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Mitsuo Ogura - One of the best experts on this subject based on the ideXlab platform.
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regulation of the response regulator gene Degu through the binding of sinr slrr and exclusion of sinr slrr by Degu in bacillus subtilis
Journal of Bacteriology, 2014Co-Authors: Mitsuo Ogura, Hirofumi Yoshikawa, Taku ChibazakuraAbstract:Bacillus subtilis Degu is a response regulator of the DegS-Degu two-component regulatory system. Phosphorylated Degu (Degu-P) controls many genes and biological processes, such as exoprotease and γ-polyglutamic acid production, in addition to the Degu gene, by binding to target gene promoters. Nonphosphorylated Degu and low levels of Degu-P are required for swarming motility and genetic competence. The DNA-binding repressors SinR and SlrR are part of a double-negative feedback loop and comprise the epigenetic switch governing biofilm formation. In this study, we found that SinR repressed Degu. Furthermore, SlrR, which interacts with SinR through protein-protein interaction, seems to have an active role in Degu expression in in vivo lacZ analysis. An in vitro transcription assay supported this observation. An electrophoretic mobility shift assay (EMSA) showed that SinR bound to the Degu promoter and that SlrR formed a complex with SinR on the Degu promoter. In EMSA, Degu-P excluded the SinR/SlrR complex but not SinR from the Degu promoter in the presence of RNA polymerase. These findings suggest that Degu-P interacts with SlrR. In support of this hypothesis, disruption of the slrR gene resulted in decreased Degu expression. This newly identified regulatory mechanism for Degu is considered to be sequential transcription factor replacement.
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Regulation of the response regulator gene Degu through the binding of SinR/SlrR and exclusion of SinR/SlrR by Degu in bacillus subtilis
Journal of Bacteriology, 2014Co-Authors: Mitsuo Ogura, Hirofumi Yoshikaw, Taku ChibazakuraAbstract:Bacillus subtilis Degu is a response regulator of the DegS-Degu two-component regulatory system. Phosphorylated Degu (Degu-P) controls many genes and biological processes, such as exoprotease and γ-polyglutamic acid production, in addition to the Degu gene, by binding to target gene promoters. Nonphosphorylated Degu and low levels of Degu-P are required for swarming motility and genetic competence. The DNA-binding repressors SinR and SlrR are part of a double-negative feedback loop and comprise the epigenetic switch governing biofilm formation. In this study, we found that SinR repressed Degu. Furthermore, SlrR, which interacts with SinR through protein-protein interaction, seems to have an active role in Degu expression in in vivo lacZ analysis. An in vitro transcription assay supported this observation. An electrophoretic mobility shift assay (EMSA) showed that SinR bound to the Degu promoter and that SlrR formed a complex with SinR on the Degu promoter. In EMSA, Degu-P excluded the SinR/SlrR complex but not SinR from the Degu promoter in the presence of RNA polymerase. These findings suggest that Degu-P interacts with SlrR. In support of this hypothesis, disruption of the slrR gene resulted in decreased Degu expression. This newly identified regulatory mechanism for Degu is considered to be sequential transcription factor replacement.
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the bacillus subtilis response regulator gene Degu is positively regulated by ccpa and by catabolite repressed synthesis of clpc
Journal of Bacteriology, 2013Co-Authors: Hiroshi Ishii, Teruo Tanaka, Mitsuo OguraAbstract:In Bacillus subtilis, the response regulator Degu and its cognate kinase, DegS, constitute a two-component system that regulates many cellular processes, including exoprotease production and genetic competence. Phosphorylated Degu (Degu-P) activates its own promoter and is degraded by the ClpCP protease. We observed induction of Degu by glucose in sporulation medium. This was abolished in two mutants: the ccpA (catabolite control protein A) and clpC disruptants. Transcription of the promoter of the operon containing clpC (PclpC) decreased in the presence of glucose, and the disruption of ccpA resulted in derepression of PclpC. However, this was not directly mediated by CcpA, because we failed to detect binding of CcpA to PclpC. Glucose decreased the expression of clpC, leading to low cellular concentrations of the ClpCP protease. Thus, Degu is induced through activation of autoregulation by a decrease in ClpCP-dependent proteolysis of Degu-P. An electrophoretic mobility shift assay showed that CcpA bound directly to the Degu upstream region, indicating that CcpA activates Degu through binding. The bound region was narrowed down to 27 bases, which contained a cre (catabolite-responsive element) sequence with a low match to the cre consensus sequence. In a footprint analysis, CcpA specifically protected a region containing the cre sequence from DNase I digestion. The induction of Degu by glucose showed complex regulation of the Degu gene.
