Growth conditions of M. acetivorans
All M. acetivorans strains (Additional file 1: Table S3) were routinely grown anaerobically as pre-cultures at 37 °C in an 80 % N2/19 % CO2/1 % H2 atmosphere with mild shaking in 10 mL HS medium  with 150 mM methanol as the carbon source. Cell growth was measured spectrophotometrically and direct cell counts were confirmed by staining cell cultures (often containing precipitates) with SYTO9 dye (Life Technologies, Carlsbad, CA, USA) and viewed microscopically using a bright-line hemocytometer (Hausser Scientific, Horsham, PA, USA) under phase-contrast and epifluorescence settings (Zeiss Axio Scope.A1, Germany). All 28-mL culture tubes (18 × 150 mm, Bellco Glass, Vinelanad, NJ, USA) were sealed by aluminum crimp seals. Plasmids were maintained with 2 µg/mL puromycin.
For long-term (ca., 40 days) growth on methane with low-density inocula (1 %), the M. acetivorans strains were grown in 5 mL HS medium in 28-mL culture tubes with additional electron acceptors at 37 °C with mild shaking; the electron acceptors tested were FeCl3, FeSO4, KNO3, NaNO3, NaNO2, and ZnSO4 (0.05–100 mM) as well as Fe2O3 (1 mM) and MnO2 (2 mM). As a frame of reference, a concentration of 0.1 mM would be 0.5 µmol in a 5-mL culture. The preferred carbon sources of M. acetivorans, methanol, trimethylamine, and acetate, were not present in the medium. The headspace of the tube was filled with methane (99.999 % purity, catalog no. ME5.0RS, Praxair, Danbury, CT), and crimped with aluminum seals. PCR was used to verify the presence of pES1-MATmcr1 in methane-grown cells after 30 days of incubation. Cells were used as genomic templates for PCR amplification of ANME-1 mcrA using primers B4-f and pES1-r (Additional file 1: Table S4). Cell morphology was examined on a transmission electron microscope (FEI Tecnai G2 Spirit BioTwin, Hillsboro, OR, USA) using uranyl acetate-stained cells.
To document cell growth and methane consumption as a function of time, 107 CFU/mL of M. acetivorans/pES1-MATmcr3 and M. acetivorans/pES1(Pmat) cells were incubated at 37 °C for 6 weeks in 8 mL HS medium and 10 mM FeCl3 in 40-mL bottles stoppered with butyl rubber stoppers and crimped with aluminum seals. Each culture and its methane in the headspace were sampled every 1–2 weeks. After each sampling, petroleum jelly was applied to the surface of the stoppers to prevent leaking from needle punctures. All culture bottles were inverted during incubation to prevent methane from escaping from the vessels, and were shaken to ensure homogenous mixing of liquid, precipitates, and cells. As a control, cultures grown on methanol in parallel reached a saturated density of (4 ± 0.3) × 108 CFU/mL.
For short-duration (ca., 5 days) growth on methane in which high cell-density inocula were used, 2 mL of each strain was pre-grown in 200 mL of HS medium with 150 mM methanol (and 2 µg/mL puromycin when plasmids were present) at 37 °C for 5 days (OD600 ~ 1.0). Cells were collected by centrifugation (5000 rpm for 20 min), and were washed three times with HS medium and puromycin alone to remove residual methanol. The final cell pellet was resuspended using 5 mL of HS medium supplemented with 0.1 or 10 mM FeCl3 and 2 µg/mL puromycin when appropriate, to yield a density of 4 × 1010 CFU/mL. After filling the headspace of each tube with methane, the tubes were incubated at 37 °C with mild shaking for 5–10 days.
