Methanothermococcus jasoni sp. nov., a novel thermophilic methanogen from subseafloor hydrothermal vent fluids of Axial Seamount, Juan de Fuca Ridge

Abstract

Low-temperature (34 °C) hydrothermal fluid was collected from Axial Seamount along the Juan de Fuca Ridge in the northeast Pacific Ocean. A thermophilic methanogen, designated strain Ax23^T, was isolated from this fluid. The archaeon was anaerobic, autotrophic and coccoidal and grew separately on H₂/CO₂ and formate. Ax23^T grew on NH₄⁺ and by fixing N₂, but did not grow on NO₃^-. The isolate grew at temperatures ranging from 33 to 75 °C (optimum 73 °C) and a minimum doubling time of 32 min, in the presence of 3–6% NaCl (optimum 3–4.5%) and pH 4.0–9.0 (optimum 6.0–8.0). It increased its growth yield (cells produced per mole of CH₄ produced) when grown on 10 kPa H₂ relative to 160 kPa H₂, suggesting that it is capable of a metabolic trade-off with varying H₂ availability. Phylogenetic analysis based on 16S rRNA gene sequence indicated that the strain was 95.6–99.7% identical to other thermophilic species of the family Methanococcaceae in the kingdom Methanobacteriati. The complete genome of Ax23^T was sequenced, which yielded a 1,662,948 bp chromosome with 1,663 protein-coding sequences and a 7,732 bp plasmid. Based on genome-relatedness index analyses and its phenotypic characteristics, strain Ax23^T represents a novel species, for which the name Methanothermococcus jasoni is proposed. The type strain is Ax23^T (=DSM 118471^T=JCM39656^T).

Introduction

Methanogens in the order Methanococcales are among the more common thermophilic chemolithoautotrophs found at deep-sea hydrothermal vents [1]. At the Marker 113 deep-sea hydrothermal vent site at Axial Seamount along the Juan de Fuca Ridge, high-temperature methanogens comprise a large portion of the total thermophilic chemolithoautotrophs present [2,4]. The hyperthermophilic methanogens Methanocaldococcus bathoardescens and Methanocaldococcus sp. FS406-22 were both isolated from this vent site [5,6]. However, DNA- and RNA-sequencing indicate that most of the methanogens present at the site belong to the genus Methanothermococcus [2,3], for which there is no cultured representative. At present, only two species of Methanothermococcus, namely Methanothermococcus thermolithotrophicus SN-1^T and Methanothermococcus okinawensis IH1^T, have been characterized [7,8]. Both are thermophilic, obligately autotrophic and use H₂/CO₂ and formate separately for growth (Table 1). The purpose of this study was to isolate and characterize a Methanothermococcus strain from Marker 113 hydrothermal fluids.

Table 1. Physiological characteristics differentiating strain Ax23^T from the closest type strains in the family Methanococcaceae.

Strains: 1, Ax23^T; 2, M. okinawensis IH1^T [8]; 3, M. thermolithotrophicus SN-1^T [7]; 4, Methanococcus aeolicus Nankai-3^T [33]; 5, Methanococcus aeolicus PL15/HP^P [33]. +, Positive; −, negative; nd, not determined. All data for Ax23^T were from this study, unless otherwise indicated.

Characteristic	1	2	3	4	5
Habitat	Hydrothermal vent, Axial Seamount, Pacific Ocean	Hydrothermal vent, Okinawa Trough, Pacific Ocean	Beach sediment, Naples, Italy	Marine sediment, Nankai Trough, Pacific Ocean	Beach sediment, Lipari Islands, Italy
Depth (m)	1,521	972	0.5	1,197	nd
16S rRNA gene identity (%)	100	99.7	95.6	96.1	96.2
OGRI analyses
ANI (%)	100	85.1	76.3	77.0	77.4
GGDC (%)	100	27.1	22.5	23.5	24.7
Morphology
Cell shape	Coccoid	Coccoid	Coccoid	Coccoid	Coccoid
Diameter (μm)	1–2	1.0–1.5	1.5	1.5–2.0	1.5–2.0
Temperature range of growth and (optimum) (°C)	33–75 (73)	40–75 (60-65)	30–70 (65)	20–55 (46)	≤20–45
pH range of growth and (optimum)	5.0–8.0 (6-8)	4.5–8.5 (6-7)	6–8 (7)	5.5–7.5 (6.5)	6.5–8.0
NaCl range of growth and (optimum) (%)	3–6 (3–4.5)	0.5–6.0 (1.5–3.0)	1.3–8.3 (4)	0.3–5.8 (1.8)	0.3–4.7
Minimum doubling time	32 min	30 min	55 min	83 min	nd
Electron donors
H2	+	+	+	+	+
Formate	+	+	+	+	+
Nitrogen
NH4+	+	+	+	+	+
NO3-	−	−	+	nd	nd
N2	+	nd	+	nd	+

