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京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第19761号 / 農博第2157号 / 新制||農||1039(附属図書館) / 学位論文||H28||N4977(農学部図書室) / 32797 / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 三上 文三, 教授 加納 健司, 教授 喜多 恵子 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

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Thesis (MSc)--University of Stellenbosch, 2000. / ENGLISH ABSTRACT: The wine yeast Saccharomyces cerevisiae has the ability to utilize several different nitrogenous compounds to fulfill its metabolic requirements. Based upon different growth rates of the yeast in a particular nitrogen source, nitrogen compounds have been classified as either good or poor nitrogen sources. In an environment which contains different quality nitrogen sources, such as grape must, the yeast first utilizes good and then the poor nitrogen sources. This discrimination between good and poor nitrogen sources is referred to as nitrogen catabolite repression (NCR). Examples of good nitrogen sources are ammonia, glutamine and asparagine. Nitrogen sources such as allantoin, y-aminobutyrate (GABA), arginine and proline are poor quality nitrogen sources. Several regulatory proteins, Ure2p, Gln3p, Da180p,Gat1pand Deh1p, mediate NCR in S. cerevisiae. These trans-acting factors regulate transcription of NCR sensitive genes. All these proteins, except Ure2p, bind cis-acting elements in the promoters of genes that are responsible for degradation of poor nitrogen sources. Gln3p is an activator of NCR sensitive genes in the absence of good nitrogen sources. The predominant mechanism by which NCR functions is by using Ure2p to inactivate the activator Gln3p in the presence of a good nitrogen source. Several research groups have studied the Ure2p, mainly due to its prion-like characteristics. The Ure2p has two domains: a prion inducing domain located in the N-terminal region and a NCR regulatory domain located in the C-terminal domain. The aims of this study were (i) to determine the part of the C-terminal domain which is responsible for NCR, (ii) to establish if ure2 deletion mutants produce less ethyl carbamate during wine fermentations and (iii) if NCR functions in industrial yeast strains. Nested deletions of the URE2 gene revealed that the NCR regulatory domain resides in the last ten amino acids of the Ure2p. This was established by Northern blot analysis on the NCR sensitive genes DAL5, CAN1, and GAP1 genes. Ethyl carbamate in wine is produced by spontaneous chemical reaction between urea and ethanol in wine. Urea is produced by S. cerevisiae during the metabolism of arginine. Arginine is degraded to ornithine and urea by arginase, the product of the CAR1 gene. Degradation of urea by S. cerevisiae is accomplished by urea amidolyase, a bi-functional enzyme and product of the DUR1,2 gene which is subject to NCR. This study investigated if a ure2 mutant strain produced less ethyl carbamate during wine fermentations. Wine fermentations were conducted with diploid laboratory strains: a ure2 mutant strain and its isogenic wild type strain. GC/MS analysis of the wine revealed that the ure2 mutant produced less ethyl carbamate but more ethanol than the wild type strain when arginine, di-ammoniumphosphate, asparagine or glutamine were added as nitrogen sources, in combinations and separately. There was no significant difference between the wild type fermentation and the ure2 mutant fermentation when no nitrogen was added. It was found that a combination between the deletion of URE2 and the addition of a good nitrogen source resulted in lower levels of ethyl carbamate. High density micro array analysis done on an industrial strain wine yeast in Chardonnay grape must revealed that the GAP1, CAN1, CAR1 and DUR1,2 genes, responsible for transport and metabolism of arginine and degradation of urea, are NCR sensitive. These data strongly suggest that NCR functions in industrial yeast strains. / AFRIKAANSE OPSOMMING: Die wyngis Saccharomyces cerevisiae kan verskillende stikstofbronne gebruik om in sy stikstofbehoeftes te voldoen. Stikstofbronne word as goeie of swak stikstofbronne geklassifiseer op grond van die groeitempo van die gis op die betrokke stikstofbron. 