The phylum 'Campylobacterota' is a phylogenetically and ecophysiologically diverse Bacterial group that consists of two classes, i.e., Desulfurellia (the former order Desulfurellales) and 'Campylobacteria' (the former class Epsilonproteobacteria). While this phylum is widely recognised as a group including pathogenic microorganisms, e.g. Helicobacter pylori and Campylobacter jejuni, and many studies have focused on these pathogens, an ever expanding number of non-pathogenic species have been identified which thrive as mesophiles or thermophiles in a wide range of natural environments (e.g. deep-sea hydrothermal fields, stratified ocean, terrestrial sulfidic caves, and oil fields) where they are recognized as important players in biogeochemical cycles. Cultivation and characterisation of these bacteria has also expanded our knowledge on the evolution and diversification of pathogenic relatives, biogeography, and the potential of biotechnological applications to mitigate global warming.
At deep-sea hydrothermal vents, Bacteria belonging to the phylum 'Campylobacterota' are known as the dominant community members, including sulphide chimney structures where they can comprise up to 85% of the microbial biomass. Taxonomically and metabolically diverse members of chemosynthetic 'Campylobacterota' are responsible for the primary production. Ever since thermophilic 'Campylobacterota' were first cultivated from hydrothermal vents, the number of culturable thermophilic members has increased with the refinement of cultivation conditions. Nevertheless, the described thermophilic species still account for only 14% of the total number of validly published species within 'Campylobacterota', and therefore there is still insufficient information on their genomes and intra-specific diversity. This also leaves the classification of thermophiles unresolved as almost all thermophilic families are composed of only a single genus, which were mostly retrieved from deepsea hydrothermal vents.
The family Nitratiruptoraceae is one of the thermophilic groups within 'Campylobacteria' that is frequently detected in deep-sea hydrothermal environments globally. This family consists so far of one validly described genus and species, Nitratiruptor tergarcus, isolated from the deep-sea hydrothermal chimney structure in the Mid-Okinawa Trough. The recently described species 'Nitratiruptor labii' was also isolated from the same deep-sea hydrothermal region. In addition to the importance of Nitratiruptoraceae species in biogeochemical cycles, their potential for industrial applications has been described. Isolation of novel Nitratiruptoraceae species and elucidation of their physiological and genomic characteristics are both necessary to help understanding the diversity of this group and the evolutionary relationships within 'Campylobacteria'.
16S rRNA gene sequences have been the universal molecular chronometer for microbial taxonomic affiliation for more than three decades, but this tool does not work well in classifying either closely related species or distantly related taxa. The rapid advances in sequencing technology over the past decade have resulted in an increase in the amount of whole genome data and have brought significant opportunities to introduce robust and accurate criteria to improve microbial taxonomy. One advance is the genome-based taxonomy based on the use of a large number of conserved core genes and indices such as in silico DNA-DNA hybridisation, average nucleotide identity, average amino acid identity, which refines phylogenetic analyses using genome sequence data. These classifiers enable the robust classification of novel species or genera, resulting in a more accurate microbial taxonomy. Genome-based methods could also be effective in classifying members within the phylum 'Campylobacterota'. However, the robust and accurate criteria using genome relatedness indices have not yet been fully evaluated for all species within 'Campylobacterota'. In order to further expand the knowledge of their phylogenetic relationships and to propose a more robust classification methodology, establishment of clear classification criteria is needed to be evaluated.
In a paper published in the journal PLoS One on 10 December 2020, Taiki Shiotani, Sayaka Mino, Wakana Sato, Sayo Nishikawa, and Masanori Yonezawa of the Laboratory of Microbiology at the Faculty of Fisheries Sciences at Hokkaido University, Stefan Sievert of the Department of Biology at the Woods Hole Oceanographic Institution, and Tomoo Sawabe, also of the Laboratory of Microbiology at the Faculty of Fisheries Sciences at Hokkaido University, report a new thermophilic Campylobacterium, strain EPR55-1T, and evaluate its taxonomic assignment using a comprehensive approach based on whole genome sequence of the phylum 'Campylobacterota'.