Methods

Sample collection

In July 2023, two 34 °C hydrothermal fluid samples were collected on separate days from the Marker 113 hydrothermal vent site at Axial Seamount (1,521 m depth) on the Juan de Fuca Ridge (TN420; R2R DOI: 10.7284/910168). The fluid samples were drawn into 5-l plastic bags (lamination of nylon-white polyethylene-aluminium foil-coex polyethylene, ProAmpac, Rochester, NY, USA) with polyethylene valves using the National Deep Submergence Facility’s Universal Fluid Obtainer attached to the remotely operated vehicle (ROV) Jason II. The temperature of the exiting hydrothermal fluid was measured using a temperature probe, and then the intake for the fluid sampler was placed next to the tip of the probe. Fluid was pumped at a rate of 500 ml min⁻¹ for ~8 min. Once shipboard, the hydrothermal fluid samples were immediately processed while minimizing exposure to air for most probable number (MPN) methanogen concentration estimates and total cell counts preserved in 3% (vol/vol) formaldehyde. Total cell counts in the original hydrothermal fluids were quantified by filtering a sample onto 0.2 µm pore size polycarbonate membrane filters prestained with Irgalan black (Whatman), staining with 0.01% (wt/vol) acridine orange [9] and counting fluorescent cells using epifluorescence microscopy (Nikon Eclipse 55i).

Growth conditions

Unless otherwise stated, the growth medium used was a modification of DSM 282 medium [2,10] and contained, as a supplement, per litre, 0.1 ml of 0.1% (wt/vol) Na₂WO₄∙2H₂O/0.1% (wt/vol) Na₂SeO₄. The medium was pH balanced to 6.00±0.05, reduced with 0.025% each of cysteine-HCl and Na₂S∙9H₂O and pressurized with 200 kPa of H₂:CO₂ headspace. Three-tube MPN analyses were performed at sea on each of the two hydrothermal fluid samples as previously described [11]. After inoculation, Balch tubes sealed with butyl rubber stoppers were incubated at 65 °C in a forced-air incubator for up to 7 days. Growth of methanogens in the tubes was confirmed using autofluorescence and phase-contrast light microscopies and by analysing for CH₄ using gas chromatography of the headspace. The proportion of the total microbial community that was coccoid and showed methanogen autofluorescence was determined using a wet mount, a fluorescence microscope (Nikon Eclipse 55i) and blue light excitation (AT480/30×, Chroma). The amount of CH₄ produced in each microcosm was determined using a gas chromatograph fitted with a flame ionization detector (SRI 8610C) and a 6′ Hayesep D packed chromatography column (Supelco).

One positive methanogen growth tube from each of the two MPN sets was used to isolate a thermophilic methanogen strain through three consecutive rounds of dilution-to-extinction incubations [12] at 65 °C. The most dilute tube to show growth on the third round of incubation was used as the pure strain. DNA from each of the two strains was extracted using a Wizard Genomic DNA Purification Kit (Promega), and a portion of their 16S rRNA gene was amplified using the polymerase chain reaction and 517F and 958R primers. The nucleotide sequences amplified from the bulk DNA of strains isolated from the two separate dives to Marker 113 showed only one nucleotide sequence per sample, suggesting they were pure cultures and were 100% identical to each other. Therefore, the strain from the first hydrothermal vent sample (dive J1501) was used as the pure strain and named Ax23^T.

