Nitrogen-fixing Ability and Nitrogen Fixation-related Genes of Thermophilic Fermentative Bacteria in the Genus Caldicellulosiruptor

Fermentative nitrogen-fixing bacteria have not yet been examined in detail in thermal environments. In the present study, we isolated the thermophilic fermentative bacterium, strain YA01 from a hot spring. This strain grew at temperatures up to 78°C. A phylogenetic analysis based on its 16S rRNA gene sequence indicated that strain YA01 belonged to the genus Caldicellulosiruptor, which are fermentative bacteria in the phylum Firmicutes, with 97.7–98.0% sequence identity to its closest relatives. Strain YA01 clearly exhibited N2-dependent growth at 70°C. We also confirmed N2-dependent growth in the relatives of strain YA01, Caldicellulosiruptor hydrothermalis 108 and Caldicellulosiruptor kronotskyensis 2002. The nitrogenase activities of these three strains were examined using the acetylene reduction assay. Similar activities were detected for all tested strains, and were slightly suppressed by the addition of ammonium. A genome analysis revealed that strain YA01, as well as other Caldicellulosiruptor, possessed a gene set for nitrogen fixation, but lacked the nifN gene, which encodes a nitrogenase iron-molybdenum cofactor biosynthesis protein that is commonly detected in nitrogen-fixing bacteria. The amino acid sequences of nitrogenase encoded by nifH, nifD, and nifK shared 92–98% similarity in Caldicellulosiruptor. A phylogenetic tree of concatenated NifHDK sequences showed that NifHDK of Caldicellulosiruptor was in the deepest clade. To the best of our knowledge, this is the first study to demonstrate the nitrogen-fixing ability of fermentative bacteria at 70°C. Caldicellulosiruptor may have retained an ancient nitrogen-fixing enzyme system.

Nitrogen is one of the most abundant and important elements for life. Nitrogen-fixing microorganisms play significant roles in converting atmospheric N 2 gas to ammonia in ecosystems. According to a review by Postgate (1998), the first nitrogen-fixing bacteria or diazotrophs were discovered by Winogradsky in 1893. Nitrogen-fixing microorganisms have been reported in 16 phyla in Bacteria and 1 phylum in Archaea from various environments (Mus et al., 2019). Aerobic free living and symbiotic Proteobacteria and phototrophs have been widely reported (Martinez-Romero, 2006;Flores et al., 2015;Wasai and Minamisawa, 2018), and the nitrogen-fixing ability of anaerobic respiratory bacteria, such as Anaeromyxobacter in soil, has recently been attracting increasing attention (Masuda et al., 2020). In 1988, fermentative nitrogen-fixing bacteria were reported in the genus Clostridium in Firmicutes (Leschine et al., 1988); however, limited information is currently available on fermentative nitrogen-fixing bacteria. Nitrogen fixation by fermentative metabolism utilizing polysaccharides (e.g., cellulose) has been suggested to play an important role in nitrogen cycles in soil and animal intestines (Monserrate et al., 2001;Yamada et al., 2007).
Nitrogen fixation is achieved by multiple proteins encoded by nif genes (Raymond et al., 2004). Phylogenetic examinations indicated that nitrogenase genes originated in archaea and were horizontally transferred to bacteria (Boyd et al., 2011a). The nifH gene encoding the nitrogenase reductase subunit of nitrogenase is widely regarded as an indicator of the existence of diazotrophs (Zehr et al., 2003). The diversity and distribution of nifH genes have been analyzed in natural ecosystems, including thermal environments (Mehta et al., 2003;Hamilton et al., 2011;Zehr, 2011;König et al., 2016;Pajares and Bohannan, 2016;Nishihara et al., 2018c). The nitrogen-fixing methanogenic archaeon, Methanocaldococcus FS406-22, was isolated from a deepsea hyperthermal vent and its nitrogen-fixing ability was demonstrated at temperatures up to 92°C (Mehta and Baross, 2006). In 1986, nitrogen-fixing ability was reported in a thermophilic cellulose-degrading fermentative bacterium that grew at 60°C (Bogdahn and Kleiner, 1986a). Nishihara et al. (2018b) recently reported the nitrogenfixing ability of H 2 -oxidizing aerobic bacteria in the genus Hydrogenobacter sp. in the deeply branching phylum Aquificae at 70°C; this is the highest temperature observed for N 2 fixation in Bacteria. However, thermophilic isolates that grow at temperatures higher than 70°C are still limited.
In the present study, we isolated thermophilic fermentative bacteria using a combined nitrogen-poor medium from microbial communities developed at approximately 80°C in Nakabusa Hot Spring and characterized their nitrogen-fixing abilities and genetic features in comparisons with their closest relatives.