'n Goeie stikstofbron laat die gis vinniger groei as wat dit op 'n swak stikstofbron sou groei. In omgewings soos druiwemos waar daar 'n verskeidenheid van stikstofbronne teenwoordig is, sal die gis eers die goeie bronne en daarna die swak bronne benut. Stikstofbronne soos ammonium, asparagien en glutamien word geklassifiseer as goeie bronne. Allantoïen, y-amino-butaraat (GABA), prolien en arginien word as swak stikstofbronne geklassifiseer. Die meganisme waarmee S. cerevisiae tussen die stikstofbronne onderskei, staan as stikstof kataboliet onderdrukking (NCR) bekend. Die proteïene wat vir verantwoordelik is NCR naamlik Ure2p, Gln3p, Gat1 p, Dal80p en Deh1 p, bind met die uitsondering van Ure2p, almal aan cis-werkende elemente in die promoters van NCR-sensitiewe gene. Die trans-werkende faktore reguleer die transkripsie van NCR-sensitiewe gene. NCR werk hoofsaaklik deur die inhibering van Gln3p deur Ure2p in die teenwoordigheid van 'n goeie stikstofbron. Die oorgrote meerderheid NCR-sensitiewe gene word deur Gln3p in die afwesigheid van 'n goeie stikstofbron geaktiveer. Heelwat navorsing is op die prionvormings vermoë van Ure2p gedoen. Ure2p het twee domeine: 'n N-terminale domein wat vir prionvorming verantwoordelik is en die C-terminale domein waar die NCR funksie van Ure2p gesetel is. Die doel van die studie was (i) om te bepaal waar in die C-terminale domein van Ure2p die NCR regulering geleë is, (ii) of ure2 delesie mutante minder etielkarbamaat tydens wynfermentasies produseer en (iii) of NCR in industriële gisrasse funksioneel is. Delesie analises van URE2 het getoon dat die NCR regulerings domein in die laaste tien aminosure gesetel is. Dit is vas gestel m.b.v. noordlike klad tegniek analises op die OALS, CAN1 en GAP1 gene.Etielkarbamaat in wyn word deur die spontane chemiese reaksie tussen ureum en alkohol geproduseer. Ureum word gedurende die metabolisme van arginien in S. cerevisiae geproduseer. Arginien word deur arginase, produk van die CAR1 geen, na ornitien en ureum afgebreek. Die bi-funksionele ureum amidoliase, gekodeer deur die DUR1,2 geen, breek ureum na CO2 en NH/ af. As gevolg van die NCRsensitiwiteit van dié gene is ondersoek ingestel na In ure2 mutant se vermoë om minder etielkarbamaat tydens wynfermentasies te produseer. Chardonnay druiwemos is met In diploiede laboratorium ras en die isogeniese ure2 mutant gefermenteer. GC/MS analise op die wyn het getoon dat die ure2 mutant minder etielkarbamaat, maar meer alkohol in vergelyking met die wilde tipe gis produseer, as arginien, di-ammoniumfosfaat, asparagien en glutamien, afsonderlik of gesamentlik byvoeg is. Daar was egter nie In merkwaardige verskil tussen die fermentasies waar geen stikstof bygevoeg is nie. Dit dui daarop dat In kombinasie van In URE2 delesie en die byvoeging van stikstof etielkarbamaat vlakke verlaag. Mikro-skyfie analise van In industriële gis in Chardonnay mos het getoon dat die GAP1, CAN1, CAR1 en DUR1,2 gene wat verantwoordelik is vir die transport en metabolisme van arginien en degradasie van ureum, wel NCR-sensitief is. Dit dui daarop dat NCRwel in industriële gisrasse funksioneel is.

L'adaptation d'une bactérie aux changements de son environnement est contrôlée par un réseau de régulation large et complexe, faisant intervenir de nombreux acteurs et modules différents. Dans ce travail, nous avons étudiés un module de régulation spécifique, contrôlant l'adaptation de la bactérie Escherichia coli à un changement de sources de carbone. Dans un milieu contenant du glucose et de l'acétate, la croissance est divisée en deux phases : les bactéries utilisent préférentiellement le glucose et commencent à métaboliser l'acétate qu'après l'épuisement du glucose. En effet, la présence du glucose réprime la transcription d'un gène nécessaire à la croissance sur acétate, le gène acs (codant pour l'acétyl-CoA synthétase). Le mécanisme régulateur fait intervenir le facteur de transcription Crp-AMPc et le système de transfert de phosphate (PTS), qui permet l'import du glucose. Plusieurs modèles décrivent en détail la cascade de réactions moléculaires à l'origine de cette « répression catabolique ». Cependant, certaines de nos observations expérimentales ne sont pas correctement prédites par les modèles actuels. Ces modèles doivent être révisés ou complétés. L'outil majeur que nous employons pour les expériences est la fusion transcriptionnelle : une région promotrice fusionnée en amont d'un gène rapporteur (GFP, luciferase). Avec ces constructions, nous mesurons la dynamique de l'expression génique dans différentes souches (mutants) et différentes conditions environnementales. Les observations à l'échelle de la population sont corroborées par des mesures similaires à l'échelle de la cellule unique. Nous utilisons cette même technologie pour construire de petits systèmes synthétiques qui sondent davantage le phénomène de répression catabolique. Nous avons ainsi créé un interrupteur génétique dont le fonctionnement est contrôlé par le flux glycolytique et nous avons construit un petit système de communication intercellulaire basé sur la molécule AMPc. Enfin, nous proposons une manière originale de mesurer l'état métabolique des cellules en utilisant la dépendance énergétique de la luciferase. / The adaptation of bacteria to changes in their environment is controlled by a large and complex regulatory network involving many different actors and modules. In this work, we have studied a specific module controlling