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swra regulates assembly of bacillus subtilis Degu via its interaction with n terminal domain of Degu
Journal of Biochemistry, 2012Co-Authors: Mitsuo Ogura, Kensuke TsukaharaAbstract:: The Bacillus subtilis response regulator Degu controls many physiological events including swarming motility and exoprotease production. Swarming motility is a multicellular movement of hyper-flagellated cells on a surface. The swarming motility regulator SwrA and Degu cooperatively drive transcription of fla/che encoding flagella components, chemotaxis constituents and motility-specific sigma factor, which is regarded as the primary event in the development of motility. We have identified ycdA involved in swarming motility, encoding a putative lipoprotein. We showed that the ycdA gene is positively regulated by Degu and SwrA. Mutational analysis of ycdA-lacZ revealed that SwrA changes the use of cis-acting sites for Degu. This suggested that SwrA operates the Degu-regulation mode through changes in the Degu assembly state. Degu binding to the ycdA-promoter region carrying an unusual arrangement of Degu-recognition sequences with low affinity was found to be stimulated by SwrA in electrophoretic mobility shift assay and DNase I footprinting. Yeast two- and three-hybrid analyses revealed that the N-terminal domain of Degu interacts with whole Degu, which is facilitated by SwrA. Together, these results demonstrate that SwrA can stabilize the binding of Degu to the ycdA promoter with low affinity. Thus, SwrA is a novel type of bacterial transcription factor in this regard.
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autoregulation of the bacillus subtilis response regulator gene Degu is coupled with the proteolysis of Degu p by clpcp
Molecular Microbiology, 2010Co-Authors: Mitsuo Ogura, Kensuke TsukaharaAbstract:Summary The response regulator Degu and its cognate kinase DegS constitute a two-component system in Bacillus subtilis that regulates many cellular processes, including exoprotease production and competence development. Using DNA footprint assay, gel shift assay and mutational analyses of P3Degu-lacZ fusions, we showed that phosphorylated Degu (Degu-P) binds to two direct repeats (DR1 and DR2) of the consensus Degu-binding sequence in the P3Degu promoter. The alteration of chromosomal DR2 severely decreased Degu expression, demonstrating its importance in positive autoregulation of Degu. Observation of Degu protein levels suggested that Degu is degraded. Western blot analysis of Degu in disruption mutants of genes encoding various ATP-dependent proteases strongly suggested that ClpCP degrades Degu. Moreover, when de novo protein synthesis was blocked, Degu was rapidly degraded in the wild-type but not in the clpC and clpP strains, and Degu with a mutated phosphorylation site was much stable. These results suggested preferential degradation of Degu-P by ClpCP, but not of unphosphorylated Degu. We confirmed that Degu-P was degraded preferentially using an in vitro ClpCP degradation system. Furthermore, a mutational analysis showed that the N-terminal region of Degu is important for proteolysis.
Luis A. Ebensperger - One of the best experts on this subject based on the ideXlab platform.
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Dustbathing and intra-sexual communication of social Degus, Octodon
2020Co-Authors: Luis A. EbenspergerAbstract:I observed the behavior of captive male and female Octodon Degus to assess if dustbathing behavior plays a role in social communication among unfamiliar, same-sex conspecifics. Degus of a first group (control responders) were individually exposed during 1 0-min tests to an arena containing loose, clean soil. I compared the latency to first dustbathing as well as the overall frequency of dustbathing events recorded to control responders with the corresponding figures recorded to a second group of Degus (experimental responders) after they were individually introduced into the same arena but with soil previously used for dustbathing by a same-sex conspecific (depositor). I also compared the location of dustbathing events by experimental responders with that of depositor individuals. Although male Degus tended to exhibit shorter latencies to first dustbathing event when in clean soil, this variable was not significantly influenced by sex of responders or the type of soil (clean or used). In contrast, a significant interaction between both factors revealed that males dustbathe at a higher rate than females when on clean soil, but similarly so when in a substratum previously dustbathed by a same-sex conspecific. The place chosen by both male and female responders to conduct their dustbathing behavior was unrelated to the presence of previous marks left by a depositor Degu. I conclude that dustbathing is involved in communication during male-male, but not during female-female, interactions in the Degu. I suggest that such male-male interactions represent competition for mates.
