Phosphorelay Signal Transduction System Research Articles

Transcriptional survey of abiotic stress response in maize (Zea mays) in the level of gene co-expression network and differential gene correlation analysis.

Abstract. Maize may be exposed to several abiotic stresses in the field. Therefore, identifying the tolerance mechanisms of natural field stress is mandatory. Gene expression data of maize upon abiotic stress were collected, and 560 differentially expressed genes (DEGs) were identified through meta-analysis. The most significant gene ontology terms in up-regulated genes were 'response to abiotic stress' and 'chitinase activity'. 'Phosphorelay signal transduction system' was the most significant enriched biological process in down-regulated DEGs. The co-expression analysis unveiled seven modules of DEGs, with a notable positive correlation between the modules and abiotic stress. Furthermore, the statistical significance was strikingly high for the turquoise, green and yellow modules. The turquoise group played a central role in orchestrating crucial adaptations in metabolic and stress response pathways in maize when exposed to abiotic stress. Within three up-regulated modules, Zm.7361.1.A1_at, Zm.10386.1.A1_a_at and Zm.10151.1.A1_at emerged as hub genes. These genes might introduce novel candidates implicated in stress tolerance mechanisms, warranting further comprehensive investigation and research. In parallel, the R package glmnet was applied to fit a logistic LASSO regression model on the DEGs profile to select candidate genes associated with abiotic responses in maize. The identified hub genes and LASSO regression genes were validated on an independent microarray dataset. Additionally, Differential Gene Correlation Analysis (DGCA) was performed on LASSO and hub genes to investigate the gene-gene regulatory relationship. The P value of DGCA of 16 pairwise gene comparisons was lower than 0.01, indicating a gene-gene significant change in correlation between control and abiotic stress. Integrated weighted gene correlation network analysis and logistic LASSO analysis revealed Zm.11185.1.S1_at, Zm.2331.1.S1_x_at and Zm.17003.1.S1_at. Notably, these 3 genes were identified in the 16 gene-pair comparisons. This finding highlights the notable significance of these genes in the abiotic stress response. Additional research into maize stress tolerance may focus on these three genes.

Transcriptome and metabolome analyses reveal the efficiency of in vitro regeneration by TDZ pretreatment in mulberry

Thidiazuron (TDZ) is a plant growth regulator that is widely used as a defoliant and cytokinin-like compound. Additionally, mulberry is an economically important crop. However, regeneration is difficult in most mulberry species, and the regulatory mechanism controlling the regeneration process remains unclear. In this work, an efficient in vitro shoot regeneration system for mulberry was established by TDZ application. The results showed that the optimum adventitious bud induction, 70.83% in tetraploid M. notabilis cv. Chuanbei1 (CB1) and 96.82% in M. abla cv. Yunhua (YH), was obtained by the shoot pretreatment method using petiolate leaves as explants. The high-frequency regeneration petiolate leaf explants of YH treated with TDZ (T8) and the control (CK) were subjected to multiomic analyses. The comparison of the TDZ-treated with the CK groups identified 494 upregulated differentially expressed genes (DEGs) and 293 downregulated DEGs. GO analysis indicated that these DEGs were related mostly to integral component of membrane, transcription factor activity, and phosphorelay signal transduction system. Metabolomic analysis identified 1,889 compounds, including 205 differentially accumulated metabolites (DAMs). Of these DAMs, the number of lipids and lipid-like molecules was highest and accounted for 27.8% of all DAMs. Transcriptome profile, metabolomic, and phytohormone integrated analyses revealed that TDZ rearranged adenine-type cytokinins (CTKs) and negatively regulated endogenous CTK signal transduction. Moreover, TDZ treatment enhanced auxin signal transduction by increasing the expression of auxin flux carrier genes and downstream regulated genes without changing auxin biosynthesis in mulberry. In summary, this study provides a new method for mulberry in vitro regeneration and contributes to a better understanding of the transcriptional and metabolic changes that occur in mulberry after TDZ treatment.

Deciphering the Dynamics of Signaling Cascades and Virulence Factors of B. cinerea during Tomato Cell Wall Degradation.

