The CYP71AZ P450 Subfamily: A Driving Factor for the Diversification of Coumarin Biosynthesis in Apiaceous Plants

International audienceThe production of coumarins and furanocoumarins (FCs) in higher plants is widely considered a model illustration of the adaptation of plants to their environment. In this report, we show that the multiplication of cytochrome P450 variants within the CYP71AZ subfamily has contributed to the diversification of these molecules. Multiple copies of genes encoding this enzyme family are found in Apiaceae, and their phylogenetic analysis suggests that they have different functions within these plants. CYP71AZ1 from Ammi majus and CYP71AZ3, 4, and 6 from Pastinaca sativa were functionally characterized. While CYP71AZ3 merely hydroxylated esculetin, the other enzymes accepted both simple coumarins and FCs. Superimposing in silico models of these enzymes led to the identification of different conformations of three regions in the enzyme active site. These sequences were subsequently utilized to mutate CYP71AZ4 to resemble CYP71AZ3. The swapping of these regions lead to significantly modified substrate specificity. Simultaneous mutations of all three regions shifted the specificity of CYP71AZ4 to that of CYP71AZ3, exclusively accepting esculetin. This approach may explain the evolution of this cytochrome P450 family regarding the appearance of FCs in parsnip and possibly in the Apiaceae

Discutindo a educação ambiental no cotidiano escolar: desenvolvimento de projetos na escola formação inicial e continuada de professores

A presente pesquisa buscou discutir como a Educação Ambiental (EA) vem sendo trabalhada, no Ensino Fundamental e como os docentes desta escola compreendem e vem inserindo a EA no cotidiano escolar., em uma escola estadual do município de Tangará da Serra/MT, Brasil. Para tanto, realizou-se entrevistas com os professores que fazem parte de um projeto interdisciplinar de EA na escola pesquisada. Verificou-se que o projeto da escola não vem conseguindo alcançar os objetivos propostos por: desconhecimento do mesmo, pelos professores; formação deficiente dos professores, não entendimento da EA como processo de ensino-aprendizagem, falta de recursos didáticos, planejamento inadequado das atividades. A partir dessa constatação, procurou-se debater a impossibilidade de tratar do tema fora do trabalho interdisciplinar, bem como, e principalmente, a importância de um estudo mais aprofundado de EA, vinculando teoria e prática, tanto na formação docente, como em projetos escolares, a fim de fugir do tradicional vínculo “EA e ecologia, lixo e horta”.Facultad de Humanidades y Ciencias de la Educació

