Characterization of NikA Histidine Kinase and Two Response Regulators with Special Reference to Osmotic Adaptation and Asexual Development in Aspergillus nidulans

Aspergillus nidulans has many histidine-to-aspartate (His-Asp) phosphorelay components, including 15 histidine kinases (HKs), four response regulators (RRs), and a histidine-containing phosphotransfer intermediate (HPt). Of these, NikA (HK) is highly conserved in many filamentous fungi. It has been found that NikA is responsible for the responses of filamentous fungi to fungicides such as iprodione and fludioxonil. Two RRs, SskA and SrrA, are also involved in the fungicide response, providing a typical example of the His-Asp phosphorelay system, in which NikA functions as a sensor upstream of SskA and SrrA in response to fungicides. To gain further insight into the physiological roles of the NikA-SskA/SrrA phosphorelay system, we constructed a pair of ΔnikAΔsskA and ΔnikAΔsrrA double mutants. Here we provide evidence regarding the crucial involvement of the NikA-SskA/SrrA phosphorelay system in both osmotic adaptation and asexual development, including conidia formation. Based on these results, a general insight into the A. nidulans His-Asp phosphorelay network is also discussed.     

An Escherichia coli protein that exhibits phosphohistidine phosphatase activity towards the HPt domain of the ArcB sensor involved in the multistep His-Asp phosphorelay

The Escherichia coli sensory kinase, ArcB, possesses a histidine-containing phosphotransfer (HPt) domain, which is implicated in the His-Asp multistep phosphorelay. We searched for a presumed phosphohistidine phosphatase, if present, which affects the function of the HPt domain through its dephosphorylation activity. Using in vivo screening, we first identified a gene that appeared to interfere with the His-Asp phosphorelay between the HPt domain and the receiver domain of OmpR, provided that the gene product was expressed through a multicopy plasmid. The cloned gene (named sixA ) was found to encode a protein consisting of 161 amino acids, which has a noticeable sequence motif, an arginine–histidine–glycine (RHG) signature, at its N-terminus. Such an RHG signature, which presumably functions as a nucleophilic phosphoacceptor, was previously found in a set of divergent enzymes, including eukaryotic fructose-2,6-bisphosphatase, E. coli periplasmic phosphatase and E. coli glucose-1-phosphate phosphatase, and ubiquitous phosphoglycerate mutase. Otherwise, the entire amino acid sequences of none of these enzymes resembles that of SixA. It was demonstrated in vitro that the purified SixA protein exhibited the ability to release the phosphoryl group from the HPt domain of ArcB, but the mutant protein lacking the crucial histidine residue in the RHG signature did not. Evidence was also provided that a deletion mutation of sixA on the chromosome affected the in vivo phosphotransfer signalling. These results support the view that SixA is capable of functioning as a phosphohistidine phosphatase that may be implicated in the His-Asp phosphorelay through regulating the phosphorylation state of the HPt domain.     

Response regulators SrrA and SskA are central components of a phosphorelay system involved in stress signal transduction and asexual sporulation in Aspergillus nidulans

Among eukaryotes, only slime molds, fungi, and plants contain signal transduction phosphorelay systems. In filamentous fungi, multiple sensor kinases appear to use a single histidine-containing phosphotransfer (HPt) protein to relay signals to two response regulators (RR). In Aspergillus nidulans, the RR SskA mediates activation of the mitogen-activated protein kinase SakA in response to osmotic and oxidative stress, whereas the functions of the RR SrrA were unknown. We used a genetic approach to characterize the srrA gene as a new member of the skn7/prr1 family and to analyze the roles of SrrA in the phosphorelay system composed of the RR SskA, the HPt protein YpdA, and the sensor kinase NikA. While mutants lacking the HPt protein YpdA are unviable, mutants lacking SskA (DeltasskA), SrrA (DeltasrrA), or both RR (DeltasrrA DeltasskA) are viable and differentially affected in osmotic and oxidative stress responses. Both RR are involved in osmostress resistance, but DeltasskA mutants are more sensitive to this stress, and only SrrA is required for H(2)O(2) resistance and H(2)O(2)-mediated induction of catalase CatB. In contrast, both RR are individually required for fungicide sensitivity and calcofluor resistance and for normal sporulation and conidiospore viability. The DeltasrrA and DeltasskA sporulation defects appear to be related to decreased mRNA levels of the key sporulation gene brlA. In contrast, conidiospore viability defects do not correlate with the activity of the spore-specific catalase CatA. Our results support a model in which NikA acts upstream of SrrA and SskA to transmit fungicide signals and to regulate asexual sporulation and conidiospore viability. In contrast, NikA appears dispensable for osmotic and oxidative stress signaling. These results highlight important differences in stress signal transmission among fungi and define a phosphorelay system involved in oxidative and osmotic stress, cell wall maintenance, fungicide sensitivity, asexual reproduction, and spore viability.     