The ascomycete Botrytis cinerea is one of the most relevant plant pathogenic fungi, affecting fruits, flowers, and greenhouse-grown crops. The infection strategy used by the fungus comprises a magnificent set of tools to penetrate and overcome plant defenses. In this context, the plant-pathogen communication through membrane receptors and signal transduction cascades is essential to trigger specific routes and the final success of the infection. In previous reports, proteomics approaches to B. cinerea signal transduction cascades changes in response to different carbon source and plant-based elicitors have been performed. Analyzing the secretome, membranome, phosphoproteome, and the phosphomembranome. Moreover, phenotypic changes in fungal biology was analyzed, specifically toxin production. To obtain the whole picture of the process and reveal the network from a system biology approach, this proteomic information has been merged with the phenotypic characterization, to be analyzed using several bioinformatics algorithms (GO, STRING, MCODE) in order to unravel key points in the signal transduction regulation crucial to overcome plant defenses, as well as new virulence/pathogenicity factors that could be used as therapeutic targets in the control of the gray mold rot disease. A total of 1721 and 663 exclusive or overexpressed proteins were identified under glucose (GLU) and deproteinized tomato cell walls (TCW), summarizing all of the protein identifications under phenotypic characterized stages. Under GO analysis, there are more biological process and molecular functions described in GLU, highlighting the increase in signaling related categories. These results agree with the high number of total identified proteins in GLU, probably indicating a more varied and active metabolism of the fungus. When analyzing only GO annotations related with signal transduction, it was revealed that there were proteins related to TOR signaling, the phosphorelay signal transduction system, and inositol lipid-mediated signaling, only under GLU conditions. On the contrary, calcium-mediated signaling GO annotation is only present between the proteins identified under TCW conditions. To establish a potential relationship between expressed proteins, cluster analyses showed 41 and 14 clusters under GLU and TCW conditions, confirming an increase in biological activity in GLU, where we identified a larger number of clusters related to transcription, translation, and cell division, between others. From these analyses, clusters related to signal transduction and clusters related to mycotoxin production were found, which correlated with the phenotypic characterization. The identification of the proteins encompassed in each condition and signal transduction cascade would provide the research community with new information about the B. cinerea infection process and potential candidates of pathogenicity/virulence factors, overcoming plant defenses, and new therapeutic targets.

General stress response or adaptation to rapid growth in Aspergillus nidulans?

Genome-wide transcriptional changes in Aspergillus nidulans induced by nine different stress conditions were evaluated to reveal the general environmental stress response gene set showing unidirectional expressional changes under various types of stress. Clustering the genes by their transcriptional changes was a useful technique for identifying large groups of co-regulated genes. Altogether, 1642 co-upregulated and 3916 co-downregulated genes were identified. Nevertheless, the co-regulated genes describe the difference between the transcriptomes recorded under the stress conditions tested and one chosen reference culture condition which is designated as the “unstressed” condition. Obviously, the corresponding transcriptional differences may be attributed to either the general stress response or the reference condition. Accordingly, reduced growth and increased transcription of certain antioxidative enzymes observed under stress may be interpreted as elements of the general stress response or as a feature of the “optimal growth” reference condition and decreased antioxidative protection due to “rapid growth” stress. Reversing the many to one comparison underlying the identification of co-regulated gene sets allows the same procedure to highlight changes under a single condition with respect to a set of other “background” conditions. As an example, we compared menadione treatment to our other conditions and identified downregulation of endoplasmic reticulum dependent processes and upregulation of iron-sulfur cluster assembly as well as glutathione-S-transferase genes as changes characteristic of MSB-treated cultures. Deletion of the atfA gene markedly altered the co-regulated gene sets primarily by changing the reference transcriptome; not by changing the stress responsiveness of genes. The functional characterization of AtfA-dependent co-regulated genes demonstrated the involvement of AtfA in the regulation of both vegetative growth and conidiogenesis in untreated cultures. Our data also suggested that the diverse effects of atfA gene deletion on the transcriptome under different stress conditions were the consequence of the altered transcription of several phosphorelay signal transduction system genes, including fphA, nikA, phkA, srrB, srrC, sskA and tcsB. Hopefully, this study will draw further attention to the importance of the proper selection of reference cultures in fungal transcriptomics studies especially when elements of specific stress responses are mapped.

In silico studies reveal RSc1154 and RhlE as temperature-related pathogenic proteins of Ralstonia solanacearum.