Functional genomics of microbial degradation of chloromethane

Le chlorométhane (CH3Cl) est un composé organique volatil essentiellement produit naturellement et responsable de 15% de la dégradation de l'ozone stratosphérique due aux composés halogénés. L'estimation du budget global du CH3Cl est incertaine et sous-estime l'importance des émissions végétales et de sa dégradation par les microorganismes.Cette thèse a eu pour but de développer la compréhension des bases moléculaires de la dégradation bactérienne du CH3Cl, avec aussi comme perspective la valorisation des ressources génétiques de l'environnement pour la dépollution. Une approche combinant génomique comparative et fonctionnelle a été développée pour étudier l'adaptation au CH3Cl chez Methylobacterium extorquens CM4, bactérie aérobie pour laquelle une voie d'utilisation du CH3Cl a été identifiée et dont le génome a été séquencé au Genoscope.L'analyse du génome de CM4 a révélé l'existence d'un plasmide de 380 kb porteur des gènes connus de la voie d'utilisation du CH3Cl et de la biosynthèse de la cobalamine et du tétrahydrofolate, les deux cofacteurs essentiels à cette voie.L'analyse différentielle du protéome de CM4 cultivé en présence et en absence de CH3Cl a confirmé la voie d'utilisation précédemment proposée, et permis d'identifier de nouvelles protéines associées au métabolisme du CH3Cl.Un dernier volet du travail a été l'étude de la diversité des bactéries CH3Cl-dégradantes associées aux plantes. Trois souches CH3Cl-dégradantes de la phyllosphère ont été isolées à partir de feuilles d'Arabidopsis thaliana, une plante connue pour émettre du CH3Cl. Les résultats de ce travail serviront de base aux études futures de la dégradation bactérienne du chlorométhane.Chloromethane (CH3Cl) is a volatile organic compound of mainly natural origin. It accounts for at least 15% of chlorine-catalysed stratospheric ozone depletion. Obtaining reliable estimates of the global CH3Cl budget is difficult due to the incomplete inventory of CH3Cl sources and sinks, including plant emissions and microbial degradation.The aim of this PhD thesis was to better understand the molecular basis of microbial degradation of CH3Cl, also with the perspective of applying microbial and genetic resources to bioremediation of polluted environments. Approaches involving comparative and functional genomics were developed to study the adaptation to CH3Cl of Methylobacterium extorquens CM4, the aerobic bacterial strain in which a degradation pathway for CH3Cl was previously identified, and whose genome sequence is now known.Analysis of the genome of strain CM4 revealed the presence of a 380 kb plasmid harbouring the already characterised genes of the known pathway for CH3Cl utilisation, as well as genes involved in the biosynthesis of cobalamin and tetrahydrofolate, two cofactors essential for CH3Cl degradation by this pathway.Differential proteomic analysis of strain CM4 grown in the presence or the absence of CH3Cl confirmed this pathway and also enabled the identification of new proteins associated with CH3Cl metabolism.In addition, the diversity of CH3Cl-degrading bacteria associated with the phyllosphere was investigated. Three CH3Cl-degrading strains of the phyllosphere were isolated from leaves of Arabidopsis thaliana, a plant known to emit CH3Cl.The obtained results will serve as the basis for future studies of bacterial CH3Cl degradation

Functional genomics of microbial degradation of chloromethane

Le chlorométhane (CH3Cl) est un composé organique volatil essentiellement produit naturellement et responsable de 15% de la dégradation de l'ozone stratosphérique due aux composés halogénés. L'estimation du budget global du CH3Cl est incertaine et sous-esChloromethane (CH3Cl) is a volatile organic compound of mainly natural origin. It accounts for at least 15% of chlorine-catalysed stratospheric ozone depletion. Obtaining reliable estimates of the global CH3Cl budget is difficult due to the incomplete in

