Pyrodictium delaneyi sp. nov., a hyperthermophilic autotrophic archaeon that reduces Fe(III) oxide and nitrate

Abstract

A hyperthermophilic, autotrophic iron and nitrate reducer, strain Su06^T, was isolated from an active deep-sea hydrothermal vent chimney on the Endeavour Segment in the north-eastern Pacific Ocean. It was obligately anaerobic, hydrogenotrophic and reduced Fe(III) oxide to magnetite and ${\mathrm{NO}}_3^{-}$ to N₂. Phylogenetic analysis based on 16S rRNA gene sequences indicated that the strain was more than 97% similar to other species of the genera Pyrodictium and Hyperthermus. Therefore, overall genome relatedness index analyses were performed to establish whether strain Su06^T represents a novel species. For each analysis, strain Su06^T was most similar to Pyrodictium occultum PL-19^T. Relative to this strain, the average nucleotide identity score for strain Su06^T was 72%, the genome-to-genome direct comparison score was 13–19% and the species identification score at the protein level was 89%. For each analysis, strain Su06^T was below the species delineation cutoff. Based on its whole genome sequence and its unique phenotypic characteristics, strain Su06^T is suggested to represent a novel species of the genus Pyrodictium, for which the name Pyrodictium delaneyi is proposed. The type strain is Su06^T (=DSM 28599^T=ATCC BAA-2559^T).

The family Pyrodictiaceae is, at the time of writing, composed of five hyperthermophilic anaerobes. Pyrodictium occultum PL-19^T (Stetter et al., 1983), Pyrodictium brockii S1^T (Stetter et al., 1983) and Pyrolobus fumarii 1A^T (Blöchl et al., 1997) are strict autotrophs that reduce sulfur compounds and grow optimally at 105–108 °C (Table 1). Pyrolobus fumarii 1A^T also reduces ${\mathrm{NO}}_3^{-}$. Pyrodictium abyssi AV2^T (Pley et al., 1991) and Hyperthermus butylicus PLM1-5^T (Zillig et al., 1990) are strict peptide-utilizing organotrophs and grow optimally near 97 °C by fermentation or sulfur reduction with concomitant production of short-chain organic acids and alcohols (Table 1). Their growth is stimulated by H₂.

Table 1. Differential characteristics between species of the family Pyrodictiaceae.

Strains: 1, Su06^T; 2, Pyrodictium occultum PL-19^T; 3, Pyrodictium brockii S1^T; 4, Pyrodictium abyssi SV2^T; 5, Hyperthermus butylicus PLM1-5^T; 6, Pyrolobus fumarii 1A^T. Reference data were obtained from Stetter et al. (1983), Zillig et al. (1990), Pley et al. (1991), Blöchl et al. (1997) and Lin et al. (2014). +, Positive; −, negative; nd, no data.

Characteristic	1	2	3	4	5	6
Morphology	Coccoid	Disc-shaped with network of fibres	Disc-shaped with network of fibres	Disc-shaped often with flat protrusions	Coccoid	Coccoid
Flagellation	Lophotrichous	–	–	–	Lophotrichous	–
Temperature for growth (°C)
Range	82–97	80–110	80–110	80–110	>75–108	90–113
Optimum	90–92	105	105	97	95–106	106
pH for growth
Range	5.0–9.0	4.5–7.2	4.5–7.2	4.7–7.1	nd	4.0–6.5
Optimum	5.0	5.5	5.5	5.5	7.0	5.5
NaCl for growth (%)
Range	0.9–3.6	0.2–12	0.2–12	0.7–4.2	< 3.0	1–4
Optimum	1.9	1.5	1.5	~2	1.7	1.7
Strictly autotrophic growth	+	+	+	−	−	+
Electron acceptors	Fe(III) oxide, ${\mathrm{NO}}_3^{-}$	S°, ${\mathrm{S}}_2{\mathrm{O}}_3^{2-}$	S°, ${\mathrm{SO}}_3^{2-}$	S°, ${\mathrm{S}}_2{\mathrm{O}}_3^{2-}$, fermentation	S°, fermentation	${\mathrm{NO}}_3^{-}$, ${\mathrm{S}}_2{\mathrm{O}}_3^{2-}$, low O2
End products	Magnetite, N2	H2S	H2S	H2S, CO2, isovalerate, isobutyrate, butanol	H2S, CO2, acetate, propionate, phenylacetate, 1-butanol	${\mathrm{NH}}_4^{+}$, H2S