The sample of a sulphide chimney structure was collected from the Bio9 deep-sea hydrothermal vent on the East Pacific Rise (9.83˚ N 104.28˚ W, water depth 2511 m) by HOV Alvin during the AT26-23 scientific cruise aboard the R/V Atlantis in 2014. The interior part of the chimney sample was mixed anaerobically with 25 ml sterilised seawater containing 0.05% (w/v) neutralized sodium sulfide in 100 ml glass bottles (Schott Glaswerke) soon after HOV Alvin was recovered.
Cells of EPR55-1T are Gram-negative rods (1.0 μm long and 0.5 μm in wide). Cells are motile by means of flagella. Spore formation has not been observed. Strain EPR55-1T grew at temperature between 50˚C and 60˚C, with optimum growth at 60˚C. No growth was observed below 40˚C or above 65˚C. Growth occurred between pH 5.4 and 8.6, with optimum growth at pH 6.6. No growth was detected below pH 3.2 or above pH 9.8. Growth was observed sodium chloride concentrations between 2.4 and 3.2% (weight/volume), with optimum growth at 2.4%. No growth was observed at concentrations below 1.6% or above 4.0%. Temperature, pH, and sodium chloride ranges for growth of strain EPR55-1T were similar to those of 'Nitratiruptor labii' strain HRV44T.
Strain EPR55-1T was only able to use molecular hydrogen as electron donor. Nitrate (0.1%, weight/volume), nitrous oxide (33%, volume/volume), thiosulphate (0.1%, weight/volume), elemental sulfur (1%, weight/volume) and molecular oxygen (0.1%, volume/volume) were able to serve as the sole electron acceptors. The isolate could not utilise any organic compounds as energy or carbon sources. These results indicated that strain EPR55-1T was a strictly hydrogen-oxidizing thermophilic chemolithoautotroph. The isolate was able to use ammonium as its sole nitrogen source and utilisation of molecular nitrogen was not observed. Strain EPR55-1T utilized thiosulphate, sulfite (0.01 to 0.001%, weight/volume) and elemental sulphur as sulphur sources. None of the chemosynthetic 'Campylobacterota' isolated so far are reported as possessing the ability to utilise sulphite as its sulphur source. Strain EPR55-1T was sensitive to ampicillin, chloramphenicol, kanamycin, streptomycin and rifampicin.
With a nearly full length of 16S rRNA gene sequence of strain EPR55-1T as a query in BLAST search, 96.0%, 94.3%, 93.0%, and 93.0% similarity were estimated with 'Nitratiruptor labii' HRV44T, Nitratiruptor sp. SB155-2, Nitratiruptor tergarcus MI55-1T, and Hydrogenimonas thermophila EP1-55-1T, respectively, indicating that strain EPR55-1T may be a new species of Nitratiruptor or even a member of newly described genus with strain HRV44T. The phylogenetic analysis showed that strain EPR55-1T was closely related to the Nitratiruptor species.
Hybrid genome assembly with Unicycler resulted in a single complete circular contig with a length of 1 807 889 base pairs. Of the 1833 genes predicted, 1783 were coding sequences, 41 tRNA genes, and 3 set of rRNA genes. These values were comparable to those of closely rerated Nitratiruptor isolates; 'Nitratiruptor labii' HRV44T (1 990 315 base pairs and 2050 coding sequences without a plasmid) Nitratiruptor tergarcus MI55-1T (1 894 691 base pairs and 1935 coding sequences) and Nitratiruptor sp. SB155-2 (1 877 931 base pairs and 1,857 coding sequences). The G + C content was 37.7%, which is similar to that of Nitratiruptor sp. SB155-2 (39.7%).