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Seasonal changes in the time budget of Degus, Octodon Degus
Behaviour, 2020Co-Authors: Luis A. Ebensperger, María José HurtadoAbstract:The activity budget of an individual is the allocation of time to all its activities, and is expected to vary in response to both internal and environmental factors that influence its energy acquisition, breeding success, and survival. We recorded seasonal variation in individual and social behaviour of a natural population of Degus (Octodon Degus), a diurnal, semi-subterranean and social rodent from central Chile. We related changes in Degu activity to differences in sex, seasonality (breeding activity, abundance of high quality food), and abundance of Degu predators. On average, Degus allocated most of their time while active above ground to foraging (46%) and alertness (32%); activities such as resting (8%), locomotor activity (7%), self-grooming (3%), burrow digging (0.2%), dust-bathing (1%), and social interactions (3%) occupied a relatively small percentage of Degus' time budget. Time spent in foraging and total vigilance did not vary seasonally, but they were inversely related, reflecting a trade-off. Degus adjusted bipedal vigilance and locomotor activity partially to the presence of predators. Sex interacted with seasonality to influence Degu behaviour. Male Degus dust-bathed more and were more aggressive toward conspecifics than females during breeding time. We hypothesize that breeding activity is a more important predictor than abundance of high quality food to account for these interactions.
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postnatal development of the Degu octodon Degus endocrine stress response is affected by maternal care
Journal of Experimental Zoology, 2016Co-Authors: Carolyn M Bauer, Luis A. Ebensperger, Loren D. Hayes, Juan Ramirezestrada, Cecilia León, Michael L RomeroAbstract:Maternal stress and care significantly affect offspring's future behavior and physiology. Studies in laboratory rats have shown that maternal stress decreases maternal care and that low rates of certain maternal behaviors cause offspring to develop hyperreactive stress responses. Plurally breeding rodents that practice communal care, such as Degus (Octodon Degus), may be able to buffer some of these effects since offspring receive care from multiple females. Directly after parturition, 0% (Control group), 50% (Mixed group), or 100% (CORT group) per cage of pair-housed female Degus were implanted with 21-day release cortisol pellets. The amount of maternal care provided by females was determined from video recordings during the next 3 weeks. Females with cortisol implants did not alter rates of maternal care. However, females recently introduced to captivity had low rates of pup contact and pup retrievals compared to females of captive origin. When pups reached 4 weeks of age, we determined their baseline and stress-induced cortisol levels, in addition to assessing their negative feedback efficacy and adrenal sensitivity. Pups from mothers recently introduced to captivity had weak negative feedback. Within captive pups, those from CORT mothers weighed less compared to pups from either Control or Mixed mothers. Captive CORT pups also had weak adrenal sensitivity compared to captive Control pups. These findings demonstrate that maternal care and glucocorticoid elevation impact certain components of the Degu pup stress response, but that plural breeding with communal care may buffer some of these effects.
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Octodon Degus kin and social structure
Journal of Mammalogy, 2015Co-Authors: Garrett T. Davis, Luis A. Ebensperger, Rodrigo A. Vásquez, Elie Poulin, Enrique A. Bazán-león, Loren D. HayesAbstract:A growing body of evidence showing that individuals of some social species live in non-kin groups suggests kin selection is not required in all species for sociality to evolve. Here, we investigate 2 populations of Octodon Degus , a widespread South American rodent that has been shown to form kin and non-kin groups. We quantified genetic relatedness among individuals in 23 social groups across 2 populations as well as social network parameters (association, strength, and clustering coefficient) in order to determine if these aspects of sociality were driven by kinship. Additionally, we analyzed social network parameters relative to ecological conditions at burrow systems used by groups, to determine if ecological characteristics within each population could explain variation in sociality. We found that genetic relatedness among individuals within social groups was not significantly higher than genetic relatedness among randomly selected individuals in both populations, suggesting that non-kin structure of groups is common in Degus. In both populations, we found significant relationships between the habitat characteristics of burrow systems and the social network characteristics of individuals inhabiting those burrow systems. Our results suggest that Degu sociality is non-kin based and that Degu social networks are influenced by local conditions. Es creciente la evidencia que apoya la ocurrencia de especies sociales donde los individuos no estan emparentados geneticamente, lo que sugiere que la seleccion de parentesco no es indispensable para la evolucion de la sociabilidad. En este estudio se examinaron dos poblaciones de Octodon Degus , un roedor sudamericano donde los grupos sociales pueden o no incluir individuos cercanamente emparentados. Se cuantifico el parentesco genetico entre individuos en 23 grupos sociales y en redes sociales de dos poblaciones para determinar si estos aspectos de la sociabilidad dependen del grado de parentesco. Ademas, se examinaron asociaciones entre los parametros cuantificados de las redes sociales (asociacion, fuerza, coeficiente de anidamiento) y las condiciones ecologicas a nivel de los sistemas de madriguera usados por cada grupo. El grado de parentesco genetico dentro de los grupos no fue distinto del grado de parentesco entre individuos de la poblacion tomados al azar, lo que apoya que una estructura de grupos no emparentada es la regla en Octodon Degus . En ambas poblaciones se registro una asociacion entre caracteristicas ecologicas de los sistemas de madriguera y atributos de las redes sociales de los individuos que usan estas estructuras. Nuestros resultados indican que la sociabilidad en Octodon Degus no esta basada en relaciones de parentesco y que las redes sociales de estos animales dependen de las condiciones ecologicas.
