High-Quality Draft Genome Sequence of “Candidatus Methanoperedens sp.” Strain BLZ2, a Nitrate-Reducing Anaerobic Methane-Oxidizing Archaeon Enriched in an Anoxic Bioreactor

We extracted DNA from a bioreactor highly enriched in “Ca. Methanoperedens sp.” strain BLZ2 using the cetyltrimethylammonium bromide (CTAB) method as described previously (1) and the Power soil DNA extraction kit (Mo Bio Laboratories, Uden, The Netherlands). To shear genomic DNA and add adapters in the same step (“tagmentation”), the Illumina Nextera XT library prep kit (Illumina, San Diego, CA, USA) was used. The library was normalized to 4 nM and sequenced with a MiSeq instrument (Illumina) using the 300 paired-end sequencing protocol. Sequencing data were quality trimmed, yielding 11.8 million paired-end reads with an average length of 268 bp. Reads were assembled de novo (CLC Genomics Workbench v9.5.2 [CLC bio, Aarhus, Denmark]; word size, 55; maximum bubble size, 5,000). Contigs were binned using MetaBAT (2), yielding a draft genome composed of 85 contigs (N₅₀ = 74.304 bp). Afterward, quality was assessed using CheckM (3). Annotation was done using Prokka (4) and also as previously described (5). Based on the presence of 228 specific marker genes, the draft genome was 99.35% complete and only 4.58% contaminated. The final sequence had a GC content of 40.3% and was composed of 3.74 Mb with 4,041 putative open reading frames.

The results of this study were largely in accordance with previous data (6, 7). The 16S rRNA gene in this genome was 100% identical to that found in “Ca. Methanoperedens” BLZ1 and 95% identical to “Ca. Methanoperedens nitroreducens” ANME2d. All genes necessary for reverse methanogenesis were present in the genome, including mer (encoding F₄₂₀-dependent 5,10-methenyl-H₄MPT reductase).

Electron carriers reduced during reverse methanogenesis include coenzyme B (CoB), coenzyme M (CoM), F₄₂₀, and ferredoxin (Fd). It has been suggested that HdrABC/FrhB catalyzes the confurcation of electrons from CoM-SH, CoB-SH, and Fd_red to F₄₂₀ (7). Interestingly, in the present data set, four copies of hdrA were identified, two of which were fused with mvhD (see also reference 8). Furthermore, a gene cluster was identified that contains hdrABC as well as frhB and two copies of mvhD, with mvhD possibly being involved in electron transfer.

Membrane-bound enzymes included Ech hydrogenase (echABCEF missing echD) and the membrane-bound heterodisulfide reductase (hdrDE). Furthermore, the complete F₄₂₀:quinone oxidoreductase (Fqo), a Rieske/cyt_b complex, and an ammonium-forming cytochrome c nitrite reductase (nrfAH) were present. Nitrate reductase was present as canonical (NarGHI) and noncanonical (NarGH, potential membrane anchors HCOIIa, HCOIIb, NapH, cytochrome subunit Orf7 and chaperone NarJ, being the similarity to “Ca. Methanoperedens” BLZ1 between 92 and 100%) versions, both encoded in two clusters present in close proximity to each other on the same 129 kb-long contig.

This work was financially supported by the Netherlands Organization for Scientific Research through the SIAM Gravitation grant 024.002.002. S.B. and C.U.W. were supported by ALWOP.293. M.S.M.J. was furthermore supported by the ERC AG EcoMoM 339880 and the NESSC Gravitation grant 024.002.001.

We thank Theo van Alen for technical assistance.