Isolation of bacteria under nitrogen-fixing conditions
Pale tan-colored microbial mats developed in hot spring water at 78.3°C were collected at Nakabusa Hot Springs (36° 23′ 20″ N 137° 44′ 22″ E), Nagano, Japan on January 8th, 2018. Hot spring water was slightly alkaline (pH 8.5-8.9) and contained 5.0-6.1 μmol L -1 of ammonia , but not nitrate or nitrite Kimura et al., 2010). Samples were immediately injected into the anoxic medium in glass vials (see below) with attempts to avoid oxygen contamination at the sampling site. The vials were stored in hot spring water at 60-75°C for 7 h during transportation to our laboratory and then incubated at 70°C.
Winogradsky's nitrogen-poor mineral medium (Tchan and New, 1984) was prepared with a slight modification and used for the cultivation and isolation of bacteria (L -1 ): 0.28 g K 2 HPO 4 , 0.053 g KH 2 PO 4 , 0.12 g MgSO 4 ·7H 2 O, 0.125 g NaCl, 0.05 g yeast extract, 0.01 g CaCl 2 ·2H 2 O, 2.5 mg FeSO 4 ·7H 2 O, 2.5 mg MnSO 4 ·5H 2 O, 2.5 mg Na 2 MoO 4 ·2H 2 O, 2.5 g glucose, 2.5 g sucrose, and 2.5 g Napyruvate. The pH of the medium was adjusted to 7.5. Twenty milliliters of the medium was placed into a 70-mL glass vial. The vial was sealed with a butyl rubber stopper and aluminum cap, and then autoclaved after the gas phase had been replaced with N 2 . In total, 0.5 mL of the culture was repetitively sub-cultured every week in fresh medium. After 10 sub-cultivations, an isolate was obtained by the twice dilution-to-extinction technique. The single morphology of microbial cells was confirmed under a phase-contrast microscope (Axio Imager 2; Carl Zeiss).

16S rRNA gene sequence analysis
Bacterial cells were collected by centrifugation and total DNA was extracted according to a method reported by Noll et al. (2005). A DNA fragment of the 16S rRNA gene was PCR-amplified using the 27F and 1492R primers (Lane et al., 1985;Lane, 1991), and amplified DNA after purification by the LaboPass PCR purification Kit (CosmoGenetech) was directly sequenced using BigDye terminator kit v3.1 on an ABI3130 Genetic Analyzer (Applied Biosystems). Sequences were compared using the BLAST program (Altschul et al., 1997) with those available in the DDBJ/EMBL/ GenBank databases.

Growth capability in nitrogen-poor media
Modified Winogradsky's nitrogen-poor mineral medium (described above) was used to assess N 2 -dependent growth. Ten milliliters of medium was prepared in 32-mL glass test tubes sealed with butyl rubber stoppers and screw caps and the gas phase of the culture tube was filled with N 2 or argon (Ar) gas. In total, 0.5 mL of bacterial cultures pre-cultivated in nitrogen-poor medium were inoculated into fresh nitrogen-poor medium. To test the growth capability of C. bescii DSM 6725, a pre-cultivation was conducted using medium supplemented with 2 mmol L -1 of NH 4 Cl. Growth in the culture was assessed by measurements of optical density (OD) at 660 nm (miniphoto 518R; Taitec). Cultivation medium containing 2 mmol L -1 NH 4 Cl was also used to compare N 2dependent growth with growth on ammonium.