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seasonal variation in the Degu octodon Degus endocrine stress response
General and Comparative Endocrinology, 2014Co-Authors: Carolyn M Bauer, Luis A. Ebensperger, Loren D. Hayes, Michael L RomeroAbstract:Many wild animals show seasonal variation in circulating levels of stress hormones. Seasonal changes in the stress response may help animals better cope with the different challenges faced during each life history stage. We determined the seasonal stress profile of wild, free-living Degus in Chile. Female Degus were sampled during non-breeding (January), mating/early gestation (July), late gestation (August), and lactation (1st litter-September, 2nd litter-January). Male Degus were sampled during the first three time-points. We measured baseline cortisol (CORT), stress-induced CORT, and negative feedback efficacy using a dexamethasone suppression test. While we found that neither males nor females showed seasonal variation in baseline CORT or negative feedback levels, we did find significant seasonal variation in stress-induced CORT levels of both sexes. Male stress-induced CORT was lowest during mating while female stress-induced CORT was highest during late gestation and lactation. Overall, females had higher stress-induced CORT compared to males. Our data suggest that stress-induced levels of CORT are highest during periods with increased chance of stressor exposure or times of positive energy balance. Consequently, CORT responses to stress appear to be regulated according to different life history needs.
Georges Rapoport - One of the best experts on this subject based on the ideXlab platform.
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characterization of a novel member of the degs Degu regulon affected by salt stress in bacillus subtilis
Journal of Bacteriology, 1998Co-Authors: Veronique Dartois, Frank Kunst, Michel Debarbouille, Georges RapoportAbstract:As a soil bacterium also found in estuarine and marine habitats, Bacillus subtilis has evolved various sensing and adaptation systems in order to face salt stress conditions. Among these regulatory mechanisms is the DegS-Degu signal transduction system, which was previously shown to be stimulated by high salt concentrations. A search for promoters regulated in response to salt stress led to the identification of wapA, encoding a wall-associated protein, which is strongly expressed at low salt concentrations and almost completely repressed in the presence of 0.7 M disodium succinate. Repression of wapA transcription by salt stress was shown to require the phosphorylated form of Degu. Moreover, Degu-mediated repression of wapA occurred only in high-salt medium. Alignment between the control region of wapA and other Degu-regulated promoters allowed the identification of a putative Degu target sequence, AGAAN11TTCAG. Mutation/deletion analyses of the wapA promoter region confirmed the role of the putative Degu control site in repression of wapA transcription at high salt concentrations and revealed a second site of repression located downstream from the transcription start site. Since residual negative control was observed at this second site in the absence of Degu, it seems likely that an additional repressor acts on the wapA control region to further downregulate wapA transcription under salt stress conditions.