Cloning ANME-1 mcrBGA
All oligonucleotides are listed in Additional file 1: Table S4. The ANME-1 mcrBGA genes (3.9 kb, locus tag fos0113c9_0022-0024, Genbank accession FP565147.1) encoding the ANME-1 Mcr whose 3D structure has been determined , was assembled from six DNA fragments of 600–700 bp (Integrated DNA Technologies) using the Gibson assembly method . Unlike the mcr locus from M. acetivorans  and other methanogenic archaea , mcrC and mcrD are not present. ANME-1 mcrBGA were cloned downstream of promoter Pcdh using the XbaI and BmtI sites of pES1 to form pES1-MATmcr1. After electroporating the plasmid into E. coli DH5α-λpir, the complete mcrBGA locus and promoter region were sequenced (via primers veri-p-f, pES1-f, MATmcrB2-f, MATmcrB3-f, MATmcrB4-r, and pES1-r) to confirm no errors were introduced during cloning. For pES1-MATmcr2, in which ANME-1 mcrBGA genes are under the transcription of mcr promoter from M. acetivorans (Pmcr_M. acetivorans
), a 419-bp DNA fragment that corresponds to Pmcr_M. acetivorans
was amplified from the genomic DNA (catalog #35395D-5, American Type Culture Collection, Manassas, VA, USA) using primers Pmcr-f2 and Pmcr-r2. Pmcr_M. acetivorans
is further fused to ANME-1 mcrBGA using overlap PCR via primers Pmcr-f2 and B6-r1. To place ANME-1 mcrBGA under control its native promoter (which we named Pmcr_ANME-1), a 237-bp DNA fragment upstream of the ANME-1 mcrBGA genes was synthesized and assembled with mcrBGA to create Pmcr_ANME-1::mcr
ANME-1 using overlap PCR (via primers Pmat-f, Pmat-r, and B6-r1). The resulting plasmid is pES1-MATmcr3. The empty plasmid harboring Pmcr_ANME-1 was created by linearizing the vector backbone of pES1-MATmcr3 using primers pES1(Matprom)-f and pES1(Matprom)-r, followed by self-ligation of the backbone to form pES1(Pmat). All plasmids were transformed into M. acetivorans using liposome-mediated transformation .
Genomic DNA was isolated using the Ultraclean® Microbial DNA Isolation Kit (MO BIO Laboratories, Carlsbad, CA, USA). After shearing the genomic DNA using a Covaris ultrasonicator, DNA fragments were barcoded using TruSeq DNA Nano (Illumina, San Diego, CA, USA). The pooled, barcoded DNA library was sequenced on a MiSeq sequencing platform (Illumina) to generate 2,597,170 paired-end reads of 150 bp for M. acetivorans (ancestral strain) grown on methanol, and 2,699,253 paired-end reads of 150 bp for M. acetivorans/pES1-MATmcr1 grown on methane and 0.1 mM FeCl3.
To identify single-nucleotide polymorphisms, insertions, and deletions between the ancestral strain and the methane-grown strain, sequencing reads of each strain were mapped to the reference genome of M. acetivorans (Genbank accession NC_003552.1) using the Burrows-Wheeler Alignment tool . Aligned reads were sorted based on mapped position in the reference genome using SortSam from the Picard tools (http://broadinstitute.github.io/picard). Misalignments caused by insertions and deletions were corrected locally using IndelRealigner from the Genome Analysis ToolKit package . Duplicated reads were marked using MarkDuplicates.jar from the Picard tools to remove sequencing bias. Unified Genotyper  was then used to call the variants of each strain after removing variants that are present in less than 40 % of the population. The differences between the ancestral strain and the methane-grown strain were identified using vcftools .
16S rDNA amplification and sequencing
To verify archaeal strains, primers ARCH109-F and ARCH934-R  were used to amplify an 0.8-kb PCR product of 16S rDNA genes. To verify the absence of bacteria, primers 27F and 1492-R  were used to amplify an 1.5-kb PCR product of 16S rDNA genes.
Total protein, cysteine, bicarbonate, and iron reduction assays
Cultures (120 µL) were centrifuged briefly to remove the supernatant, and the cell pellets were resuspended with 10–24 µL of sterile water to lyse the cells. The total protein concentration of these cell suspensions was determined using the Bradford assay (Bio-Rad Laboratories, Hercules, CA, USA).
The concentration of total cysteine (reduced and oxidized forms) in HS media (in which cells showed methane consumption) was determined spectrophotometrically using ninhydrin which reacts specifically with l-cysteine, even in the presence of other thiols, to form a pink-colored product (εmax 560 nm) under acidic conditions . The concentration of bicarbonate of HS media (in which cells showed methane consumption) was measured spectrophotometrically using the MaxDiscovery™ Carbon Dioxide Enzymatic Assay Kit (Bioo Scientific, Austin, TX, USA). Filtered supernatant of each culture was used as a sample solution.