For kinetic experiments, strain Ax23^T was grown at 70 °C as described above unless otherwise noted, in triplicate, at temperatures ranging from 28 to 78 °C; at pH 3.3 and 4 (no buffer), pH 5 and 6 (5 mM MES buffer), pH 7 and 8 (20 mM PIPES buffer), pH 8 and 9 (100 mM EPPS buffer) and pH 10 (80 mM glycine-NaOH buffer); and 2.25% (wt/vol) to 7.5% NaCl to determine their effect on growth. To test alternative carbon and electron donor sources, strain Ax23^T was grown separately, each with a 200 kPa N₂ headspace, on 0.1% (wt/vol) sodium formate, 0.1% (wt/vol) sodium acetate, 0.1% (wt/vol) sodium propionate, 0.5% (wt/vol) glucose, 0.5% (wt/vol) tryptone and 0.1% (wt/vol) yeast extract. To test alternative nitrogen sources, strain Ax23^T was grown on 0.1% (wt/vol) KNO₃ in a medium lacking NH₄Cl and in a medium lacking NH₄Cl with a 200 kPa H₂:N₂:CO₂ (40%:40%:20%) headspace. For all kinetic experiments, an aliquot from each serum bottle was removed at various time points, and the concentration of cells was determined using a Petroff–Hausser counting chamber and phase-contrast light microscopy (Nikon Eclipse 55i). The specific growth rate of the culture was determined by a best-fit curve through the logarithmic portion of the growth data.

Genome sequence analyses

Following genomic DNA extraction as described above, a combination of Illumina short-read sequencing and Oxford Nanopore long-read sequencing was used for whole-genome sequencing of Ax23^T. For Illumina sequencing, library construction was performed using a NexteraXT DNA library prep kit (Illumina) per the manufacturer’s protocol. Both Illumina library construction and sequencing were performed by Azenta Life Sciences (South Plainfield, NJ, USA). The DNA was sequenced using a MiSeq instrument (Illumina) with 2×150 bp chemistry. Trimmomatic version 0.36 [13] was used to trim the last 8 bp of each sequence and regions with low-quality Q scores (Q<30). For Nanopore sequencing, sample libraries were prepared using the PCR-free Ligation Sequencing kit (Oxford Nanopore Technologies) with the NEBNext Companion Module (New England BioLabs) per the manufacturer’s protocol. Both Nanopore library construction and sequencing were performed by SeqCenter (Pittsburgh, PA, USA). Nanopore sequencing was performed on an Oxford Nanopore GridION sequencer using R10.4.1 flow cells in one or more multiplexed shared-flow-cell runs. Run design utilized the 400 bp sequencing mode with a minimum read length of 200 bp. Adaptive sampling was not enabled. Guppy version 6.5.7 (https://pypi.org/project/ont-pyguppy-client-lib/6.5.7) was used for super-accurate basecalling, demultiplexing and adapter removal. No quality trimming was performed.

De novo genome assemblies were generated from the Nanopore read data with Flye [14] under the nano-hq (ONT high-quality reads) model. Additional Flye options initiated the assembly by first using reads longer than an estimated N₅₀ based on a genome size of 6 Mbp. Subsequent polishing used the Illumina read data with Pilon [15] under default parameters. To reduce erroneous assembly artefacts caused by low-quality Nanopore reads, long-read contigs with an average short-read coverage of 15× or less were removed from the assembly. Assembled contigs were evaluated for circularization via Circlator [16] using the Nanopore long reads. The completeness of the genome sequence was determined using CheckM [17]. ORFs were identified and annotated using the National Center for Biotechnology Information (NCBI) Prokaryotic Genome Annotation Pipeline version 6.7. The whole-genome shotgun sequence project was deposited at DDBJ/ENA/GenBank under accession numbers CP157522 and CP157523. The raw reads were deposited in the Sequence Read Archive under BioProject number PRJNA1116089.

A 16S rRNA gene phylogenetic tree was constructed to determine the closest phylogenetic relatives of Ax23^T using sequences from NCBI, the maximum likelihood method and the Tamura–Nei model [18]. Ribosomal multilocus sequence typing (rMLST) analyses [19] were performed to confirm phylogenetic relationships by concatenating 56 ribosomal protein sequences from 18 whole-genome sequences in NCBI and aligning them using the maximum-likelihood method and a Jones–Taylor–Thornton matrix-based model [20]. Evolutionary analyses were conducted in mega X [21]. For overall genome relatedness index (OGRI) analyses, the blast-based average nucleotide identity (ANI) score was calculated using the JSpeciesWS program, version 3.2.2 [22]. Genome-to-genome direct comparison (GGDC) analyses were performed using Equation 2 in the GGDC program, version 3.0 [23]. Default parameters were used for all software analyses.

Results and discussion

Thermophilic methanogens in hydrothermal fluid

The average number of total cells and the average number of culturable thermophilic methanogens based on MPN estimates in the two hydrothermal vent samples from Marker 113 were 1.4×10⁸ cells l⁻¹ and 5,370 cells l⁻¹, respectively.

