Etude de 4 gènes paralogues de Bacillus subtilis codant des enzymes maliques

Diplôme : Dr. d'UniversiteFour paralogous genes have been identified in the Bacillus subtilis genome (maeA et malS, ytsJ et mleA), whose products are homologous to malic enzymes, that catalyze the interconversion of malate and pyruvate. The purified proteins do possess a malic enzyme activity, but have various cofactor specificities. MleA is strictly NAD-dependent, MaeA and MalS have a catalytic efficacity four to ten fold higher in the presence of NAD, and YtsJ has a catalytic efficacity seventy fold higher in the presence of NADP. The genes have various transcription profils: ytsJ is constitutively expressed, malS is poorly expressed in all conditions tested, mleA is weakly expressed in minimal medium, but strongly expressed in rich medium, and maeA transcription is specifically induced by the presence of malate. Genetic and biochemical anlysis showed that the unkown two-component systems MalK/MalR directly control the activation of maeA transcription in response to the presence of malate. The phenotypic analysis of mutants of these genes showed that ytsJ plays a major role in the use of malate for the growth, whereas maeA, malS and mleA play a minor role, different to tha of ytsJ, for which they can substitute one for another. A ytsJ knock-out can not be compensated for by the overexpression of maeA, malS or mleA nor by the overexpression of the two Escherichia coli malic enzymes, SfcA and MaeB, respectively NAD- and NADP-dependent. The incomplete complementation by the overxpression of E. coli transhydrogenase UdhA indicates that the specific role of YtsJ is linked to the metabolism of NADP.Bacillus subtilis possède quatre gènes paralogues (maeA, malS, ytsJ et mleA), dont les produits sont homologues à des enzymes maliques, catalysant l'interconversion du malate et du pyruvate. Les protéines purifiées possèdent une activité enzyme malique, et présentent des spécificités de cofacteurs distinctes. MleA est strictement dépendante du NAD, MaeA et MalS ont une efficacité catalytique quatre à dix fois supérieure en présence de NAD, et YtsJ a une efficacité catalytique soixante dix fois supérieure en présence de NADP. Leurs profils d'expression sont différents : ytsJ est transcrit constitutivement ; malS est faiblement transcrit dans les conditions testées ; mleA est faiblement transcrit en milieu minimun mais fortement transcrit en milieu complexe ; et la transcription de maeA est spécifiquement induite par la présence de malate. Des approches génétiques et biochimiques ont montré que le système MalK/MalR contrôle directement l'activation de la transcription de maeA en réponse à la présence de malate. L'analyse phénotypique des mutants de ces gènes a montré que ytsJ joue un rôle physiologique majeur dans l'utilisation du malate pour la croissance et que les trois autres gènes maeA, malS et mleA jouent un rôle mineur, distinct de celui d'ytsJ. L'inactivation d'ytsJ ne peut être compensée par la surexpression de malS, maeA ou mleA, ni par celle des enzymes maliques de Escherichia coli, SfcA et MaeB, respectivement NAD- et NADP-dépendantes. La complémentation partielle par la surexpression de la transhydrogénase UdhA d'E. coli indique que le rôle spécifique d'YtsJ est lié au métabolisme du NADP

Etude de 4 gènes paralogues de Bacillus subtilis codant des enzymes maliques

Diplôme : Dr. d'UniversiteFour paralogous genes have been identified in the Bacillus subtilis genome (maeA et malS, ytsJ et mleA), whose products are homologous to malic enzymes, that catalyze the interconversion of malate and pyruvate. The purified proteins do possess a malic enzyme activity, but have various cofactor specificities. MleA is strictly NAD-dependent, MaeA and MalS have a catalytic efficacity four to ten fold higher in the presence of NAD, and YtsJ has a catalytic efficacity seventy fold higher in the presence of NADP. The genes have various transcription profils: ytsJ is constitutively expressed, malS is poorly expressed in all conditions tested, mleA is weakly expressed in minimal medium, but strongly expressed in rich medium, and maeA transcription is specifically induced by the presence of malate. Genetic and biochemical anlysis showed that the unkown two-component systems MalK/MalR directly control the activation of maeA transcription in response to the presence of malate. The phenotypic analysis of mutants of these genes showed that ytsJ plays a major role in the use of malate for the growth, whereas maeA, malS and mleA play a minor role, different to tha of ytsJ, for which they can substitute one for another. A ytsJ knock-out can not be compensated for by the overexpression of maeA, malS or mleA nor by the overexpression of the two Escherichia coli malic enzymes, SfcA and MaeB, respectively NAD- and NADP-dependent. The incomplete complementation by the overxpression of E. coli transhydrogenase UdhA indicates that the specific role of YtsJ is linked to the metabolism of NADP.Bacillus subtilis possède quatre gènes paralogues (maeA, malS, ytsJ et mleA), dont les produits sont homologues à des enzymes maliques, catalysant l'interconversion du malate et du pyruvate. Les protéines purifiées possèdent une activité enzyme malique, et présentent des spécificités de cofacteurs distinctes. MleA est strictement dépendante du NAD, MaeA et MalS ont une efficacité catalytique quatre à dix fois supérieure en présence de NAD, et YtsJ a une efficacité catalytique soixante dix fois supérieure en présence de NADP. Leurs profils d'expression sont différents : ytsJ est transcrit constitutivement ; malS est faiblement transcrit dans les conditions testées ; mleA est faiblement transcrit en milieu minimun mais fortement transcrit en milieu complexe ; et la transcription de maeA est spécifiquement induite par la présence de malate. Des approches génétiques et biochimiques ont montré que le système MalK/MalR contrôle directement l'activation de la transcription de maeA en réponse à la présence de malate. L'analyse phénotypique des mutants de ces gènes a montré que ytsJ joue un rôle physiologique majeur dans l'utilisation du malate pour la croissance et que les trois autres gènes maeA, malS et mleA jouent un rôle mineur, distinct de celui d'ytsJ. L'inactivation d'ytsJ ne peut être compensée par la surexpression de malS, maeA ou mleA, ni par celle des enzymes maliques de Escherichia coli, SfcA et MaeB, respectivement NAD- et NADP-dépendantes. La complémentation partielle par la surexpression de la transhydrogénase UdhA d'E. coli indique que le rôle spécifique d'YtsJ est lié au métabolisme du NADP

