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

Abstract

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 (cmu) was characterized in Methylobacterium extorquens 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 cmuA- and cmuB-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 cmu 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 M. extorquens strains unable to grow with chloromethane showed that plasmid pCMU01 harbors unique genes without homologs in the compared genomes (bluB2, btuB, cobA, cbiD), 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 (folC2). 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 M. extorquens 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.

Introduction

Chloromethane (CH₃Cl) is a volatile organic compound emitted by oceans, plants, wood-rotting fungi and biomass burning, estimated to account for 17% of chlorine-catalyzed ozone degradation in the stratosphere [1]. Chloromethane-utilizing bacteria have been isolated from a wide variety of environments such as seawater, soil, sludge, and recently from plant leaf surfaces [2], and represent a potential biotic filter for chloromethane emissions. Many chloromethane degraders are facultative methylotrophic Proteobacteria [3] growing in aerobiosis with chloromethane and other C₁ carbons such as methanol as unique source of carbon and energy. Complete and assembled genomes of two chloromethane-utilizing strains, Methylobacterium extorquens strain CM4 and Hyphomicrobium sp. strain MC1, are available [4], [5]. The only known microbial aerobic utilization pathway for chloromethane is tetrahydrofolate (H₄F)-dependent [6]. This pathway was identified in the alpha-Proteobacterium M. extorquens CM4 using minitransposon random mutagenesis [7] and its chloromethane dehalogenase activity characterized in detail [8], [9]. The first step of the cmu (chloromethane utilization) pathway is catalyzed by the two-domain methyltransferase/corrinoid-binding CmuA protein that transfers the methyl group from chloromethane to a corrinoid cofactor [9], [10]. The methylcobalamin:H₄F methyltransferase CmuB enzyme subsequently catalyzes the transfer of the methyl group from the corrinoid cofactor to H₄F [8]. The H₄F-bound C₁ moiety of chloromethane, methylene-H₄F (CH₂ = H₄F) is oxidized to carbon dioxide via formate to produce energy, or funneled into the serine pathway for biomass synthesis (Fig. 1). Evidence that H₄F is an essential cofactor of the cmu-dependent degradation of chloromethane was obtained from mutant analyses in M. extorquens CM4, which identified metF (encoding methylene-H₄F reductase) and purU (encoding formyl-H₄F hydrolase) as essential genes for growth with chloromethane [10]. The pathway for chloromethane utilization in Methylobacterium is thus completely different from that for dichloromethane (CH₂Cl₂), which involves DcmA, a cytoplasmic dichloromethane dehalogenase/glutathione S-transferase yielding the central intermediate of methylotrophic metabolism formaldehyde (HCHO) [11].

Figure 1. Methylotrophic metabolism and chloromethane utilization pathway in Methylobacterium extorquens CM4.

The left-hand scale indicates carbon oxidation state. The chloromethane utilization cmu 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) [67]. Spontaneous condensation of HCHO with H₄F or H₄MPT, and formaldehyde oxidation to methylene-H₄F are shown with broken line. In the cmu 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 [12]. 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 [68].

Known pathways for tetrahydromethanopterin (H₄MPT)- and H₄F-dependent C₁ substrate oxidation in Methylobacterium strains are compared in Figure 1.When growing on methanol, M. extorquens CM4 uses the H₄MPT formaldehyde oxidation pathway first discovered in M. extorquens AM1 [12] and subsequently found to be widespread among methylotrophs.

Growth with chloromethane depends on the presence of cobalt in the medium [9] since CmuA methyltransferase activity requires a vitamin B₁₂-related corrinoid cofactor that incorporates cobalt. As described for adenosylcobalamin (AdoCbl), the corrinoid cofactor may be synthesized de novo by one of Nature’s most complex metabolic pathways requiring around 30 enzyme-mediated steps [13], [14]. Of those, only cobUQD genes found adjacent to cmu genes have been described in M. extorquens CM4 [10]. Many microorganisms synthesize vitamin B₁₂-related compounds from imported corrinoid intermediates [14] or from precursors such as dimethylbenzimidazole (DMB) [15] by pathways that have not been identified in chloromethane-degrading bacteria.

In this work, combined experimental and bioinformatics analysis was performed to gain a better understanding of the genes and proteins specifically associated with chloromethane utilization in M. extorquens CM4. A differential proteomic approach compared M. extorquens CM4 proteins under methylotrophic growth conditions with either chloromethane or methanol as the sole carbon and energy source. Gene clusters specific to the chloromethane response were identified, and compared to previously published clusters involved in the response of M. extorquens DM4 to dichloromethane [16], or involved in the methylotrophic growth of M. extorquens AM1 to methanol [17]. We found that growth with chloromethane elicits a specific adaptive response in M. extorquens CM4. In addition, the genome sequence of the chloromethane-degrading strain CM4 was compared to available complete sequences of other M. extorquens strains unable to grow on chloromethane (strains AM1, PA1, BJ001 and DM4; [5], [11]). Genomic analysis revealed that additional gene homologs of chromosome-encoded cognate genes for coenzyme biosynthesis, as well as specific genes such as bluB2, which is predicted to be involved in both H₄F and vitamin B₁₂ cofactor biosynthesis, were found nearby previously characterized genes cmuA and cmuB on a 380 kb plasmid.

Materials and Methods

Manual Gene Annotation and Bioinformatic Analysis

Comparative analyses were performed using the fully sequenced genomes of four representatives of the M. extorquens species; strain CM4 (GenBank accession no. CP001298, CP001299, CP001300), strain AM1 (GenBank accession no. CP001511, CP001512, CP001513 and CP001514), strain DM4 (GenBank accession no. FP103042 and FP103043 and FP103044), strain PA1 (GenBank accession no. CP000908) and strain BJ001 (GenBank accession no. CP001029, CP001030 and CP001031) [5]. Putative orthology relationships were operationally defined by gene pairs from different genomes satisfying an alignment threshold of at least 40% amino acid sequence identity (aa Id) over at least 80% of the length of the smallest encoded protein. The search for conserved genes clusters was performed as previously described [11]. Manual validation of automatic gene annotations on pCMU01 plasmid (also known as pMCHL01) was performed using the relational database [18] Microscope web interface (MethyloScope) https://www.genoscope.cns.fr/agc/mage/wwwpkgdb/Login/log.php?pid=26). Insertion sequence (IS) annotations were done as previously described [11]. IS elements were given names of type “ISMch3”, with “Mch” for Methylobacterium extorquens degrading chloromethane.

Biological Materials, Media and Growth Conditions

The composition of Methylobacterium mineral medium M3 was adapted from that given in Vannelli et al. [7] with 0.2 g.L⁻¹ (NH₄)₂SO₄ final concentration and substitution of ZnCl₂ by ZnSO₄ in the trace element solution. M. extorquens CM4 was grown aerobically at 30°C either with chloromethane or with methanol as carbon substrate, on a rotary shaker (140 rpm) in 1.2-liter Erlenmeyer flasks containing 200 mL M3 medium, closed with gas-tight screw caps with mininert valves (Supelco). Methanol (sterile-filtered) was added to a final concentration of 40 mM. Chloromethane gas was added to a final concentration of 15 mM in the liquid phase, assuming a Henry constant of 0.0106 m³.atm.mol⁻¹ at 30°C [19], as previously described [6]. Acetone was added to a final concentration of 5 mM from a sterile-filtered solution at 250 mM. Chloromethane and acetone degradation were quantified using a CP 3800 gas chromatograph connected to a flame ionization detector (GC-FID; Varian, USA) equipped with a GC column (CP-Sil 5 CB, length 15 m; Varian).