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a signal transduction network in bacillus subtilis includes the degs Degu and comp coma two component systems
1995Co-Authors: Tarek Msadek, Frank Kunst, Georges RapoportAbstract:Soil bacteria such as Bacillus subtilis are subject to drastic variations of environmental conditions such as temperature, humidity, and nutrient source availability. At the onset of the stationary phase, faced with a depletion of essential nutrients, B. subtilis can adopt several responses, including synthesis of macromolecule-degrading enzymes, competence for genetic transformation, increased motility and chemotaxis, antibiotic production, and finally, sporulation. Regulation by the B. subtilis two-component systems presents several original features. Some of these original features are discussed, within the framework of the DegS/Degu and ComP/ComA signal transduction network. The chapter describes degradative enzyme synthesis. Sequence similarities with two-component systems suggest the conserved His-189 residue of the DegS protein kinase and Asp-56 residue of the Degu response regulator as likely candidates for the respective phosphorylation sites of the two proteins. The chapter discusses competence gene expression, and signal transduction network. Both the ComP/ComA and DegS/Degu two-component systems control the expression of late competence genes; however, they seem to act through two different branches in the competence regulatory pathway that intersect to allow expression of comK. An exciting area of future research will be to identify the types of signals involved in regulation by each of these two-component systems and by the other regulators such as MecB/MecA and the ComQ-ComX-Spo0K pathway, as well as determining how these regulators interact within the signal transduction network.
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the phosphorylation state of the Degu response regulator acts as a molecular switch allowing either degradative enzyme synthesis or expression of genetic competence in bacillus subtilis
Journal of Biological Chemistry, 1992Co-Authors: M K Dahl, Tarek Msadek, F Kunst, Georges RapoportAbstract:Abstract Two classes of mutations were identified in the degS and Degu regulatory genes of Bacillus subtilis, leading either to deficiency of degradative enzyme synthesis (degS or Degu mutations) or to a pleiotropic phenotype which includes overproduction of degradative enzymes and the loss of genetic competence (degS(Hy) or Degu(Hy) mutations). We have shown previously that the DegS protein kinase and the Degu response regulator form a signal transduction system in B. subtilis. We now demonstrate that the DegS protein kinase also acts as a Degu phosphatase. We present evidence that the Degu response regulator has two active conformations: a phosphorylated form which is necessary for degradative enzyme synthesis and a nonphosphorylated form required for expression of genetic competence. The Degu146-encoded response regulator, allowing expression of genetic competence, has been purified and seems to be modified within the putative phosphorylation site (D56----N) since it is no longer phosphorylated by DegS. Both the Degu146 mutation as well as the degS220 mutation, which essentially abolishes DegS protein kinase activity, lead to deficiency of degradative enzyme synthesis, indicating the requirement of phosphorylated Degu for the expression of this phenotype. We also purified the Degu32(Hy)-encoded protein and showed that this response regulator is phosphorylated by the DegS protein kinase in vitro. In addition, the phosphorylated form of the Degu32(Hy)-encoded protein presented a strongly increased stability as compared with the wild type Degu protein, thus leading to hyperproduction of degradative enzymes in vivo.
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degs Degu and comp coma modulator effector pairs control expression of the bacillus subtilis pleiotropic regulatory gene degq
Journal of Bacteriology, 1991Co-Authors: Tarek Msadek, Frank Kunst, A Klier, Georges RapoportAbstract:Production of a class of both secreted and intracellular degradative enzymes in Bacillus subtilis is regulated at the transcriptional level by a signal transduction pathway which includes the DegS-Degu two-component system and at least two additional regulatory genes, degQ and degR, encoding polypeptides of 46 and 60 amino acids, respectively. Expression of degQ was shown to be controlled by DegS-Degu. This expression is decreased in the presence of glucose and increased under any of the following conditions: growth with poor carbon sources, amino acid deprivation, phosphate starvation, and growth in the presence of decoyinine, a specific inhibitor of GMP synthetase. In addition, expression of degQ is shown to be positively regulated by the ComP-ComA two-component system. Separate targets for regulation of degQ gene expression by DegS-Degu and ComP-ComA were located by deletion analysis between positions -393 and -186 and between positions -78 and -40, respectively. Regulation of degQ expression by amino acid deprivation was shown to be dependent upon ComA. Regulation by phosphate starvation, catabolite repression, and decoyinine was independent of the two-component systems and shown to involve sequences downstream from position -78. The ComP-ComA and DegS-Degu two-component systems seem to be closely related, sharing several target genes in common, such as late competence genes, as well as the degQ regulatory gene. Sequence analysis of the degQ region revealed the beginning of an open reading frame directly downstream from degQ. Disruption of this gene, designated comQ, suggests that it also controls expression of degQ and is required for development of genetic competence. Images