Roles of the His-Asp phosphorelay signal transduction system in controlling cell growth and development in Aspergillus nidulans

The His-Asp phosphorelay signal transduction system has been identified in most organisms, including bacteria, yeasts, fungi, and plants, except for animals. This system is important in adaptation to stress, control of cell growth, and induction of development in response to environmental changes. On the basis of genomic information, it has been found that Aspergillus nidulans, a model species of fungi, includes 15 histidine kinases (HKs), one histidine-containing phosphotransmitter protein (HPt), and four response regulators (RRs) as factors related to the signal transduction system. In this review, it is explain that the His-Asp phosphorelay system is important in controlling cell growth (responses to fungicides, the induction of asexual and sexual development, and so on) under different growth conditions with reference to A. nidulans.     

The SskA and SrrA response regulators are implicated in oxidative stress responses of hyphae and asexual spores in the phosphorelay signaling network of Aspergillus nidulans

Histidine-to-Aspartate (His-Asp) phosphorelay (or two-component) systems are common signal transduction mechanisms implicated in a wide variety of cellular responses to environmental stimuli in both prokaryotes and eukaryotes. For a model filamentous fungi, Aspergillus nidulans, in this study we first compiled a complete list of His-Asp phosphorelay components, including 15 genes for His-kinase (HK), four genes for response regulator (RR), and only one for histidine-containing phosphotransfer intermediate (HPt). For these RR genes, a set of deletion mutants was constructed so as to create a null allele for each. When examined these mutant strains under various conditions stressful for hyphal growth and asexual spore development, two of them (designated DeltasskA and DeltasrrA) showed a marked phenotype of hypersensitivity to oxidative stresses (particularly, to hydrogen peroxide). In this respect, expression of the vegetative-stage specific catB catalase gene was severely impaired in both mutants. Furthermore, conidia from DeltasskA were hypersensitive not only to treatment with H(2)O(2), but also to treatment at aberrantly low (4 degrees C) and high (50 degrees C) temperatures, resulting in reduced germination efficiency. In this respect, not only the catA catalase gene specific for asexual development, but also a set of genes encoding the enzymes for synthesis of certain stress tolerant compatible solutes, such as trehalose and glycerol, were markedly downregulated in conidia from DeltasskA. These results together are indicative of the physiological importance of the His-Asp phosphorelay signaling network involving the SskA and SrrA response regulators.     

Compilation and characterization of Arabidopsis thaliana response regulators implicated in His-Asp phosphorelay signal transduction.

His-Asp phosphorelays are evolutionary-conserved powerful biological tactics for intracellular signal transduction. Such a phosphorelay is generally made up of "sensor histidine (His)-kinases", "response regulators", and "histidine-containing (HPt) phosphotransmitters". In the higher plant, Arabidopsis thaliana, results from recent intensive studies suggested that His-Asp phosphorelays may be widely used for propagating environmental stimuli, such as phytohormones (e.g., ethylene and cytokinin). In this study, we first inspected extensively the occurrence of Arabidopsis response regulators in order to compile and characterize them. The results showed that this higher plant has, at least, 14 members of the family of response regulators that can be classified into two distinct subtypes (type-A and type-B), as judged from their structural designs, biochemical properties, and expression profiles. Comparative studies were conducted for each representative (ARR3 and ARR4 for type-A, and ARR10 for type-B). It was suggested that expression of the type-A response regulator is cytokinin-inducible, while that of the type-B response regulator appears to be not. Results from yeast two-hybrid analyses suggested that the type-B response regulator may have an ability to stably interact with a set of HPt phosphotransmitters (AHPs). These and other results will be discussed with special reference to the His-Asp phosphorelay signaling network in Arabidopsis thaliana.     