The in silico DNA-DNA hybridisation values of strain EPR55-1T against 'Nitratiruptor labii' HRV44T, Nitratiruptor tergarcus MI55-1T and Hydrogenimonas thermophila EP1-55-1%T were 18.7%, 18.1% and 17.4%, respectively, well below a threshold of 70% in silico DNA-DNA hybridisation used for the definition of Bacterial species. In addition, average nucleotide identity values of strain EPR55-1T against 'Nitratiruptor labii' HRV44T, Nitratiruptor tergarcus MI55-1T and Hydrogenimonas thermophila EP1-55-1%T were 77.5%, 71.4% and 70.4%, respectively, well below the species threshold (95.0%). These results support the proposal that the isolate is a novel species within the class 'Campylobacteria'. Average amino acid identity values of the novel isolate against 'Nitratiruptor labii' HRV44T, Nitratiruptor tergarcus MI55-1T, Nitratiruptor sp. SB155-2 and Hydrogenimonas thermophila EP1-55-1%T were 69.9%, 64.1%, 63.7% and 59.4%, respectively, which fall within the threshold for genus-level differentiation (60–80%). The genome-wide average nucleotide identity and alignment fractions of strain EPR55-1T against the closely related species were 73.85% and 0.53 to 'Nitratiruptor labii' HRV44T, 71.45% and 0.38 to Nitratiruptor tergarcus MI55-1T, 71.32% and 0.38 to Nitratiruptor sp. SB155-2, respectively. These values below the genus level threshold (genome-wide average nucleotide identity value of 73.98 (mean) and 73,11 (median), alignment fraction value of 0.33 (mean) and median (0.345)) are indicative of genus-level differentiation of strain EPR55-1T with strain HRV44T, though percentage of conserved proteins values of strain EPR55-1T against the all three relative strains were over 70.1%, higher than genus threshold (above 50%).
A Venn-diagram showed the presence of a conserved core set of 1270 gene clusters that are shared by all Nitratiruptor genomes, representing more than a half of the proteins in each strain. In addition, strain EPR55-1T, HRV44T, MI55-1T and SB155-2 possessed 215, 345, 250 and 245 singletons, respectively.
Average amino acid identity analysis between 160 genomes, which vary extensively within 'Campylobacterota, illustrates that there are genera, which need to be considered reclassification. Average amino acid identity, genome-wide average nucleotide identity, alignment fractions, and percentage of conserved proteins values between type strains of the family Nautiliaceae, Nautilia profundicola AmHT, Caminibacter mediatlanticus TB-2T, Cetia pacifica TB-6T, and Lebetimonas natsushimae HS1857T, were 71.6–74.2%, 76.1–78.0%, 0.52–0.81, and 76.02–84.0%, respectively, within or higher than the genus demarcation given by previous studies. The phylogenomic analyses based on both single-copy core genes and multilocus sequence analysis genes also showed these strains could be regarded as one clade. Although the genome-based taxonomy indicated these strains could be considered to the species belonging the same genus, the current intergenus 16S rRNA gene sequence identities of these strains were below 94.5% with the exception of Cetia pacifica TB-6T which showed more than 95% identity to all type strains of the genus Caminibacter.
In addition to the thermophilic taxa, some differences compared to the current classification were observed for the genus Helicobacter and the family Arcobacteraceae. Helicobacter pametensis and Helicobacter cholecystus, Helicobacter brantae showed low average amino acid identity values against other Helicobacter species (48.5–55.8%). Similarly, Helicobacter equorum and Helicobacter himalayensis, and Helicobacter anseris and Helicobacter mustelae showed average amino acid values below the genus threshold against other Helicobacter species (50.0–59.9% and 49.8–55.9%, respectively). Phylogenomic analyses also showed that these six species formed new three clades (Helicobacter E F, and G). Additionally, Helicobacter pylori, Helicobacter acinonychis, and Helicobacter cetorum, currently belonging to the Helicobacter clade, showed lower average amino acid identity values than the genus threshold against other current Helicobacter clade species (57.0–58.1%). In the both neighbour-joining and maximum likelihood phylogenomic trees on the basis on 139 single-copy core genes, the current Helicobacter clade branched to two clades (Helicobacter I and II). Same branching patterns were also observed in the neighbour-joining tree based on amino acid sequences of multilocus sequence analysis genes. In the genera of the family Arcobacteraceae, average amino acid identity values between species belonging to different genera Aliarcobacter, Poseidonibacter, Malaciobacter, Arcobacter, Halarcobacter were 61.9–78.7%, indicating they could be regarded as different species of the same genus. The large clade consisting of these genera was also identified by phylogenomic analyses based on both single-copy core genes and multilocus sequence analysis genes. However, some species showed the inter-genus 16S rRNA gene sequence identities below 94.5%, indicating differentiation at genus level. Average amino acid identity values among Campylobacter B were relatively lower (57.02–65.86%), possibly due to the low degree of relatedness between Campylobacter B species (less than 94.5% 16S rRNA gene similarities).