Protein Extraction

Triplicates of M. extorquens CM4 cultures were harvested by centrifugation (10 min at 10,000 g) in mid-exponential growth phase of chloromethane- and methanol-grown cultures, using 100 mL at OD₆₀₀ of 0.2 and 33 mL at OD₆₀₀ at 0.6, respectively. Cell pellets were resuspended in 10 mM Tris, 1 mM EDTA buffer pH 7.6 (TE buffer), washed once and resuspended in 400 µL of the same buffer in the presence of benzonase (250 units; GE Healthcare) and 4 µL protease inhibitor mix 100× (GE Healthcare). Cells were disrupted using glass beads (0.1 mm in diameter, 1 g per 0.4 mL extract) in a MM2 mixer mill (Retsch Haan, Germany) at maximal speed for 6 cycles of 30 sec, and then placed on ice for one hour. Cell debris and beads were removed by centrifugation at 14,000 g for 15 min at 4°C, and the supernatant was centrifuged again at 14,000 g for one hour at 4°C. Protein concentration in the supernatant was assayed using a commercial Bradford assay (Biorad) with bovine serum albumin as a standard, and subsequently adjusted to 1 mg/mL in TE buffer.

Two-dimensional Gel Electrophoresis (2D–E)

Protein extracts (100 µg) were precipitated overnight with 9 vol. of acetone at 4°C, centrifuged at 10,000 g for 10 min at 4°C, washed three times with 80% acetone. Proteins were resuspended in 350 µL of purified rehydration buffer (RB). RB buffer (7 M urea, 2 M thiourea) was purified by mixing for one hour with 10 g.L⁻¹ Amberlite IRN-150L (GE Healthcare), 2.5% wt/vol CHAPS (3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate), 0.6% immobilized pH gradient (IPG) buffer (pH range 4–7 or 3–10, respectively), 65 mM dithiothreitol (DTT), and 0.002% wt/vol bromophenol blue. Proteins (80 µg) were loaded on 18-cm IPG strips (linear gradient pH 4–7 and 3–10) with the IPGphor 3 isoelectric focusing (IEF) system, as recommended by the manufacturer (GE Healthcare). Rehydration (6 h at 0 V and 6 h at 30 V) was followed by four two-hour increments during which the voltage was increased stepwise from 150 V, 500 V, 1000 V, to 3000 V. Finally, separation was obtained using 8,000 V until a minimum of 45,000 V.h⁻¹ was reached, and strips were stored at –80°C. Before use, strips were thawed at room temperature, and placed in equilibration solution (50 mM Tris-HCl, pH 8.8, 6 M urea, 30% glycerol, 2% SDS, and 0.002% bromophenol blue) twice for 15 min, first with 10 mg/mL DTT and then with 25 mg/mL iodoacetamide. The second dimension of electrophoretic separation was performed by 11.5% SDS-PAGE using the Ettan DALTII system (GE Healthcare). Gels were fixed during 1 h in a 40% ethanol, 7% acetic acid solutions. Proteins were stained overnight with Brilliant Blue G-colloidal (Sigma). Scanning of gels was performed on an Image Scanner (GE Healthcare) with LabScan software (GE Healthcare).

Two-dimensional Fluorescence Differential Gel Electrophoresis (2D-DIGE)

Samples were labeled with CyDye DIGE Fluor minimal dyes (Cy2, Cy3 or Cy5, GE Healthcare) according to the manufacturer’s instructions. After acetone precipitation, the resulting protein pellet was resuspended in DIGE rehydration buffer (DRB) (7 M urea, 2 M thiourea, 4% wt/vol CHAPS, Tris 20 mM, pH 8.5). The protein concentration was quantified using a slightly modified Bradford method (as described above except for use of DRB), standardized with known concentrations of bovine serum albumin, and adjusted to 2 mg/mL using DRB. For each DIGE experiment, 8 samples were labeled, corresponding to four independent cultures for each condition. Labeling was performed by mixing 50 µg total protein from each of the four samples with either Cy3 (two samples) or Cy5 (two other samples) DIGE minimal dye (400 pmol) (GE Healthcare), to take into account biases resulting from different labeling efficiency. A pooled set of internal standards, comprising 25 µg aliquots from each of the 8 samples (200 µg total), was labeled with Cy2 DIGE minimal dye (1600 pmol total). Labeling was performed for 30 minutes on ice and in the dark, and quenched by the addition of 10 mM lysine (1 µL for Cy3- or Cy5-labeled extracts, and 4 µL for Cy2-labeled extracts, respectively). Samples were incubated for 10 min on ice in the dark. Finally, protein samples that were separated on the same gel were mixed (one Cy3- and one Cy5-labeled sample each together with one-fourth volume of the pooled set of internal standards), and supplemented with 0.6% IPG pH 3–10 NL (non-linear) and 65 mM DTT. Buffer RB was added to each mix to final volume of 350 µL before IEF separation. Proteins were loaded on 18-cm IPG strips (non-linear gradient pH 3–10) and submitted to separation steps as described above. Gels were fixed and proteins stained as described above. Scanning of gels was performed with an Ettan DIGE Imager (GE Healthcare).

Proteome Image Analysis

Differential analysis was performed using ImageMaster 2D Platinum software (v. 6.0, GE Healthcare). Six gels were grouped in two classes of three independent gels depending on the two compared conditions (chloromethane or methanol growth conditions). Gels were matched with one reference gel (master gel) following spot detection. For each spot, the relative volume corresponded to the normalized volume of the spot compared to the normalized volume of the entire gel coloration. Statistical analysis was performed by calculating the Student t value for each spot, as well as a ratio value defined as the mean of the relative volume of the spot obtained in the different replicates for growth with chloromethane divided by the mean of the relative volumes obtained for growth with methanol. Spots with a Student t value higher than 1.9 (corresponding to a p-value of <0.1) and ratios ≥2.0 or ≤−2.0 were analyzed by mass spectrometry. DIGE images were analyzed with DeCyder software (v. 7.0, GE Healthcare). A total of twelve images obtained from 4 gels (three images each) were analyzed. Student’s t test was used to determine differential abundance of proteins. In this procedure, the p-values were corrected for false discovery rate [20]. Spots with a p-value <0.01 and ratios ≥2.0 or ≤−2.0 were considered to be differentially abundant.

Mass Spectrometry Protein Identification

The procedure of Muller et al. [16] was followed for spot identification, with minor adjustments. Mass spectrometry analyses were performed in reflector positive mode on a Biflex III (Bruker-Daltonik GmbH, Bremen, Germany) matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (MALDI-TOF TOF) and on an Autoflex III Smartbeam (Bruker-Daltonik GmbH, Bremen, Germany) matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (MALDI-TOF TOF). A saturated solution of α-cyano-4-hydroxycinnamic acid in 50% water/50% acetonitrile was used as matrix for MALDI mass measurement on the Biflex III. Peptide mass fingerprinting data (PMF) and peptide fragment fingerprinting data (PFF) were combined by Biotools 3 software (Bruker Daltonik) and transferred to the search engine MASCOT (Matrix Science, London, UK). Peptide mass error was limited to 100 ppm for the Biflex III and to 50 ppm for the Autoflex III Smartbeam. Proteins were identified by searching against the NCBI non-redundant protein sequence database and the predicted proteins of strain CM4 (GenBank accession no. CP001298, CP001299, CP001300).

Results

The 380 kb Episome in M. extorquens CM4 Harbors cmu Genes and Associated Genes

The repertoire of known cmu genes and genes conserved in chloromethane-degrading strains includes genes essential for dehalogenation of chloromethane (cmuA, cmuB), genes essential for growth with chloromethane as the sole carbon and energy source (cmuC, metF, purU), and genes found in the vicinity of genes cmuA, cmuB and cmuC in methylotrophic chloromethane-degrading strains (fmdB, paaE, hutI, and cbiD) [2]. All these genes co-localize on a 138 kb region flanked by transposable elements nested within the 380 kb plasmid pCMU01 in M. extorquens CM4. This plasmid encodes proteins associated with growth on chloromethane, such as the enzymes for chloromethane degradation and for metabolism of the two essential dehalogenase cofactors AdoCbl and H₄F, as well as transport proteins for coenzyme B₁₂ precursors (Table 1). Thus, plasmid pCMU01 can be designated as a chloromethane catabolic plasmid that harbors the cognate essential genes for growth on chloromethane.

Table 1. Analysis of the theoretical proteome of plasmid pCMU01.

