion of electron flow to oxygen in E. coli and in Paracoccus denitrificans (4, 33). Despite these studies, an experimental method for the measurement of posttranslational regulation by oxygen has been lacking.In the present study, the effects of oxygen and nitrate on the expression of the narK1K2GHJI operon (27) were examined during aerobic or anaerobic growth with and without nitrate. In addition, a P LAC element was inserted upstream of the respiratory nitrate reductase genes (narK2GHJI) of P. aeruginosa to overcome transcriptional regulation of the nar operon by oxygen and nitrate. The levels of transcription, respiratory nitrate reductase activity, and whole-cell physiological reduction of nitrate to nitrite were measured under both aerobic and anaerobic conditions, thus allowing quantitative assessment of posttranslational regulation by oxygen.The bacterial strains and plasmids used in this study are listed in Table 1. All bacteria were grown at 37°C from singlecolony isolates or overnight cultures in Luria-Bertani (LB) broth (Fisher Scientific, Pittsburgh, PA). The medium was supplemented with 1% (wt/vol) KNO 3 (LB-NO 3 ) when indicated. Aerobic cultures were set up as 50-ml volumes of LB or LB-NO 3 in a 500-ml Erlenm...