The Myxococcus xanthus asgA gene encodes a novel signal transduction protein required for multicellular development.

The Myxococcus xanthus asgA gene is one of three known genes necessary for the production of extracellular A-signal, a cell density signal required early in fruiting body development. We determined the DNA sequence of asgA. The deduced 385-amino-acid sequence of AsgA was found to contain two domains: one homologous to the receiver domain of response regulators and the other homologous to the transmitter domain of histidine protein kinases. A kanamycin resistance (Kmr) gene was inserted at various positions within or near the asgA gene to determine the null phenotype. Those strains with the Kmr gene inserted upstream or downstream of asgA are able to form fruiting bodies, while strains containing the Kmr gene inserted within asgA fail to develop. The nature and location of the asgA476 mutation were determined. This mutation causes a leucine-to-proline substitution within a conserved stretch of hydrophobic residues in the N-terminal receiver domain. Cells containing the insertion within asgA and cells containing the asgA476 substitution have similar phenotypes with respect to development, colony color, and expression of an asg-dependent gene. An analysis of expression of a translational asgA-lacZ fusion confirms that asgA is expressed during growth and early development. Finally, we propose that AsgA functions within a signal transduction pathway that is required to sense starvation and to respond with the production of extracellular A-signal.     

Four signalling domains in the hybrid histidine protein kinase RodK of Myxococcus xanthus are required for activity

In prokaryotes, the principal signal transduction systems operating at the level of protein phosphorylation are the two-component systems. A number of hybrid histidine protein kinases in these systems contain several receiver domains, however, the function of these receiver domains is unknown. The RodK kinase in Myxococcus xanthus has an unconventional domain composition with a putative N-terminal sensor domain followed by a histidine kinase domain and three receiver domains. RodK is essential for the spatial coupling of the two morphogenetic events underlying fruiting body formation in M. xanthus, aggregation of cells into nascent fruiting bodies and the subsequent sporulation of these cells. RodK kinase activity is indispensable for RodK activity. By systematically substituting the conserved, phosphorylatable aspartate residues in the three receiver domains, genetic evidence is provided that each receiver domain is important for RodK function and that each receiver domain has a distinct function, which depends on phosphorylation. Biochemical analyses provided indirect evidence for phosphotransfer from the RodK kinase domain to the third receiver domain. This is the first example of a hybrid histidine protein kinase in which four signalling domains have been shown to be required for full activity.     

Intra- and interprotein phosphorylation between two-hybrid histidine kinases controls Myxococcus xanthus developmental progression

Histidine-aspartate phosphorelay signaling systems are used to couple stimuli to cellular responses. A hallmark feature is the highly modular signal transmission modules that can form both simple "two-component" systems and sophisticated multicomponent systems that integrate stimuli over time and space to generate coordinated and fine-tuned responses. The deltaproteobacterium Myxococcus xanthus contains a large repertoire of signaling proteins, many of which regulate its multicellular developmental program. Here, we assign an orphan hybrid histidine protein kinase, EspC, to the Esp signaling system that negatively regulates progression through the M. xanthus developmental program. The Esp signal system consists of the hybrid histidine protein kinase, EspA, two serine/threonine protein kinases, and a putative transport protein. We demonstrate that EspC is an essential component of this system because ΔespA, ΔespC, and ΔespA ΔespC double mutants share an identical developmental phenotype. Neither substitution of the phosphoaccepting histidine residue nor deletion of the entire catalytic ATPase domain in EspC produces an in vivo mutant developmental phenotype. In contrast, substitution of the receiver phosphoaccepting residue yields the null phenotype. Although the EspC histidine kinase can efficiently autophosphorylate in vitro, it does not act as a phosphodonor to its own receiver domain. Our in vitro and in vivo analyses suggest the phosphodonor is instead the EspA histidine kinase. We propose EspA and EspC participate in a novel hybrid histidine protein kinase signaling mechanism involving both inter- and intraprotein phosphotransfer. The output of this signaling system appears to be the combined phosphorylated state of the EspA and EspC receiver modules. This system regulates the proteolytic turnover of MrpC, an important regulator of the developmental program.     

