Biogeochemical Implications of N2O-Reducing Thermophilic Campylobacteria in Deep-Sea Vent Fields, and the Description of Nitratiruptor labii sp. nov.

Nitrous oxide (N2O) is a potent greenhouse gas and has significantly increased in the atmosphere. Deep-sea hydrothermal fields are representative environments dominated by mesophilic to thermophilic members of the class Campylobacteria that possess clade II nosZ encoding nitrous oxide reductase. Here, we report a strain HRV44T representing the first thermophilic campylobacterium capable of growth by H2 oxidation coupled to N2O reduction. On the basis of physiological and genomic properties, it is proposed that strain HRV44T (=JCM 34002 = DSM 111345) represents a novel species of the genus Nitratiruptor, Nitratiruptor labii sp. nov. The comparison of the N2O consumption ability of strain HRV44T with those of additional Nitratiruptor and other campylobacterial strains revealed the highest level in strain HRV44T and suggests the N2O-respiring metabolism might be the common physiological trait for the genus Nitratiruptor. Our findings provide insights into contributions of thermophilic Campylobacteria to the N2O sink in deep-sea hydrothermal environments.

Introduction

Nitrous oxide (N2O) is the one of the gaseous nitrogen compounds that is the third most important long-lived greenhouse gas and a major ozone-depleting substance (Crutzen, 1970; Ravishankara et al., 2009; Ciais et al., 2013). Atmospheric N2O level has increased and its concentration reached 330 ppb in 2018, which is an increase of 23% in comparison with the pre-industrial era (Machida et al., 1995). Construction of a scheme for N2O mitigation and control of N2O emissions is therefore an essential and urgent global goal. N2O is emitted from natural sources (e.g., oceans, forests, and savannas) and anthropogenic sources (e.g., agriculture, biomass burning, power plants, and wastewater treatment plants). The ocean is considered to be the third largest source of N2O that accounts for average 21% of combined natural and anthropogenic N2O sources (Ciais et al., 2013). Ongoing environmental changes (e.g., ocean warming, acidification, and eutrophication) may significantly alter the global oceanic N2O emissions; therefore, a global survey of oceanic N2O flux must be accurately assessed (Bange et al., 2019). Most of the atmospheric N2O is produced through the microbial processes of ammonia oxidation coupled with NO2- reduction and denitrification, driven by metabolically versatile microorganisms (Bange et al., 2010; Thomson et al., 2012; Hu et al., 2015). Furthermore, N2O production by dissimilatory nitrate reduction to ammonium (DNRA), nitrite oxidation, anaerobic methane oxidation pathways have also been reported, although their magnitudes for global N2O budget are unclear (Hallin et al., 2018).

In strong contrast to the manifold N2O-producing processes, there is only one known N2O sink in the biosphere: microbial N2O reduction to harmless N2 gas. This pathway is catalyzed by N2O reductase (NosZ) implementing two copper centers (CuA and CuZ), encoded by nosZ gene (Zumft and Kroneck, 2007; Pauleta et al., 2013). NosZ encoded by a typical (clade I) nosZ gene had been accepted as the only enzyme catalyzing N2O reduction for a long time (Zumft and Kroneck, 2007). However, an unprecedented nos gene cluster with a novel nosZ containing an additional c-type heme domain at the C terminus was discovered in Wolinella succinogenes (Simon et al., 2004). The novel nosZ is currently termed atypical (clade II) nosZ, and clade II nosZ has been identified in a broad range of microbial taxa extending beyond bacteria to even archaea (Sanford et al., 2012; Jones et al., 2013). The sequences of clade II nosZ have been detected in similar abundance to those of clade I nosZ in various environments (Sanford et al., 2012; Jones et al., 2013). Recent biochemical approaches for two types of NosZ revealed relatively lower whole-cell half-saturation constants for N2O with clade II bacteria compared with those with clade I bacteria (Yoon et al., 2016; Suenaga et al., 2019), suggesting a high affinity for N2O in clade II nosZ organisms. In addition, ecological niche partitioning between two nosZ clade microorganisms has been demonstrated by culture-dependent and -independent analyses (Dini-Andreote et al., 2016; Graf et al., 2016; Wittorf et al., 2016; Juhanson et al., 2017). Although these previous studies have emphasized the large contributions of clade II nosZ microorganisms to the global N2O sink, understanding of the genomics, physiology, and ecology of microorganisms with clade II nosZ gene is still limited to several model microorganisms.

