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

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 “D”.</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, 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

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-H₄F (CH₂ = H₄F), while the methanol (CH₃OH) oxidation pathway operates with formaldehyde (HCHO) as a metabolic intermediate (grey arrows). H₄F- and H₄MPT-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 H₄F or H₄MPT, and formaldehyde oxidation to methylene-H₄F are shown with broken line. In the <i>cmu</i> pathway, the methyl group enters a specific H₄F-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:H₄F methyltransferase; Fae, formaldehyde activating enzyme; Fch, methenyl-H₄F cyclohydrolase; FDHs, formate dehydrogenases; Fhc, formyltransferase-hydrolase complex; FolD, bifunctional methylene-H₄F dehydrogenase/cyclohydrolase; FtfL, formate-H₄F ligase; Gck, glycerate kinase; GcvT, H₄F-dependent aminomethyltransferase; HprA, hydroxypyruvate reductase; MDH, methanol dehydrogenase; MetF, methylene-H₄F reductase; MtdA, bifunctional NAD(P)-dependant methylene-H₄F and methylene-H₄MPT dehydrogenase; MtdB, NAD(P)-dependent methylene-H₄MPT dehydrogenase; Mch, methenyl-H₄MPT cyclohydrolase; MtkA, malate thiokinase large subunit; MxaF, MDH alpha subunit, PurU, 10-formyl-H₄F 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 H₄F, 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

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

Doubling times (hours ± 1σ) of <i>M</i>. <i>extorquens</i> DM4 strains on succinate in optimized media.

<p>Mean of three replicates per strain/substrate combination.</p

Sensitivity of strains to toxic compounds in disk diffusion assays.

<p>Sensitivity of strains to toxic compounds in disk diffusion assays.</p

Hopanoid-free <i>Methylobacterium extorquens</i> DM4 overproduces carotenoids and has widespread growth impairment

<div><p>Hopanoids are sterol-like membrane lipids widely used as geochemical proxies for bacteria. Currently, the physiological role of hopanoids is not well understood, and this represents one of the major limitations in interpreting the significance of their presence in ancient or contemporary sediments. Previous analyses of mutants lacking hopanoids in a range of bacteria have revealed a range of phenotypes under normal growth conditions, but with most having at least an increased sensitivity to toxins and osmotic stress. We employed hopanoid-free strains of <i>Methylobacterium extorquens</i> DM4, uncovering severe growth defects relative to the wild-type under many tested conditions, including normal growth conditions without additional stressors. Mutants overproduce carotenoids–the other major isoprenoid product of this strain–and show an altered fatty acid profile, pronounced flocculation in liquid media, and lower growth yields than for the wild-type strain. The flocculation phenotype can be mitigated by addition of cellulase to the medium, suggesting a link between the function of hopanoids and the secretion of cellulose in <i>M</i>. <i>extorquens</i> DM4. On solid media, colonies of the hopanoid-free mutant strain were smaller than wild-type, and were more sensitive to osmotic or pH stress, as well as to a variety of toxins. The results for <i>M</i>. <i>extorquens</i> DM4 are consistent with the hypothesis that hopanoids are important for membrane fluidity and lipid packing, but also indicate that the specific physiological processes that require hopanoids vary across bacterial lineages. Our work provides further support to emerging observations that the role of hopanoids in membrane robustness and barrier function may be important across lineages, possibly mediated through an interaction with lipid A in the outer membrane.</p></div