Specificity of the BvgAS and EvgAS phosphorelay is mediated by the C-terminal HPt domains of the sensor proteins

Despite the presence of highly conserved signalling modules, significant cross-communication between different two-component systems has only rarely been observed. Domain swapping and the characterization of liberated signalling modules enabled us to characterize in vitro the protein domains that mediate specificity and are responsible for the high fidelity in the phosphorelay of the unorthodox Bvg and Evg two-component systems. Under equimolar conditions, significant in vitro phosphorylation of purified BvgA and EvgA proteins was only obtained by their histidine kinases, BvgS and EvgS respectively. One hybrid histidine kinase consisting of the BvgS transmitter and HPt domains and of the EvgS receiver domain (BvgS-TO-EvgS-R) was able to phosphorylate BvgA but not EvgA. In contrast, the hybrid protein consisting of the BvgS transmitter and the EvgS receiver and HPt domains (BvgS-T-EvgS-RO) was unable to phosphorylate BvgA but efficiently phosphorylated EvgA. These results demonstrate that the C-terminal HPt domains of the sensor proteins endow the unorthodox two-component systems with a high specificity for the corresponding regulator protein. In the case of the response regulators, the receiver but not the output domains contribute to the specific interaction with the histidine kinases, because a hybrid protein consisting of the EvgA receiver and the BvgA output domain could only be phosphorylated by the EvgS protein.     

A new class of signal transducer in His-Asp phosphorelay systems

Nitrate transport activity of the LtnT permease of the cyanobacterium Synechococcus elongatus is activated when LtnA, a response regulator without an effector domain, is phosphorylated by LtnB, a hybrid histidine kinase. We identified a protein (LtnC) that is required for activation of LtnT. LtnC consists of an N-terminal histidine-containing phosphoacceptor (HisKA) domain, a receiver domain, and a unique C-terminal domain found in some cyanobacterial proteins. Because LtnC lacks an ATP-binding kinase domain of a histidine kinase, it is incapable of autophosphorylation, but LtnC is phosphorylated by LtnA. The histidine residue in the HisKA domain but not the aspartate residue in the receiver domain is essential for phosphorylation of LtnC and activation of LtnT. LtnC phosphorylation leads to oligomerization of the protein. Fusion of the C-terminal domain of LtnC to glutathione S-transferase, which forms oligomers, also activates LtnT, suggesting that oligomerization of the LtnC C-terminal domain causes LtnT activation. These results indicate that the C-terminal domain of LtnC acts as an effector domain that directs the output of the signal from the phosphorelay system. The two-step (His-Asp-His) phosphorelay system, composed of the LtnB, LtnA, and LtnC proteins, is distinct from the known phosphorelay systems, namely, the typical two-component system (His-Asp) and the multistep phosphorelay system (His-Asp-His-Asp), because the HisKA domain of LtnC is the terminal phosphoacceptor that determines the signal output. LtnC is a new class of signal transducer in His-Asp phosphorelay systems that contains a HisKA domain and an effector domain.     

The receiver domain of FrzE, a CheA-CheY fusion protein, regulates the CheA histidine kinase activity and downstream signalling to the A- and S-motility systems of Myxococcus xanthus

The Frz chemosensory system is a two-component signal transduction pathway that controls cell reversals and directional movements for the two motility systems in Myxococcus xanthus. To trigger cell reversals, FrzE, a hybrid CheA-CheY fusion protein, autophosphorylates the kinase domain at His-49, and phosphoryl groups are transferred to aspartate residues (Asp-52 and Asp-220) in the two receiver domains of FrzZ, a dual CheY-like protein that serves as the pathway output. The role of the receiver domain of FrzE was unknown. In this paper, we characterize the FrzE protein in vitro and show that the receiver domain of FrzE negatively regulates the autophosphorylation activity of the kinase domain of FrzE. Unexpectedly, it does not appear to play a direct role in phospho-relay as in most other histidine kinase receiver domain hybrid systems. The regulatory role of the FrzE receiver domain suggests that it may interact with or be phosphorylated by an unknown protein. We also show the dynamics of motility system-specific marker proteins in FrzE mutants as cells move forward and reverse. Our studies indicate that the two motility systems are functionally co-ordinated and that any system-specific branching of the pathway most likely occurs downstream of FrzE.     

Conserved sequence motifs in the unorthodox BvgS two-component sensor protein ofBordetella pertussis

