SI Materials and Methods

General procedures. Oligonucleotide primers were synthesized by Integrated DNA Technologies (Coralville, IA). Genomic DNA was extracted using the UltraClean Microbial DNA Isolation Kit (MO BIO Laboratories, Carlsbad, CA). Recombinant plasmid DNA was purified with a QIAGEN Plasmid Mini Kit (Qiagen, Valencia, CA). Purification of PCR reactions and gel extraction of DNA fragments for restriction endonuclease clean up were performed using an Illustra GFX PCR DNA and Gel Band Purification Kit from GE Healthcare or a QIAquick Gel Extraction Kit (Qiagen). DNA sequencing was performed by Beckman Coulter Genomics (Danvers, MA). Nickel-nitrilotriacetic acid-agarose (Ni-NTA) resin was purchased from Qiagen. SDS-PAGE gels were purchased from Bio-Rad. Protein concentrations were determined according to the method of Bradford using bovine serum albumin (BSA) as a standard (1). Optical densities of E. coli cultures were determined with a DU 730 Life Sciences UV/Vis spectrophotometer (Beckman Coulter, Brea, CA) by measuring absorbance at 600 nm.

All chemicals were purchased from Sigma-Aldrich, except for (trimethyl-d9)-choline chloride (Cambridge Isotope Laboratories, Tweksbury, MA), NAD+ (MP Biomedicals), NADH (Calbiochem/EMD Millipore, Billerica, MA), and NADPH (Enzo Lab Sciences, Farmingdale, NY). Methanol and water used for liquid chromatography-mass spectrometry (LC-MS) were B&J Brand high purity solvents (Honeywell Burdick & Jackson, Muskegon, MI).

LC-MS/MS analysis was performed in the Small Molecule Mass Spectrometry Facility at Harvard University on an Agilent 6460 Triple Quadrupole Mass Spectrometer with Agilent 1290 uHPLC (Agilent Technologies, Wilmington, DE) (for derivatized TMA, derivatized TMA-d9, acetyl-CoA and propionyl-CoA detection) or in our research labs in the Department of Chemistry and Chemical Biology, Harvard University, on an Agilent 6410 Triple Quadrupole LC/MS instrument (Agilent Technologies, Wilmington, DE) (for underivatized TMA-d9). Samples and blanks were introduced via an electrospray ionization (ESI) source. The mass spectrometers were operated in multiple reaction monitoring (MRM) mode. The capillary voltage was set to 4.0 kV and the fragmentor voltage to 110 V (underivatized and derivatized TMA and TMA-d9), 200 V (propionyl-CoA), or 220 V (acetyl-CoA). The drying gas temperature was maintained at 350 °C (Agilent 6460) or 200 °C (Agilent 6410) with a flow rate of 12 L/min (Agilent 6460) or 10 L/min (Agilent 6460) and a nebulizer pressure of 25 psi (derivatized TMA and TMA-d9) or 35 psi (acetyl-CoA and propionyl-CoA) or 45 psi (underivatized TMA-d9). The precursor-product ion pairs used in MRM mode were: m/z 69.1→m/z 51 (underivatized TMA-d9), m/z 155.1→ m/z 66.2 (derivatized d9-TMA), m/z 146.1→ m/z 58.1 (derivatized TMA), m/z 808.1→ m/z 79 (acetyl-CoA), m/z 822.1→ m/z 79 (propionyl-CoA). The collision energies for the precursor-product ion pairs were 21 V (underivatized TMA-d9), 43 V (derivatized TMA and d9-TMA), 81 V (acetyl-CoA), and 85 V (propionyl-CoA). MS1 resolution was set to wide, MS2 resolution was set to unit, the time filter width used was 0.07 min, and the ΔEMV (Electron Multiplier Voltage) was 400 V. Data analysis was performed with Mass Hunter Workstation Data Acquisition software (Agilent Technologies).

For derivatized trimethylamine detection, the LC and data analyses were performed as previously described (2). For underivatized TMA-d9, the LC analysis was performed in positive mode, using a Kinetex (Phenomenex) HILIC column (2.6 μm, 30 mm × 2.1 mm, 100 Å), preceded by a C4 precolumn (3.5 μm, 2.0 mm × 20 mm). The LC conditions were: 80% B for 1.6 min, a gradient decreasing to 40% B over 2.4 min, 40% B for 2 min, a gradient increasing to 80% B over 2 min, and 80% B for 3 min (solvent A = 5 mM ammonium formate in water with 0.1% formic acid, solvent B = 100% acetonitrile). The flow rate was maintained at 0.3 mL/min for each run. Run time per sample was 9 min, and the first 1.4 min of flow through was sent to waste. The injection volume was 3 μL for both standards and samples. For quantification of TMA-d9, an external standard curve was prepared in BHI media supplemented with 1 mM choline-d9 and analyzed at concentrations ranging from 104 pg/mL to 627 pg/mL TMA-d9.