Functional class	Occurrence in sequenced Methylobacterium extorquens genomesa
	Unique	Common	Occasional
Chloromethane degradation	6(CmuA, CmuB, CmuC, CmuC2b, HutI, PaaE-like)	0	0
Cobalamin metabolism	3(BluB2, CbiD, CobA)	13(Cob protein)	0
H4F and C1 metabolism	3(FolD, MetF2 c, PurU)	4(FolC2, Hss2, SerC2, XoxF2)	0
Acetone degradation	3(AcxA, AcxB, AcxC)	0	0
Other metabolisms	7	10 (Gck2, Shc2)	19 (GdhA, IspF, SorA, SorB)
Stress	0	1 (UspA fragment)	1 (Usp) d
Plasmid-related function	3 (MmeI)	1 (ArdC)	19 (DotABC-like, IcmBCEKL, TraGDCA, RepABC)
Transporter	3	10 (BtuC, BtuF, BtuD, ClcA, ModA2, ModB2, ModC2, Mop2)	7 (BtuB, CzcBA2 e)
Regulator	14 (AcxR, FmdB)	8	12 (CzcSR e)
Mobile element-related	25	3	43
Unknown	90	6	72

^aCompared predicted proteome sizes are, M. extorquens 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 M. extorquens genomes including that of strain CM4; Occasional, 173 pCMU01 plasmid-encoded proteins with homologs in at least one of the 5 M. extorquens genomes. Plasmid pCMU01 and plasmid p1METDI of strain DM4 share 56 homologs localized on three gene clusters. Selected examples are indicated when relevant.

^bCmuC/CmuC2 homologs share less homologies between them (31% aa Id) than with homologs found in other chloromethane-degrading Hyphomicrobium strains: 40% with strain CM2 CmuC [71] and 37% aa Id with strain MC1 CmuC [4]. M. extorquens CM4 is the only chloromethane-degrading strain so far which contains two methyltransferase-encoding cmuC genes of unknown function. Transposon insertion in gene cmuC was previously demonstrated to prevent strain CM4 growth with chloromethane [10].

^cpCMU01 plasmid encoded protein MetF2 (Mchl_5726) previously demonstrated to be essential for chloromethane utilization [6] encodes a protein with only 25% aa Id to E. coli MetF. It is more distantly related to the canonical MetF than its chromosomal homolog (Mchl_1881, 56% aa Id to E. coli MetF).

^dPutative universal stress protein (Mchl_5472) also found in the DCM-dehalogenating M. extorquens DM4 only (METDI4473).

^eClose homologs (>65% Id aa) located in synteny on the 1.26 Mb megaplasmid of strain AM1.

Overview of Plasmid pCMU01 Gene Content

Plasmid pCMU01 is characterized by a somewhat lower GC content (66.3%) than the chromosome of strain CM4 (68.2%). To a large extent (41%), it features unique genes encoding predicted proteins without close homologs (>40% aa Id, >80% of the protein length) in the genomes of four other M. extorquens strains unable to degrade chloromethane [5] (Table 1). Of its 386 predicted CDS, 56% belong to at least one COG group [21] related to metabolism (enzymes, 69 CDS; transporters, 20 CDS), plasmid functions (23 CDS), adaptive response (regulation, 34 CDS; stress, two CDS), and genomic plasticity (mobile DNA elements, 71 CDS with a total of 18 identified IS elements representing 4% of the predicted CDS of the plasmid). Pseudogenes may account for 9% of the total predicted CDS on the plasmid with 35 pseudogenes detected.

Chloromethane is not the only organic molecule for which the plasmid allows to transform for growth. A complete acetone-catabolic gene cluster encoding the acetone carboxylase subunits (β subunit, acxA; α subunit, acxB; γ subunit, acxC) and its cognate transcriptional activator (gene acxR) was found. Acetone carboxylase is the key enzyme of bacterial acetone metabolism in Xanthobacter autotrophicus strain Py2, catalyzing the ATP-dependent carboxylation of acetone to form acetoacetate [22]. High sequence conservation was found between the acxRABC gene clusters of strains CM4 and X. autotrophicus Py2 (>82% aa Id for enzyme subunits and 53% for the regulator). The ability of M. extorquens CM4 to degrade acetone was tested in aerobic liquid cultures in M3 medium. When 5 mM acetone was supplied as the unique source of carbon and energy, strain CM4 grew up to an OD₆₀₀ of 0.4 at stationary growth phase, with total degradation of acetone as measured using GC-FID. No acetone degradation was observed in the abiotic control or in cultures of M. extorquens DM4 lacking the acx cluster under the same conditions. Thus, the plasmid pCMU01-encoded acx cluster seems to be functional in strain CM4.

Evidence of the mode of replication, maintenance, and conjugation of plasmid pCMU01 was suggested from sequence similarity searches. A combined replication and partitioning repABC unit (Mchl_5615–5617) including the incompatibility antisense RNA (ctRNA) between the repB-repC genes was found [23]. The gene products of repA, repB and repC share at least 43% aa Id with the corresponding proteins of the characterized Rhizobium etli p42d plasmid repABC cassette [24]. In a recent review, plasmid pCMU01 was classified within the RepABC family plasmids of large low-copy-number plasmids found exclusively in Alphaproteobacteria [25]. The plasmid harbors components of a core type IVB secretion/conjugation system complex often used for horizontal propagation, including the gene encoding the conserved central component of the DNA transport activity core complex (Mchl_5595), and traD (Mchl_5572) which lies within a traGDCA gene cluster (Mchl_5572–5575) conserved in other Alphaproteobacteria plasmids including Agrobacterium tumefaciens Ti plasmids (encoded proteins TraG, D, C and A sharing 43, 53, 38 and 44% aa Id, respectively). Finally, a putative restriction- modification system encoding protein Mchl_5634 shares 48% aa Id with a bifunctional DNA methyltransferase/type II restriction endonuclease MmeI [26] (Table 1). Taken together, the described genomic features indicate that plasmid pCMU01 represents a low-copy plasmid, vertically transmitted via a RepABC replication and partitioning unit, and most probably able to propagate by horizontal transfer within Alphaproteobacteria.

Extensive Plasmid-encoded Gene Redundancy Associated with Vitamin B₁₂ Metabolism

M. extorquens CM4 is able to synthesize coenzyme B₁₂, a cofactor essential for activity of chloromethane dehalogenase CmuAB [9]. A complete set of cob genes homologous to those described in P. denitrificans for the aerobic biosynthesis pathway of AdoCbl [14] is found on the chromosome of CM4 as well as on the chromosomes of four other M. extorquens strains (Table 2). Remarkably, strain CM4 also contains plasmid-borne copies of 13 cob genes and genes coding for cobalt and preformed corrinoid transporters beyond to the close chromosomal homologs of these genes shared by M. extorquens strains. These include the putative cobalt transporter CzcA-related RND transporter [27] (the plasmid-borne gene product Mchl_5715 displays 43% aa Id with the chromosome-encoded Mchl_1072; Table 2), and a homolog of the preformed corrinoid specific transporter Btu [28]. Unlike the plasmid-borne btu gene cluster, the chromosome-encoded btuFCD cluster lacks the btuB gene preceded by a cobalamin riboswitch [29] (Mchl_misc_RNA_1, Table 2), suggesting that expression of the plasmid-borne btu gene cluster is controlled by cobalamin in its coenzyme form (AdoCbl).

Table 2. Gene redundancy for cobalamin and tetrahydrofolate metabolism in M. extorquens CM4.