This study aimed to explore the temperature-related pathogenic mechanism of Ralstonia solanacearum infection in tomato (Lycopersicon esculentum Mill.). Based on bioinformatics analysis of microarray dataset (GSE33657), the co-differentially expressed genes (co-DEGs) ribonucleic acids were identified in R. solanacearum GMI1000-infected L. esculentum Mill., which was cultured at 20°C and 28°C, in rich medium containing casamino acids, peptone, and glucose (CPG) and planta. In total, 63 upregulated co-DEGs and 57 downregulated co-DEGs were identified between 20°C and 28°C in the CPG and planta groups. Protein-protein interaction network revealed 70 protein interaction pairs and 59 nodes. Notably, iolG, iolE, ioll and RSc1248 played critical roles in the network. The subcellular localization and functional annotation showed that the increased expressed proteins were mainly localized in the inner cell membrane, while those with decreased expression were localized in the cytoplasm. Furthermore, these proteins were mainly enriched in regulation of DNA-templated transcription. RSc1154 and RhlE were predicted to be temperature-related pathogenic genes for R. solanacearum in tomato. Furthermore, phosphorelay signal transduction system function might play an important role in R. solanacearum infection. The candidate genes were verified by quantitative real-time PCR, and the results were consistent with gene expression profile.

Transcriptomic profiling and analysis of differentially expressed genes in asparagus bean (Vigna unguiculata ssp. sesquipedalis) under salt stress.

Asparagus bean (Vigna unguiculata ssp. sesquipedalis) is a warm season legume which is widely distributed over subtropical regions and semiarid areas. It is mainly grown as a significant protein source in developing countries. Salinity, as one of the main abiotic stress factors, constrains the normal growth and yield of asparagus bean. This study used two cultivars (a salt-sensitive genotype and a salt-tolerant genotype) under salt stress vs. control to identify salt-stress-induced genes in asparagus bean using RNA sequencing. A total of 692,086,838 high-quality clean reads, assigned to 121,138 unigenes, were obtained from control and salt-treated libraries. Then, 216 root-derived DEGs (differentially expressed genes) and 127 leaf-derived DEGs were identified under salt stress between the two cultivars. Of these DEGs, thirteen were assigned to six transcription factors (TFs), including AP2/EREBP, CCHC(Zn), C2H2, WRKY, WD40-like and LIM. GO analysis indicated four DEGs might take effects on the “oxidation reduction”, “transport” and “signal transduction” process. Moreover, expression of nine randomly-chosen DEGs was verified by quantitative real-time-PCR (qRT-PCR) analysis. Predicted function of the nine tested DEGs was mainly involved in the KEGG pathway of cation transport, response to osmotic stress, and phosphorelay signal transduction system. A salt-stress-related pathway of “SNARE interactions in vesicular transport” was concerned. As byproducts, 15, 321 microsatellite markers were found in all the unigenes, and 17 SNP linked to six salt-stress induced DEGs were revealed. These candidate genes provide novel insights for understanding the salt tolerance mechanism of asparagus bean in the future.

The phosphorelay signal transduction system in Candida glabrata: an in silico analysis.

Signaling systems allow microorganisms to sense and respond to different stimuli through the modification of gene expression. The phosphorelay signal transduction system in eukaryotes involves three proteins: a sensor protein, an intermediate protein and a response regulator, and requires the transfer of a phosphate group between two histidine-aspartic residues. The SLN1-YPD1-SSK1 system enables yeast to adapt to hyperosmotic stress through the activation of the HOG1-MAPK pathway. The genetic sequences available from Saccharomyces cerevisiae were used to identify orthologous sequences in Candida glabrata, and putative genes were identified and characterized by in silico assays. An interactome analysis was carried out with the complete genome of C. glabrata and the putative proteins of the phosphorelay signal transduction system. Next, we modeled the complex formed between the sensor protein CgSln1p and the intermediate CgYpd1p. Finally, phosphate transfer was examined by a molecular dynamic assay. Our in silico analysis showed that the putative proteins of the C. glabrata phosphorelay signal transduction system present the functional domains of histidine kinase, a downstream response regulator protein, and an intermediate histidine phosphotransfer protein. All the sequences are phylogenetically more related to S. cerevisiae than to C. albicans. The interactome suggests that the C. glabrata phosphorelay signal transduction system interacts with different proteins that regulate cell wall biosynthesis and responds to oxidative and osmotic stress the same way as similar systems in S. cerevisiae and C. albicans. Molecular dynamics simulations showed complex formation between the response regulator domain of histidine kinase CgSln1 and intermediate protein CgYpd1 in the presence of a phosphate group and interactions between the aspartic residue and the histidine residue. Overall, our research showed that C. glabrata harbors a functional SLN1-YPD1-SSK1 phosphorelay system.

Role of the inner-membrane histidine kinase RcsC and outer-membrane lipoprotein RcsF in the activation of the Rcs phosphorelay signal transduction system in Escherichia coli.