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mutational analysis of the bacillus subtilis Degu regulator and its phosphorylation by the degs protein kinase
Journal of Bacteriology, 1991Co-Authors: M K Dahl, Tarek Msadek, Frank Kunst, Georges RapoportAbstract:The DegS-Degu protein kinase-response regulator pair controls the expression of genes encoding degradative enzymes as well as other cellular functions in Bacillus subtilis. Both proteins were purified. The DegS protein was autophosphorylated and shown to transfer its phosphate to the Degu protein. Phosphoryl transfer to the wild-type Degu protein present in crude extracts was shown by adding 32P-labeled DegS to the reaction mixture. Under similar conditions, the modified proteins encoded by the Degu24 and Degu31 alleles presented a stronger phosphorylation signal compared with that of the wild-type Degu protein. This may suggest an increased phosphorylation of these modified proteins, responsible for the hyperproduction of degradative enzymes observed in the Degu24 and Degu31 mutants. However, the Degu32 allele, which also leads to hyperproduction of degradative enzymes, encodes a modified Degu response regulator which seems not to be phosphorylatable. The expression of the hyperproduction phenotype of the Degu32 mutant is still dependent on the presence of a functional DegS protein. DegS may therefore induce a conformational change of the Degu32-encoded response regulator enabling this protein to stimulate degradative enzyme synthesis. Two alleles, Degu122 and Degu146, both leading to deficiency of degradative enzyme synthesis, seem to encode phosphorylatable and nonphosphorylatable Degu proteins, respectively.
Taku Chibazakura - One of the best experts on this subject based on the ideXlab platform.
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regulation of the response regulator gene Degu through the binding of sinr slrr and exclusion of sinr slrr by Degu in bacillus subtilis
Journal of Bacteriology, 2014Co-Authors: Mitsuo Ogura, Hirofumi Yoshikawa, Taku ChibazakuraAbstract:Bacillus subtilis Degu is a response regulator of the DegS-Degu two-component regulatory system. Phosphorylated Degu (Degu-P) controls many genes and biological processes, such as exoprotease and γ-polyglutamic acid production, in addition to the Degu gene, by binding to target gene promoters. Nonphosphorylated Degu and low levels of Degu-P are required for swarming motility and genetic competence. The DNA-binding repressors SinR and SlrR are part of a double-negative feedback loop and comprise the epigenetic switch governing biofilm formation. In this study, we found that SinR repressed Degu. Furthermore, SlrR, which interacts with SinR through protein-protein interaction, seems to have an active role in Degu expression in in vivo lacZ analysis. An in vitro transcription assay supported this observation. An electrophoretic mobility shift assay (EMSA) showed that SinR bound to the Degu promoter and that SlrR formed a complex with SinR on the Degu promoter. In EMSA, Degu-P excluded the SinR/SlrR complex but not SinR from the Degu promoter in the presence of RNA polymerase. These findings suggest that Degu-P interacts with SlrR. In support of this hypothesis, disruption of the slrR gene resulted in decreased Degu expression. This newly identified regulatory mechanism for Degu is considered to be sequential transcription factor replacement.
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Regulation of the response regulator gene Degu through the binding of SinR/SlrR and exclusion of SinR/SlrR by Degu in bacillus subtilis
Journal of Bacteriology, 2014Co-Authors: Mitsuo Ogura, Hirofumi Yoshikaw, Taku ChibazakuraAbstract:Bacillus subtilis Degu is a response regulator of the DegS-Degu two-component regulatory system. Phosphorylated Degu (Degu-P) controls many genes and biological processes, such as exoprotease and γ-polyglutamic acid production, in addition to the Degu gene, by binding to target gene promoters. Nonphosphorylated Degu and low levels of Degu-P are required for swarming motility and genetic competence. The DNA-binding repressors SinR and SlrR are part of a double-negative feedback loop and comprise the epigenetic switch governing biofilm formation. In this study, we found that SinR repressed Degu. Furthermore, SlrR, which interacts with SinR through protein-protein interaction, seems to have an active role in Degu expression in in vivo lacZ analysis. An in vitro transcription assay supported this observation. An electrophoretic mobility shift assay (EMSA) showed that SinR bound to the Degu promoter and that SlrR formed a complex with SinR on the Degu promoter. In EMSA, Degu-P excluded the SinR/SlrR complex but not SinR from the Degu promoter in the presence of RNA polymerase. These findings suggest that Degu-P interacts with SlrR. In support of this hypothesis, disruption of the slrR gene resulted in decreased Degu expression. This newly identified regulatory mechanism for Degu is considered to be sequential transcription factor replacement.