Members of the class Campylobacteria (former class Epsilonproteobacteria) are considered as the predominant bacterial group and play a significant role as primary producers in global deep-sea hydrothermal environments (Nakagawa et al., 2005a; Huber et al., 2007; Akerman et al., 2013; Muto et al., 2017). Nitrate respiration is a widespread energy metabolism in chemosynthetic Campylobacteria; more than 70% of campylobacterial species from deep-sea hydrothermal environments possess the capability to utilize nitrate as a sole electron acceptor (Sievert and Vetriani, 2012; Vetriani et al., 2014). Culture-dependent studies using several mesophilic and thermophilic isolates have demonstrated their ability to mediate complete nitrate reduction to dinitrogen, suggesting the capability of N2O as an electron acceptor. In the genomes of campylobacterial isolates from deep-sea hydrothermal environments, clade II nosZ genes have been identified (Inagaki et al., 2003, 2004; Nakagawa et al., 2005b; Giovannelli et al., 2016). Recently, the capability of exogeneous N2O as a sole electron acceptor has been characterized for the first time using an enrichment culture dominated by a novel mesophilic campylobacterial species, which possesses the clade II nosZ gene on its genome (Mino et al., 2018). Although diverse Campylobacteria with clade II nosZ possibly contribute to the nitrogen cycle as N2O reducers in natural environments (Fortunato and Huber, 2016; Mino et al., 2018), their ability to utilize N2O has yet to be fully elucidated. In particular, little is known about N2O-reducing thermophilic Campylobacteria.

Here, in this study, a novel thermophilic campylobacterium strain HRV44T is described that was isolated from a deep-sea hydrothermal vent in the Mid-Okinawa Trough. The genomic and physiological characteristics of strain HRV44T are further determined, and Nitratiruptor labii sp. nov. is proposed as type strain HRV44T. In addition, we compare the nosZ gene cluster and the capability to reduce exogenous N2O among campylobacterial strains. Our results provide insight into the contribution of thermophiles as N2O reducers to nitrogen cycles in deep-sea hydrothermal environments.

Results

Isolation and Phylogenetic Analysis Based on 16S rRNA Gene Sequences of N2O-Reducing Microorganisms

Six isolates (i.e., strains No.9, No.33, No.34, No.37, No.41, and HRV44T) were obtained from deep-sea hydrothermal samples using HNN medium containing H2 and N2O as a sole electron donor and acceptor, respectively, at 55°C. Since the unusual insertion sequence (IS) was found within the 16S rRNA gene sequence of strain HRV44T, the sequence similarities were calculated after the removal of IS. 16S rRNA gene sequences of strain HRV44T and other five strains were most closely related to Nitratiruptor sp. EPR55-1 (95.2%), which was isolated from a deep-sea hydrothermal vent in the East Pacific Rise (unpublished data), and Nitratiruptor sp. SB155-2 (99.5–99.9%) (Nakagawa et al., 2007), respectively. The sequence similarity between HRV44T and the only formally described Nitratiruptor species, Nitratiruptor tergarcus, was lower than the threshold for distinct species (98.7%–99.0%) (Stackebrandt and Ebers, 2006). Phylogenetic analysis based on 16S rRNA gene sequences also indicated that all of the six isolates belonged to the genus Nitratiruptor (Figure 1).

Figure 1

Phylogenetic Relationship of the Isolates Obtained in This Study and Related Campylobacteria

The phylogenetic tree was constructed using the maximum-likelihood method based on 1,048 nucleotide positions of 16S rRNA gene sequences. Numbers at nodes represent bootstrap values (%) (1,000 replicates).