Nitrogenase activity by the acetylene reduction assay
Nitrogenase activity was detected using the acetylene reduction assay method (Leschine et al., 1988). In total, 0.5 mL of bacterial pre-cultures in modified Winogradsky's nitrogen-poor mineral medium was inoculated into 10 mL of the same medium in 25-mL glass vials and cultivated under a N 2 gas atmosphere. At the exponential growth phase, a portion (0.5 mL) of the culture solution was removed and mixed with 0.05 mL of 10% Formalin Neutral Buffer Solution (pH 7.4-7.5, Fujifilm Wako Pure Chemical) to fix cells for the cell number count. The gas phase of culture vials was then replaced with N 2 gas and 1.5 mL of 99.9999% acetylene gas was injected into each vial. Vials were incubated at 70°C and, after a 24-h incubation, 1 mL of 37% neutralized formaldehyde was added to stop the reaction. The production of ethylene by the reduction of acetylene was quantified using a GC-2014 gas chromatograph equipped with a flame ionization detector (Shimadzu) and 80/100 Porapak T (GL Science) column. Analysis conditions were as follows; carrier gas, N 2 gas; column temperature, 70°C; injection temperature, 100°C; detector temperature, 100°C. Fresh medium containing no bacterial cells was prepared in the vial as a negative control to confirm abiotic ethylene production under the same conditions.

Nucleotide sequence accession number
The 16S rRNA gene sequence was deposited in the DDBJ/ EMBL/GenBank databases with the accession number LC603168. The accession numbers of the genomic sequences of strain YA01 were AP024480 (chromosome) and AP024481 and AP024482 (two plasmids).

Bacterial isolate from the hot spring under anaerobic nitrogen-fixing conditions
Pale tan-colored microbial mats collected at 78.3°C from Nakabusa Hot Spring were directly inoculated into glass vials with modified Winogradsky's nitrogen-poor mineral medium and anaerobically incubated at 70°C. After several sub-cultivations at one-week intervals, a stable enrichment culture was obtained. A pure culture containing cells of a single morphotype, i.e., short rods (Fig. S1), was obtained by dilution-to-extinction and the isolate was designated as strain YA01. Strain YA01 grew at temperatures up to 78°C. The 16S rRNA gene sequence of strain YA01 (1,474 bp) showed 98.0, 97.7, and 97.7% identities to those of its closest relatives, C. hydrothermalis 108, C. bescii DSM 6725, and C. kronotskyensis 2002, respectively. This result indicated that strain YA01 was a species of the genus Caldicellulosiruptor.
Nitrogen-fixation related genes in Caldicellulosiruptor DNBSEQ-G400 and GridION runs resulted in the generation of approximately 12,782,840 reads with a total of 1,917 Mbp and 155,919 reads with a total of 255 Mbp, respectively. The complete genome of strain YA01 consisted of a single chromosome with a length of 2,592,764 bp and two plasmids with lengths of 3,514 and 1,547 bp. The G+C content of the genome was 34.8%. Coding potential predictions identified 2,412 protein-coding genes, three rRNA operons, and 47 tRNA genes. Three 16S rRNA genes had the same sequence, which was identical to the 16S rRNA gene sequence amplified by PCR. Average nucleotide identity (ANI) between strain YA01 and its close relatives (C. hydrothermalis 108, C. bescii DSM 6725, and C. kronotskyensis 2002) calculated using an ANI calculator (http://enve-omics.ce.gatech.edu/ani/) (Rodriguez-R, L.M., and Konstantinidis, K.T. 2016 The enveomics collection: a toolbox for specialized analyses of microbial genomes and metagenomes. PeerJ Preprints 4: e1900v1) ranged between 90.31 and 91.10%.
The amino acid sequences of the nitrogenase structural proteins were similar among the genus (NifH, 98.37±0.41%; NifD, 96.58±1.54%; NifK, 92.05±5.26%). A concatenated NifHDK phylogenetic tree was constructed for all Caldicellulosiruptor species possessing nitrogen fixationrelated genes (Fig. 2). Strain YA01 clustered with all other members of the genus Caldicellulosiruptor within the cluster Nif-C (Fig. 2). The NifHDK sequences of Caldicellulosiruptor formed a monophyletic lineage, were placed in the deepest clade in the branch of the Anf/Vinf/ Nif-D/Nif-C/Unknown lineage (Boyd and Peters, 2013; Poudel et al., 2018;Garcia et al., 2020), and were distantly related to those in other thermophilic bacteria, such as Thermoanaerobacterium thermosaccharolyticum (Nif-A lineage) and Hydrogenobacter sp. (Nif-B lineage) (Boyd and Peters, 2013). The thermophilic features of NifHDK did not appear to correlate with the primary structure.