Teruo Tanaka - One of the best experts on this subject based on the ideXlab platform.
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the bacillus subtilis response regulator gene Degu is positively regulated by ccpa and by catabolite repressed synthesis of clpc
Journal of Bacteriology, 2013Co-Authors: Hiroshi Ishii, Teruo Tanaka, Mitsuo OguraAbstract:In Bacillus subtilis, the response regulator Degu and its cognate kinase, DegS, constitute a two-component system that regulates many cellular processes, including exoprotease production and genetic competence. Phosphorylated Degu (Degu-P) activates its own promoter and is degraded by the ClpCP protease. We observed induction of Degu by glucose in sporulation medium. This was abolished in two mutants: the ccpA (catabolite control protein A) and clpC disruptants. Transcription of the promoter of the operon containing clpC (PclpC) decreased in the presence of glucose, and the disruption of ccpA resulted in derepression of PclpC. However, this was not directly mediated by CcpA, because we failed to detect binding of CcpA to PclpC. Glucose decreased the expression of clpC, leading to low cellular concentrations of the ClpCP protease. Thus, Degu is induced through activation of autoregulation by a decrease in ClpCP-dependent proteolysis of Degu-P. An electrophoretic mobility shift assay showed that CcpA bound directly to the Degu upstream region, indicating that CcpA activates Degu through binding. The bound region was narrowed down to 27 bases, which contained a cre (catabolite-responsive element) sequence with a low match to the cre consensus sequence. In a footprint analysis, CcpA specifically protected a region containing the cre sequence from DNase I digestion. The induction of Degu by glucose showed complex regulation of the Degu gene.
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involvement of nitrogen regulation in bacillus subtilis Degu expression
Journal of Bacteriology, 2008Co-Authors: Ayako Yasumura, Teruo TanakaAbstract:The bacterial two-component regulatory system is a device to cope with changing environments around the cell. One such example is the Bacillus subtilis DegS-Degu pair, which controls various cellular processes. These include competence development (6, 10, 11, 25), swarming, flagellum formation (1, 16), biofilm formation (15, 35), osmotic response (29), poly-γ-glutamic acid synthesis (35), salt resistance (19), antibiotic synthesis (17), and the synthesis of extracellular degradative enzymes (21). It has been proposed that some of the events regulated by the DegS-Degu system are manifested by different extents of phosphorylation of the response regulator Degu by the DegS kinase (16, 40). Thus, the wide variety of processes regulated by the DegS-Degu system suggest that the signals activating Degu are transduced to DegS via different mechanisms. Changes in nitrogen availability lead to the alteration of gene expression in B. subtilis (8). This nitrogen regulation is mediated by two transcriptional regulators, GlnR and TnrA. GlnR is a repressor of the ureABC, glnR-glnA, and tnrA genes, whereas TnrA is a global regulator that exerts positive and negative regulation of many genes (3, 7, 8, 42, 47). In nitrogen-rich environments, glutamine synthetase (GS), the gene product of glnA, is feedback inhibited, resulting in repression of glnA expression via GlnR. On the other hand, feedback-inhibited GS inhibits the global regulator TnrA by protein-protein interaction (44). Since GS is the only means for ammonium assimilation in B. subtilis (2) and inactivating mutations in this enzyme result in high-level expression of glnRA in medium containing excess nitrogen, GS has been proposed to be a monitor for the nitrogen status of the cell (31, 33). Thus, the genes under nitrogen regulation are regulated by either GlnR or TnrA through GS, which senses the nitrogen status in the cell (8, 42). Production of the extracellular neutral and alkaline proteases, encoded by nprE and aprE, respectively, is subject to regulation by the DegS-Degu two-component system. Since the degradation products of these proteases could supply the cells with a nitrogen source, it may be possible that the expression of the degS and/or Degu gene is subject to regulation by nitrogen metabolism. To investigate whether the degS-Degu two-component system is influenced by nitrogen availability, we examined the effect of deletion of the glnA gene, whose gene product (glutamine) supplies nitrogen for the synthesis of about 25% of nitrogen-containing compounds in the cell (28). In this report, we show that there are two promoters before the Degu gene, namely, the upstream P2 and the downstream P3 promoters, and that they are under the regulation of GlnA-TnrA and DegS-Degu, respectively.