N2O Consumption of the Isolates

Comparison of headspace N2O consumption and cell growth during 72-h cultivation revealed that N2O consumption rates and growth rates varied markedly among the six isolates. Strain HRV44T consumed approximately 97% of initial headspace N2O with a 10-fold increase in the total cell counts after 24 h (Figure 2). Maximum N2O consumption rate of strain HRV44T was about 9.9 ± 1.4 μmol h−1 per mL of culture, which was the highest among the six isolates (Figure 2). Higher N2O consumption and cell growth were observed in HRV44T than in the other strains (Figure S1). Production of N2 during the cultivation was also confirmed in all of the strains (Figure S2).

Figure 2

N2O Consumption of Six Isolates

(A) N2O consumption for 72-h cultivation of six isolates obtained in this study are shown. Error bars represent standard errors (n = 3). Different small letters above the bars indicate significant differences assessed by Tukey's HSD test (p < 0.05). The cell yields of strains are shown below the bars.

(B) Time course changes in headspace N2O concentration and the growth of strain HRV44T are shown in filled circles with solid line and open circles with dotted line, respectively.

Characteristics of Strain HRV44T

Strain HRV44T was rod shaped and motile with a polar flagellum (Figure S3). Cells of the strain often formed aggregates. Strain HRV44T grew at 45°C–60°C, showing optimum growth at 53°C. The strain grew at pH 5.4–6.4, showing optimum growth at pH 6.0. Optimum NaCl concentration for growth of strain HRV44T was 2.5% (w/v), and growth range was 2.0%–4.0% (w/v) (Figure S4). The doubling time under the optimum condition was 1.17 h. Strain HRV44T utilized H2 as a sole electron donor and nitrate, N2O, elemental sulfur, and oxygen (up to 1.0% (v/v) in the headspace gas) as a sole electron acceptor. The maximum cell yield of strain HRV44T was approximately 3.6×108 cells mL−1 in HNN medium. Strain HRV44T was able to use none of the tested carbon sources other than carbon dioxide. The strain was able to utilize nitrate and ammonium as sole nitrogen sources. Sulfate and elemental sulfur were utilized as sole sulfur sources of the strain (Table 1). When comparing the growth ranges of pH and NaCl among related Campylobacteria, those of strain HRV44T were relatively narrower than those of mesophiles and other thermophiles as well as other Nitratiruptor species (Figure S5).

Table 1

Comparison of the Major Physiological Characteristics of Strain HRV44T with Those of Members of the Genus Nitratiruptor Isolated from Deep-Sea Hydrothermal Fields

Characteristics	Nitratiruptor labii HRV44T (This Study)	Nitratiruptor sp. EPR55-1 (Unpublished Data)	Nitratiruptor tergarcus MI55-1T (Nakagawa et al., 2005b)	Nitratiruptor sp. SB155-2 (Nakagawa et al., 2007)
Origin	MOT	EPR	MOT	MOT
Temperature range (˚C)	45–60	50–60	40–55	37–65
Optimum temperature (˚C)	53	60	55	55
pH range	5.4–6.4	5.4–8.6	5.4–6.9	ND
Optimum pH	6.0	6.6	6.4	ND
NaCl range (% w/v)	2.0–4.0	2.4–3.2	1.5–4.0	ND
Optimum NaCl (% w/v)	2.5	2.4	2.5	ND
Electron donors	H2	H2	H2	H2, S2−, S0, S2O32-
Electron accepters	NO3-, N2O, S0,O2 (up to 1.0%, v/v)	NO3-, S2O32−, S0, O2	NO3-, S0a,O2 (up to 0.7%, v/v)	NO3-, O2
Nitrogen sources	NO3-, NH4+	NH4+	NO3-, NH4+	ND
Carbon sources	CO2	CO2	CO2	ND
DNA G + C content (%)	33.4	37.7	29.6b	39.7

ND, not determined. MOT, Mid-Okinawa Trough; EPR, East Pacific Rise.

S0 could not serve as a sole electron acceptor to support growth.

The G + C content in mol% of DNA.