Growth capability under nitrogen-fixing conditions
Strain YA01 and its three relatives, C. hydrothermalis 108, C. bescii DSM 6275, and C. kronotskyensis 2002, were cultivated in nitrogen-poor medium and ammoniumcontaining medium under the N 2 or argon (Ar) gas phase (Fig. 3). C. bescii, which did not possess nitrogen-fixing genes, did not grow in nitrogen compound-free medium (Fig. 3D). Strain YA01, C. hydrothermalis 108, and C. kronotskyensis 2002 showed marked increases in OD in nitrogen compound-free medium under N 2 gas, but not under the Ar gas phase (Fig. 3A, B, and C). These three strains reached the stationary phase within 2 to 3 days and final OD were 0.05 to 0.14 under N 2 -fixing conditions, corresponding to 1.06×10 7 to 2.27×10 7 cells mL -1 . In ammonium-containing medium, the growth of all these strains was faster than in the absence of ammonium and final OD were 0.20 to 0.35. In C. hydrothermalis 108 and C. kronotskyensis 2002, growth yields in ammoniumcontaining medium were slightly higher under N 2 gas than under Ar gas.

Nitrogenase activity
To test nitrogenase activity, strain YA01 and its relatives C. hydrothermalis 108 and C. kronotskyensis 2002 were cultivated to the exponential growth phase in nitrogen-poor medium and ammonium-containing medium under the N 2 gas phase. Acetylene was injected into the vials and incubated at 70°C. The results of ethylene production after a 24-h incubation are summarized in Table 1. Ethylene production was observed in all tested strains, even in the presence of ammonium. Strain YA01 showed the highest value among the three strains in the absence of ammonium. The amount of ethylene produced in the presence of ammonium for 24 h was lower than that in its absence in all strains. The suppressive effects of ammonium on C. kronotskyensis  were weak (approximately 60% of that in its absence). Acetylene-reducing activities were also observed under an incubation at 78°C for all strains: 9.50±5.05, 8.37±5.53, and 15.0±12.1 nmol C 2 H 4 10 6 cells -1 24 h -1 by strain YA01, C. hydrothermalis 108, and C. kronotskyensis 2002, respectively. Activities at 78°C were weaker than those at 70°C.