Function	Gene in strain CM4				Occurrence in M. extorquens a	MaGe annotationb		EC n°		Plasmid pCMU01 identifier
	Chromosome	Plasmid pCMU01	Paralog aa Id (%)
Cobalamin metabolism
Aerobic AdoCbl biosynthesis from precursors c
	bluB	bluB2	38.4		core	5,6-dimethylbenzimidazole synthase (flavin destructase), putative cob(II)yrinic acid a,c-diamide reductase			1.16.8.1		Mchl_5732
	/d	cbiD	/		CM4 specific	Cobalamin biosynthesis protein, putative cobalt-precorrin-6A synthase [deacetylating]			2.1.1.-		Mchl_5729
	/	cobA e	/		CM4 specific	S-adenosyl-L-methionine-dependent uroporphyrinogen III methylase (SUMT)			2.1.1.107		Mchl_5731
	cobB	/	/		core	Cobyrinic acid a,c-diamide synthase					/
	cobC	cobC2 e	51.2		core	L-threonine-O-3-phosphate decarboxylase			4.1.1.81		Mchl_5730
	cobD	cobD2	76.4		core	Cobalamin biosynthesis protein CobD					Mchl_5724
	cobE	cobE2	64.4		core	Cobalamin biosynthesis protein CobE					Mchl_5686
	cobF	/	/		core	Precorrin-6A synthase			2.1.1.152		/
	cobG	/	/		core	Putative precorrin-3B synthase CobG			1.14.13.83		/
	cobH	cobH2	86.2		core	Precorrin-8X methylmutase			5.4.1.2		Mchl_5691
	cobI	cobI2	80.7		core	Precorrin-2 C(20)-methyltransferase			2.1.1.130		Mchl_5690
	cobJ	cobJ2	79.1		core	Precorrin-3B C(17)-methyltransferase			2.1.1.131		Mchl_5689
	cobK	cobK2	63.4		core	Precorrin-6A reductase			1.3.1.54		Mchl_5688
	cobL	cobL2	76.0		core	Precorrin-6Y C(5,15)-methyltransferase			2.1.1.132		Mchl_5687
	cobM	cobM2	82.7		core	Precorrin-4 C(11)-methyltransferase			2.1.1.133		Mchl_5685
	cobN	/	/		core	Putative cobaltochelatase, CobN-related			6.6.1.2		/
	cobO	cobO2	83.5		core	Cob(I)yrinic acid a,c-diamide adenosyltransferase			2.5.1.17		Mchl_5722
	cobP	cobP2	73.6		core	Bifunctional adenosylcobalamin biosynthesis protein CobP			2.7.7.62		Mchl_5721
	cobQ	cobQ2	75.2		core	Cobyric acid synthase					Mchl_5723
	cobS	/	/		core	Aerobic cobaltochelatase subunit CobS			6.6.1.2		/
	cobT	/	/		core	Aerobic cobaltochelatase subunit CobT			6.6.1.2		/
	cobU	cobU2	54.5		core	Nicotinate-nucleotide–dimethylbenzimidazole phosphoribosyltransferase			2.4.2.21		Mchl_5702
	cobV	/	/		core	Cobalamin synthase			2.-.-.-		/
	cobW	/	/		core	Cobalamin biosynthesis protein CobW					/
	cobW	/	/		core	Putative cobalamin biosynthesis protein CobW					/
	cobW	/	/		core	Putative cobalamin biosynthesis protein CobW					/
	cysG	/e	/		core	Siroheme synthase			2.1.1.107/1.3.1.76/4.99.1.4		/
Cobalt and cobalamin transporters
	/	btuB	/		accessory	Putative vitamin B12 outer membrane transporter BtuB					Mchl_5676
	btuC	btuC2	73.5		core	Putative vitamin B12 import system permease protein BtuC					Mchl_5678
	btuD	btuD2	66.0		core	Putative vitamin B12 transport system BtuD, ATPase component					Mchl_5679
	btuF	btuF2	61.0		core	Putative vitamin B12-binding protein BtuF			1.16.8.1		Mchl_5677
	cbtA	/	/		core	Putative cobalt transporter, subunit CbtA					/
	cbtB	/	/		core	Putative cobalt transporter, subunit CbtB					/
	corA	/	/		core	Putative cobalt transporter CorA					/
	czcA	czcA2	43.4		core	RND efflux transporter, membrane component, cobalt-zinc-cadmium resistance protein					Mchl_5715
	/	czcB	/		accessory	RND efflux transporter, membrane fusion protein, putative CzcB protein					Mchl_5714
	exbB	/	/		core	Transport protein ExbB					/
	exbD	/	/		core	Transport protein ExbD					/
	icuA	/	/		core	TonB-dependent outer membrane transporter associated to improved cobalt uptake					/
	icuB	/	/		core	Periplasmic binding protein associated to improved cobalt uptake					/
	icuC	/	/		core	Putative periplasmic binding protein; improves cobalt uptake when overexpressed					/
	tolQ	/	/		core	Transport protein ExbB/TolQ					/
	tolR	/	/		core	Transport protein ExbD/TolR					/
	tonB	/	/		core	Putative TonB family protein					/
Tetrahydrofolate metabolism
de novo tetrahydrofolate biosynthesis f
	dmrA	/	/		core	Dihydromethanopterin reductase, putative dihydrofolate reductase					/
	folA	/	/		core	Dihydrofolate reductase (also called dfrA)			1.5.1.3		/
	folB	/	/		core	Dihydroneopterin aldolase			4.1.2.25		/
	folC	folC2	46.3		core	Bifunctional folylpolyglutamate synthase/dihydrofolate synthase			6.3.2.17/6.3.2.12		Mchl_5701
	folE	/	/		core	GTP cyclohydrolase I			3.5.4.16		/
	folK	/	/		core	2-amino-4-hydroxy-6-hydroxymethyldihydropteridin pyrophosphokinase			2.7.6.3		/
	folP	/	/		core	Dihydropteroate synthase			2.5.1.15		/
	Mchl_0356	/	/		core	NUDIX hydrolase (NudG), putative dihydroneopterin triphosphate pyrophosphatase (NtpA-like)					/
	pabA	/	/		core	Aminodeoxychorismate synthase subunit II, p-aminobenzoate synthase component			2.6.1.85		/
	pabB	/	/		core	Para-aminobenzoate synthase component I			2.6.1.85		/
	pabC	/	/		core	Putative 4-amino-4-deoxychorismate lyase component of para-aminobenzoate synthase			4.1.3.38		/
D-erythrose-4P to chorismate
	aroA	/	/		core	3-enolpyruvylshikimate-5-phosphate synthetase			2.5.1.19		/
	aroC	/	/		core	Chorismate synthase			4.2.3.5		/
	aroE	/	/		core	Putative shikimate 5-dehydrogenase			1.1.1.25		/
	aroG	/	/		core	2-dehydro-3-deoxyphosphoheptonate aldolase			4.1.2.54		/
	aroK	/	/		core	Putative transcriptional regulator (N-terminal)/shikimate kinase (C-terminal)			2.7.1.71		/
	aroQ	/	/		core	3-dehydroquinate dehydratase, type II			4.2.1.10		/
	Mchl_1923	/	/		core	Bifunctional shikimate kinase (AroK)/dehydroquinate synthase (AroB)			4.2.3.4		/
D-erythrose-4P synthesis from sugars
	cbbA	/	/		core	Fructose-bisphosphate aldolase			4.1.2.13		/
	fbp	/	/		core	Fructose-1,6-bisphosphatase I			3.1.3.11		/
	glpX	/	/		core	Fructose 1,6-bisphosphatase II			3.1.3.11		/
	tpiA	/	/		core	Triosephosphate isomerase			5.3.1.1		/
Tetrahydrofolate interconversion
	/	folD	/	CM4 specific			Bifunctional methylene-H4F dehydrogenase/methenyl-H4F cyclohydrolase		1.5.1.5/3.5.4.9		Mchl_5700
	ftfL	/	/	core			Formate-H4F ligase		6.3.4.3		/
	gcvH	/	/	core			Glycine cleavage complex protein H				/
	lpd f	/	/	core			Glycine-cleavage complex protein L (dihydrolipoamide dehydrogenase)		1.8.1.4		/
	gcvP	/	/	core			Glycine cleavage complex protein P, PLP-dependent glycine dehydrogenase		1.4.4.2		/
	gcvT	/	/	core			Glycine cleavage complex protein T, H4F-dependent aminomethyltransferase		2.1.2.10		/
	glyA	/	/	core			Serine hydroxymethyltransferase		2.1.2.1		/
	metF	metF2	26	core			5,10-methylene-H4F reductase		1.5.1.20		Mchl_5726
	purH	/	/	core			Bifunctional IMP cyclohydrolase/phosphoribosyl-aminoimidazolecarboxamide formyltransferase		3.5.4.10/2.1.2.3		/
	purN	purU e	32	core			Phosphoribosylglycinamide formyltransferase 1		2.1.2.2		Mchl_5699

^aHomologs with >90% aa Id (with mentioned exceptions) found in the chromosome of all M. extorquens 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 btuB homolog (Mpop_3807, 65% aa Id) in strain BJ001. For strain AM1, a putative dihydrofolate reductase dfrB 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 czcA2) are found.