Whole-Genome Sequencing of Strain HRV44T

We obtained high-quality sequencing reads using PacBio and Illumina sequencing. The de novo assembly using both PacBio and Illumina reads resulted in the acquisition of the complete genome of strain HRV44T, composed of a chromosome and a plasmid. The sizes of chromosome and plasmid are 1,990,315 bp with G + C content of 33.46% and 102,672 bp with G + C content of 33.01%, respectively. The numbers of CDS of chromosome and plasmid were 2,050 and 128, respectively, of which 34.2% and 78.1% were annotated as hypothetical genes, respectively. Three rRNA operons organized by 5S, 16S, and 23S rRNA gene and 41 tRNA genes were identified on the chromosome (Table S1). The three copies of 16S rRNA gene with IS were all completely identical. Several CDSs of plasmid were annotated as genes involved in plasmid replication, DNA replication, and plasmid conjugation. The 41 plasmid CDSs of strain HRV44T shared 37%–86% identities with plasmid CDSs of Campylobacter iguaniorum, isolated from reptiles (Gilbert et al., 2015) (Table S2). No known antibiotic resistance genes were found on the plasmid. A combination of the Oxford Nanopore Technologies (ONT) and Illumina data also yielded two circular units, a large circular chromosome of 2,021,942 bp, and a plasmid of 102,626 bp.

Description of IS and Prediction of the 16S rRNA Secondary Structure of Strain HRV44T

Insertion sequence located within the V2 region of the 16S rRNA gene sequence of strain HRV44T was detected, and its size was determined to be 268 bp. There was no ORF in IS. The secondary structure prediction of the 16S rRNA showed IS constructed bifurcated structure at the tip of the stem, and two branched stems had internal and hairpin loops (Figure S6). 16S rRNA secondary structure with IS did not greatly differ from that lacking IS. Identical IS was retrieved from all three copies of 16S rRNA genes.

Calculation of Genome Sequence Similarities and Phylogenomic Analysis

On the basis of the complete genome of strain HRV44T, in silico DNA-DNA hybridization (in silico DDH), average nucleotide identity (ANI), and average amino acid identity (AAI) values were calculated against genomes of related species. In silico DDH values (Formula 2, recommended) of strain HRV44T against Nitratiruptor tergarcus MI55-1T, Nitratiruptor sp. SB155-2, Nitratiruptor sp. EPR55-1, and Hydrogenimonas thermophila EP1-55-1%T were 20.2%, 20.4%, 18.7%, and 17.1%, respectively, further distinguishing strain HRV44T from those species (Wayne et al., 1987). ANI values of the novel strain against Nitratiruptor tergarcus MI55-1T, Nitratiruptor sp. SB155-2, Nitratiruptor sp. EPR55-1, and Hydrogenimonas thermophila EP1-55-1%T were 77.1%, 76.4%, 77.5%, and 72.7%, respectively, well below the threshold for the species circumscription (95.0%–96.0%) (Goris et al., 2007; Richter and Rosselló-Móra, 2009). AAI values of HRV44T against Nitratiruptor tergarcus MI55-1T, Nitratiruptor sp. SB155-2, Nitratiruptor sp. EPR55-1, and Hydrogenimonas thermophila EP1-55-1%T were 66.3%, 66.2%, 69.9%, and 58.1%, respectively, which fall within the circumscription for genus-level differentiation (Rodriguez-R and Konstantinidis, 2014).

Phylogenomic analysis of HRV44T and representative Campylobacteria strains was performed based on 127 conserved single core protein sequences among the genomes (Figure S7). Strain HRV44T clustered with the other strains belonging to the genus Nitratiruptor. The topology of the phylogenomic tree differed from the 16S rRNA-based tree.