Strain Su06^T was isolated from an actively venting deep-sea hydrothermal chimney collected from the Sully hydrothermal edifice at a depth of 2197 m in 2006 at the Endeavour Segment hydrothermal vent field in the northeastern Pacific Ocean (GPS position; 47° 56.9' N 129° 5.9' W) (Ver Eecke et al., 2009). Cells were regular to irregular shaped coccoids 0.5–0.8 μm in diameter with lophotrichous flagellation (Fig. 1). Strain Su06^T was a mildly acidophilic (optimum pH 5), obligately hydrogenotrophic autotroph with optimal growth temperatures of 90–92 °C (Lin et al., 2014). Based on Mössbauer spectroscopy of the mineral phases, it reduced laboratory-synthesized ferrihydrite [Fe₁₀ O₁₄(OH)₂] to nanophase (<12 nm) magnetite (Fe₃O₄) in a growth-dependent manner with no detectable mineral intermediates (Lin et al., 2014). It also reduced KNO₃ to N₂ based on GC analyses and did not produce ${\mathrm{NH}}_4^{+}$ based on colorimetric analysis (Grasshoff et al., 1983). The washed insoluble protein fraction contained nitrate reductase activity according to the method of Afshar et al. (1998). No growth was observed when individual amino acids, sugars, formate, butyrate, malate or succinate was used as the sole electron donor and carbon source, although yeast extract stimulated growth in the presence ofH₂ and CO₂ (Lin et al., 2014). It was unable to grow without the addition of an added terminal electron acceptor, nor on S°, ${\mathrm{S}}_2{\mathrm{O}}_3^{2-}$, O₂ or commercially synthesized goethite, haematite, maghaemite or lepidocrocite (Lin et al., 2014). Its phenotypic characteristics were most similar to members of the family Pyrodictiaceae (Table 1).

Fig. 1.

Transmission electron micrograph of strain Su06^T using 1–2% uranyl acetate as a negative stain showing lophotrichous flagellation of a coccoid-shaped cell. Bar, 500 nm.

The phylogenetic relatedness of strain Su06^T to other species of the family Pyrodictiaceae was determined using 16S rRNA gene sequences obtained from the Ribosomal Database Project (Cole et al., 2007) and comparing them via megablast (McGinnis & Madden, 2004). Strain Su06^T showed 99.5% 16S rRNA gene sequence similarity with Pyrodictium brockii S1^T, 99.4% with Pyrodictium abyssi AV2^T, 99.3% with H. butylicus PLM1-5^T, 99.1% with Pyrodictium occultum PL-19^T and 97.4% with Pyrolobus fumarii 1A^T. We aligned sequences representing the order Desulfurococcales using the default settings for clustal w (Larkin et al., 2007) in mega6 software (Tamura et al., 2013). We reconstructed neighbour-joining phylogenetic trees in mega6 with the Jukes-Cantor model and bootstrap values obtained from 500 replicate trees (Fig. 2). According to this alignment, the closest relatives to strain Su06^T were Pyrodictium abyssi AV2^T and H. butylicus PLM1-5^T.

Fig. 2.

Neighbour-joining tree showing the position of strain Su06^T within the order Desulfurococcales based on sequences of the 16S rRNA gene (1305 nt). GenBank/EMBL/DDBJ accession numbers are included in parentheses. The topology of the tree was estimated by bootstrap analysis based on 500 replications. Numbers at branch points are the percentage support by bootstrap analysis. Bar, 1% sequence divergence.

As the 16S rRNA gene sequences of species of the family Pyrodictiaceae show more than 97 % similarity across the family (Fig. 2), the complete genome sequence of strain Su06^T was compared with the complete and draft genome sequences of its closest phylogenetic relatives using overall genome relatedness index (OGRI) analyses (Chun & Rainey, 2014). All genome sequences were obtained from the Gen-Bank sequence database (Table 2). We calculated the blast-based average nucleotide identity (ANI) score using the JSpeciesWS program with the default parameters (Richter et al., 2015). Genome-to-genome direct comparison (GGDC) analyses were performed using all three equations in the GGDC program, version 2.0 (Meier-Kolthoff et al., 2014). Forty marker proteins defined in the species identification (SpecI) program (Mende et al., 2013) were manually compared using blast-p. The SpecI program did not function using Pyrodictiaceae genomes.

Table 2. ANI, GGDC and SpecI-type analyses of genomic DNA and predicted protein sequences from strain Su06^T and related species of the family Pyrodictiaceae.

Data in bold type indicate the closest relatives. DDH, DNA-DNA homology.