^bMaGe annotation (https://www.genoscope.cns.fr/agc/microscope).

^cPrecursors are uroporphyrinogen III and 5,6-dimethylbenzimidazole.

^dn.d., not detected.

^eEncode for homologs of different length: CobA (267 aa)/CysG (485 aa); CobC2 (519 aa)/CobC (338 aa); PurU (287 aa)/PurN (219 aa).

^fIn M. extorquens strains, H₄F is synthesized either de novo or salvaged from 5,10-methenyl-H₄F, or 5- or 10-formyl-H₄F [11], [72], [73].

Experimental Identification of Gene Clusters Specific of the Chloromethane Response

Differential analyses of proteins extracted from chloromethane- and methanol-grown cultures of M. extorquens CM4 were performed using 2D-E and 2D-DIGE. Overall, 88 protein spots showing differences in abundance between the two compared conditions were detected, resulting in the identification of 49 proteins (Table 3; Fig. S1). In total, 33 proteins were specific of chloromethane-grown cultures, whereas sixteen proteins were more abundant in methanol-grown cultures.

Table 3. Proteomic analysis of differentially expressed proteins in chloromethane- and methanol-grown cultures of M. extorquens CM4.

Protein	Identifiera	Gene	Protein parameters			Mass spectrometry identification data b of different pI ranges tested
			Ratio c CH3Cl/CH3OH	M r (kDa)	pI	4–7			3–10			3–10 NL d
						Score	Error (ppm)	Coverage (%)	Score	Error (ppm)	Coverage (%)	Score	Error (ppm)	Coverage (%)
Chloromethane utilization
CmuA, two-domain methyltransferase/corrinoid binding protein	Mchl_5697	cmuA e	CH3Cl f	67.0	5.5	223	54	33	203	37	50	224	47	48
CmuB, methylcobalamin:H4F methyltransferase (EC 2.1.1.86)	Mchl_5727	cmuB e	CH3Cl f	33.3	5.1	203	21	57	142	27	47	130	33	49
CobH2, precorrin-8X methylmutase (EC 5.4.1.2)	Mchl_5691g	cobH2 e	CH3Cl	22.0	5.1	203	28	85	n.d. h	n.d.	n.d.	n.d.	n.d.	n.d.
MetF, 5,10-methylene-H4F reductase (EC 1.5.1.20)	Mchl_1881	metF	CH3Cl	34.1	6.6	n.d.	n.d.	n.d.	170	63	47	n.d.	n.d.	n.d.
PaaE-like, oxidoreductase FAD/NAD(P)-binding domain protein	Mchl_5717	paaE e	CH3Cl/+++ f	40.2	4.7	258	27	66	271	51	64	216	10	65
PurU, formyl-H4F hydrolase (EC 3.5.1.10)	Mchl_5699	purU e	CH3Cl f	32.8	6.6	n.d.	n.d.	n.d.	238	40	66	147	13	61
Methylotrophy
Fae, formaldehyde-activating enzyme (EC 4.3.−. − )	Mchl_2169	fae	− −	18.1	5.7	108	47	42	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.
Fch, methenyl-H4F cyclohydrolase (EC 3.5.4.9)	Mchl_2134	fch	− − f	21.7	4.8	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	116	18	55
FtfL, formate-H4Fligase (EC 6.3.4.3)	Mchl_0447	ftfL	+++	59.5	6.8	n.d.	n.d.	n.d.	348	46	58	n.d.	n.d.	n.d.
Hpr, hydroxypyruvate reductase, NAD(P)H-dependent (EC 1.1.1.29)	Mchl_2132	hprA	− −	34.2	5.2	152	46	39	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.
MauB, methylamine dehydrogenase (EC 1.4.99.3) large subunit	Mchl_0565	mauB	CH3Cl/+++	44.7	7.2	n.d.	n.d.	n.d.	270	49	66	168	11	49
MtdA, bifunctional protein [NADP-dependent methylene-H4MPT/methylene-H4Fdehydrogenase] (EC 1.5.1.−/1.5.1.5)	Mchl_2133	mtdA	− −	29.7	7.0	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	121	43	34
MtkA, malate thiokinase large subunit (EC 6.2.1.9)	Mchl_2135	mtkA	− −	42.0	5.8	n.d.	n.d.	n.d.	278	51	75	n.d.	n.d.	n.d.
MxaF, methanol dehydrogenase (EC 1.1.99.8) large subunit	Mchl_4518	mxaF	CH3Cl/+++ f	68.4	5.9	129	16	19	364	63	54	82	8	20
Sga, serine glyoxylate aminotransferase (EC 2.6.1.45)	Mchl_2131	sga	− − f	43.2	6.9	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	283	24	78
Central metabolism
Acs, acetyl-CoA synthetase (EC 6.2.1.1)	Mchl_2785	acs	CH3Cl/+++ f	72.2	5.6	413	38	53	374	53	55	231	16	41
CbbA, fructose-bisphosphate aldolase (EC 4.1.2.13)	Mchl_2646	cbbA	CH3Cl	38.6	5.5	92	37	28	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.
CysK, cysteine synthase A and O-acetylserine sulfhydrolase A subunit (EC 2.5.1.47)	Mchl_0937	cysK	CH3Cl	34.5	5.9	186	37	64	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.
EtfA, electron transfer flavoprotein subunit alpha	Mchl_1823	etfA	− −	32.4	5.0	n.d.	n.d.	n.d.	190	57	76	n.d.	n.d.	n.d.
EtfB, electron transfer flavoprotein subunit beta	Mchl_1822	etfB	− −	26.7	7.9	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	90	7	37
FumC, fumarase C (EC 4.2.1.2)	Mchl_2891	fumC	CH3Cl	49.8	5.6	129	81	51	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.
GcvT, H4F-dependent aminomethyltransferase, glycine cleavage complex subunit (T protein) (EC 2.1.2.10)	Mchl_0814	gcvT	CH3Cl	40.3	6.0	n.d.	n.d.	n.d.	189	42	55	n.d.	n.d.	n.d.
GlpX, fructose 1,6-bisphosphatase, class II (EC 3.1.3.11)	Mchl_2242	glpX	+++	34.6	5.4	146	44	41	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.
HisA, phosphoribosylformimino-5-aminoimidazole carboxamide ribotide isomerase	Mchl_2774	hisA	CH3Cl	26.7	5.4	183	27	42	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.
HisD, bifunctional histidinal dehydrogenase and histidinol dehydrogenase (EC 1.1.1.23)	Mchl_2261	hisD	CH3Cl	45.4	5.0	n.d.	n.d.	n.d.	200	34	58	n.d.	n.d.	n.d.
Hss, homospermidine synthase (EC 2.5.1.44)	Mchl_5462 i	hss2 e	− −	53.2	5.3	246	37	40	n.d.	n.d.	n.d.	103	9	25
Lpd, dihydrolipoamide dehydrogenase (EC 1.8.1.4), glycine cleavage complex	Mchl_1930	lpd	− − −	49.0	5.7	81	26	20	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.
MetK, S-adenosylmethionine synthetase (EC 2.5.1.6)	Mchl_3629	metK	− −	41.8	5.4	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	227	19	76
NAD(P)H:quinone oxidoreductase (EC 1.6.5.2)	Mchl_4391	qorB	CH3Cl	38.2	7.8	254	31	63	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.
NuoE, NADH-quinone oxidoreductase, chain E (EC 1.6.5.3)	Mchl_1210	nuoE	++	44.6	4.8	n.d.	n.d.	n.d.	139	57	65	n.d.	n.d.	n.d.
NuoF, NADH-quinone oxidoreductase, chain F (EC 1.6.5.3)	Mchl_1209	nuoF	CH3Cl	47.4	6.4	n.d.	n.d.	n.d.	217	30	49	n.d.	n.d.	n.d.
PntAA, NAD(P)+ transhydrogenase, subunit alpha part 1 (EC 1.6.1.2)	Mchl_2986	pntAA	CH3Cl/+++ f	39.6	5.6	177	35	45	n.d.	n.d.	n.d.	256	18	83
ureidoglycolate lyase (EC 4.3.2.3)	Mchl_4377	j	CH3Cl	31.4	5.3	70 l	24 l	25 l	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.
putative homoserine O-acetyltransferase (EC 2.3.1.31)	Mchl_4434	j	++	42.1	5.9	n.d.	n.d.	n.d.	279	27	66	n.d.	n.d.	n.d.
Adaptation to stress
KatA, catalase (hydroperoxidase II) (EC 1.11.1.6)	Mchl_3534	katA	CH3Cl/+++ f	60.0	5.9	394	23	63	324	35	57	256	6	56
MdoG, periplasmic glucan biosynthesis protein	Mchl_2321	mdoG	CH3Cl	58.7	5.6	n.d.	n.d.	n.d.	323	47	56	n.d.	n.d.	n.d.
RfbC, dTDP-4-dehydrorhamnose 3,5-epimerase (EC 5.1.3.13)	k	rfbC	+++	19.8	5.5	102	25	50	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.
SufS, selenocysteine lyase (EC 4.4.1.16)	Mchl_4348	sufS	+++	45.6	5.9	223	33	65	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.
SurE, 5′-nucleotidase (EC 3.1.3.5)	Mchl_4603	surE	CH3Cl	27.3	5.4	112	27	46	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.
UspA-like, putative universal stress protein	Mchl_1555	uspA	+++	29.3	6.1	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	79	10	32
putative manganese catalase (EC 1.11.1.6)	Mchl_3002	j	CH3Cl	31.0	4.9	144	36	42	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.
Protein biosynthesis and modification
AspS, aspartyl-tRNA synthetase (EC 6.1.1.12)	Mchl_4374	aspS	CH3Cl	67.2	5.5	n.d.	n.d.	n.d.	245	16	48	n.d.	n.d.	n.d.
ClpP, ATP-dependent Clp protease, proteolytic subunit (EC 3.4.21.92)	Mchl_2679	clpP	− −	23.1	5.8	n.d.	n.d.	n.d.	181	40	71	n.d.	n.d.	n.d.
EF-Ts, protein chain elongation factor	Mchl_2348	tsf	− −	32.3	5.5	n.d.	n.d.	n.d.	214	27	70	n.d.	n.d.	n.d.
EF-Tu, protein chain elongation factor, GTP-binding factor	Mchl_2438	tufB	− − f	43.1	5.4	n.d.	n.d.	n.d.	86	32	28	n.d.	n.d.	n.d.
Other functional classes
ABC transporter, sulfate/thiosulfate transporter periplasmic protein	Mchl_0592	cysP	CH3Cl	30.9	5.1	n.d.	n.d.	n.d.	254	31	63	n.d.	n.d.	n.d.
ABC transporter, putative periplasmic substrate-binding protein	Mchl_0388	j	CH3Cl	69.0	6.7	288	45	52	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.
ABC transporter, putative substrate-binding protein, aliphatic sulphonates	Mchl_0381	j	− −	34.3	8.1	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	120	8	47
conserved protein of unknown function	Mchl_4437	j	− −	16.1	5.5	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	234 l	21 l	26 l