For acetyl-CoA and propionyl-CoA detection, the LC analysis was performed in negative ion mode, with an Eclipse XDB-C18 (Agilent Technologies) column (5 μm, 4.6 x 150 mm), at a flow rate of 0.4 mL/min. The LC conditions were: 30% B for 4 min, a gradient increasing to 100% B over 4 min, 100% B for 1 min, a gradient decreasing to 30% B over 1 min, and 30% B for 2 min (solvent A = 1 mM ammonium bicarbonate in 10% methanol, solvent B = 1 mM ammonium bicarbonate in 100% methanol). The injection volume was 20 mL for both standards and samples, the run time was 12 min, and first 2 min of flow through was sent to waste. For quantification of acetyl-CoA, an external standard curve was prepared and analyzed at concentrations ranging from 100 pg/mL to 10000 pg/mL acetyl-CoA, with propionyl-CoA as an internal standard at 1000 pg/mL.

GC-MS experiments for ethanol detection were conducted using a Waters Quattro micro GC Mass Spectrometer (Waters, Milford, MA, USA) equipped with a Combi PAL headspace autosampler (CTC Analytics, Zwingen, Swtizerland) and a split/splitless injector. The column used was a DB-624UI, 30 m x 0.32 mm x 1.80 µm (Agilent, Santa Clara, CA) and the inlet liner was a 1 mm straight single taper Ultra Inert liner (Agilent). The carrier gas was helium, held at 2.3 mL/min constant flow. Samples were incubated at 37 °C for 30 min, with an agitator speed of 500 rpm. The GC injection port was set at 220 °C, the syringe temperature to 120 °C, and the needle flush time was 120 s. A headspace sample volume of 1 mL was injected into the inlet with a split ratio of 20:1. The column temperature was initially maintained at 30 °C for 3 min, then increased to 250 °C at a rate of 50 °C/min with a total run time of 6.2 min. The GC was coupled to a mass spectrometer, equipped with an electron ionization source and a triple quadrupole mass analyzer with acquisition in positive mode. The GC interface temperature was maintained at 220 °C, and the ion source temperature at 200 °C. The mass analyzer was set to scan over a 45–100 m/z range for 0–1.3 min and a 20–100 m/z range for 1.3–6.2 min of the run. The Selected Ion Recording (SIR) function at m/z 31 was used for detection of ethanol over the length of the run. GC-MS data analysis was performed with the MassLynx software package.

RNA isolation and quantitative RT-PCR. D. desulfuricans ATCC 27774 cultures were grown in six 18 x 160 mm modified Hungate tubes containing 10 mL of lactate sulfate (LS) media and six tubes containing choline sulfate (CS) media with argon in the headspace as previously described (2). Media was inoculated with 0.1 mL of a two day-old LS starter culture. LS cultures were incubated at 21 °C for 10 hours and then at 37 °C for 30 additional hours. CS cultures were incubated at 37 °C for 40 hours. Upon incubation, the headspace of all cultures was flushed with argon for 30 min in order to remove any accumulated hydrogen sulfide and TMA. Two volumes of argon-sparged RNAprotect Bacteria Reagent (Qiagen) were added directly to one volume of each culture. RNA was extracted under anaerobic conditions using the RNeasy Protect Bacteria Kit (Qiagen) using chemical, enzymatic, and mechanical lysis with acid-washed <1-6 μm glass beads (Sigma Aldrich, St. Louis, MO). Residual DNA in the samples was removed by incubating 32 μL sample with 4 μL 10X DNase buffer and 4 μL RNase-free DNase (2 U/μL) for 1 hour at 37 °C, followed by RNA purification using the RNeasy Protect Bacteria Kit.