Strain	ANI	GGDC DDH 1	GGDC DDH 2	GGDC DDH 3	‘SpecI’
Pyrodictium occultum PL-19T	72	14/13	19/17	14/13	89
Hyperthermus butylicus PLM1-5T	69	13	22	13	85
Pyrolobus fumarii 1AT	67	13	26	13	74

For each OGRI analysis, strain Su06^T was most closely related to Pyrodictium occultum PL-19^T (Table 2). The ANI score for strain comparisons between Su06^T and Pyrodictium occultum PL-19^T was 72% and between Su06^T and H. butylicus PLM1-5^T was 69%, which were both below the 96% cut-off value for species determination by this approach. The GGDC calculations with blast+ for strain Su06^T and Pyrodictium occultum PL-19^T gave DNA-DNA homology values of 13 and 14 %, 17 and 19 %, and 13 and 14 % for the three equations in the program and the two contigs in the Pyrodictium occultum PL-19^T draft genome sequence, which were all below the 60 % cut-off for delineating species by this approach (Table 2). The SpecI-type protein analysis for strain Su06^Tgave values of 89% for Pyrodictium occultum PL-19^T and 85 % for H. butylicus PLM1-5^T, which are below the 96.5 % cut-off for delineating species by this approach (Table 2). Therefore, all three OGRI analyses indicated that strain Su06^T represents a novel species. Therefore, based on its whole genome sequence and phenotypic differences, strain Su06^T is suggested to represent a novel species of the genus Pyrodictium, for which the name Pyrodictium delaneyi sp. nov. is proposed.

The physiological mechanism of iron reduction in hyper-thermophilic archaea is unknown. In mesophilic bacteria, iron reduction by Geobacter and Shewanella species is dependent upon polyheme c-type cytochrome proteins for electron transfer across the cell wall (Weber et al., 2006). A survey for c-type cytochrome proteins in the genome sequences of strain Su06^T showed eight proteins containing one or two CXXCH motifs, and nine proteins containing three or more CXXCH motifs. Five of the genes for these putative cytochrome proteins are arranged in neighbouring six- and three-gene operons (Pyrde_0256–0264) encoding proteins with membrane signal peptide sequences based on SignalP 4.1 analysis (Petersen et al., 2011). These genes have no known function and there are no homologues of these genes in any other Pyrodictiaceae genome. Three genes for putative cytochrome proteins in strain Su06^T are in an egene operon (Pyrde_0485–0492) encoding a putative membrane-bound sulfide reductase complex, with homologous genes found in Pyrodictium occultum PL-19^T, H. butylicus PLM1–5^T and Pyrolobus fumarii 1A^T. Two putative cytochrome proteins with no known function in strain Su06^T are in an 11-gene operon (Pyrde_0777–0787) and contain membrane signal peptide sequences with homologous genes in Pyrolobus fumarii 1A^T. It remains to be determined if any of the putative c-type cytochrome proteins in strain Su06^T are involved in iron reduction.

Description of Pyrodictium delaneyi sp. nov.

Pyrodictium delaneyi (de.la′ney.i; delaneyi named for Dr John R. Delaney, co-discoverer of the Endeavour Segment hydrothermal vent field, co-founder of the RIDGE hydrothermal vent programme, early advocate for the use of astrobiology and cabled ocean observatories).

Cells are lophotrichously flagellated regular to irregular cocci. Cell diameter is approximately 0.8 μm. Obligate anaerobe. Optimal growth occurs at 90–92 °C (range 82–97 °C), at pH 5.0 (range pH 5.0–9.0) and with 1.9% total salts (range 0.9–3.6%). Autotrophic growth occurs via poorly crystalline Fe(III) oxide reduction to magnetite and ${\mathrm{NO}}_3^{-}$ reduction to N₂ using H₂ and CO₂. No growth is observed when individual amino acids, sugars, formate, butyrate, malate or succinate is used as the electron donor and carbon source, although yeast extract stimulates growth in the presence of H₂ and CO₂. No growth on S°, ${\mathrm{S}}_2{\mathrm{O}}_3^{2-}$, O₂, goethite, haematite, maghaemite or lepidocrocite, or purely by fermentation.

The type strain is Su06^T (=DSM 28599^T=ATCC BAA-2559^T), isolated from an active hydrothermal vent sulfide chimney (Sully) on the Endeavour Segment, Juan de Fuca Ridge, in the north-eastern Pacific Ocean. The genomic DNA G+C content of the type strain is 53.9 mol% based on total genome calculations.

Abbreviations:

ANI

average nucleotide identity

GGDC

genome-to-genome direct comparison

OGRI

overall genome relatedness index