The unorthodox two-component sensor protein BvgS ofBordetella pertussis contains several interesting sequence motifs of unknown functional relevance, such as a histidine motif in its output domain, which is conserved among several unorthodox sensor proteins, a putative nucleotide binding site [Walker box type A] in its linker region, and a region in its periplasmic domain with significant homology to the TonB protein ofEscherichia coli. We investigated potential functions of these sequences by constructingB. pertussis strains that express mutant BvgS derivatives. The His₁₁₇₂ residue in the output domain was exchanged for Gln, and the Walker motif was mutated either by the replacement of Lys₆₂₅ by Arg, or of Gly₆₂₄ by Val and Lys₆₂₅ by Leu. To analyse the TonB motif, the periplasmic domain of BvgS was replaced with the corresponding domain of EvgS, anE. coli sensor that is highly homologous to BvgS but lacks the similarity with TonB. All mutations except the conservative Lys/Arg exchange in the Walker box caused the inactivation of BvgS, indicating the functional importance of the conserved motifs. The activity of the mutant proteins could be restored by complementation in trans with various separately expressed, truncated parts of BvgS. Mutations in the BvgS receiver domain could be complemented not only by a construct expressing the wild-type receiver and output domains, but also by the derivative containing the His-Gln exchange. Therefore, the histidine motif, although important for BvgS function, is not essential for complementation of BvgS mutants. The mutations in the Walker box and in the periplasmic domain could be complemented by a truncated BvgS derivative lacking the receiver and output domains. The characterization of a spontaneous revertant of the strain expressing the originally inactive EvgS/BvgS hybrid protein revealed the presence of a mutation in the BvgS linker region, conferring constitutive activity on the protein. As TonB energizes transport processes across the outer membrane ofE. coli, the strain expressing the constitutive EvgS/BvgS hybrid protein lacking the TonB motif was used in preliminary investigations of a possible direct involvement of BvgS in transport processes.     

Conserved sequence motifs in the unorthodox BvgS two-component sensor protein ofBordetella pertussis

The unorthodox two-component sensor protein BvgS ofBordetella pertussis contains several interesting sequence motifs of unknown functional relevance, such as a histidine motif in its output domain, which is conserved among several unorthodox sensor proteins, a putative nucleotide binding site [Walker box type A] in its linker region, and a region in its periplasmic domain with significant homology to the TonB protein ofEscherichia coli. We investigated potential functions of these sequences by constructingB. pertussis strains that express mutant BvgS derivatives. The His₁₁₇₂ residue in the output domain was exchanged for Gln, and the Walker motif was mutated either by the replacement of Lys₆₂₅ by Arg, or of Gly₆₂₄ by Val and Lys₆₂₅ by Leu. To analyse the TonB motif, the periplasmic domain of BvgS was replaced with the corresponding domain of EvgS, anE. coli sensor that is highly homologous to BvgS but lacks the similarity with TonB. All mutations except the conservative Lys/Arg exchange in the Walker box caused the inactivation of BvgS, indicating the functional importance of the conserved motifs. The activity of the mutant proteins could be restored by complementation in trans with various separately expressed, truncated parts of BvgS. Mutations in the BvgS receiver domain could be complemented not only by a construct expressing the wild-type receiver and output domains, but also by the derivative containing the His-Gln exchange. Therefore, the histidine motif, although important for BvgS function, is not essential for complementation of BvgS mutants. The mutations in the Walker box and in the periplasmic domain could be complemented by a truncated BvgS derivative lacking the receiver and output domains. The characterization of a spontaneous revertant of the strain expressing the originally inactive EvgS/BvgS hybrid protein revealed the presence of a mutation in the BvgS linker region, conferring constitutive activity on the protein. As TonB energizes transport processes across the outer membrane ofE. coli, the strain expressing the constitutive EvgS/BvgS hybrid protein lacking the TonB motif was used in preliminary investigations of a possible direct involvement of BvgS in transport processes.     

Biochemical properties of CikA,an unusual phytochrome-like histidine protein kinase that resets the circadian clock in Synechococcus elongatus PCC 7942

We recently described the cikA (circadian input kinase A) gene, whose product supplies environmental information to the circadian oscillator in the cyanobacterium Synechococcus elongatus PCC 7942. CikA possesses three distinct domains: a GAF, a histidine protein kinase (HPK), and a receiver domain similar to those of the response regulator family. To determine how CikA functions in providing circadian input, we constructed modified alleles to tag and truncate the protein, allowing analysis of each domain individually. CikA covalently bound bilin chromophores in vitro, even though it lacks the expected ligand residues, and the GAF domain influenced but did not entirely account for this function. Full-length CikA and truncated variants that carry the HPK domain showed autophosphorylation activity. Deletion of the GAF domain or the N-terminal region adjacent to GAF dramatically reduced autophosphorylation, whereas elimination of the receiver domain increased activity 10-fold. Assays to test phosphorelay from the HPK to the cryptic receiver domain, which lacks the conserved aspartyl residue that serves as a phosphoryl acceptor in response regulators, were negative. We propose that the cryptic receiver is a regulatory domain that interacts with an unknown protein partner to modulate the autokinase activity of CikA but does not work as bona fide receiver domain in a phosphorelay.     