the adaptation of Escherichia coli to a change in carbon sources. In a medium containing glucose and acetate, growth is divided into two phases : the bacteria preferentially use glucose and start to metabolize acetate only after glucose exhaustion. Indeed, the presence of glucose represses the transcription of a gene needed for growth on acetate : the acs gene (coding for acetyl-CoA synthetase). The regulatory mechanism involves the Crp-cAMP regulator and the phosphate transfer system (PTS), which is responsible for glucose import. Several models describe the cascade of molecular reactions responsible for this « catabolite repression ». However, our work shows that many of our experimental observations are incorrectly predicted by current models. These models have to be amended.We use transcriptional fusion, i.e., the fusion of a promoter region upstream of a reporter gene (GFP, luciferase), to measure the dynamics of gene expression in different genetic backgrounds and environmental conditions. Observations at the population level are corroborated by similar measurements at the single cell level. We use this same technology to construct small synthetic systems that probe further aspects of the phenomenon of catabolite repression. We have thus created a genetic toggle switch controlled by the glycolytic flux and we have built an inter-cellular communication system mediated by cAMP. Finally, we propose a novel way to measure the metabolic state of cells by using the energy dependence of the luciferase enzyme.

Clostridium perfringens is a versatile pathogen capable of causing a wide array of diseases, ranging from clostridial food poisoning to tissue infections such as gas gangrene. An important factor in virulence as well as in the distribution of C. perfringens is its ability to form an endospore. The symptoms of C. perfringens food poisoning are directly correlated to the release of an enterotoxin at the end of the sporulation process. The sporulation process in C. perfringens is subject to carbon catabolite repression (CCR) by sugars, especially glucose. CCR is a regulatory pathway that alters transcription based on carbon source availability. In Gram-positive bacteria, the HPr kinase/phosphatase is responsible for this nutritional sensing by phosphorylating or dephosphorylating the serine-46 residue of HPr. HPr-Ser-P then forms a complex with the transcriptional regulator CcpA to regulate transcription. We were able to show here that purified recombinant C. perfringens HPr kinase/phosphatase was able to phosphorylate the serine-46 residue of HPr. When the codon for this serine residue is mutated through PCR mutagenesis to encode alanine, phosphorylation could not take place. We have also shown that in gel retardation assays, CcpA and HPr-Ser-P were able to bind to two DNA fragments containing putative C. perfringens CRE-sites, sequences where CcpA binds to regulate transcription. The genome sequence of a food poisoning strain of C. perfringens was searched for potential CRE-sites using degenerate sequences designed to match those CRE-sites CcpA was shown to bind. DNA fragments containing these newly identified CRE-sites were then used in gel retardation assays to determine whether CcpA binds to these CRE-sites, making them candidates for CCR regulation. These results, combined with comparisons of metabolic characteristics of a ccpA- strain versus wild-type C. perfringens, provide evidence that CcpA participates in the regulation of carbon catabolite repression in the pathogenic bacterium C. perfringens / Master of Science

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Carbon catabolite repression in A. nidulans is a regulatory system which allows the organism to utilize the most preferable carbon source by repressing the expression of genes encoding enzymes utilizing alternative carbon sources. A ubiquitination pathway was shown to be one of the key mechanisms which regulate carbon source utilization, when creB was found to encode a deubiquitinating enzyme. Strains containing mutations in creB show loss of repression for some metabolic pathways in carbon catabolite repressing conditions, and also grow very poorly on several sole carbon sources such as quinate and proline, suggesting CreB plays multiple roles in the cell. This work describes the analysis of the interaction of CreB with CreA, and with PrnB and QutD. Various epitope-tagged versions of CreA were expressed in A. nidulans, and an internally located HA-epitope tag was found to allow detection of CreA using Western analysis. A diploid strain was constructed between strains containing HA-tagged CreA and FLAG-tagged CreB. When CreB was immunoprecipitated, HA-tagged CreA was also precipitated in the diploid, indicating that CreA and CreB are present in a complex in vivo. To determine whether CreA is a ubiquitinated