RNA was converted to cDNA using Superscript II reverse transcriptase from Invitrogen. Reactions containing 3 μg random hexamers (Invitrogen, Carlsbad, CA), 10 μmol dNTPs, and 3.5 μg RNA in a final volume of 13 μL were incubated at 65 °C for 5 min and then chilled on ice. 4 μL of 5X First-Strand Buffer, 1 μL of 0.1 M DTT, and 1.5 μL Superscript II reverse transcriptase (200 U/μL) were added to the reaction mixtures followed by incubation at 25 °C for 7.5 min, 42 °C for 75 min, and 70 °C for 15 min. The resulting cDNA was stored at –20 °C. Specific primers for qRT-PCR, listed in Table S1, were designed using Primer3 (3). The cutC gene was amplified by PCR from D. desulfuricans ATCC 27774 genomic DNA and gel purified. DNA was quantified by measuring absorbance at 260 nm, and then serially diluted to construct a standard curve. PCR reactions contained 10 ng of cDNA, 0.07 μM of each primer, and 12.5 μL of ABsolute qPCR SYBR Green Mix (Thermo Scientific, Waltham, MA), and 25 nM of ROX reference dye, in a total volume of 25 μL. Thermocycling was carried out on a Mx3000P qPCR System instrument (Stratagene/Agilent) with the following amplification conditions: 95 °C for 15 min, 40 cycles of 95 °C for 30 s, 54 °C for 45 s, and 72 °C for 1 min, followed by a dissociation curve from 30 s at 55 °C to 30 s at 95 °C. All samples were measured in duplicate, and multiple plate analysis used the standard curve as an inter-run calibrator. No template, no primer, and no reverse transcriptase controls were included. Transcription levels of the genes analyzed were normalized to 16S rRNA levels. The relative fold change of RNA transcription (choline/lactate) was determined using the 2−ΔΔCt method (4).

Cloning, expression, and purification of CutF and CutO. The cutF and cutO genes were PCR amplified from Desulfovibrio alaskensis G20 genomic DNA using the primers listed in Table S2. All PCR reactions (set up in triplicate) contained Phusion High-Fidelity PCR Master Mix (New England Biolabs, Ipswitch, MA), 1.3 ng DNA template, and 200 pmoles of each primer in a total volume of 20 mL. Thermocycling was carried out in a MyCycler gradient cycler (Bio-Rad, Hercules, CA) using the following parameters: denaturation for 30 s at 98 °C, followed by 35 cycles of 10 s at 98 °C, 30 s at 59.5 °C for cutO and 57.7 °C for cutF, 105 s at 72 °C, and a final extension time of 5 min at 72 °C. PCR reactions were analyzed by agarose gel electrophoresis with ethidium bromide staining, pooled, and purified. Amplified fragments were digested with NdeI and XhoI (New England Biolabs) for 2.5 hours at 37 °C. Digests contained 1 mL of water, 3 mL of NEB Buffer 4 (10X), 3 mL of BSA (10X), 20 mL of PCR product, 1.5 mL of NdeI (20 U/mL), and 1.5 mL of XhoI (20 U/mL). Restriction digests were purified directly using agarose gel electrophoresis and then ligated into a linearized expression vector using T4 DNA ligase (New England Biolabs). The cutF gene was ligated into the pET-29b vector to encode a C-terminal His6-tagged construct and cutO was cloned into the pET-28a vector to encode an N-terminal His6-tagged construct. Ligations were incubated at 16 °C overnight and contained 3 mL water, 1 μL T4 Ligase Buffer (10X), 1 mL digested vector, 3 mL digested insert DNA, and 2 mL T4 DNA ligase (400 U/ml). 9 mL of each ligation were used to transform a single tube of E. coli TOP10 cells (Invitrogen). The identities of the resulting pET-29b-CutF and pET-28a-CutO constructs were confirmed by sequencing of purified plasmid DNA. These constructs were transformed into chemically competent E. coli BL21 (DE3) cells (Invitrogen) and stored at –80 °C as frozen LB/glycerol stocks.