^aMaGe database (http://www.genoscope.cns.fr/agc/mage).

^bProbability-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.

^cSpots indicated as “CH₃Cl” were only detected in the proteome of M. extorquens 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:++(−−) 2- to 5-fold;+++(−−−) more than 5-fold.

^dNL, non linear pI range used in 2D-DIGE experiments.

^eOnly found in strain CM4 (among the 8 Methylobacterium strains for which the complete genome sequence is known; [5], [11]) and localized on plasmid pCMU01.

^fMultiple spots detected.

^gMass spectrometry used to discriminate from Mchl_1712 displaying 86% sequence identity at the protein level.

^hn.d., not detected.

ⁱMass spectrometry used to discriminate from Mchl_2317 displaying 96% sequence identity at the protein level.

^jNo assigned gene name.

^kMass spectrometry data did not allow us to discriminate between two homologs with 99% sequence identity (Mchl_2669/Mchl_4004).

^lTandem mass spectrometry identification.

Many of the identified proteins with differential abundance have known or suspected roles in chloromethane utilization and methylotrophy (Table 3). Many of these proteins allowed to define chloromethane-specific clusters encoding proteins more abundant during growth with chloromethane (Fig. 2, Clusters A–F), clusters responding both to chloromethane and methanol (Clusters G–H), and or to methanol only (Clusters I–J). The two-domain methyltransferase/corrinoid binding protein CmuA, the methylcobalamin:H₄F methyltransferase CmuB, and the formyl-H₄F hydrolase PurU shown to be essential for chloromethane metabolism in strain CM4, were identified in the chloromethane proteome only (Fig. S1) as expected [6], [9], [10]. Experimental evidence for chloromethane-enhanced expression of a protein involved in cobalamin biosynthesis (precorrin-8X methylmutase CobH2), and of a putative oxidoreductase with FAD/NAD(P)-binding domain encoded by a paaE-like gene often associated with cmu genes [2], was obtained here for the first time. Overall, only cluster A encoding proteins more abundant during growth with chloromethane (CmuA; CmuB; CobH2; PaaE-like; PurU; Hss2; Table 3) was localized on plasmid pCMU01.

Figure 2. Gene clusters associated with the chloromethane response.

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 M. extorquens DM4 grown with dichloromethane compared to methanol [16], are indicated with circles labeled by a “D”.

Proteomic Identification of Stress-related Proteins

Upon dehalogenation, each mole of chloromethane yields one mole of hydrochloric acid with concomitant decrease in pH and increase in chloride concentration [7]. Chloromethane-associated proteins with homologs characterized in E. coli for their role in osmoprotection were identified (Proteomic data, Table 3). Among these, protein MdoG may be associated with metabolism of osmoregulated periplasmic glucans [30], the putative dTDP-4-dehydrorhamnose 3,5-epimerase RfbC may be involved in the synthesis of surface polysaccharides [31], and the putative nucleotidase SurE may be associated with survival at high NaCl concentrations, as observed in E. coli [32], where the corresponding gene lies within a survival operon conserved in Gram-negative bacteria [33].

Production of reactive oxygen species also seems associated with chloromethane utilization. One representative of each class of catalases known to catalyze disproportionation of hydrogen peroxide (H₂O₂) [34] was more abundant in the chloromethane proteome. Mchl_3002 is a putative non-haem manganese-containing catalase. Mchl_3534 is a KatA-like protein, whose gene is found next to a putative H₂O₂ activator gene sharing 43% aa Id with E. coli OxyR. In E. coli, OxyR induces the Suf system (sulfur mobilization [Fe-S] cluster) to combat inactivation of the [Fe-S] Isc assembly system by H₂O₂ [35]. Moreover, E. coli mutants lacking the Suf machinery are hypersensitive to cobalt at high concentrations of 200 µM [36]. In this study, SufS, a selenocysteine lyase homolog, was found more abundant in the chloromethane proteome. Similarly, the CysK cysteine synthase homolog more abundant in chloromethane cultures suggests the probable importance of reactivation systems to maintain chloromethane dehalogenase activity under aerobic conditions, as cysteine is involved in maintaining the catalytic activity and structure of many proteins with [Fe-S] clusters including ferredoxins [37].