Discussion
In the present study, we isolated a bacterial strain by cultivation in nitrogen-poor medium from Nakabusa Hot Spring, Japan. The results of the phylogenetic analysis based on the 16S rRNA gene sequence suggested that this isolate, strain YA01, is a new species in the genus Caldicellulosiruptor and is closely related to C. hydrothermalis, C. bescii, and C. kronotskyensis. The results of the genomic analysis indicated that strain YA01 as well as C. hydrothermalis 108 and C. kronotskyensis 2002 possess a set of nitrogen fixationrelated genes (Fig. 1) and their NifHDK formed a monophyletic lineage in the deeply branching group of the NifHDK tree (Fig. 2). Growth capability with N 2 gas as the sole nitrogen source and acetylene-reducing activity were successfully demonstrated for the new isolate, C. hydrothermalis 108, and C. kronotskyensis 2002 ( Fig. 3 and Table 1). To the best of our knowledge, this is the first study to detect nitrogen-fixing ability in the genus Caldicellulosiruptor. The nitrogenase activities of bacteria were previously reported at temperatures up to 70°C by Nishihara et al. (2018b) in the chemolithoautotrophic bacteria, Hydrogenobacter sp. in the phylum Aquificae. The nitrogenase activities of Caldicellulosiruptor were detected at temperatures higher than 70°C, i.e., 78°C, which was the maximum growth temperature of strain YA01.
The nif gene operons of strain YA01 and its relatives, C. hydrothermalis 108 and C. kronotskyensis 2002 basically comprised nifHDKEB (Fig. 1). Commonly known nif gene operons contain the additional gene, nifN; however, a homologous gene to nifN was not identified in Caldicellulosiruptor. nifN encodes subunits of the tetrameric protein NifEN (2NifE 2NifN), which is required for the biosynthesis of the iron molybdenum co-factor of Motype nitrogenase (Hu et al., 2005(Hu et al., , 2008Corbett et al., 2006;Burén et al., 2020). Evolutionary studies based on molecular phylogram and comparative analyses of amino acid sequences suggested that nifN and nifK are paralogous genes that were derived through gene duplication (Raymond et al., 2004;Boyd et al., 2011b). Similar to Caldicellulosiruptor, the diazotrophic archaeon, Methanocaldococcus sp. FS406-22 also lacks nifN (Mehta and Baross, 2006). This finding indicates that the protein coded by nifK in these thermophiles performs the same function as NifN. Alterna-tively, NifE may work without an NifN subunit, as suggested by Garcia et al. (2020), because NifE in Caldicellulosiruptor showed low similarity with other known NifE (Fig. S2). The phylogenetic trees of NifHDK (Fig. 2) and NifE (Fig. S2) indicated that Caldicellulosiruptor has an ancient nitrogen-fixing enzyme system. As proposed in the phylogenetic study of nitrogen fixation-related genes by Garcia et al. (2020), Monitrogenase in the genus Caldicellulosiruptor may have emerged earlier and then evolved into modern nitrogenases in wide lineages of prokaryotes.
The nitrogenase activities of the Caldicellulosiruptor strains were not completely suppressed by the addition of ammonium (Table 1); however, the inhibition of nitrogenase activity by ammonium has been traditionally reported in most diazotrophic bacteria (Dixon and Kahn, 2004). In cellulolytic fermentative diazotrophic bacteria, Clostridium sp. in Firmicutes, acetylene-reducing activity decreased under the detection limit when ammonium was added (Bogdahn andKleiner, 1986a, 1986b). However, this activity was not suppressed by ammonium for the thermophilic relative, Clostridium thermocellum (now Hungateiclostridium thermocellum) (Bogdahn and Kleiner, 1986a;Tindall, 2019). Although the protein, NifA has been shown to regulate the expression of nif genes in nitrogen-fixing aerobes in Proteobacteria (Merrick, 1992), most anaerobic diazotrophs, including Caldicellulosiruptor and Clostridium, do not possess the nifA gene (Boyd et al., 2015). The evolution of Nif regulation systems from anaerobic to aerobic metabolism is still debatable (Boyd et al., 2015). Further transcriptional and enzymological studies are required to elucidate responses to ammonium in these thermophilic diazotrophs.
Caldicellulosiruptor are frequently detected from microbial mats in geothermal springs (Lee et al., 2018;Blumer-Schuette, 2020). Microbial mats are stratified communities of microorganisms with thicknesses of 3 to 5 mm and thermophilic microbial mats have been utilized as a model microbial community to investigate the development and maintenance of ecosystems (Taffs et al., 2009;Klatt et al., 2013;Kim et al., 2015;Lindemann et al., 2016;Bernstein et al., 2017;Haruta, 2020). Previous studies focused on primary production in communities in hot spring streams and reported a spatial and temporal distribution and the cooccurrence of carbon-fixing metabolism via oxygenic and anoxygenic photosynthesis, aerobic chemosynthesis (e.g., H 2 and sulfide oxidation), and anaerobic chemosynthesis (e.g., sulfur disproportionation) (Rothschild and Mancinelli, 1990;Kimura et al., 2010;Kojima et al., 2016;Tamazawa et al., 2016;Sharrar et al., 2017;Gutiérrez-Preciado et al., 2018;Kawai et al., 2019). Dinitrogen fixation is also required for community development in spring waters that are poor in nitrogen compounds Steunou et al., 2006;Kimura et al., 2010;Hamilton et al., 2011;Loiacono et al., 2012). However, possible thermophilic diazotrophs at temperatures higher than 70°C in terrestrial springs have not been clarified. The present results provide important insights into the development of microecosystems in thermal environments. Caldicellulosiruptor may utilize organic compounds derived from primary producers at the anoxic layer of microbial mats and provide ammonium to the communities. Caldicellulosiruptor possessing the ancient type of nitrogenase may play important roles in carbon and nitrogen cycles not only in modern thermal springs, but also in the early Earth.