The Rcs phosphorelay signal transduction system of Escherichia coli controls genes for capsule production and many other envelope-related functions and is implicated in biofilm formation. The outer-membrane lipoprotein RcsF is an essential component of the Rcs system. Mislocalization of RcsF to the periplasm or the cytoplasmic membrane leads to high activation of the Rcs system, suggesting that RcsF functions by interacting with the cytoplasmic membrane component(s) of the system in activating the system. This is consistent with the result reported by Cho et al. (Cell159, 1652-1664, 2014) showing that RcsF interacts with the periplasmic domain (YrfFperi) of the inner-membrane protein YrfF (IgaA in Salmonella enterica serovar Typhimurium), which is a negative regulator of the Rcs system. In this study we show that RcsF also interacts with the periplasmic domain of the innermembrane-localized histidine kinase RcsC (RcsCperi). RcsCperi, which was secreted to the periplasm by fusion to maltose-binding protein, titrated RcsF's activating effect. A bimolecular fluorescence complementation experiment showed interaction of RcsF with RcsCperi, as well as with YrfFperi. We conclude that RcsF interacts with the periplasmically exposed region of RcsC, as well as with that of YrfF.

Evolution of a multi-step phosphorelay signal transduction system in Ensifer: recruitment of the sigma factor RpoN and a novel enhancer-binding protein triggers acid-activated gene expression.

Most Ensifer strains are comparatively acid sensitive, compromising their persistence in low pH soils. In the acid-tolerant strain Ensifer medicae WSM419, the acid-activated expression of lpiA is essential for enhancing survival in lethal acidic conditions. Here we characterise a multi-step phosphorelay signal transduction pathway consisting of TcsA, TcrA, FsrR, RpoN and its cognate enhancer-binding protein EbpA, which is required for the induction of lpiA and the downstream acvB gene. The fsrR, tcrA, tcsA and rpoN genes were constitutively expressed, whereas lpiA and acvB were strongly acid-induced. RACE mapping revealed that lpiA/acvB were co-transcribed as an operon from an RpoN promoter. In most Ensifer species, lpiA/acvB is located on the chromosome and the sequence upstream of lpiA lacks an RpoN-binding site. Nearly all Ensifer meliloti strains completely lack ebpA, tcrA, tcsA and fsrR regulatory loci. In contrast, E. medicae strains have lpiA/acvB and ebpA/tcrA/tcsA/fsrR co-located on the pSymA megaplasmid, with lpiA/acvB expression coupled to an RpoN promoter. Here we provide a model for the expression of lpiA/acvB in E. medicae. This unique acid-activated regulatory system provides insights into an evolutionary process which may assist the adaptation of E. medicae to acidic environmental niches.

Crystal structure of nonphosphorylated receiver domain of the stress response regulator RcsB from Escherichia coli.

RcsB, the transcription-associated response regulator of the Rcs phosphorelay two-component signal transduction system, activates cell stress responses associated with desiccation, cell wall biosynthesis, cell division, virulence, biofilm formation, and antibiotic resistance in enteric bacterial pathogens. RcsB belongs to the FixJ/NarL family of transcriptional regulators, which are characterized by a highly conserved C-terminal DNA-binding domain. The N-terminal domain of RcsB belongs to the family of two-component receiver domains. This receiver domain contains the phosphoacceptor site and participates in RcsB dimer formation; it also contributes to dimer formation with other transcription factor partners. Here, we describe the crystal structure of the Escherichia coli RcsB receiver domain in its nonphosphorylated state. The structure reveals important molecular details of phosphorylation-independent dimerization of RcsB and has implication for the formation of heterodimers.

Stress sensitivity of a fission yeast strain lacking histidine kinases is rescued by the ectopic expression of Chk1 from Candida albicans

The development of new drugs against the pathogenic yeast Candida albicans is compelling and the evolution of relevant bioassays is important to achieve this goal. Promising drug targets are proteins that lack human counterparts which are true for the His-to-Asp phosphorelay signal transduction systems, important for stress sensing in bacteria, fungi, and plants. In the pathogenic yeast, Candida albicans, the CaChk1 histidine kinase is a trigger of the pathway that leads to a switch from yeast to hyphal growth necessary for invasion. Intriguingly, the model yeast Schizosaccharomyces pombe has a similar phosphorelay system, with three histidine kinases named Mak1, Mak2, and Mak3, which are important for the prevention of aberrant mating and sporulation on rich media. This study uncovered distinct functions for the three histidine kinases; Mak1 alone or Mak2 and Mak3 together were sufficient for the repression of the meiotic cycle when nutrients were available. Moreover, strains lacking histidine kinase genes were sensitive to various types of stress conditions in an auxotrophic strain background, while the stress sensitivity was lost in prototrophic strains. Finally, the stress sensitivity of a S. pombe strain that lacks endogenous histidine kinases could be complemented by the ectopic expression of the CaChk1 histidine kinase from C. albicans. This finding opens up for the possibility to perform a drug screen with a biological read-out in S. pombe to find inhibitors of CaChk1.