Comparison of nos Gene Cluster, Putative Transcription Regulators, and N2O-Reducing Ability among Related Campylobacteria Isolated from Deep-Sea Hydrothermal Fields

We reconstructed nos gene clusters from genomes of the clade II nosZ-possessing Campylobacteria isolated from deep-sea hydrothermal environments. The composition of nos gene cluster organized by 11 genes (nosZ, -B, -D, -G, -C1, -C2, -H, -F, -Y, -L, and a hypothetical gene) was well conserved in the related campylobacterial members, with an exception of Sulfurimonas autotrophica, which lacked one hypothetical gene (Figure 3). Using Campylobacter jejuni NssR (Nitrosative stress sensing Regulator; Cj0466) as database search template, two putative transcription regulators of the Crp-Fnr superfamily were found to be encoded in strain HRV44T (Figure S8) (Kern et al., 2011). Notably, Nitratiruptor species encoded a Nss-type regulator upstream of the nor gene cluster in reverse orientation altogether, whereas in other mesophilic species the regulators were located upstream the nap gene cluster. The three putative Nss-binding sites were found upstream of nosZ of strain HRV44T (Figure S8). In addition, genes necessary for denitrification are clustered together in Nitratiruptor species, although mesophiles appeared to form three separate loci, the nap, nir-nor, and nos gene clusters.

Figure 3

Comparison of nos Gene Cluster and of N2O-Reducing Ability between Campylobacteria Isolated from Deep-Sea Hydrothermal Environments

(A) ML tree based on 127 conserved protein sequences with schematic of nos gene cluster. Numbers at nodes represent bootstrap values (%) (1,000 replicates). nosZ and its accessary genes are colored according to homology across the different species.

(B) Results of N2O-respiring cultivation test. +; positive, -; negative, NT; not tested.

(C) Maximum N2O-reducing rate of N2O-respiring-positive strains. Error bars represent standard errors (n = 3). Different small letters indicate significant differences assessed by Tukey's HSD test (p < 0.001).

Growth under N2O-respiring condition was observed in all strains belonging to the thermophilic genus Nitratiruptor. However, the other mesophilic strains (i.e., Sulfurimonas autotrophica, Sulfurovum lithotrophicum, Sulfurovum riftiae, Sulfurovum sp. NBC37-1, Nitratifractor salsuginis) showed no growth under the same condition, even though they had the nosZ gene on their genomes. Further evaluation of N2O consumption abilities of the N2O-respiring strains (i.e., Nitratiruptor tergarcus, Nitratiruptor sp. SB155-2, Nitratiruptor sp. EPR55-1) revealed that 4.3%–80% of initial headspace N2O was consumed along with an increase in cell numbers (Figure S1).

Multiple Alignment of NosZ Primary Structure and Phylogenetic Analysis Based on Genes within the nos Gene Cluster

Multiple alignment of NosZ primary structures that contained strain HRV44T and other clade I and clade II sequences showed that nosZ amino acid sequences of all campylobacterial strains conserved important structural motifs such as seven histidine ligands of CuZ center, two cysteine and three other ligands of CuA center, and the heme c-binding motif (Cys-Xaa-Xaa-Cys-His) at the C terminus (Figure S9). In addition, the five calcium-binding sites were conserved among most campylobacterial strains except for Sulfurimonas autotrophica, Nitratifractor salsuginis, and Nitratiruptor sp. SB155-2.

In order to understand the evolutionary relationship of NosZ and its accessary proteins, phylogenetic analyses were performed based on each gene within nos gene cluster. The strains belonging to the genera Nitratiruptor and Sulfurovum were respectively clustered according to their genus (Figure S10). Mesophilic Sulfurimonas autotrophica was often placed in the outermost Campylobacteria from deep-sea hydrothermal environments, which is contrary to its phylogenetic position inferred by genome sequences (Figure S7). The tree topologies were relatively conserved among all genes with the exception of nosC1, whose topology was consistent with both genome- and 16S rRNA gene-based phylogeny.

Discussion

Physiological and Genomic Characteristics of Strain HRV44T

The physiological characteristics of strain HRV44T indicated that the strain is a facultatively anaerobic chemolithoautotroph. 16S rRNA gene sequence analyses indicated that strain HRV44T was most closely related to Nitratiruptor sp. EPR55-1, although its physiological characteristics were more similar to that of Nitratiruptor tergarcus. Although it was difficult to determine whether strain HRV44T belongs to the genus Nitratiruptor based solely on 16S rRNA gene sequences, combination of AAI and genome-based phylogeny suggested that strain HRV44T represents a novel species of the genus Nitratiruptor.