Phenotypic characteristics of Ax23^T

Strain Ax23^T is a thermophilic, anaerobic autotroph that produces CH₄. Phase-contrast and fluorescence microscopies revealed coccoid cells 1–2 µm in diameter that showed autofluorescence under blue light typical of methanogens. Growth on H₂ and CO₂ was observed between 33 and 75 °C with an optimum of 73 °C (Fig. 1a), between pH 4.0 and 9.0 with an optimum of pH 6.0–8.0 (Fig. 1b) and between 3 and 6% NaCl with an optimum of 3–4.5% (Fig. 1c). Its minimum doubling time was 32 min. When grown at 70 °C on H₂ and CO₂, the growth rate of Ax23^T was 0.88 h⁻¹ ±0.08 h⁻¹ (± 95% confidence interval), and up to 28 mmol of CH₄ was produced per litre of growth medium. Ax23^T grew at 70 °C on formate with 200 kPa N₂ in the headspace at a rate of 0.59 h⁻¹±0.11 h⁻¹. It did not grow on acetate, propionate, glucose, tryptone or yeast extract with 200 kPa N₂ in the headspace. However, growth at 70 °C with H₂ and CO₂ was stimulated when 0.1% yeast extract was added with a growth rate of 1.33 h⁻¹±0.23 h⁻¹. Ax23^T also grew on 80 kPa H₂, 80 kPa N₂ and 40 kPa CO₂ in growth medium lacking NH₄Cl with a growth rate of 0.25 h⁻¹±0.04 h⁻¹ but did not grow in control bottles lacking NH₄Cl with 160 kPa H₂ and 40 kPa CO₂, suggesting that it can fix N₂ for growth. There was no growth on H₂ and CO₂ when NH₄Cl was replaced with KNO₃. These phenotypic characteristics were most similar to those found for other thermophilic methanogens in the family Methanococcaceae (Table 1).

Fig. 1. Specific growth rates for strain Ax23^T across its growth range of (a) temperature at pH 6 and 3% NaCl, (b) pH at 70 °C and 3% NaCl and (c) chlorinity at 70 °C and pH 6. The error bars represent 95% confidence intervals.

The growth yield (biomass produced per mole of CH₄ produced) of hydrogenotrophic methanogens increases upon H₂ limitation [24,27]. This is due to a growth rate-growth yield metabolic trade-off where organisms can either grow slowly with high growth yields when resources are scarce or grow fast with low growth yields when resources are replete [28]. Strain Ax23^T also showed an average increase in cell yield from 1.2×10¹² cells/mol CH₄ (SD=0.6×10¹² cells/mol CH₄, n=5) at 160 kPa H₂ to 2.0×10¹² cells/mol CH₄ (SD=0.5×10¹² cells/mol CH₄, n=5) at 10 kPa H₂. The cell yield for strain Ax23^T was significantly higher (P<0.05) when grown on 10 kPa H₂ relative to 160 kPa H₂ using an unpaired t-test.

16S rRNA gene and rMLST phylogenies

Based on its 16S rRNA gene sequence obtained from the whole-genome sequence, strain Ax23^T was most closely related to members of the family Methanococcaceae in the kingdom Methanobacteriati [29] (Fig. 2). It showed the highest sequence identities to M. okinawensis IH1^T (99.7%), Methanococcus aeolicus PL15/HP^P (96.2%), M. aeolicus Nankai-3^T (96.1%) and M. thermolithotrophicus SN-1^T (95.6%) (Table 1). M. aeolicus PL15/HP^P, M. aeolicus Nankai-3^T and M. thermolithotrophicus SN-1^T sequence identities were below the value of 98.7%, suggesting that Ax23^T is a different species from these known organisms [30]. The rMLST phylogenetic tree confirmed that Ax23^T was most closely related to M. okinawensis IH1^T (Fig. 3).

Fig. 2. Phylogenetic tree based on the 16S rRNA gene showing the position of strain Ax23^T within the order Methanococcales relative to other type strains. After 1,000 bootstrap constructions, the tree with the highest log likelihood (−6054.62) is shown, with values next to nodes indicating the percentage of reconstructions in which the topology was preserved (values <70% are omitted for clarity). There were a total of 1,519 nt positions in the final dataset. Branch lengths are to scale and indicate the number of substitutions per site. The outgroup is Methanopyrus kandleri AV19^T from the order Methanopyrales. GenBank/EMBL/DDBJ accession numbers are included in parentheses.