Production of reactive oxygen species in <i>Aspergillus nidulans</i> through the function of His-Asp phosphorelay signal transduction system

Production of reactive oxygen species in <i>Aspergillus nidulans</i> through the function of His-Asp phosphorelay signal transduction system

Importance of the proline-rich region for the regulatory function of RcsF, an outer membrane lipoprotein component of the Escherichia coli Rcs signal transduction system

The outer membrane lipoprotein RcsF is an essential component of the Rcs phosphorelay signal transduction system in Escherichia coli. It senses stresses imposed on the cell envelope and conveys the information to histidine kinase RcsC in the cytoplasmic membrane. Mislocalization of RcsF to the periplasm, effected by fusing it to the periplasmic maltose-binding protein, or to the cytoplasmic membrane, brought about by changing the lipoprotein sorting signal, leads to high activation of the Rcs system, suggesting that RcsF functions as a ligand for RcsC in activating the system. Here, we focus on the proline-rich region (PRR) in the N-terminal half of RcsF, a region which also contains many basic amino acid residues. Deletion of the PRR in the mislocalized RcsF resulted in even higher activation of the Rcs system. The same deletion in wild-type RcsF lipoprotein that is correctly localized to the outer membrane, however, blocked activation of the system under stresses that normally should activate it. It is highly likely that the PRR plays an important role in the regulation of the function of RcsF in activating the Rcs system.

Activation of the Cpx phosphorelay signal transduction system in acidic phospholipid-deficient pgsA mutant cells of Escherichia coli

The pgsA gene encodes the enzyme for the committed step in the synthesis of acidic phospholipids in Escherichia coli, and the pssA gene does the same for zwitterionic phospholipid. It has been reported that the Rcs and Cpx phosphorelay signal transduction systems are activated in pgsA- and pssA-defective mutants, respectively. In this study, we show that the Cpx system is activated also in a pgsA mutant, whereas the Rcs system was not activated in a pssA mutant. Lack of phosphatidylglycerol in pgsA mutants causes inadequate modification of lipoproteins, resulting in poor localization to the outer membrane. The outer membrane lipoprotein RcsF is necessary for the response of the Rcs system to various stimuli, and Rcs activation in pgsA mutants involves inner membrane mislocalization of this lipoprotein. The outer membrane lipoprotein NlpE, however, while necessary for the surface adhesion-induced Cpx response, was not involved in Cpx activation in the pgsA mutant.

The Yersinia pestis Rcs Phosphorelay Inhibits Biofilm Formation by Repressing Transcription of the Diguanylate Cyclase Gene hmsT

Yersinia pestis, which causes bubonic plague, forms biofilms in fleas, its insect vectors, as a means to enhance transmission. Biofilm development is positively regulated by hmsT, encoding a diguanylate cyclase that synthesizes the bacterial second messenger cyclic-di-GMP. Biofilm development is negatively regulated by the Rcs phosphorelay signal transduction system. In this study, we show that Rcs-negative regulation is accomplished by repressing transcription of hmsT.

Exploring the relationship between lipoprotein mislocalization and activation of the Rcs signal transduction system in Escherichia coli

The Rcs phosphorelay signal transduction system controls genes for capsule production and many other envelope-related functions and is implicated in biofilm formation. We investigated the activation of the Rcs system in a pgsA null mutant of Escherichia coli, which completely lacks the major acidic phospholipids phosphatidylglycerol and cardiolipin. We found that the Rcs activation, and consequent thermosensitivity, were suppressed by overexpression of the lgt gene, encoding diacylglyceryltransferase, which catalyses the modification of prolipoproteins that is the first step in the maturation and localization process of lipoproteins, and is a prerequisite for the later steps. The outer-membrane lipoprotein RcsF is an essential component of Rcs signalling. This lipoprotein was poorly localized to the outer membrane in the pgsA null mutant, probably because of the absence of phosphatidylglycerol, the major donor of diacylglycerol in the Lgt reaction. Even in a pgsA(+) background, the Rcs system was activated when RcsF was mislocalized to the inner membrane by alteration of the residues at positions 2 and 3 of its mature form to inner-membrane retention signals, or when it was mislocalized to the periplasm by fusing the mature form to maltose-binding protein. These results suggest that RcsF functions as a ligand for RcsC in activating Rcs signalling. Mislocalized versions of RcsF still responded to mutations pgsA, mdoH and tolB, further activating the Rcs system, although the rfaP mutation barely caused activation. It seems that RcsF must be localized in the outer membrane to respond effectively to stimuli from outside the cell.