Strain HRV44T showed a relatively narrow growth range when compared with phylogenetically related campylobacterial strains, implying a limited niche of strain HRV44T. The strain consumed N2O more than three times as fast as the mesophilic N2O-reducing campylobacteria (Mino et al., 2018). Although previous studies evaluated microbial N2O reduction under pH above 7.0 because low pH potentially inhibits NosZ activities in some bacterial species (Bergaust et al., 2012a; Domeignoz-Horta et al., 2016), strain HRV44T showed high N2O-reducing activity at around pH 6.0. This study is the first report on a microorganism possessing the N2O-reducing ability under high temperature (>50°C) and low pH condition. In addition, the doubling time of strain HRV44T under the N2O-respiring condition was shorter than those of Nitratiruptor tergarcus (2.0 h), Nitratiruptor sp. EPR55-1 (3.5 h) under their optimum conditions. Thus, strain HRV44T might adapt its metabolism that facilitates highly efficient utilization of N2O in limited habitats. We could not directly compare the N2O-reducing rate of strain HRV44T with those of other clade I and clade II bacteria since initial N2O concentration and culture conditions varied between studies. Additionally, cell numbers used for calculating cell-specific N2O consumption rate are possibly retrieved from different growth phase of growing cells among studies, which might lead to the misevaluation of N2O-reducing ability of clade I and clade II microorganisms. Evaluation of N2O-reducing rate at single-cell level and enzymatic characterizations of NosZ may allow for a fair comparison with other N2O-reducing microorganisms.

Two assembly approaches using PacBio, ONT, and Illumina reads clearly showed the presence of a plasmid of strain HRV44T. Although there are few studies on plasmids isolated from (hyper)thermophiles living in deep-sea hydrothermal vents (Lossouarn et al., 2015), there is only a report of the presence of plasmid in Campylobacteria from deep-sea hydrothermal environments. The difference of G + C content between the chromosome and the plasmid in strain HRV44T was smaller than that in other deep-sea thermophiles (Table S3), suggesting the long-term persistence of the plasmid (Lawrence and Ochman, 1998; Rocha and Danchin, 2002), but its role in host physiology is still unclear. In addition, several plasmid CDSs including tra genes showed homologies to genomes of Campylobacter iguaniorum (Gilbert et al., 2015) as well as of other terrestrial and deep-sea hydrothermal vent campylobacterial species. Genomic traits imply the evolutionary link between terrestrial nonpathogenic Campylobacteria and their deep-sea chemolithoautotrophic relatives as suggested in previous studies (Nakagawa et al., 2007; Pérez-Rodríguez et al., 2015). Further efforts in describing novel strains with acquisition of complete genome sequences and genome comparison might provide insights into the ecological role of plasmids in deep-sea hydrothermal environments and contribute to the understanding of evolutionary links among Campylobacteria.