The reduction of iron was measured by employing the ferrozine method for quantification of only reduced iron in the form of Fe2+, as described previously . A 100-µL of culture was immediately mixed with 33 µL of 2 N HCl. Then, 10 µL of the acidified mixture was mixed with 190 µL of 1 mg/mL ferrozine in 100 mM HEPES (pH 7.0), and absorbance at 562 nm was measured. Actual Fe2+ concentrations were calculated by comparing results with those from standard solutions of Fe2+.
Gas chromatography (GC) and high-performance liquid chromatography (HPLC)
GC analyses were conducted for quantifying methane in the culture headspace. Aliquots of 50 or 100 µL volumes were passed through a 6890 N Agilent gas chromatograph equipped with a 60/80 Carboxen-1000 column (4600 × 2.1 mm, Supelco catalog no. 12390-U) and a thermal conductivity detector. The injector, column, and detector were maintained at 150, 180, and 240 °C, respectively. Carrier gas flow (nitrogen) was kept at 20 mL/min, and reference gas flow (also nitrogen) for the detector at 20 mL/min as well. Gases were identified according to their retention times and their concentrations were determined according to comparisons with standards.
HPLC analyses were conducted for the detection and quantification of three organic acids under investigation (acetic acid, formic acid, and pyruvic acid). All samples were filtered through a 0.22 µm polyvinylidene fluoride membrane before diluting 1:6 in running buffer (0.0025 M sulfuric acid in water), then 60 µL of the 1:6 dilution was fractionated by HPLC (Waters 717 autosampler with a model 515 pump, and a 2996 photodiode array detector) with a reversed-phase column [Phenomenex Rezex ROA-Organic Acid H + (8 %) (300 × 7.8 mm)]. Separations were conducted using an isocratic flow rate of 0.4 mL/min 0.0025 M sulfuric acid in water. Absorbance at 210 nm was used to detect all compounds. Chemicals used as standards for comparisons are glacial acetic acid (EMD Millipore, catalog no. AX0073-6), sodium formate (catalog no. BP356-100, Fisher Scientific, Hampton, NH, USA), and sodium pyruvate (catalog no. S648-500, Fisher Scientific). Peaks corresponding to those of pyruvic acid, formic acid, and acetic acid were confirmed by retention time, co-elution with standards, and by comparing absorbance spectra with those from the standards. Total quantities of the compounds were calculated by comparing peak areas with standard curves made by running chemical standards.
To demonstrate production of ANME-1 Mcr, a FLAG epitope tag was introduced into the carboxy terminus of ANME-1 McrA encoded by mcrA in pES1-MATmcr1, pES1-MATmcr2, and pES1-MATmcr3. The DNA encoding the FLAG tag was incorporated into primer B6-r-flag, which was used along with the respective forward primers to create ANME-1 mcrA-flag. After transformation, M. acetivorans harboring pES1-MATmcr1-flag, pES1-MATmcr2-flag, and pES1-MATmcr3-flag were grown on 200 mL HS-methanol for 5 days, and used for short-duration growth experiments. Methane consumption was measured after 5 days, and cells were harvested by centrifugation. Each cell pellet was resuspended in 2 mL Lysis Buffer [20 mM Tris–HCl, 0.1 mM EDTA, 500 mM ε-aminocaproic acid, 10 % glycerol, 1 µL protease inhibitor cocktail (Sigma)]. Cells were sonicated on ice at a power level of 10 for 150 s (30 cycles of 5 s each, 60 Sonic Dismembrator, Fisher Scientific). Total proteins were resolved via 12 % Tris–glycine-SDS gels. Western blots were performed with monoclonal horseradish peroxidase-conjugated antibodies raised against a FLAG epitope tag (Thermo Scientific, Waltham, MA, USA). Blotted proteins were detected using the chemiluminescence reagents from the SuperSignal West-Pico Chemiluminescence kit (Thermo Scientific).
