Preparation of cell free crude extracts
Thermoanaerobacter kivui LKT-1 (DSM 2030) was cultivated in 1-l flasks (Müller-Krempel, Bülach, Switzerland) containing 500 ml of complex medium previously described . The media were prepared using anaerobic techniques as described [32, 33]. A temperature of 66 °C was routinely used for the cultivation. Sterile glucose, formate, or pyruvate was added as carbon source from an anoxic stock solution. Respectively, H2 + CO2 was used as an electron donor and carbon source. Therefore, 1-l flasks were pressurized with 1 bar H2 + CO2 (80:20 [v/v]) containing only 250 ml complex medium to increase the gas-to-liquid ratio. Growth was determined by measuring the optical density at 600 nm with a UV/vis spectrophotometer. All buffers used in the following preparation procedure and the subsequent enzyme activity measurements contained 2 mm dithioerythritol and 4 μm resazurin. All steps were performed under strictly anoxic conditions at room temperature in an anaerobic chamber (Coy Laboratory Products, Grass Lake, MI) filled with a gas composition of 95–98% N2 and 2–5% H2 as described . For the preparation of cell free extracts, the cells were harvested in the late exponential growth phase and were subsequently washed with buffer A (25 mM Tris/HCl, 20 mM MgSO4, 20% glycerin [v/v], pH 7.5). Afterwards, the cells were resuspended in 3 ml buffer A including 0.5 mM PMSF and a spade point DNaseI. Cells were disrupted by a single passage through a French pressure cell press (SLM Aminco, SLM Instruments, USA) at 97 MPa. Cell debris was removed by centrifugation at 2000×g for 20 min. The supernatant was immediately used for the measurement of the hydrogen production from formate in the way described below.
Purification of the HDCR complex
Cells of T. kivui LKT-1 (DSM 2030) were grown heterotrophically with pyruvate as substrate at 66 °C under anoxic conditions in 20-l flasks (Glasgerätebau Ochs; Bovenden-Lenglern, Germany). All purification steps of the HDCR complex were performed under strictly anoxic conditions at room temperature in an anaerobic chamber as described before. Cells of T. kivui were harvested at an OD 600 of around 1.3 and were washed twice with buffer A (25 mM Tris/HCl, 20 mM MgSO4, 20% glycerin [v/v], pH 7.5). Afterwards, the cells were resuspended in 15 ml buffer A including 0.5 mM PMSF and a spade point DNaseI. Cells were disrupted by a single passage through a French pressure cell press (SLM Aminco, SLM Instruments, USA) at 110 MPa. Cell debris was removed by centrifugation at 23,700×g for 20 min at 4 °C. To separate the membranes from the cytoplasmic fraction, an ultracentrifugation at 184,000×g for 45 min was performed. The supernatant containing the cytoplasmic fraction with around 1800 mg of protein was used for the further purification.
Ammonium sulfate (0.4 M) was added to the cytoplasmic fraction and the sample was loaded onto a Phenyl-Sepharose high-performance column (2.6 cm × 6.4 cm) equilibrated with buffer B (25 mM Tris/HCl, 20 mM MgSO4, 20% glycerin [v/v], 0.4 M (NH4)2SO4, pH 7.5). Methylviologen-dependent formate dehydrogenase activity eluted at (NH4)2SO4 concentrations below 0.4 M in a linear gradient over 120 ml from 0.4 M to 0 M (NH4)2SO4. The fractions with Methylviologen-dependent formate dehydrogenase activity were pooled and the sample was diluted to a conductivity of 14 mS/cm with buffer A. Then, the sample was applied to a Q-Sepharose high-performance column (1.6 cm × 11.9 cm) equilibrated with buffer A. The formate dehydrogenase activity was found in the flow through. The pooled fractions were concentrated by using ultrafiltration in 100-kDa VIASPIN tubes (Sartorius Stedim Biotech GmbH, Germany). Half of the concentrated sample was loaded on a Superose 6 10/300 GL prepacked column (GE Healthcare Life Sciences, Little Chalfont, UK) equilibrated with buffer C (25 mM Tris/HCl, 20 mM MgSO4, 20% glycerin [v/v], 150 mM NaCl, pH 7.5) and eluted at a flow rate of 0.5 ml/min. Formate dehydrogenase activity eluted not in a defined single peak but spread over a wide elution volume. This step was repeated with the second half of the concentrated sample in a separate run to achieve a better separation. The formate dehydrogenase activity was enriched by 55-fold from the cytoplasm resulting in 1.5–2 mg of homogeneous protein from 36 g of wet cell mass. The fractions were pooled and stored at 4 °C.