Coupling of multicellular morphogenesis and cellular differentiation by an unusual hybrid histidine protein kinase in Myxococcus xanthus

We describe an unusual hybrid histidine protein kinase, which is important for spatially coupling cell aggregation and sporulation during fruiting body formation in Myxococcus xanthus. A rodK mutant makes abnormal fruiting bodies and spores develop outside the fruiting bodies. RodK is a soluble, cytoplasmic protein, which contains an N-terminal sensor domain, a histidine protein kinase domain and three receiver domains. In vitro phosphorylation assays showed that RodK possesses kinase activity. Kinase activity is essential for RodK function in vivo. RodK is present in vegetative cells and remains present until the late aggregation stage, after which the level decreases in a manner that depends on the intercellular A-signal. Genetic evidence suggests that RodK may regulate multiple temporally separated events during fruiting body formation including stimulation of early developmental gene expression, inhibition of A-signal production and inhibition of the intercellular C-signal transduction pathway. We speculate that RodK undergoes a change in activity during development, which is reflected in changes in phosphotransfer to the receiver domains.     

Myxococcus xanthus mokA encodes a histidine kinase-response regulator hybrid sensor required for development and osmotic tolerance

A gene, mokA, encoding a protein with similarities to histidine kinase-response regulator hybrid sensor, was cloned from a Myxococcus xanthus genomic library. The predicted mokA gene product was found to contain three domains: an amino-terminal input domain, a central transmitter domain, and a carboxy-terminal receiver domain. mokA mutants placed under starvation conditions exhibited reduced sporulation. Mutation of mokA also caused marked growth retardation at high osmolarity. These results indicated that M. xanthus MokA is likely a transmembrane sensor that is required for development and osmotic tolerance. The putative function of MokA is similar to that of the hybrid histidine kinase, DokA, of the eukaryotic slime mold Dictyostelium discoideum.     

Structural Model of the Cytosolic Domain of the Plant Ethylene Receptor 1 (ETR1)

Ethylene initiates important aspects of plant growth and development through disulfide-linked receptor dimers located in the endoplasmic reticulum. The receptors feature a small transmembrane, ethylene binding domain followed by a large cytosolic domain, which serves as a scaffold for the assembly of large molecular weight complexes of different ethylene receptors and other cellular participants of the ethylene signaling pathway. Here we report the crystallographic structures of the ethylene receptor 1 (ETR1) catalytic ATP-binding and the ethylene response sensor 1 dimerization histidine phosphotransfer (DHp) domains and the solution structure of the entire cytosolic domain of ETR1, all from Arabidopsis thaliana. The isolated dimeric ethylene response sensor 1 DHp domain is asymmetric, the result of different helical bending angles close to the conserved His residue. The structures of the catalytic ATP-binding, DHp, and receiver domains of ethylene receptors and of a homologous, but dissimilar, GAF domain were refined against experimental small angle x-ray scattering data, leading to a structural model of the entire cytosolic domain of the ethylene receptor 1. The model illustrates that the cytosolic domain is shaped like a dumbbell and that the receiver domain is flexible and assumes a position different from those observed in prokaryotic histidine kinases. Furthermore the cytosolic domain of ETR1 plays a key role, interacting with all other receptors and several participants of the ethylene signaling pathway. Our model, therefore, provides the first step toward a detailed understanding of the molecular mechanics of this important signal transduction process in plants.     

Role of the Two Structural Domains from the Periplasmic Escherichia coli Histidine-binding Protein HisJ

Escherichia coli HisJ is a type II periplasmic binding protein that functions to reversibly capture histidine and transfer it to its cognate inner membrane ABC permease. Here, we used NMR spectroscopy to determine the structure of apo-HisJ (26.5 kDa) in solution. HisJ is a bilobal protein in which domain 1 (D1) is made up of two noncontiguous subdomains, and domain 2 (D2) is expressed as the inner domain. To better understand the roles of D1 and D2, we have isolated and characterized each domain separately. Structurally, D1 closely resembles its homologous domain in apo- and holo-HisJ, whereas D2 is more similar to the holo-form. NMR relaxation experiments reveal that HisJ becomes more ordered upon ligand binding, whereas isolated D2 experiences a significant reduction in slower (millisecond to microsecond) motions compared with the homologous domain in apo-HisJ. NMR titrations reveal that D1 is able to bind histidine in a similar manner as full-length HisJ, albeit with lower affinity. Unexpectedly, isolated D1 and D2 do not interact with each other in the presence or absence of histidine, which indicates the importance of intact interdomain-connecting elements (i.e. hinge regions) for HisJ functioning. Our results shed light on the binding mechanism of type II periplasmic binding proteins where ligand is initially bound by D1, and D2 plays a supporting role in this dynamic process.     