13C-labeled methane-grown cultures, 13C-labeled bicarbonate-grown cultures, 13C NMR, and GC/MS
Starter cultures (200 mL) of M. acetivorans/pES1(Pmat) and M. acetivorans/pES1-MATmcr3 were used for short-duration growth experiments. For cultures incubated with 13C-labeled methane, the headspace was filled with 13C-labeled methane (99 % 13C atom, Sigma). HS medium for cultures incubated with 13C-labeled bicarbonate was prepared using 13C-labeled sodium bicarbonate (99 % 13C atom, Cambridge Isotope Laboratories, Tewksbury, MA, USA). All cultures were incubated at 37 °C for 10 days. Acetate was measured using an Agilent 7890A/5975C GC/MSD using a Nukol (Supelco) capillary column (30 × 0.32, 0.25 μm phase thickness comprised of a bonded polyethylene glycol). Incorporation was determined by integrating the peaks areas corresponding to 12C and 13C acetate. NMR experiments were conducted on a Brüker Avance III HD spectrometer operating at 500.20 MHz and 125.78 MHz for 1H and 13C nuclei, respectively, using H2O:D2O as a solvent. 13C and DEPT-135 spectra were recorded with a spectral width of 220 ppm, using 64 K data points, a 90° excitation pulse (11 µs) and relaxation delay of 5 s. 1 k scans were collected and spectra zero-filled to 128 K. For all FIDs, line broadening of 1 Hz was applied prior to Fourier transform. Chemical shifts are reported in ppm from DSS (δ = 0). The gradient-selected 1H-13C heteronuclear multiple bond correlation (gHMBC) experiment was performed using a low-pass J-filter (3.4 ms) and delays of 65 and 36 ms to observe long-range C–H couplings with 256 increments and 64 transients of 2048 data points. The relaxation delay was 2.0 s. Zero-filling to a 2 K × 2 K matrix and π/2-shifted sine square bell multiplication was performed prior to Fourier transform. Heteronuclear Single Quantum Coherence (gHSQC) spectra were recorded with 256 increments in F1 and 32 scans per increment, using the standard hsqcetgpsisp.2 Brüker pulse sequence. Relaxation delay of 2 s and 2 K data points was used for spectral width of 10 ppm in the proton dimension, whereas the spectral width in the carbon dimension was 180 ppm.
Differential gene analysis of two growth conditions (three biological replicates each) was performed: (1) M. acetivorans/pES1-MATmcr3 contacted with methane and (2) M. acetivorans/pES1-MATmcr3 grown on methanol. All starter cultures (200 mL) were grown on methanol for 5 days, and harvested by centrifugation. Cell pellets were washed three times with HS medium, and resuspended using 5 mL HS medium, 2 µg/mL puromycin, and 0.1 mM FeCl3. For condition (1), methane was filled into the headspace of the cultures. For condition (2), 150 mM methanol was added. All cultures were incubated at 37 °C for 5 days, followed by rapid centrifugation in the presence of 50 µL RNAlater solution (Ambion, Austin, TX, USA) per mL of culture. Total RNA isolated using the RNeasy Mini kit (Qiagen, Valencia, CA, USA) was digested with terminator 5′-phosphate-dependent exonuclease (Epicentre, Madison, WI, USA) to partially remove ribosomal RNA. Digested RNA was cleaned using AgenCourt RNAClean XP beads (AgenCourt Bioscience, Beverly, MA, USA) and used for cDNA library construction using the TruSeq Stranded mRNA Library kit (Illumina). The pooled and barcoded cDNA library was sequenced on a HiSeq sequencing platform (Illumina). Obtained reads were mapped to the reference genome of M. acetivorans (Genbank accession NC_003552.1) and plasmid pES1-MATmcr3 using STAR . The mapped reads were assembled using Cufflink v2.2.1  to identify potential novel transcripts. Assembled, unannotated novel transcripts for all the strains were combined with the list of known genes. Differential expression of genes and potential novel transcripts were determined using Cuffdiff  at a significance cutoff at q < 0.07 with a false discovery rate of 0.05. Expression levels of gene transcripts are expressed as fragments per kilobase of transcript per million mapped fragments (FPKM) , and expression changes are determined by the ratio of FPKM of culture replicates grown on methane to FPKM of culture replicates grown on methanol. Gene expression data have been deposited in the Gene Expression Omnibus under accession code GSE66445.