Measurement of enzyme activity
All enzyme assays, unless otherwise stated, were performed in 1.8 ml anaerobic cuvettes (Glasgerätebau Ochs, Bovenden-Lenglern, Germany) sealed by rubber stoppers at 60 °C and filled with 1 ml buffer. In all enzyme assays, the buffer was pre-incubated at the temperature of interest.
Methylviologen-dependent formate dehydrogenase activity was measured with formate (10 mM) as electron donor and methylviologen (10 mM) as electron acceptor in 1 ml buffer D (100 mM HEPES/NaOH, 2 mM DTE, pH 7.0) and a gas phase of 100% N2 at a pressure of 1.1 × 105 Pa. The reduction of Methylviologen was monitored at 604 nm by UV/Vis spectrophotometry (ε = 13.9 mM−1 cm−1).
Measurements of Methylviologen-dependent hydrogenase activity were performed under the same conditions except that the gas phase was 100% H2 at a pressure of 1.1 × 105 Pa. Formate was omitted in the enzyme assay and Methylviologen reduction was measured at 604 nm as described before. For KM determination, the H2 concentration in the gas phase was manually adjusted using a gas tight syringe. The concentrations of H2 in the liquid phase were calculated according to Henry’s law.
For the determination of the pH and temperature profile, the enzyme was preincubated 10 min at the pH or temperature indicated, respectively. The reaction temperature for the determination of the pH optima was set to 60 °C. The buffer used for the pH optima determination was 50 mM MES, 50 mM MOPS, 50 mM HEPES, 50 mM EPPS, 50 mM CHES, 2 mM DTE, and the pH as indicated.
Hydrogen-dependent carbon dioxide reductase activity was measured in a two-step enzyme assay. In the first part, the hydrogen-dependent CO2-reduction was started by using a gas phase of 80% H2 + 20% CO2 [v/v] at a pressure of 1.1 × 105 Pa in 120-ml serum bottles (Glasgerätebau Ochs, Bovenden-Lenglern, Germany) sealed by rubber stoppers, containing 5 ml buffer D at 60 °C in a shaking water bath. At defined time points, a sample was taken from the reaction mixture and stored on ice. Afterwards, the determination of the formate concentration of all samples was carried out by using a commercially available formic acid-kit (Boehringer Mannheim/R-Biopharm AG, Mannheim/Darmstadt, Germany). It contained a formate dehydrogenase as a reporter enzyme and the formation of NADH was monitored at 340 nm.
Hydrogen production from formate was measured in 9-ml serum vials (Glasgerätebau Ochs, Bovenden-Lenglern, Germany) sealed by rubber stoppers, containing 1 ml buffer D at 60 °C in a shaking water bath. The gas phase in the serum vials was 100% N2 at atmospheric pressure. Before each gas sample was taken, the overpressure in the serum vials due to the H2 production from formate oxidation was determined and adapted to ambient pressure. Gas samples of 50 µl volume were withdrawn with a gas-tight syringe (Hamilton CO. glass syringe, Reno, USA). The concentrations of H2 were determined by using a gas chromatograph (Clarus 580 GC; PerkinElmer, Waltham, MA, USA) with a Shin Carbon ST 80/100 column (Restek GmbH, Bad Homburg, Germany) kept at 40 °C. Nitrogen was used as carrier gas, respectively, with a head pressure of 400 kPa and a split flow of 30 ml/s. H2 was detected with a thermal conductivity detector kept at 100 °C.
The protein concentration was measured according to Bradford . Proteins were separated in 12% polyacrylamide gels and stained with Coomassie brilliant blue G250.
All DNA and protein sequences were retrieved from the National Center for Biotechnology Information database. Homology searches were performed using BLASTp with default settings (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Protein sequences were analyzed for conserved domains and functional sites by InterProScan 5 . For pairwise sequence alignment, we used EMBOSS Water using default settings . Novel putative HDCR gene cluster was identified using MultiGeneBlast with the amino acid sequence of FdhF1, HycB1, HycB3, and HydA2 from A. woodii as input . Minimal percent identity was set to 30, minimal sequence coverage to 25 and maximum distance of the genes to 20 kB. We searched against all available completed bacterial RefSeq genomes (1543 genomes in total).
All chemicals were supplied by Sigma-Aldrich Chemie GmbH (Munich, Germany) and Carl Roth GmbH & Co KG (Karlsruhe, Germany). All gases were supplied by Praxair (Düsseldorf, Germany).