Lactoferricin B Inhibits the Phosphorylation of the Two-Component System Response Regulators BasR and CreB

Natural antimicrobial peptides provide fundamental protection for multicellular organisms from microbes, such as Lactoferricin B (Lfcin B). Many studies have shown that Lfcin B penetrates the cell membrane and has intracellular activities. To elucidate the intracellular behavior of Lfcin B, we first used Escherichia coli K12 proteome chips to identify the intracellular targets of Lfcin B. The results showed that Lfcin B binds to two response regulators, BasR and CreB, of the two-component system. For further analysis, we conducted several in vitro and in vivo experiments and utilized bioinformatics methods. The electrophoretic mobility shift assays and kinase assays indicate that Lfcin B inhibits the phosphorylation of the response regulators (BasR and CreB) and their cognate sensor kinases (BasS and CreC). Antibacterial assays showed that Lfcin B reduced E. coli's tolerance to environmental stimuli, such as excessive ferric ions and minimal medium conditions. This is the first study to show that an antimicrobial peptide inhibits the growth of bacteria by influencing the phosphorylation of a two-component system directly.

Roles of the His-Asp Phosphorelay Signal Transduction System in Controlling Cell Growth and Development inAspergillus 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.

Functional Role for a Conserved Aspartate in the Spo0E Signature Motif Involved in the Dephosphorylation of the Bacillus subtilis Sporulation Regulator Spo0A

Sporulation is a complex developmental system characterizing Gram-positive bacteria of the genus Bacillus and Clostridium. In Bacillus subtilis the phosphorelay signal transduction system regulates the initiation of sporulation by integrating a myriad of positive and negative signals through the action of histidine sensor kinases and aspartyl phosphate phosphatases. The Spo0E family of phosphatases dephosphorylates the Spo0A response regulator and transcription factor of the phosphorelay. In this study we analyzed the role of the Spo0E signature motif in protein activity. This family is characterized by a conserved signature motif centered around the sequence "SQELD." Alanine scanning mutagenesis was carried out on the T(35)IXXSQ ELDCLI(46) residues of B. subtilis Spo0E and in vivo and in vitro activities were analyzed. The ability of the mutant proteins to interact with Spo0A approximately P was assayed by fluorescence resonance energy transfer spectroscopy. The results suggested that aspartate 43 has a critical role in Spo0E catalytic activity, whereas the other residues have a role in protein conformation and/or interaction with Spo0A. Residues Thr(35) and Cys(44) did not seem to have any critical functional or structural role. We propose that Asp(43) of Spo0E may function in a manner similar to the one proposed for the catalytic mechanisms of nucleotidase members of the haloacid dehalogenase family. These proteins use an aspartyl nucleophile as their common catalytic strategy and the active site of haloacid dehalogenase proteins shares a common geometry and identity of conserved amino acids with the active site of response regulators ( Ridder, I. S., and Dijkstra, B. W. (1999) Biochem. J. 339, 223-226 ).

Functional characterization of the phosphorelay protein Mpr1p fromSchizosaccharomyces pombe

Histidine-containing phosphotransfer (HPt) proteins play an essential role in multistep histidine-aspartate phosphorelay signal transduction systems in prokaryotes and eukaryotes. The putative HPt protein in Schizosaccharomyces pombe, Mpr1p (also known as Spy1p), is a 295 amino acid protein that appears to be composed of more than one functional domain. The amino acid sequence of the N-terminal region of Mpr1p lacks homology to other known proteins, whereas the C-terminal domain is predicted to have structural similarity to the Ypd1p HPt protein from Saccharomyces cerevisiae. This study provides both in vitro and in vivo evidence that the C-terminal domain of Mpr1p indeed functions as an HPt protein in shuttling phosphoryl groups from one response regulator domain to another. Furthermore, we find that various deletions of the N-terminal region diminish both the phosphotransfer activity of Mpr1p and its affinity for response regulator domains, suggesting a possible role for the N-terminal domain in HPt-response regulator domain interactions.