8 research outputs found
The 380 kb pCMU01 Plasmid Encodes Chloromethane Utilization Genes and Redundant Genes for Vitamin B<sub>12</sub>- and Tetrahydrofolate-Dependent Chloromethane Metabolism in <i>Methylobacterium extorquens</i> CM4: A Proteomic and Bioinformatics Study
<div><p>Chloromethane (CH<sub>3</sub>Cl) 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 B<sub>12</sub> 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 B<sub>12</sub>-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
Analysis of the theoretical proteome of plasmid pCMU01.
a<p>Compared predicted proteome sizes are, <i>M. extorquens</i> strains AM1, 6531 proteins (genome sequence accession no NC_012808); DM4, 5773 proteins (NC_012988); PA1, 5357 proteins (NC_01017); CM4, 6454 proteins (NC_011757); BJ001, 6027 proteins (NC_010725). Homologous proteins were defined as proteins with at least 40% identity covering over 80% of the sequence. Three classes of proteins were considered: Unique, 157 pCMU01 plasmid-encoded proteins without homologs in any of the compared genomes, including the chromosome and the second plasmid p2MCHL of strain CM4; Common, 56 pCMU01 plasmid-encoded proteins with homologs on the chromosome of all 5 <i>M. extorquens</i> genomes including that of strain CM4; Occasional, 173 pCMU01 plasmid-encoded proteins with homologs in at least one of the 5 <i>M. extorquens</i> genomes. Plasmid pCMU01 and plasmid p1METDI of strain DM4 share 56 homologs localized on three gene clusters. Selected examples are indicated when relevant.</p>b<p>CmuC/CmuC2 homologs share less homologies between them (31% aa Id) than with homologs found in other chloromethane-degrading <i>Hyphomicrobium</i> strains: 40% with strain CM2 CmuC <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056598#pone.0056598-Borodina1" target="_blank">[71]</a> and 37% aa Id with strain MC1 CmuC <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056598#pone.0056598-Vuilleumier1" target="_blank">[4]</a>. <i>M. extorquens</i> CM4 is the only chloromethane-degrading strain so far which contains two methyltransferase-encoding <i>cmuC</i> genes of unknown function. Transposon insertion in gene <i>cmuC</i> was previously demonstrated to prevent strain CM4 growth with chloromethane <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056598#pone.0056598-Vannelli2" target="_blank">[10]</a>.</p>c<p>pCMU01 plasmid encoded protein MetF2 (Mchl_5726) previously demonstrated to be essential for chloromethane utilization <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056598#pone.0056598-Studer1" target="_blank">[6]</a> encodes a protein with only 25% aa Id to <i>E</i>. <i>coli</i> MetF. It is more distantly related to the canonical MetF than its chromosomal homolog (Mchl_1881, 56% aa Id to <i>E. coli</i> MetF).</p>d<p>Putative universal stress protein (Mchl_5472) also found in the DCM-dehalogenating <i>M. extorquens</i> DM4 only (METDI4473).</p>e<p>Close homologs (>65% Id aa) located in synteny on the 1.26 Mb megaplasmid of strain AM1.</p
Gene clusters associated with the chloromethane response.
