Hydrogenase

The database was constructed using the amino acid sequences of all curated non-redundant 3248 hydrogenase catalytic subunits represented in the NCBI RefSeq database in August 20142 (link) (Dataset S1). In order to test the classification tool, additional sequences from newly-sequenced archaeal and bacterial phyla were retrieved from the Joint Genome Institute’s Integrated Microbial Genomes database43 (link).

The hydrogenase gene hydA1 from C. reinhardtii as well as the nucleotide sequences hydGx and hydEF encoding the S. oneidensis hydrogenase maturases were PCR amplified from the pK7 shydA1 and pACYCDuet-1 hydGxEF plasmids [14] (link) using Platinum ® Taq DNA Polymerase High Fidelity (Invitrogen). PCR products were digested with restriction enzymes and inserted into expression vectors between a T7 RNA polymerase promoter and terminator using T4 DNA ligase (New England Biolabs). The pY71 vector was used as the parent plasmid for construction of C. reinhardtii shydA1 expression vectors. The plasmid pY71 cat encoding the chloramphenicol acetyl transferase enzyme was synthesized by first PCR amplifying the replication origin from the pUC19 plasmid (Invitrogen), the kanamycin resistance gene from pK7 cat[35] (link), and the nucleotide fragment from pK7 cat containing the cat gene flanked by the T7 RNA polymerase promoter and terminator sequences. These three fragments were ligated using overlapping PCR. The linear PCR product (≈2.5 kb) was digested with BamHI and ligated to form pY71 cat. Next, the shydA1 gene was cloned into the pY71 vector, from which the cat gene had been removed. The first 8 codons of shydA1 were conservatively changed to ATG GCA GCA CCA GCA GCA GAA GCG for reduced secondary structure as predicted using Mfold software to improve in vitro translation (shydA1*). pY71 shydA1* was used for addition of an N-terminal 6x-histidine tag, and the N-his₆-shydA1* insert was cloned back into the pY71 vector. Synthesis of the expression vector containing the S. oneidensis maturase genes was carried out in two parts. First, the hydGx gene segment was cloned into multiple cloning site I of the pACYCDuet-1™ expression vector (Novagen). Next, the hydEF gene segment was cloned into multiple cloning site II of pACYCDuet-1–hydGx. All expression vectors were confirmed by DNA sequencing and transformed into E. coli strain BL21(DE3) (Invitrogen). Transformed cells were selected against kanamycin resistance (40 mg L⁻¹) for pK7 and pY71 plasmids, and against chloramphenicol resistance (25 mg L⁻¹) for pACYCDuet-1 plasmids.

The E. coli strains used included MC4100 (F−, araD139, ∆(argF‐lac)U169, λ⁻, rpsL150, relA1, deoC1, flhD5301, ∆(fruK‐yeiR)725(fruA25), rbsR22, ∆(fimB‐fimE)) [22 (link)], and its isogenic derivative DHP‐D (ΔhypD) [23 (link)]. E. coli XL1‐Blue (Stratagene (Group), La Jolla, CA, USA) was used for standard cloning procedures. The plasmids used are listed in Table 1. Strains were grown on LB‐agar plates or in LB‐broth at 37 °C [24 ] for routine microbiological and molecular biological experiments, including cloning. Anaerobic cultivation of strains for hydrogenase enzyme assays and in‐gel enzyme activity staining after native PAGE, or for western blotting experiments, was performed at 37 °C as standing liquid cultures in the buffered rich medium TGYEP (1% w/v tryptone, 0.5% w/v yeast extract, 0.8% w/v glucose, 100 mm potassium phosphate, pH 6.5) [25 ], supplemented with trace element solution SLA [26 ]. Cells were harvested anaerobically by centrifugation at 5000 g for 15 min, at 4 °C, when cultures had reached an optical density at 600 nm (OD₆₀₀) of between 0.8 and 1.2. Cell pellets were generally used immediately for further experiments or were frozen at −20 °C until required.
For the purification of Strep‐tagged HybG‐HypD scaffold complexes, strains transformed with the appropriate plasmid were cultivated anaerobically at 37 °C as static cultures in modified TB medium (2.4% w/v yeast extract, 1.2% w/v peptone from casein, 0.04% w/v glycerol, 0.4% w/v glucose and 0.003% w/v magnesium sulfate heptahydrate) [10 (link)], containing 100 μg mL⁻¹ of ampicillin. Cultures were incubated until an OD₆₀₀ of 0.4 was reached and then plasmid‐based gene expression was induced by the addition of 0.1 mm isopropyl β‐d‐1‐thiogalactopyranoside (IPTG). Incubation of the cultures was continued at 30 °C for a further 3 h, and cells were subsequently harvested by centrifugation at 5000 g for 15 min at 4 °C. Cell pellets were either used immediately or stored at −20 °C until use.

Because only two of the five genomes of ‘Candidatus Methanospirare jalkutatii’ have an operon encoding a hydrogenase, we performed additional analysis to better understand this intraspecies distribution. On the one hand, we mapped the metagenomic reads from samples with genomes of ‘Candidatus Methanospirare jalkutatii’ (12019, FW4382_bin126, NA091.008, PR1007, PR1031B) to the MAGs containing the hydrogenase operon (FW4382_bin126, NA091.008_bin1) to check if reads mapping this operon are also present in samples from where the MAGs without the hydrogenase were recovered. For mapping the reads, we used bowtie2 v.2.4.2 (ref. 62 (link)) then transformed the sam files to bam using samtools (http://www.htslib.org/) and extracted the coverage depth for each position. Additionally, we performed a genomic comparison of the genomes with a hydrogenase operon (FW4382_bin126, NA091.008_bin1) with the genome FWG175 that was assembled into a single scaffold. For this, we used the genome-to-genome aligner Sibelia v.3.0.7 (ref. 63 ) and we visualized the results using Circos (http://circos.ca/).