IS within the 16S rRNA Gene of Strain HRV44T

Insertion sequences within 23S and 16S rRNA genes have been detected in some bacterial genera to date (Linton et al., 1994; Rainey et al., 1996; Selenska-Pobell and Döring, 1998; Pabbaraju et al., 2000) and are known to be excised during rRNA maturation processes (Pronk and Sanderson, 2001; Salman et al., 2012) by self-splicing caused by catalytic RNA (Salman et al., 2012) or RNase-III-mediated mechanism (Evguenieva-Hackenberg and Klug, 2000). Despite strain HRV44T having ISs within all three copies of the 16S rRNA genes, it shows robust growth under HNN medium, reaching 3.69×108 cells/mL after 24 h. Thus, ISs might be selectively neutral when present within 16S rRNA, as they are apparently not detrimental to growth as observed in Escherichia and Salmonella strains (Mattatall and Sanderson, 1998), but we could not evaluate the growth of ISs-deficient strain HRV44T here. Additionally, no ORFs found within ISs supports the rarity of ISs encoding ORFs in bacteria outside the several taxa (Brown et al., 2015). Although little is known about the biological role and evolutionary history of ISs in deep-sea hydrothermal environments, some positive influences of ISs have been reported in both eukaryotic and prokaryotic cells (Hsu et al., 1994; Cheng and Deutscher, 2003; Parenteau et al., 2019; Morgan et al., 2019). Insertion sequences therefore might be advantageous genomic traits for strain HRV44T. The taxonomic diversity of organisms possessing IS within rRNA genes is fundamental information toward understanding its evolutional and physiological functions; however, such microbes might be overlooked using meta 16S rRNA sequencing, a general method for the evaluation of bacterial and archaeal diversities (Brown et al., 2015). Metagenomic and cultivation approaches are likely to contribute toward revealing its ecophysiological advantages, coupled with enhanced success in discovery of microorganisms possessing ISs.

N2O-Reducing Ability among Campylobacteria

All Nitratiruptor spp. strains tested here grew under the N2O-respiring condition, suggesting that N2O-respiring metabolism is one of the common physiological traits in the genus Nitratiruptor. In addition to the highly conserved primary structure of the NosZ, organization of the nos gene cluster was remarkably conserved among nosZ-possessing Campylobacteria including the genus Nitratiruptor, as described in previous studies (Sanford et al., 2012; Mino et al., 2018). Phylogenetic analyses based on each gene within the nos gene cluster imply that nosC1 encoding the mono-heme cytochrome c and other genes have undergone respective evolutionary histories. These results from comparative genomics were not strong enough to explain the difference in N2O-reducing abilities demonstrated here. It is of interest that the significant difference in the rate of N2O reduction was observed among strains presenting similar genetic characteristics. W. succinogenes, a model microorganism for clade II NosZ (Kern and Simon, 2009), is known to employ Nss-type transcriptional regulatory proteins (Kern and Simon, 2016) to mediate the upregulation of NosZ. Members of Campylobacteria we studied are found to encode regulators of the Crp-Fnr superfamily on their genomes as well. However, their positions were different from W. succinogenes and highly diverse in genus. Further transcriptomic analyses might be useful in providing more insights into the mechanisms producing the difference in the rate of N2O reduction.

Incompatibility between genetic and physiological traits has been reported in nitrate reduction metabolism in campylobacterial species such as Sulfurimonas autotrophica, Nautilia profundicola, and Hydrogenimonas sp. BAL40 (Inagaki et al., 2003; Smith et al., 2008; Mino et al., 2018). In this study, mesophiles, i.e., Sulfurimonas autotrophica, Sulfurovum lithotrophicum, Sulfurovum riftiae, and Sulfurovum sp. NBC37-1, did not show N2O-respiring ability despite possessing the nos gene cluster on their genome. N2O-respiring metabolism among Campylobacteria is possibly influenced by environmental factors as clade I denitrifying bacteria regulate their N2O reductase in response to the presence of nitrate, nitrite, NO, and/or oxygen (Bergaust et al., 2012b; Bueno et al., 2012).

Biogeochemical Features of Strain HRV44T and N2O Emission from Deep-Sea Hydrothermal Environments

Denitrification has been recognized as a modular pathway that is mediated by microorganisms both possessing and lacking nosZ genes (Zumft, 1997; Jones et al., 2008; Graf et al., 2014). In addition to taxonomically diverse denitrifiers (Fortunato and Huber, 2016; Pjevac et al., 2018), microorganisms with other nitrogen metabolisms can contribute to N2O emission and mitigation in deep-sea hydrothermal environments. For example, thermophilic Campylobacteria such as Hydrogenimonas thermophila, Nautilia profundicola, Caminibacter mediatlanticus, (Voordeckers et al., 2005), Cetia pacifica (Grosche et al., 2015), and Lebetimonas natsushimae (Nagata et al., 2017) can indirectly relate to the denitrification process as DNRA microorganisms, causing decrease in nitrate, the first substrate of denitrification. The ability of strain HRV44T to utilize not only nitrate but N2O might be one of the niche partitioning strategies for environmental adaptation to deep-sea hydrothermal environments where many chemolithoautotrophs utilize nitrate as electron acceptors.