A 50 mL starter culture of E. coli BL21 pET-29b-CutF was inoculated from a freshly streaked plate and grown overnight at 37 °C in LB medium supplemented with 50 mg/mL kanamycin. The overnight culture was diluted 1:50 into 2 L of LB medium containing 50 mg/mL kanamycin. The culture was incubated at 37 °C with shaking at 175 rpm until OD600= 0.2 – 0.3 and was then transferred to an incubator at 25 °C with shaking at 175 rpm. At OD600 = 0.5 – 0.6, cells were induced with 500 μM IPTG and incubated for ~16 h. Cells from a 2 L culture were pelleted by centrifugation (6,730 g x 10 min) and resuspended in 40 mL of lysis buffer (50 mM Tris-HCl, 100 mM NaCl, 10 mM MgCl2, pH 8.0) supplemented with Pierce Protease Inhibitor Tablets (Thermo Scientific). The cells were lysed by passage through a cell disruptor (Avestin EmulsiFlex-C3, Ottawa, Canada) twice at 10,000 psi, and the lysate was clarified by centrifugation (24,610 g x 30 min). The supernatant was incubated with 2 mL of Ni-NTA resin slurry and 5 mM imidazole for 2 hours at 4 °C. The mixture was centrifuged (1,811 g x 5 min) and the unbound fraction discarded. The resin was resuspended in 5 mL of elution buffer (50 mM Tris-HCl, 100 mM NaCl, 10 mM MgCl2, 5 mM imidazole, pH 8.0) and loaded into a glass column. Protein was eluted from the column using a stepwise imidazole gradient in elution buffer collecting the following volumes: 25 mM (5 mL), 50 mM (5 mL), 75 mM (2 mL), 100 mM (2 mL), 125 mM (2 mL), 150 mM (2 mL), and 200 mM (4 mL). SDS-PAGE analysis (4-15% Tris-HCl gel) was employed to ascertain the presence and purity of protein in each fraction. Fractions containing the desired protein were combined and dialyzed twice against 2 L of storage buffer (50 mM Tris-HCl, 100 mM NaCl, 10 mM MgCl2, pH 8.0). Protein was concentrated using a 30,000 Da molecular weight cut-off Spin-X UF Concentrator (Corning, Tweksbury, MA) and was further purified by anion exchange chromatography using a UNO Q1 column (Bio-Rad) on a BioLogic DuoFlow Chromatography System (Bio-Rad). This procedure afforded yields of 0.22 mg/L of C-His6-tagged CutF. Concentrations of CutF C-His6 protein stock solutions were 49 mM.

A 50 mL starter culture of E. coli BL21 pET-28a-CutO was inoculated from a freshly streaked plate and grown overnight at 37 °C in LB medium supplemented with 50 mg/mL kanamycin. The overnight culture was diluted 1:50 into 2 L of LB medium containing 0.5% (w/v) glucose and 50 mg/mL kanamycin. The culture was incubated at 37 °C with shaking at 175 rpm until OD600= 0.3 – 0.4 and then sodium fumarate (20 mM) and iron ammonium sulfate (50 μM) were added. The culture was sparged with nitrogen until it reached an OD600 of 0.6, and IPTG was added to a final concentration of 50 μM. The flask was capped and sealed with electrical tape to preserve anoxic conditions. The culture was incubated for an additional 18 h at 15 °C with 175 rpm of shaking. All of the purification steps were performed in an anaerobic chamber at 17 °C (Coy Labs glovebox), with the exception of cell lysis and centrifugation. Cells from a 2 L culture were harvested by centrifugation (6,730 g x 10 min) and resuspended in 40 mL of anoxic lysis buffer (50 mM KH2PO4, 300 mM NaCl, 5 mM b-mercaptoethanol, pH 8.5) supplemented with a Pierce protease inhibitor tablet (Thermo Scientific, Waltham, MA). The cells were lysed by passage through a cell disruptor (Avestin EmulsiFlex-C3) twice at 10,000 psi while maintaining a nitrogen line above the receiving tube and one above the cell disruptor reservoir, and the lysate was clarified by centrifugation (24,610 g x 30 min). The supernatant was incubated with 2 mL of Ni-NTA resin slurry and 5 mM imidazole for 2 hours at 4 °C. The mixture was centrifuged (1,811 g x 5 min) and the unbound fraction discarded. The resin was resuspended in 5 mL of elution buffer (50 mM KH2PO4, 300 mM NaCl, 5 mM b-mercaptoethanol, pH 8.5) and loaded into a glass column inside the anaerobic chamber. Protein was eluted from the column using a stepwise imidazole gradient in elution buffer collecting the following volumes: 25 mM (5 mL), 50 mM (5 mL), 75 mM (5 mL), 100 mM (2 mL), 125 mM (2 mL), 150 mM (2 mL), and 200 mM (6 mL). SDS-PAGE analysis (4-15% Tris-HCl gel) was used to ascertain the presence and purity of protein in each fraction. This procedure afforded yields of 0.15 mg/L for N-His6 CutO. Concentrations of N-His6 CutO protein stock solutions were 1.5 mM.