Génomique fonctionnelle de la dégradation microbienne du chlorométhane

Le chlorométhane (CH3Cl) est un composé organique volatil essentiellement produit naturellement et responsable de 15% de la dégradation de l ozone stratosphérique due aux composés halogénés. L estimation du budget global du CH3Cl est incertaine et sous-estime l importance des émissions végétales et de sa dégradation par les microorganismes.Cette thèse a eu pour but de développer la compréhension des bases moléculaires de la dégradation bactérienne du CH3Cl, avec aussi comme perspective la valorisation des ressources génétiques de l environnement pour la dépollution. Une approche combinant génomique comparative et fonctionnelle a été développée pour étudier l adaptation au CH3Cl chez Methylobacterium extorquens CM4, bactérie aérobie pour laquelle une voie d utilisation du CH3Cl a été identifiée et dont le génome a été séquencé au Genoscope.L analyse du génome de CM4 a révélé l existence d un plasmide de 380 kb porteur des gènes connus de la voie d utilisation du CH3Cl et de la biosynthèse de la cobalamine et du tétrahydrofolate, les deux cofacteurs essentiels à cette voie.L analyse différentielle du protéome de CM4 cultivé en présence et en absence de CH3Cl a confirmé la voie d utilisation précédemment proposée, et permis d identifier de nouvelles protéines associées au métabolisme du CH3Cl.Un dernier volet du travail a été l étude de la diversité des bactéries CH3Cl-dégradantes associées aux plantes. Trois souches CH3Cl-dégradantes de la phyllosphère ont été isolées à partir de feuilles d Arabidopsis thaliana, une plante connue pour émettre du CH3Cl. Les résultats de ce travail serviront de base aux études futures de la dégradation bactérienne du chlorométhane.Chloromethane (CH3Cl) is a volatile organic compound of mainly natural origin. It accounts for at least 15% of chlorine-catalysed stratospheric ozone depletion. Obtaining reliable estimates of the global CH3Cl budget is difficult due to the incomplete inventory of CH3Cl sources and sinks, including plant emissions and microbial degradation.The aim of this PhD thesis was to better understand the molecular basis of microbial degradation of CH3Cl, also with the perspective of applying microbial and genetic resources to bioremediation of polluted environments. Approaches involving comparative and functional genomics were developed to study the adaptation to CH3Cl of Methylobacterium extorquens CM4, the aerobic bacterial strain in which a degradation pathway for CH3Cl was previously identified, and whose genome sequence is now known.Analysis of the genome of strain CM4 revealed the presence of a 380 kb plasmid harbouring the already characterised genes of the known pathway for CH3Cl utilisation, as well as genes involved in the biosynthesis of cobalamin and tetrahydrofolate, two cofactors essential for CH3Cl degradation by this pathway.Differential proteomic analysis of strain CM4 grown in the presence or the absence of CH3Cl confirmed this pathway and also enabled the identification of new proteins associated with CH3Cl metabolism.In addition, the diversity of CH3Cl-degrading bacteria associated with the phyllosphere was investigated. Three CH3Cl-degrading strains of the phyllosphere were isolated from leaves of Arabidopsis thaliana, a plant known to emit CH3Cl.The obtained results will serve as the basis for future studies of bacterial CH3Cl degradation.STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF

The 380 kb pCMU01 Plasmid Encodes Chloromethane Utilization Genes and Redundant Genes for Vitamin B₁₂- and Tetrahydrofolate-Dependent Chloromethane Metabolism in <i>Methylobacterium extorquens</i> CM4: A Proteomic and Bioinformatics Study

<div><p>Chloromethane (CH₃Cl) is the most abundant volatile halocarbon in the atmosphere and contributes to the destruction of stratospheric ozone. The only known pathway for bacterial chloromethane utilization (<i>cmu</i>) was characterized in <i>Methylobacterium extorquens</i> CM4, a methylotrophic bacterium able to utilize compounds without carbon-carbon bonds such as methanol and chloromethane as the sole carbon source for growth. Previous work demonstrated that tetrahydrofolate and vitamin B₁₂ are essential cofactors of <i>cmuA</i>- and <i>cmuB</i>-encoded methyltransferases of chloromethane dehalogenase, and that the pathway for chloromethane utilization is distinct from that for methanol. This work reports genomic and proteomic data demonstrating that cognate <i>cmu</i> genes are located on the 380 kb pCMU01 plasmid, which drives the previously defined pathway for tetrahydrofolate-mediated chloromethane dehalogenation. Comparison of complete genome sequences of strain CM4 and that of four other <i>M. extorquens</i> strains unable to grow with chloromethane showed that plasmid pCMU01 harbors unique genes without homologs in the compared genomes (<i>bluB2</i>, <i>btuB</i>, <i>cobA</i>, <i>cbiD</i>), as well as 13 duplicated genes with homologs of chromosome-borne genes involved in vitamin B₁₂-associated biosynthesis and transport, or in tetrahydrofolate-dependent metabolism (<i>folC2</i>). In addition, the presence of both chromosomal and plasmid-borne genes for corrinoid salvaging pathways may ensure corrinoid coenzyme supply in challenging environments. Proteomes of <i>M. extorquens</i> CM4 grown with one-carbon substrates chloromethane and methanol were compared. Of the 49 proteins with differential abundance identified, only five (CmuA, CmuB, PurU, CobH2 and a PaaE-like uncharacterized putative oxidoreductase) are encoded by the pCMU01 plasmid. The mainly chromosome-encoded response to chloromethane involves gene clusters associated with oxidative stress, production of reducing equivalents (PntAA, Nuo complex), conversion of tetrahydrofolate-bound one-carbon units, and central metabolism. The mosaic organization of plasmid pCMU01 and the clustering of genes coding for dehalogenase enzymes and for biosynthesis of associated cofactors suggests a history of gene acquisition related to chloromethane utilization.</p></div