<p>Sequence positions are indicated for each gene cluster. All but cluster A are located on the chromosome. Some DNA segments are omitted for clarity (double slashes), with their size indicated in kb. Gene arrows are drawn according to functional category: transport (dots); regulation, sensing or signaling (stripes); unknown (white). Protein products more abundant in cultures grown with chloromethane (C labeled circles) or with methanol (M labeled circles) are indicated, with black or white symbols used for those proteins observed exclusively or more abundant in one condition, respectively. Proteins homologous to induced genes, or proteins more abundant in a previous study of <i>M. extorquens</i> DM4 grown with dichloromethane compared to methanol <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056598#pone.0056598-Muller1" target="_blank">[16]</a>, are indicated with circles labeled by a âDâ.</p
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 âCH<sub>3</sub>Clâ were only detected in the proteome of <i>M. extorquens</i> CM4 grown with chloromethane. Spots indicated as â+â were more abundant in chloromethane-grown cultures (or less abundant in methanol-grown cultures). Spots indicated as âââ were more abundant in methanol-grown cultures (i.e. less abundant in chloromethane-grown cultures). Factors of differential abundance were defined as follows:++(<b>ââ</b>) 2- to 5-fold;+++(<b>ââ</b>â) 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>o<sup>2+</sup>/<u>Z</u>n<sup>2+</sup>/<u>C</u>d<sup>2+</sup> 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 H<sub>4</sub>F <i>de novo</i> synthesis.</p
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, H<sub>4</sub>F is synthesized either <i>de novo</i> or salvaged from 5,10-methenyl-H<sub>4</sub>F, or 5- or 10-formyl-H<sub>4</sub>F <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
Methylotrophic metabolism and chloromethane utilization pathway in <b><i>Methylobacterium extorquens</i></b><b> CM4.</b>
<p>The left-hand scale indicates carbon oxidation state. The <u>c</u>hloro<u>m</u>ethane <u>u</u>tilization <i>cmu</i> pathway (bold arrows) funnels the chloromethane-derived methyl group into central metabolism via methylene-H<sub>4</sub>F (CH<sub>2</sub>â=âH<sub>4</sub>F), while the methanol (CH<sub>3</sub>OH) oxidation pathway operates with formaldehyde (HCHO) as a metabolic intermediate (grey arrows). H<sub>4</sub>F- and H<sub>4</sub>MPT-dependent enzyme-mediated steps are depicted in blue and pink, respectively. Carbon assimilation operates via the serine cycle (Ser) coupled with the ethylmalonyl-CoA pathway (EMCP) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056598#pone.0056598-Peyraud2" target="_blank">[67]</a>. Spontaneous condensation of HCHO with H<sub>4</sub>F or H<sub>4</sub>MPT, and formaldehyde oxidation to methylene-H<sub>4</sub>F are shown with broken line. In the <i>cmu</i> pathway, the methyl group enters a specific H<sub>4</sub>F-oxidation pathway for energy production driven by the FolD and PurU enzymes. Protein-encoded genes or genes located on plasmid pCMU01 are shown in bold. Boxes and circles highlight proteins more abundant in chloromethane- and methanol grown-cultures, respectively. CmuA, methyltransferase/corrinoid-binding two-domain protein; CmuB, methylcobalamin:H<sub>4</sub>F methyltransferase; Fae, formaldehyde activating enzyme; Fch, methenyl-H<sub>4</sub>F cyclohydrolase; FDHs, formate dehydrogenases; Fhc, formyltransferase-hydrolase complex; FolD, bifunctional methylene-H<sub>4</sub>F dehydrogenase/cyclohydrolase; FtfL, formate-H<sub>4</sub>F ligase; Gck, glycerate kinase; GcvT, H<sub>4</sub>F-dependent aminomethyltransferase; HprA, hydroxypyruvate reductase; MDH, methanol dehydrogenase; MetF, methylene-H<sub>4</sub>F reductase; MtdA, bifunctional NAD(P)-dependant methylene-H<sub>4</sub>F and methylene-H<sub>4</sub>MPT dehydrogenase; MtdB, NAD(P)-dependent methylene-H<sub>4</sub>MPT dehydrogenase; Mch, methenyl-H<sub>4</sub>MPT cyclohydrolase; MtkA, malate thiokinase large subunit; MxaF, MDH alpha subunit, PurU, 10-formyl-H<sub>4</sub>F hydrolase; Sga, serine-glyoxylate aminotransferase <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056598#pone.0056598-Chistoserdova1" target="_blank">[12]</a>. Plasmid pCMU01 encoded proteins with predicted functions include putative uncharacterized methyltransferases CmuC and CmuC2, the putative PaaE-like oxidoreductase, and the putative PQQ-linked dehydrogenase of unknown specificity XoxF2. GvcT may serve to transfer methyl groups from a wide range of substrates to H<sub>4</sub>F, as proposed for members that belong to the COG0354-related enzymes such as YgfZ <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056598#pone.0056598-Halsey1" target="_blank">[68]</a>.</p
Presentation_1_The CYP71AZ P450 Subfamily: A Driving Factor for the Diversification of Coumarin Biosynthesis in Apiaceous Plants.pptx
<p>The 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.</p