N2O emission from the environments is a gross result of the balance between its production and consumption. Although redox gradient around deep-sea hydrothermal vent is conductive to microbial denitrification and nitrification, its contribution to N2O level at deep sea is quite low (Bange and Andreae, 1999). Our results imply members of the genus Nitratiruptor can significantly contribute to the capacity of N2O mitigation of the environments and the magnitude of contribution on the total N2O sink might differ at species or strain levels and be influenced by environmental factors. In addition, not all denitrifying microorganisms might account for the N2O emission as members of Nitratiruptor can utilize exogenous N2O in this study. In deep-sea hydrothermal environments, denitrifying mesophiles and nitrifying microorganisms likely appeared to be the biological sources of excess N2O observed in previous biogeochemical studies (Lilley et al., 1982; Kawagucci et al., 2010). Further comprehensive omics and physiological analysis of deep-sea hydrothermal vent microorganisms will lead us to evaluate the accurate N2O flux in these environments and their contribution to mediate climate change.

Description of Nitratiruptor labii sp. nov.

Nitratiruptor labii (la.bi'i. L. neut. gen. n. labii, flange structure from which the strain was isolated).

Weak motile cells with polar flagellum, approximately 2.0 μm long and 0.5 μm wide were frequently observed. Aggregated cells were often observed. The temperature range for growth is 45°C–60°C (optimum 53°C). The pH range was 5.4–6.4 (optimum 6.0). The NaCl requirement is 2.0%–4.0% (w/v) (optimum 2.5% (w/v)). Strictly chemolithoautotrophic growth occurs with molecular hydrogen as a sole electron donor and with nitrate, nitrous oxide, oxygen (up to 1.0%), elemental sulfur as a sole electron accepter. Nitrous oxide was reduced with construction of the pink colored pericle-like structure at gas liquid interface in HNN medium. Elemental sulfur or sulfate was used as a sole sulfur source. Nitrate or ammonium was utilized as a sole nitrogen source. The size of the genome, including megaplasmid and the calculated G + C content was 2,092,987 bp and 33.4%, respectively.

The type strain, HRV44T (=JCM 34002 = DSM 111345), was isolated from a deep-sea hydrothermal flange structure at the Iheya North hydrothermal field in the Mid-Okinawa Trough, Japan.

Limitations of the Study

This study reports the first evidence of Nitratiruptor labii sp. nov, isolated from the deep-sea hydrothermal vent in the Mid-Okinawa Trough, as a N2O-reducing thermophile coupling with H2 as an electron donor, and describes N2O-reducing ability of different campylobacterial strains from deep-sea hydrothermal environments. It should be noted that N2O-reducing rate at single-cell level has not been elucidated in this study because HRV44T forms the pericle-like structure under N2O-reducing condition, that might affect to the ability to reduce N2O. Furthermore, no data on in situ N2O-reducing rate of campylobacterial members was available. These limitations could be addressed by the use of comprehensive analysis including enzymatic analysis of NosZ, in situ transcriptome and measurement of N2O reduction in deep-sea hydrothermal environments.

Resource Availability

Lead Contact

Further information and requests for resources should be directed to and will be fulfilled by the Lead Contact, Sayaka Mino (sayaka.mino@fish.hokudai.ac.jp).

Materials Availability

This study did not generate new unique reagents.

Data and Code Availability

This project has been deposited at DDBJ/EMBL/GenBank under the BioProject PRJDB9307. Sequences for complete genome and 16S rRNA gene of strain HRV44T are available with DDBJ/EMBL/GenBank AP022826, AP0228267, and LC528620, respectively. The 16S rRNA sequences of strains No.9, No.33, No.34, No.37, No.41 are available with DDBJ/EMBL/GenBank LC533966 to LC533970, respectively.

Methods

All methods can be found in the accompanying Transparent Methods supplemental file.