Proteomic analysis of differentially expressed proteins in chloromethane- and methanol-grown cultures of <i>M. extorquens</i> CM4.

a<p>MaGe database (<a href="http://www.genoscope.cns.fr/agc/mage" target="_blank">http://www.genoscope.cns.fr/agc/mage</a>).</p>b<p>Probability-based mowse score calculated using MASCOT software (Matrix Science, London, UK); error refers to mass accuracy; coverage refers to the percentage of the protein sequence covered by the matched peptides.</p>c<p>Spots indicated as “CH₃Cl” were only detected in the proteome of <i>M. extorquens</i> CM4 grown with chloromethane. Spots indicated as “+” were more abundant in chloromethane-grown cultures (or less abundant in methanol-grown cultures). Spots indicated as “−” were more abundant in methanol-grown cultures (i.e. less abundant in chloromethane-grown cultures). Factors of differential abundance were defined as follows:++(<b>−−</b>) 2- to 5-fold;+++(<b>−−</b>−) more than 5-fold.</p>d<p>NL, non linear p<i>I</i> range used in 2D-DIGE experiments.</p>e<p>Only found in strain CM4 (among the 8 <i>Methylobacterium</i> strains for which the complete genome sequence is known; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056598#pone.0056598-Marx1" target="_blank">[5]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056598#pone.0056598-Vuilleumier2" target="_blank">[11]</a>) and localized on plasmid pCMU01.</p>f<p>Multiple spots detected.</p>g<p>Mass spectrometry used to discriminate from Mchl_1712 displaying 86% sequence identity at the protein level.</p>h<p>n.d., not detected.</p>i<p>Mass spectrometry used to discriminate from Mchl_2317 displaying 96% sequence identity at the protein level.</p>j<p>No assigned gene name.</p>k<p>Mass spectrometry data did not allow us to discriminate between two homologs with 99% sequence identity (Mchl_2669/Mchl_4004).</p>l<p>Tandem mass spectrometry identification.</p

Gene redundancy in the biosynthesis of cofactors required for chloromethane utilization in <b><i>Methylobacterium extorquens</i></b><b> CM4.</b>

<p>Cbi, cobinamide; Cbl, cobalamin; Ado, adenosyl; DMB, dimethylbenzimidazole; NaMN, nicotinate mononucleotide. AdoCbl and tetrahydrofolate are essential cofactors of the <i>cmu</i> pathway <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056598#pone.0056598-Studer1" target="_blank">[6]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056598#pone.0056598-Studer3" target="_blank">[9]</a>. Transport and enzymatic reactions are shown with dotted and full arrows, respectively. Genes indicated in bold are located on the 380 kb plasmid pCMU01. Circled gene names encode proteins more abundant in chloromethane cultures. AdoCbl can be synthesized <i>de novo</i> by an aerobic biosynthesis pathway that incorporates cobalt (diamond), or obtained from a salvage pathway after internalization of preformed Cbi or Cbl. In prokaryotes, the cobalt needed for corrin ring synthesis may be incorporated into cells using the CorA transport system <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056598#pone.0056598-Niegowski1" target="_blank">[69]</a>, the putative transmembrane proteins CbtA and CbtB <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056598#pone.0056598-Rodionov1" target="_blank">[14]</a>, the Resistance-Nodulation-Division (RND)-type <u>C</u>o²⁺/<u>Z</u>n²⁺/<u>C</u>d²⁺ efflux system CzcA <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056598#pone.0056598-Goldberg1" target="_blank">[27]</a>, or the Icu transporter <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056598#pone.0056598-Chou1" target="_blank">[70]</a>. The TonB-dependent Btu system imports preformed corrinoid compounds <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056598#pone.0056598-Cadieux1" target="_blank">[28]</a>. We hypothesize that BluB-related proteins link AdoCbl and H₄F <i>de novo</i> synthesis.</p