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binding of response regulator Degu to the apre promoter is inhibited by rapg which is counteracted by extracellular phrg in bacillus subtilis
Molecular Microbiology, 2003Co-Authors: Mitsuo Ogura, Naotake Ogasawara, Kana Shimane, Kei Asai, Teruo TanakaAbstract:Summary We screened the putative rap-phr ( r esponse regulator a spartyl-phosphate p hosphatase- ph osphatase r egulator) systems identified in the Bacillus subtilis genome for a rap gene that affects aprE ( alkaline protease gene) expression by using a multicopy plasmid. We found that rapG was involved in the regulation of aprE , which belongs to the regulon of Degu, the response regulator of the DegS-Degu two-component system. Disruption of rapG and phrG resulted in enhancement and reduction of aprE-lacZ expression, respectively, suggesting that PhrG inhibits RapG activity. Addition of 1‐30 nM of a synthetic pentapeptide (PhrG; NH 2 -EKMIG-COOH) to the phrG disruptant completely rescued aprE-lacZ expression, indicating that the PhrG peptide is indeed involved in aprE-lacZ expression. Surprisingly, either introduction of multicopy phrG or addition of the PhrG peptide at high concentrations (100‐300 nM) to the phrG cells decreased aprE-lacZ expression. These results are reminiscent of the previous observation that at higher concentrations the PhrC peptide inhibits srfA-lacZ expression directed by ComA, the regulator of the ComP-ComA two-component system. Because the Rap proteins belong to a family of aspartyl protein phosphatases, we tried to investigate the possible influence of RapG on dephosphorylation of Degu-P (phosphorylated Degu) in vitro . RapG, however, did not affect dephosphorylation of Degu-P under the adopted experimental conditions. Therefore, we hypothesized that RapG might inhibit the binding activity of Degu to the target promoters. We analysed the interaction of Degu and RapG using the aprE promoter and another target, a comK promoter. Gel shift analysis revealed that RapG served as the inhibitor of Degu binding to the promoter regions of aprE and comK and that this inhibition was counteracted by the PhrG peptide.
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dna microarray analysis of bacillus subtilis Degu coma and phop regulons an approach to comprehensive analysis of b subtilis two component regulatory systems
Nucleic Acids Research, 2001Co-Authors: Mitsuo Ogura, Hirotake Yamaguchi, Kenichi Yoshida, Yasutaro Fujita, Teruo TanakaAbstract:We have analyzed the regulons of the Bacillus subtilis two-component regulators Degu, ComA and PhoP by using whole genome DNA microarrays. For these experiments we took the strategy that the response regulator genes were cloned downstream of an isopropyl-β-d-thiogalactopyranoside-inducible promoter on a multicopy plasmid and expressed in disruptants of the cognate sensor kinase genes, degS, comP and phoR, respectively. The feasibility of this experimental design to detect target genes was demonstrated by the following two results. First, expression of lacZ fusions of aprE, srfA and ydhF, the target genes of Degu, ComA and PhoP, respectively, was stimulated in their cognate sensor kinase-deficient mutants upon overproduction of the regulators. Secondly, by microarray analysis most of the known target genes for the regulators were detected and, where unknown genes were found, the regulator dependency of several of them was demonstrated. As the mutants used were deficient in the kinase genes, these results show that target candidates can be detected without signal transduction. Using this experimental design, we identified many genes whose dependency on the regulators for expression had not been known. These results suggest the applicability of the strategy to the comprehensive transcription analysis of the B.subtilis two-component systems.
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bacillus subtilis Degu acts as a positive regulator for comk expression
FEBS Letters, 1996Co-Authors: Mitsuo Ogura, Teruo TanakaAbstract:Bacillus subtilis ComK plays a critical role in competence development. We report that B. subtilis degR, a positive regulator for exoenzyme production, is negatively regulated by overproduced ComK caused by a mecA null mutation. To identify a positive regulator for comK expression in the mecA background, mutations that allowed the degR gene to be expressed were screened in Tn10 transposon insertion mutants. As a result, we identified Degu insertion mutations as those having such a property. The Degu mutation reduced comK-lacZ expression in a competence medium in both the wild-type and mecA cells in sporulation and competence media. These results indicate that the Degu gene product acts as a positive regulator for comK expression even under the condition where the negative regulation of comK by MecA is released.