Taken together, these data suggest that growth with chloromethane may elicit stress responses, and in particular an oxidative stress response.

Discussion

This work reports genomic and proteomic data demonstrating that cmuA and cmuB genes are plasmid-borne, and that plasmid pCMU01drives the previously defined pathway for H₄F-mediated chloromethane dehalogenation [6]. Specifically, plasmid pCMU01 harbors cognate genes involved in chloromethane-associated H₄F metabolism not found in other M. extorquens genomes (folC2, folD, metF2 and purU; Table 1) [6], [10].

H₄F metabolism is likely to be strongly modulated during growth on chloromethane since proteins linked to H₄F such as CmuA, CmuB, MetF and PurU were exclusively detected during growth with chloromethane (Table 3), whereas proteins associated to methanol oxidation with the metabolic intermediate formaldehyde and the C₁ carrier H₄MPT were more abundant during growth with methanol (proteins Fae, Fch and MtdA; Fig. 1).

Here, the interplay of chloromethane and other methylotrophic pathways was evidenced for the first time. Two components of the glycine cleavage complex involved in the conversion of H₄F and glycine to 5,10-methylene-H₄F [38], the key C₁ intermediate for entry in the serine cycle, were either more abundant with chloromethane or with methanol (GcvT and Lpd, respectively; Table 3). This suggests that enzymes implied in central metabolism such as the glycine cleavage complex might be involved in integrating contradictory signals during growth with C₁ compounds, to fine-tune metabolic conditions required for growth, and to even out variations in available carbon sources.

Our proteomic study also revealed that essential serine cycle enzymes (Sga, HprA and MtkA) were more abundant in methanol-grown cultures (Table 3). These enzymes are encoded by a chromosomal region (Fig. 2, cluster J), highly conserved in Methylobacterium [11]. Acetyl-CoA, glyoxylate and NADP⁺ have been demonstrated to decrease binding of QscR, a key regulator of C₁ metabolism [39] to the sga promoter, thereby inhibiting transcription of the major operon of the serine cycle (sga- hpr-mtdA-fch, [40]). The higher level of acetyl-CoA synthetase in chloromethane-grown cultures (Table 3) may explain the observed lower abundance of five enzymes encoded by cluster J.

PaaE-like Oxidoreductase, PntAA, MetF and Acs are Proteins with Predicted Functions for Growth with Chloromethane

Proteomic data provided first experimental evidence for the involvement of four previously undetected proteins, identified here as more abundant during growth with chloromethane, in chloromethane utilization.

The PaaE-like protein encoded by plasmid pCMU01 features a ferredoxin reductase-type FAD binding domain and a 2Fe-2S ferredoxin-type iron-sulfur binding domain. The PaaE-like protein is the only iron-sulfur enzyme more abundant in the chloromethane proteome. This PaaE-like oxidoreductase was suggested to be responsible for the observed methanethiol oxidase activity in the chloromethane-degrading strain, Aminobacter lissarensis CC495 [41], [42]. It is also conceivable that the PaaE-like protein acts in the reactivation of the corrinoid cofactor from the inactive Co(II) to the Co(I) form (Fig. 1), as corrinoid-dependent methyltransferases are prone to inactivation by oxidation, and bacteria often require an efficient reactivation system to maintain such proteins in an active form [43], [44]. The implication of PaaE in chloromethane utilization may be linked to the detection of genes associated with the oxidation stress response in chloromethane-grown M. extorquens CM4.

The transhydrogenase protein PntAA (Fig. 2 cluster D) couples the transfer of reducing equivalents between NAD(H) and NADP(H) to the translocation of protons across the membrane [45]. Previous transcriptomic and proteomic studies showed that PntAA was up-regulated in succinate- vs methanol-grown cultures of M. extorquens AM1 [46], [47], indicating possible differences in energy and reducing equivalent production occurring in M. extorquens strains grown on different carbon sources. Here, the higher abundance of the PntAA complex may be the consequence of higher requirements for reducing equivalents coupled to proton extrusion during chloromethane assimilation.

The 5,10-methylene-H₄F reductase MetF identified in the chloromethane-proteome is the chromosome-encoded protein and not the plasmid-borne protein MetF2 previously shown to be essential for chloromethane utilization [6]. The protein product of metF2 with a calculated pI of 9.5 is at the limit of the pH range studied in our experiments, which may explain why MetF2 was not detected here. In E. coli, MetF provides one-carbon precursors for methionine synthesis [48] and operates in the opposite direction of the chloromethane degradation pathway. If both MetF and MetF2 homologs share the same metabolites as substrates and products, regulatory processes in the expression of the corresponding genes arising from differences in availability of metabolites may explain the observed increased abundance of MetF with chloromethane. Further experiments are required to clarify the implications of MetF homolgs in chloromethane metabolism.

Identification of the chromosome-encoded acetyl-CoA synthetase Acs as more abundant in the chloromethane proteome was initially surprising. This protein is predicted to catalyze ATP-dependent conversion of acetate to acetyl-CoA (78% aa Id with the characterized Bradyrhizobium japonicum Acs enzyme, [49]). Acetyl-CoA is a key metabolite at the interface of C₁ and multicarbon interconversions involving H₄MPT-dependent C₁ subtrate oxidation pathways, the serine cycle, and the ethylmalonyl-CoA pathway essential for growth with methanol [50]. The interconversion of central intermediates such as acetyl-CoA could thus be modulated upon growth with the H₄F-dependent chloromethane oxidation pathway compared to growth with methanol, resulting in higher abundance of Acs in cells grown with chloromethane.

Plasmid-encoded BluB2: a Potential Link between H₄F and AdoCbl Cofactors of Chloromethane Utilization

BluB was demonstrated to catalyze two distinct enzymatic reactions of the AdoCbl biosynthetic pathway: i) conversion of cobinamide to Cbl as a cob(II)yrinic acid a,c-diamide reductase (EC 1.16.8.1) [51]; ii) synthesis of the lower ligand of AdoCbl, dimethylbenzimidazole in Alphaproteobacteria Sinorhizobium meliloti [52]and Rhodospirillum rubrum [53]. Strain CM4 harbors two bluB homologs: bluB, conserved in all investigated M. extorquens chromosomes, and bluB2 located on plasmid pCMU01 (38% aa Id between BluB and BluB2; Table 2). BluB2 is highly similar to a characterized enzyme [54] that triggers the oxygen-dependent transformation of reduced flavin mononucleotide (FMNH₂) in dimethylbenzimidazole and D-erythrose 4-phosphate, a key precursor of chorismate and an intermediate in H₄F biosynthesis (Fig. 3). Considering the strong level of sequence conservation with proteins of known function (62% aa Id with S. meliloti BluB), we speculate that gene bluB2 may be central for chloromethane assimilation by providing precursors for biosynthesis of essential cofactors of this metabolism.

Figure 3. Gene redundancy in the biosynthesis of cofactors required for chloromethane utilization in Methylobacterium extorquens CM4.

Cbi, cobinamide; Cbl, cobalamin; Ado, adenosyl; DMB, dimethylbenzimidazole; NaMN, nicotinate mononucleotide. AdoCbl and tetrahydrofolate are essential cofactors of the cmu pathway [6], [9]. 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 de novo 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 [69], the putative transmembrane proteins CbtA and CbtB [14], the Resistance-Nodulation-Division (RND)-type Co²⁺/Zn²⁺/Cd²⁺ efflux system CzcA [27], or the Icu transporter [70]. The TonB-dependent Btu system imports preformed corrinoid compounds [28]. We hypothesize that BluB-related proteins link AdoCbl and H₄F de novo synthesis.

Which Role for the Observed Vitamin B₁₂-related Gene Redundancy in Chloromethane Metabolism?