Characterization of the NikA histidine kinase implicated in the phosphorelay signal transduction of Aspergillus nidulans, with special reference to fungicide responses

We recently compiled a complete list of phosphorelay signal transduction components in the model filamentous fungus Aspergillus nidulans. In this study, we characterized a histidine protein kinase (designated NikA) that is found in many fungi, with special reference to responses to potent fungicides (iprodione and fludioxonil). We provided evidence that not only NikA, but also two downstream response regulators (SskA and SrrA) are crucially implicated in the mode of action of these fungicides, and also that the further downstream HogA-MAPK cascade is exaggerated abnormally (or ectopically) in hyphae by the fungicides in a manner dependent on the NikA-SskA phosphorelay.     

Biochemical characterization of a putative cytokinin-responsive His-kinase, CKI1, from Arabidopsis thaliana

His-Asp phosphorelays are evolutionary-conserved powerful biological tactics for intracellular signal transduction. Such a phosphorelay is generally made up of "sensor histidine (His)-kinases", "response regulators", and "histidine-containing (HPt) phosphotransmitters". Results from recent intensive studies suggested that in the higher plant Arabidopsis thaliana, His-Asp phosphorelays may be widely used for propagating environmental stimuli, such as phytohormones (e.g., ethylene and cytokinin). In this study, we characterized, in vitro, the putative cytokinin-responsive CKI1 His-kinase, in terms of His-Asp phosphorelays. It was demonstrated for the first time that the receiver domain in this sensor exhibits a strong phosphohistidine phosphatase activity toward some Arabidopsis HPt phosphotransmitters (AHP1 and AHP2), suggesting the functional importance of the receiver domain for a resumed interaction of the sensor His-kinase with other His-Asp phosphorelay components.     

A novel device of bacterial signal transducers.

The osmoregulatory expression of ompC and ompF in Escherichia coli is mediated by a pair of bacterial signal transduction proteins, EnvZ (sensory kinase) and OmpR (response regulator). We isolated previously multicopy suppressors which can complement a defect in the phosphotransfer signal transduction caused by an envZ deletion mutation. Among such suppressors, arcB and barA are of particular interest because these gene products are unique in the sense that they contain both an autophosphorylated histidine site (or transmitter module) and a phospho-accepting aspartate site (or receiver module) in their primary amino acid sequences. Here we report that ArcB and BarA possess in the C-terminal region a phosphorylated histidine site which has never been noticed, in addition to the authentic one identified previously. This newly identified histidine in ArcB and BarA was demonstrated to play a crucial role in the observed multicopy suppression. Furthermore, it was demonstrated in vivo and in vitro for ArcB that the C-terminal domain containing the histidine can function as an alternative phosphodonor (or transmitter). This novel type of sensory kinase was therefore revealed to contain two independent phosphodonor sites, together with a phospho-accepting site. These findings suggest that this unique feature of ArcB and BarA, in terms of the signaling modules, make it possible for these sensory kinases to function as dual-signaling transducers.     

Domain Analysis of ArcS,the Hybrid Sensor Kinase of the Shewanella oneidensis MR-1 Arc Two-Component System,Reveals Functional Differentiation of Its Two Receiver Domains

In all species of the genus Shewanella, the redox-sensing Arc two-component system consists of the response regulator ArcA, the sensor kinase ArcS, and the separate phosphotransfer protein HptA. Compared to its counterpart ArcB in Escherichia coli, ArcS has a significantly different domain structure. Resequencing and reannotation revealed that in the N-terminal part, ArcS possesses a periplasmic CaChe-sensing domain bracketed by two transmembrane domains and, moreover, that ArcS has two cytoplasmic PAS-sensing domains and two receiver domains, compared to a single one of each in ArcB. Here, we used a combination of in vitro phosphotransfer studies on purified proteins and phenotypic in vivo mutant analysis to determine the roles of the different domains in ArcS function. The analysis revealed that phosphotransfer occurs from and toward the response regulator ArcA and involves mainly the C-terminal RecII domain. However, RecI also can receive a phosphate from HptA. In addition, the PAS-II domain, located upstream of the histidine kinase domain, is crucial for function. The results support a model in which phosphorylation of RecI stimulates histidine kinase activity of ArcS in order to maintain an appropriate level of phosphorylated ArcA according to environmental conditions. In addition, the study reveals some fundamental mechanistic differences between ArcS/HptA and ArcB with respect to signal perception and phosphotransfer despite functional conservation of the Arc system in Shewanella and E. coli.     