Fig. 3. Phylogenetic tree of strain Ax23^T as inferred using rMLST of 56 concatenated ribosomal protein amino acid sequences. After 1,000 bootstrap constructions, the tree with the highest log likelihood (−103,422.95) is shown. The percentage of trees in which the associated taxa cluster together is shown next to the branches (values <70% are omitted for clarity). The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. There was a total of 8,556 positions in the final dataset. The outgroup is Methanopyrus kandleri AV19^T from the order Methanopyrales. GenBank/EMBL/DDBJ accession numbers are included in parentheses.

Genome features

Whole-genome sequencing generated a total of 24,895,368 raw paired-end reads and 7,469 Mb of sequenced bases using MiSeq short-read sequencing and 438,399 trimmed reads and 894 Mb of sequenced bases using Oxford Nanopore long-read sequencing. Genome assembly resulted in one complete circular genome sequence that was 1,662,948 bp long and one complete circular plasmid sequence that was 7,732 bp long. The average coverage was >5,000-fold. The annotation resulted in 1,663 protein-coding genes in the chromosome and a G+C content of 31.85%. Three copies of the 5S rRNA gene, two copies each of the 16S and 23S rRNA genes and 38 tRNA genes were identified in the chromosome.

OGRI analyses also showed that strain Ax23^T was most closely related to M. okinawensis IH1^T (Table 1). The ANI scores for strain comparisons between Ax23^T and M. okinawensis IH1^T, M. thermolithotrophicus DSM 2095^T, M. aeolicus Nankai-3^T and M. aeolicus PL15/HP^P (Table 1) were all below the 95–96% cut-off for species determination [30]. Similarly, the GGDC scores for strain comparisons between Ax23^T and M. okinawensis IH1^T, M. thermolithotrophicus DSM 2095^T, M. aeolicus Nankai-3^T and M. aeolicus PL15/HP^P (Table 1) were all below the 70% cut-off for delineating species by this approach [30]. Therefore, both OGRI analyses indicated that strain Ax23^T represents a novel species.

The Ax23^T genome contained putative genes that, based on sequence identity at the amino acid level, encode for a membrane-bound Eha-type [NiFe] ferredoxin-dependent hydrogenase (group 4d, EC 1.12.7.2, Mjas_00775-Mjas_00860), two cytoplasmic Frh-type [NiFe] coenzyme F₄₂₀-reducing hydrogenases (group 3a, EC 1.12.98.1, Mjas_04535-Mjas_04550 and Mjas_06645-Mjas_06660) and two cytoplasmic Vhu-type [NiFe] H₂:CoB-CoM heterodisulphide and ferredoxin reductases (group 3c, EC 1.8.98.5, Mjas_04020-Mjas_04040 and Mjas_06625-Mjas_06640) using the classification system of Greening et al. [31]. It had putative genes in a single operon for a cytoplasmic Fdh-type formate dehydrogenase (EC 1.8.98.6) and a membrane-bound formate transporter (Mjas_08265-Mjas_08285), which together are necessary for growth on formate [32]. It also had putative genes for Nif-type nitrogen fixation (EC 1.18.6.1, Mjas_05505-Mjas_05525) [5].

Description of Methanothermococcus jasoni sp. nov.

Methanothermococcus jasoni (ja.so’ni. N.L. gen. n. jasoni, pertaining to the ROV Jason II).

Cells are cocci (1–2 µm diameter), autofluorescent, and occur singly and in pairs. Growth occurs between 33 and 75 °C, with an optimum at 73 °C, between pH 4 and 9, with an optimum at pH 6–8, and between 3 and 6% NaCl, with an optimum of 3.0–4.5% NaCl. Anaerobic and chemolithoautotrophic. Uses H₂ and CO₂ as well as formate separately as energy and carbon sources to produce methane. Capable of N₂ fixation. The G+C content is 31.85 mol%.

The type strain, Ax23^T (=DSM 118471^T=JCM39656^T), was isolated from 34 °C hydrothermal vent fluid from the Marker 113 vent field located at Axial Seamount along the Juan de Fuca Ridge in the northeast Pacific Ocean. The GenBank accession numbers for the genome and the 16S rRNA gene sequence are CP157522-CP157523 and PX508301, respectively. The raw sequence reads of the genome are stored in Short Read Archive under BioProject PRJNA1116089.

Abbreviations

ANI

average nucleotide identity

GGDC

genome-to-genome direct comparison

MPN

most probable number

OGRI

overall genome relatedness index

rMLST

ribosomal multilocus sequence typing

ROV

remotely operated vehicle