Interference amplified SERS on (multi)layered substrates

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Interference amplified SERS on (multi)layered substrates

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YtsJ Has the Major Physiological Role of the Four Paralogous Malic Enzyme Isoforms in Bacillus subtilis

The Bacillus subtilis genome contains several sets of paralogs. An extreme case is the four putative malic enzyme genes maeA, malS, ytsJ, and mleA. maeA was demonstrated to encode malic enzyme activity, to be inducible by malate, but also to be dispensable for growth on malate. We report systematic experiments to test whether these four genes ensure backup or cover different functions. Analysis of single- and multiple-mutant strains demonstrated that ytsJ has a major physiological role in malate utilization for which none of the other three genes could compensate. In contrast, maeA, malS, and mleA had distinct roles in malate utilization for which they could compensate one another. The four proteins exhibited malic enzyme activity; MalS, MleA, and MaeA exhibited 4- to 90-fold higher activities with NAD(+) than with NADP(+). YtsJ activity, in contrast, was 70-fold higher with NADP(+) than with NAD(+), with K(m) values of 0.055 and 2.8 mM, respectively. lacZ fusions revealed strong transcription of ytsJ, twofold higher in malate than in glucose medium, but weak transcription of malS and mleA. In contrast, mleA was strongly transcribed in complex medium. Metabolic flux analysis confirmed the major role of YtsJ in malate-to-pyruvate interconversion. While overexpression of the NADP-dependent Escherichia coli malic enzyme MaeB did not suppress the growth defect of a ytsJ mutant on malate, overexpression of the transhydrogenase UdhA from E. coli partially suppressed it. These results suggest an additional physiological role of YtsJ beyond that of malate-to-pyruvate conversion

Improved bioluminescent detection of pesticides

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Modélisation d'un spectromètre compact basé sur la transformée de Fourier d'ondes stationnaires en optique intégrée

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The Bacillus subtilis ywkA gene encodes a malic enzyme and its transcription is activated by the YufL/YufM two-component system in response to malate

International audienceA transcriptome comparison of a wild-type Bacillus subtilis strain growing under glycolytic or gluconeogenic conditions was performed. In particular, it revealed that the ywkA gene, one of the four paralogues putatively encoding a malic enzyme, was more transcribed during gluconeogenesis. Using a lacZ reporter fusion to the ywkA promoter, it was shown that ywkA was specifically induced by external malate and not subject to glucose catabolite repression. Northern analysis confirmed this expression pattern and demonstrated that ywkA is cotranscribed with the downstream ywkB gene. The ywkA gene product was purified and biochemical studies demonstrated its malic enzyme activity, which was 10-fold higher with NAD than with NADP ( k cat / K m 102 and 10 s −1 mM −1 , respectively). However, physiological tests with single and multiple mutant strains affected in ywkA and/or in ywkA paralogues showed that ywkA does not contribute to efficient utilization of malate for growth. Transposon mutagenesis allowed the identification of the uncharacterized YufL/YufM two-component system as being responsible for the control of ywkA expression. Genetic analysis and in vitro studies with purified YufM protein showed that YufM binds just upstream of ywkA promoter and activates ywkA transcription in response to the presence of malate in the extracellular medium, transmitted by YufL. ywkA and yufL / yufM could thus be renamed maeA for ma lic e nzyme and malK / malR for mal ate k inase sensor/ mal ate response r egulator, respectively

Bimodal Near-infrared Optical and Photoacoustic Imaging Agents Based on Low Energy Absorbing Lanthanide Complexes.

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The First Generation of β-Galactosidase-Responsive Prodrugs Designed for the Selective Treatment of Solid Tumors in Prodrug Monotherapy†

International audienceGalactoside prodrugs have been designed that can be selectively activated by lysosomal β-galactosidase located inside cancer cells expressing a specific tumor-associated receptor. This efficient enzymatic process triggers a potent cytotoxic effect, releasing the potent antimitotic agent MMAE and allowing the destruction of both receptor-positive and surrounding receptor-negative tumor cells