Based on results of phylogenomic analyses, the average amino acid identity values between species belonging to same clades or the different clades were evaluated. The minimum average amino acid value between species belonging to same clades was 59.7% (Helicobacter muridarum ST1T vs Helicobacter saguini MIT 97-6194T), and the maximum average amino acid identity value between species belonging to different clades was 61.9% (Campylobacter geochelonis RC20T vs Campylobacter hominis ATCC BAA-381T) with the exception of Nitratiruptoraceae, Nautiliaceae, and Arcobacteraceae whose 16S rRNA gene sequence similarities were not reflected in the genome relatedness. These values indicated the average amino acid identity threshold for genus demarcation of 'Campylobacterota' was about 60–62%, though there are some exceptions.
Strain EPR55-1T was the first Nitratiruptoraceae species isolated from the East Pacific Rise. The strain shows some physiological differences from other Nitratiruptoraceae isolates, and represents the only Nitratiruptoraceae species which is able to utilise thiosulphate and sulphite as its sole electron acceptor and sulfur source, respectively. The ability to utilise sulphite has also never been reported in any other thermophilic Campylobacterial species. The strain EPR55-1T possessed lophotrichous flagella, unlike the monotrichous and amphitrichous flagella of 'Nitratiruptor labii' and Nitratiruptor tergarcus, respectively. 16S rRNA gene sequence similarities,genome-wide average nucleotide identity and alignment fraction values of the strain against closely related species suggested that strain EPR55-1T designate the strain as a novel genus with strain HRV44T. The strain EPR55-1T therefore represents a novel genus within a new genus of the family Nitratiruptoraceae.
Strain EPR55-1T is therefore described as a new species, Nitrosophilus alvini, where 'Nitrosophilus' derives from 'nitrosus', full of natron, intended to mean nitrate and nitrous oxide, and 'philos', loving, friendly to, and 'alvini' derives from the name of the HOV Alvin which collected the deep-sea hydrothermal samples harbouring this strain).
Cells are rod-shaped, motile and stain Gram-negative. Anaerobic to microaerobic. Strictly chemolithoautotrophic. Thermophilic, adapted to the salinity of the ocean. On the basis of 16S rRNA gene and single-copy core-gene analyses, the genus Nitrosophilus belongs to the family Nitratiruptoraceae within the class 'Campylobacteria'.
The temperature range for growth is at 50– 60˚C (optimum 60˚C). The pH range for growth is pH 5.4–8.6 (optimum 6.6). Sodium Chloride concentration range for growth is 2.4–3.2% (weight/volume) (optimum 2.4%). Strain EPR55-1T is hydrogen-oxidising, facultatively anaerobic and chemolithoautotrophic with molecular hydrogen as its sole electron donor and with nitrate, nitrous oxide, thiosulfate, molecular oxygen or elemental sulphur as its sole electron acceptors. Ammonium is utilised as its sole nitrogen source. Thiosulphate, sulphite or elemental sulphur are utilised as its sole sulphur source. The complete genome size is 1 807 889 base pairs. The guanine + cytosine content of DNA is 37.7%. The type strain, EPR55-1T (otherwise known as JCM 32893T and KCTC 15925T), was isolated from a deep-sea hydrothermal vent in the East Pacific Rise.
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