Draft Genome Sequence of "Candidatus Bathyarchaeota" Archaeon BE326-BA-RLH, an Uncultured Denitrifier and Putative Anaerobic Methanotroph from South Africa's Deep Continental Biosphere.

Metagenomic sequencing of fracture fluid from South Africa recovered a nearly complete "Candidatus Bathyarchaeota" archaeon genome. The metagenome-assembled genome of BE326-BA-RLH contains genes involved in methane metabolism and dissimilatory nitrate reduction. This study presents the first genomic evidence for potential anaerobic methane oxidation in the phylum "Ca. Bathyarchaeota."

ANNOUNCEMENT

The uncultured “Candidatus Bathyarchaeota” is a deeply branching and diverse phylum of deep-biosphere inhabitants whose recently inferred role in methanogenesis supports an early evolution of biogenic methane cycling (1, 2). We report here a near-complete genome sequence of “Ca. Bathyarchaeota” archaeon BE326-BA-RLH assembled from metagenomic data obtained from a subsurface chemolithoautotrophic microbial ecosystem (SLiME) (3) in oligotrophic fracture fluid (33.2°C; pH 7.5; reduction potential [pe], –0.48) from 1.34 km below land surface (kmbls) at the Beatrix Gold Mine in South Africa. The genomic DNA (gDNA) was extracted following established procedures (4, 5). The metagenomic library was prepared using the PrepX DNA library kit and an automated Apollo 324 system (WaferGen Biosystems, Inc., Fremont, CA). Paired-end (2 × 100-nucleotide [nt]) metagenomic sequencing was performed at the Marine Biological Laboratory (Woods Hole, MA) using the HiSeq 2000 platform (Illumina, Inc., San Diego, CA).

Tools on the Galaxy Web platform at Princeton University (https://usegalaxy.org) (6) were used to quality filter 43,742,580 reads. Filter FASTQ v.1.1.1 removed reads for which 90% of the bases had a Phred quality score of <30. Remove Sequencing Artifacts v.1.0.1 removed homopolymers. Trim Galore! v.0.4.3.1 removed reads that matched the Illumina universal adapter sequence anywhere with a maximum error rate of 0.1, a match time of 1 nt, and a minimum overlap length of 20 nt, including ambiguous bases (Ns) as matches. Trim v.0.0.1 removed 5 nt from the 3′ end and then reads shorter than 50 nt and those containing Ns. The resulting 34,820,059 reads were assembled using SPAdes v.3.11.0 (–meta option) (7). Scaffolds were binned using MetaBAT v.2.11.2 (8). Reads were mapped back to the metagenome assembly using Bowtie2 v.2.3.2 (default –very-sensitive mode options) (9).

A near-complete (estimated at 89.79%) low-contamination (3.74%) bin with 0% strain heterogeneity was identified as an unknown archaeon using CheckM v.1.0.7 (10). Metagenome reads that mapped to the scaffolds in this initial bin were reassembled using SPAdes v.3.11.0 (default option). The resulting draft genome (2.09 Mb, 44.9% GC content) comprised 227 contigs with an N50 length of 14,564 bp (Table 1). Protein-encoding genes were identified using Prodigal v.2.6.3 (11) and annotated using NCBI BLAST v.2.2.29+ (12), Prokka v.1.13 (13), GraftM v.0.11.1 (14), and HHpred v.3.0.0beta (15). The final draft genome sequence of BE326-BA-RLH has an 86% coding density and contains 23 30S and 29 50S ribosomal proteins, a single copy of 16S, 23S, and 5S rRNA genes, and 91 tRNA genes. A BLASTn search determined that the 16S rRNA gene (1,139 bp) shares 97% similarity to uncultured “Ca. Bathyarchaeota” (GenBank accession numbers EU559699, EU155992, and EU155991) and 87% and 86% similarity to “Ca. Bathyarchaeota” methanogens BA2 (GenBank accession number LIHK01000010) and BA1 (GenBank accession number LIHJ01000085), respectively (1).

TABLE 1

Statistics summary of methane-metabolizing “Ca. Bathyarchaeota” genomes

Statistic	BE326-BA-RLH (this study)	BA1a	BA2a
Completeness (%)	89.8b	91.6	93.8
Contamination (%)	3.7	2.8	3.7
Total length (bp)	2,097,091	1,931,714	1,455,689
GC content (%)	44.9	47.1	44.2
No. of contigs	227	96	58
N50 value of contigs (bp)	14,564c	32,677	43,519
No. of coding sequences	2,229	2,403	1,761
Coding density (%)	86.0	80.8	83.6
Avg coverage (×)	21.1	35.8	49.8
Relative abundance (%)	0.36d	0.92	1.03

Source: Evans et al. (1).

Based on lineage-specific marker genes determined via CheckM (9).

Calculated from Prodigal (10).

Estimated from the percentage of reads mapped back to the metagenome assembly.

BE326-BA-RLH encodes proteins for hydrogenotrophic, acetoclastic, and methylotrophic methanogenesis and for carbon fixation via the reductive acetyl-CoA pathway. A partial methyl-coenzyme M reductase subunit A (mcrA) sequence was identified in a 265-bp-long contig in the metagenome assembly, which shared 70% identity to an amino acid sequence of an uncultured archaeon (GenBank accession number AGA20295) based on a BLASTp search. A maximum likelihood (ML) tree was constructed using RAxML v.8.2.11 (PROTGAMMAILGF mode) (16) for this and for 175 mcrA protein sequences obtained from GenBank. The ML tree placed this mcrA as a deep branch rooting the “Ca. Bathyarchaeota” clade. The GC content and tetranucleotide frequency variance of this mcrA were consistent with those of BE326-BA-RLH, but the sequence was too short to be added to the bin.

BE326-BA-RLH contains genes encoding periplasmic nitrate reductase (narH) and nitrite reductase (nrfHA). Formate dehydrogenase (fdhAD) was found adjacent to tungsten-containing formylmethanofuran dehydrogenase (fwdDACB), suggesting that formate may serve as a possible electron shuttle between reverse methanogenesis and denitrification. To our knowledge, BE326-BA-RLH is the first described “Ca. Bathyarchaeota” genome encoding genes that may couple anaerobic methane oxidation (AOM) to known oxidants. This study provides further support showing that members of the “Ca. Bathyarchaeota” may perform AOM (1, 17, 18).

Data availability.

The BE326 BH2 whole-metagenome shotgun and draft genome sequences of “Ca. Bathyarchaeota” BE326-BA-RLH have been deposited at NCBI GenBank under the accession numbers QZGF00000000 (SRA number SRR7867194) and QYYE00000000 (SRA number SRR7866305), respectively. The version described in this paper is version number QYYE01000000.