Spatial expression and characterization of a putative ethylene receptor protein NTHK1 in tobacco

A putative ethylene receptor gene NTHK1 encodes a protein with a putative signal peptide, three transmembrane segments, a putative histidine kinase domain and a putative receiver domain. The receiver domain was expressed in an Escherichia coli expression system, purified and used to generate polyclonal antibodies for immunohistochemistry analysis. The spatial expression of the NTHK1 protein was then investigated. We found that NTHK1 was abundant during flower and ovule development. It was also expressed in glandular hairs, stem, and in leaves that had been wounded. The NTHK1 gene was further introduced into the tobacco plant and we found that, in different transgenic lines, the NTHK1 gene was transcribed to various degrees. Upon ACC treatment, the etiolated transgenic seedlings showed reduced ethylene sensitivity when compared with the control, indicating that NTHK1 is a functional ethylene receptor in plants.     

Mammalian PASKIN, a PAS-serine/threonine kinase related to bacterial oxygen sensors.

The PAS domain is a versatile protein fold found in many archaeal, bacterial, and plant proteins capable of sensing environmental changes in light intensity, oxygen concentration, and redox potentials. The oxygen sensor FixL from Rhizobium species contains a heme-bearing PAS domain and a histidine kinase domain that couples sensing to signaling. We identified a novel mammalian PAS protein (PASKIN) containing a domain architecture resembling FixL. PASKIN is encoded by an evolutionarily conserved single-copy gene which is ubiquitously expressed. The human PASKIN and mouse Paskin genes show a conserved intron-exon structure and share their promoter regions with another ubiquitously expressed gene that encodes a regulator of protein phosphatase-1. The 144-kDa PASKIN protein contains a PAS region homologous to the FixL PAS domain and a serine/threonine kinase domain which might be involved in signaling. Thus, PASKIN is likely to function as a mammalian PAS sensor protein.     

Latent nitrate transport activity of a novel sulfate permease-like protein of the cyanobacterium Synechococcus elongatus

The Synechococcus elongatus mutant lacking the nrtABCD gene cluster (NA3) is defective in active nitrate transport and requires high nitrate concentrations (>30 mm) for sustained growth. Prolonged incubation of NA3 in medium containing 2 mm nitrate led to isolation of a pseudorevertant (NA3R) capable of transport of millimolar concentrations of nitrate, from which three mutants with improved affinity for nitrate were obtained. We identified three genes responsible for the latent transport activity for nitrate: ltnA, which encodes a response regulator with no effector domain; ltnB, which encodes a hybrid histidine kinase with two receiver domains; and ltnT, which encodes a sulfate permease-like protein with a putative cyclic nucleoside monophosphate (cNMP)-binding domain. Missense mutations of the high affinity derivatives of NA3R were found in ltnT, verifying that LtnT acts as the transporter. Overexpression of truncated LtnT lacking the cNMP-binding domain (but not full-length LtnT) conferred nitrate transport activity on NA3, suggesting that the cNMP-binding domain inhibits transport under normal conditions. A nonsense mutation in ltnB that resulted in elimination of the receiver domains of the encoded protein was responsible for expression of nitrate transport activity in NA3R. Expression of LtnB derivatives lacking the receiver domains also conferred low affinity nitrate transport activity on NA3. The phosphoryl group of the histidine kinase domain of LtnB was transferred to Asp(52) of LtnA in vitro. Overexpression of LtnA (but not LtnA(D52E)) led to manifestation of the latent nitrate transport activity in NA3, indicating involvement of phosphorylated LtnA in activation of the novel transporter.     

The RdeA-RegA system, a eukaryotic phospho-relay controlling cAMP breakdown.

The regA and rdeA gene products of Dictyostelium are involved in the regulation of cAMP signaling. The response regulator, RegA, is composed of an N-terminal receiver domain linked to a C-terminal cAMP-phosphodiesterase domain. RdeA may be a phospho-transfer protein that supplies phosphates to RegA. We show genetically that phospho-RegA is the activated form of the enzyme in vivo, in that the predicted site of aspartate phosphorylation is required for full activity. We show biochemically that RdeA and RegA communicate, as evidenced by phospho-transfer between the two proteins in vitro. Phospho-transfer is dependent on the presumed phospho-accepting amino acids, histidine 65 of RdeA and aspartate 212 of RegA, and occurs in both directions. Phosphorylation of RegA by a heterologous phospho-donor protein activates RegA phosphodiesterase activity at least 20-fold. Our results suggest that the histidine phosphotransfer protein, RdeA, and the response regulator, RegA, constitute two essential elements in a eukaryotic His-Asp phospho-relay network that regulates Dictyostelium development and fruiting body maturation.