Half of the genomes of sequenced prokaryotes that contain homologs of cobalt-utilizing enzymes also possess the AdoCbl biosynthetic pathway, while 90% of the remaining acquire external vitamin B₁₂ via the BtuFCD transport system [55]. The genome of M. extorquens CM4 contains chromosomal- and plasmid-borne genes for both AdoCbl biosynthesis and corrinoid salvaging via the Btu transporter (Table 2). In addition, plasmid pCMU01 potentially encodes the capacity to remodel exogenous corrinoids, as suggested from the presence of gene cobO2 encoding the cob(I)yrinic acid a,c-diamide adenosyltransferase enzyme [14] and of gene cobU2 encoding nicotinate-nucleotide-dimethylbenzimidazole phosphoribosyltransferase (Fig. 3). We speculate that the presence of a variety of corrinoid salvaging pathways, possibly with different substrate affinities and expression profiles (e. g. in response to oxygen, cobalt, vitamin B₁₂ or dimethylbenzimidazole availability), may supply M. extorquens CM4 with corrinoid coenzyme required for efficient chloromethane dehalogenation in different environments.

Gene duplications are known to correlate with adaptive interactions in prokaryotes by providing competitive advantages for adaptation on specific environmental conditions [56]. Here, cobA-encoded S-adenosyl-L-methionine-dependent uroporphyrinogen III methylase (SUMT) may catalyze the transformation of uroporphyrinogen III into precorrin-2 [57] (Fig. 3). SUMT activity may be provided by either CobA and CysG [58], but only CysG is conserved in Methylobacterium sequenced strains. Plasmid-borne CobA protein, as a key branch point enzyme in the biosynthesis of modified tetrapyrroles, may favor synthesis of AdoCbl over siroheme driven by chromosome-encoded CysG, control flux to AdoCbl precursor synthesis, and consequently facilitate growth of M. extorquens CM4 with chloromethane.

Plasmid pCMU01 encodes a full complement of genes for the corrinoid salvaging pathway, but contains only an incomplete set of genes for the oxygen-dependent AdoCbl de novo biosynthesis pathway. Genes cobB, cobF, cobG, cobN, cobS, cobT and cobV are missing compared to the canonical pathway characterized in P. denitrificans (Fig. 3; Table 2; [14]). However, other yet uncharacterized protein-encoding genes of plasmid pCMU01 may substitute for missing AdoCbl biosynthetic genes. For instance, as previously suggested in Streptomyces [59], cbiD located next to cmuBC encodes a typical oxygen-independent AdoCbl biosynthetic enzyme which, as the missing CobF, features a S-adenosylmethionine binding site. Protein CbiD was reported as a cobalt-precorrin-6A synthase in the anaerobic AdoCbl biosynthesis pathway [60], but its potential role in the corresponding aerobic pathway remains to be investigated.

Suprisingly, out of more than 20 putative vitamin B₁₂-related proteins encoded by plasmid pCMU01, only CobH2 was more abundant in the chloromethane proteome (Table 2). A possible explanation for this observation is that vitamin B₁₂-related proteins are also required during growth with methanol, e.g. as cobalamin is an essential cofactor for MeaA (ethylmalonyl-CoA mutase) activity in the ethylmalonyl-CoA pathway for glyoxylate regeneration [61].Whether redundant genes encoding vitamin B₁₂-related protein in M. extorquens CM4 are expressed and functional remains to be evidenced.

Plasmid pCMU01 provides another example of bacterial clustering of genes encoding functional enzymes and cognate genes for cofactor biosynthesis (Fig. 2A). Such a genetic linkage was described for the insect symbiont Hogdkinia which has one of the smallest genomes, but dedicates 7% of its proteome to cobalamin synthesis [62]. Similarly, Lactobacillus reuteri possesses a metabolic genomic island involved in 3-hydroxypropionaldehyde biosynthesis which associates cobalamin biosynthetic genes and genes of the anaerobic glycerol metabolism [63], most probably reflecting a cobalamin requirement for glycerol dehydratase activity [64], and the polyketide synthesizing bacterium Streptomyces sp. DSM4137 has AdoCbl biosynthetic genes adjacent to a putative elaiophylin biosynthetic gene cluster that includes a gene encoding AdoCbl-dependent methylmalonyl-CoA mutase [59]. The genetic linkage of cmu and cob genes is likely to provide an evolutionary advantage for efficient bacterial growth with chloromethane by the cmu pathway.

Origin and Evolution of Plasmid pCMU01

The mosaic organization of plasmid pCMU01 suggests a history of carbon utilization-related gene acquisition for chloromethane and acetone. Multistep assembly of various genetic elements dedicated to chloromethane utilization in the cmu plasmid is supported by several observations: i) A 137 kb segment flanked by IS elements (ISMch8 and ISMch3 of IS5 and IS3 family, respectively) contains all hitherto identified chloromethane utilization genes; ii) A 33 kb segment between cmuBC and cmuA contains remnants of transposase genes (Mchl_5703 and Mchl_5711) and genes involved in corrinoid compound metabolism such as the cobU2 gene and the putative cobalt heavy metal efflux transporter Czc cluster (Fig. 2A); iii) Atypical genes encoding enzymes relevant to growth on chloromethane such as genes bluB2, cbiA, and cobA are found (Table 1; [65]); iv) The acxCABR cluster encoding acetone carboxylase for carbon assimilation is flanked by mobile elements (Table 1 and data not shown); v) 10% of the predicted plasmid CDS are mobile element-related sequences (Table 1); vi) Relics of genes driving plasmid replication (Mchl_5589, 50 residues share 70% aa Id with the N-terminal part of the replication protein RepA, Mchl_5615, 408 residues) suggests that plasmid pCMU01 was assembled by acquisition of parts of different episomes.

Concluding Comments

Our proteomic analysis showed that the adaptive response of M. extorquens CM4 to chloromethane mostly involves functions which are common to M. extorquens strains, as observed previously for adaptation of M. extorquens DM4 to dichloromethane [16]. Indeed, out of five identified gene clusters responding specifically to chloromethane (Fig. 2), only the catabolic gene cluster essential for growth with chloromethane is encoded by plasmid pCMU01. When these five gene clusters responding specifically to chloromethane in M. extorquens CM4 were compared to the seven gene clusters responding specifically to dichloromethane in M. extorquens DM4, only one chromosomal gene cluster common to chloromethane-degrading strain CM4 and dichloromethane-degrading strain DM4 was identified (cluster B in Fig. 2; cluster C in [16], suggested to be involved in cell structure). Based on these findings, the adaptive response to growth with chloromethane is clearly quite different from that for growth with dichloromethane, in line with the completely different pathways for metabolism of these two halogenated C₁ compounds.

On a final note, it is striking that the organization of the cmu genes on plasmid pCMU01 of M. extorquens CM4 is different from that found in bacteria utilizing the cmu pathway known to date [2]. M. extorquens CM4 constitutes the first representative strain of the M. extorquens species for which a plasmid-encoded carbon utilization function has been clearly established, and its plasmid pCMU01features the only known catabolic gene cluster for the metabolism of halogenated methanes that is not chromosome-borne [4], [11], [16], [66]. The state of plasmid pCMU01 as an autonomous replicating episome may have enabled the efficient acquisition of relevant resources for growth with chloromethane, and the shaping of unique genetic features not observed in other genomes of chloromethane-degrading bacteria.

Supporting Information

Figure S1

2D-DIGE master gel image of total protein extracts from chloromethane- and methanol-grown M. extorquens CM4 labeled with Cy2 (internal standard). Highlighted spots (circles) displayed differential abundance between chloromethane and methanol conditions, and were identified by mass spectrometry. 1, CmuA; 2, CmuB; 3, PurU; 4, PaaE-like oxidoreductase; 5, Fch; 6, Sga; 7, MtdA; 8, putative UspA-like protein; 9, KatA; 10, MetK; 11, Hss; 12, Acs; 13, PntAA; 14, putative endoribonuclease (Mchl_4437) (See Table 3 and Fig. 1 legend).

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Funding Statement

S. Roselli was supported by a PhD grant from the French ministry of research and higher education. Support of this work is by REALISE, the Alsace Network for Engineering and Environmental Sciences (http://realise.u-strasbg.fr), and by the EC2CO program of Institut National des Sciences de l’Univers, Centre national de la recherche scientifique. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.