GFP-tagged expression analysis revealed that some histidine kinases of Aspergillus nidulans show temporally and spatially different expression during the life cycle

His-Asp phosphorelays are signal transduction mechanisms widely found in both prokaryotes and eukaryotes. The phosphorelay comprises three types of signal transducers: a sensor with histidine kinase (HK), a response regulator containing a phospho-accepting receiver (RR), and a histidine-containing phosphotransmitter (HPt). In this study, we examined HK expression using a green fluorescent protein (GFP) reporter driven by HK promoters in Aspergillus nidulans. All the transformants showed fluorescence derived from GFP in a submerged culture, although some of them were very weak, indicating that all the promoters were active. As judged by the fluorescence of transformants grown on a culture plate on which sexual development was induced, promoters of fphA, hk-8-2, and hk-8-5 preferentially functioned in conidial heads, the promoter of phkA preferentially functioned in cleistothecia, and the promoters of tcsB and nikA function in both conidial heads and cleistothecia. These results indicate that at least some HKs of A. nidulans showed temporally and spatially different expression during the cell cycle.     

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 core dimerization domains of histidine kinases contain recognition specificity for the cognate response regulator

Histidine kinases DivJ and PleC initiate signal transduction pathways that regulate an early cell division cycle step and the gain of motility later in the Caulobacter crescentus cell cycle, respectively. The essential single-domain response regulator DivK functions downstream of these kinases to catalyze phosphotransfer from DivJ and PleC. We have used a yeast two-hybrid screen to investigate the molecular basis of DivJ and PleC interaction with DivK and to identify other His-Asp signal transduction proteins that interact with DivK. The only His-Asp proteins identified in the two-hybrid screen were five members of the histidine kinase superfamily. The finding that most of the kinase clones isolated correspond to either DivJ or PleC supports the previous conclusion that DivJ and PleC are cognate DivK kinases. A 66-amino-acid sequence common to all cloned DivJ and PleC fragments contains the conserved helix 1, helix 2 sequence that forms a four-helix bundle in histidine kinases required for dimerization, autophosphorylation and phosphotransfer. We present results that indicate that the four-helix bundle subdomain is not only necessary for binding of the response regulator but also sufficient for in vivo recognition specificity between DivK and its cognate histidine kinases. The other three kinases identified in this study correspond to DivL, an essential tyrosine kinase belonging to the same kinase subfamily as DivJ and PleC, and the two previously uncharacterized, soluble histidine kinases CckN and CckO. We discuss the significance of these results as they relate to kinase response regulator recognition specificity and the fidelity of phosphotransfer in signal transduction pathways.     

Genome-wide comparison of the His-to-Asp phosphorelay signaling components of three symbiotic genera of Rhizobia.

Histidine-to-aspartate (His-Asp) phosphorelay (or two-component) systems are very common signal transduction mechanisms that are implicated in a wide variety of cellular responses to environmental stimuli. The His-Asp phosphorelay components include "sensor histidine kinase (HK)", "phosphotransfer intermediate (HPt)", and "response regulator (RR)". With special reference to three bacterial species (Mesorhizobium loti, Bradyrhizobium japonicum, Sinorhizobium meliloti), each of which belongs to a different genera of Rhizobia, here we attempted to compile all of the His-Asp phosphorelay components in order to reveal a comparative genome-wide overview as to the His-Asp phosphorelay. It was revealed that M. loti has 47 HKs, 1 HPts, and 58 RRs; B. japonicum has 80 HKs, 3 HPts, and 91 RRs; whereas S. meliloti has 40 HKs, 1 HPt, and 58 RRs. These His-Asp phosphorelay components were extensively compiled and characterized. The resulting overview as to the His-Asp phosphorelay of Rhizobia will provide us with a basis for understanding of the fundamental mechanisms underlying interactions between plants and microorganisms (including symbiosis), as well as nitrogen fixation.     

His-Asp phosphorelay signaling: a communication avenue between plants and their environment

His-Asp phosphorelay systems have been recently discovered in plants and have emerged as some of the most important signaling systems. The phosphorelay systems in plants include components with sensor (His-protein kinase) domains, His-containing phosphotransfer (HPt) domains, and receiver (response regulator) domains. Recent studies implicate phosphorelay systems in sensing and propagating signals from a wide variety of external and/or internal stimuli such as ethylene, cytokinin, and osmolarity. In maize and Arabidopsis, some response regulators are up-regulated by both cytokinins and nitrate. These findings imply that the His-Asp phosphorelay may operate in an inorganic nitrogen-signaling pathway mediated by cytokinin in plants.