Detection and isolation of chloromethane-degrading bacteria from the Arabidopsis thaliana phyllosphere, and characterization of chloromethane utilisation genes

Chloromethane gas is produced naturally in the phyllosphere, the compartment defined as the aboveground parts of vegetation, which hosts a rich bacterial flora. Chloromethane may serve as a growth substrate for specialized aerobic methylotrophic bacteria, which have been isolated from soil and water environments, and use cmu genes for chloromethane utilization. Evidence for the presence of chloromethane-degrading bacteria on the leaf surfaces of Arabidopsis thaliana was obtained by specific quantitative PCR of the cmuA gene encoding the two-domain methyltransferase corrinoid protein of chloromethane dehalogenase. Bacterial strains were isolated on a solid mineral medium with chloromethane as the sole carbon source from liquid mineral medium enrichment cultures inoculated with leaves of A. thaliana. Restriction analysis-based genotyping of cmuA PCR products was used to evaluate the diversity of chloromethane-degrading bacteria during enrichment and after strain isolation. The isolates obtained, affiliated to the genus Hyphomicrobium based on their 16S rRNA gene sequence and the presence of characteristic hyphae, dehalogenate chloromethane, and grow in a liquid culture with chloromethane as the sole carbon and energy source. The cmu genes of these isolates were analysed using new PCR primers, and their sequences were compared with those of previously reported aerobic chloromethane-degrading strains. The three isolates featured a colinear cmuBCA gene arrangement similar to that of all previously characterized strains, except Methylobacterium extorquens CM4 of known genome sequence

Gene redundancy for cobalamin and tetrahydrofolate metabolism in <i>M. extorquens</i> CM4.

a<p>Homologs with >90% aa Id (with mentioned exceptions) found in the chromosome of all <i>M. extorquens</i> strains AM1, BJ001, DM4, and PA1 (common core genome), in one of the strains (shared accessory genome), or none of these strains (CM4 specific CDS). The accessory genome includes a <i>btuB</i> homolog (Mpop_3807, 65% aa Id) in strain BJ001. For strain AM1, a putative dihydrofolate reductase <i>dfrB</i> gene (META2_0242, 34 and 28% aa Id with DmrA and DfrA, respectively) is found in addition to the chromosomal gene; moreover, homologs to Mchl_1923 (META2_0462, 33% aa Id with the N-terminal domain), and CzcA2 (META2_1026, 85% aa Id with pCMU01 plasmid <i>czcA2</i>) are found.</p>b<p>MaGe annotation (<a href="https://www.genoscope.cns.fr/agc/microscope" target="_blank">https://www.genoscope.cns.fr/agc/microscope</a>).</p>c<p>Precursors are uroporphyrinogen III and 5,6-dimethylbenzimidazole.</p>d<p>n.d., not detected.</p>e<p>Encode for homologs of different length: CobA (267 aa)/CysG (485 aa); CobC2 (519 aa)/CobC (338 aa); PurU (287 aa)/PurN (219 aa).</p>f<p>In <i>M. extorquens</i> strains, H₄F is synthesized either <i>de novo</i> or salvaged from 5,10-methenyl-H₄F, or 5- or 10-formyl-H₄F <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056598#pone.0056598-Vuilleumier2" target="_blank">[11]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056598#pone.0056598-Maden1" target="_blank">[72]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056598#pone.0056598-Vorholt1" target="_blank">[73]</a>.</p