protein, a version of CreA that was tagged with both an HA epitope and a His-tag was expressed in A. nidulans, and protein extracts were precipitated with an UbiQapture™-Q matrix. Western analysis was used to show that CreA was present in the precipitate. These findings suggest that CreA is a ubiquitinated protein, and a target of the CreB deubiquitination enzyme. To determine whether the proline permease (PrnB) is a direct substrate of CreB, plasmids to express epitope-tagged versions of PrnB were constructed and introduced into the prnB mutant strain. No tagged protein could be detected by Western analysis, even when these constructs were over-expressed from the gpdA promoter. However, a construct to express an HA epitope tagged version of quinate permease (QutD) fully complemented the qutD mutant strain, and HA-tagged QutD could be easily detected in Western analysis when probed with the anti-HA monoclonal antibody. A diploid strain was made between a complementing transformant and a strain expressing a FLAG-tagged CreB construct. When QutDHA was immunoprecipitated, CreBFLAG was detected in the immunoprecipitate of the diploid. A proportion of QutDHA was also co-precipitated in the diploid when CreBFLAG was immunoprecipitated. Thus, CreB is present in a complex with QutD in vivo. Further results showed that the concentration of QutD in the cell is lower in a creB null mutant background than in the wild-type background, indicating that deubiquitination is required to prevent protein turnover. Northern analysis of mRNA showed that the failure of creB mutant strains to grow on quinate medium was not due to a failure of transcriptional induction of qutD, as the amount of mRNA was not lower in a creB1937 mutant background compared to the wild-type. Furthermore, experiments were undertaken that showed that QutD is a ubiquitinated protein. These findings suggest that quinate permease is regulated through deubiquitination involving the CreB deubiquitination protein in A. nidulans. In addition to the candidate protein approach asking whether CreA is a substrate of CreB, a proteomics approach was also used to identify proteins that interact with CreA. However, no clear interacting proteins were identified using this approach. / Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2008

Polycyclic aromatic hydrocarbons, produced by anthropological and natural activities, are hazardous through formation of oxidative radicals and DNA adducts. Growth of Mycobacterium sp. strain KMS, isolated from a contaminated soil, on the model hydrocarbon pyrene induced specific proteins. My work extends the study of isolate KMS to the gene level to understand the pathways and regulation of pyrene utilization. Genes encoding pyrene-induced proteins were clustered on a 72 kb section on the KMS chromosome but some also were duplicated on plasmids. Skewed GC content and presence of integrase and transposase genes suggested horizontal transfer of pyrene-degrading gene islands that also were found with high conservation in five other pyrene-degrading Mycobacterium isolates. Transcript analysis found both plasmid and chromosomal genes were induced by pyrene. These processes may enhance the survival of KMS in hydrocarbon-contaminated soils when other carbon sources are limited. KMS also grew on benzoate, confirming the functionality of an operon containing genes distinct from those in other benzoate-degrading bacteria. Growth on benzoate but not on pyrene induced a gene, benA, encoding a benzoate dioxygenase α-subunit, but not the pyrene-induced nidA encoding a pyrene dioxygenase α-subunit; the differential induction correlated with differences in promoter sequences. Diauxic growth occurred when pyrene cultures were amended with benzoate or acetate, succinate, or fructose, and paralleled delayed expression of nidA. Single phase growth and normal expression of benA was observed for benzoate single and mixed cultures. The nidA promoters had potential cAMP-CRP binding sites, suggesting that cAMP could be involved in carbon repression of pyrene metabolism. Growth on benzoate and pyrene requires gluconeogenesis. Intermediary metabolism in isolate KMS involves expression from genes encoding a novel malate:quinone oxidoreductase and glyoxylate shunt enzymes. Generation of C3 structures involves transcription of genes encoding malic enzyme, phosphoenolpyruvate carboxykinase, and phosphoenolpyruvate synthase. Carbon source modified the transcription patterns for these genes. My findings are the first to show duplication of pyrene-degrading genes on the chromosome and plasmids in Mycobacterium isolates and expression from a unique benzoate-degrading operon. I clarified the routes for intermediary metabolism leading to gluconeogenesis and established a potential role for cAMP-mediated catabolite repression of pyrene utilization.