Isolation and maintenance of strains
The strains J5BT and M52T were isolated from a microbial mat obtained from a hot spring in Japan (42° 57′ 53″ N 141° 09′ 47″ E). They were isolated into pure cultures under nitrate-reducing conditions, by using bicarbonate and thiosulfate as sole carbon source and electron donor (Watanabe et al, 2019; Ospino et al., 2019). The strains were maintained in the laboratory with a bicarbonate-buffered medium referred to as “S5 medium” (Kojima et al., 2017), supplemented with 10 mM nitrate. Purity and identity of the cultures were periodically checked by microscopic observation and direct sequencing of the 16S rRNA gene.
Cellular fatty acid analysis
For fatty acid analysis, cells of strains J5BT and M52T were grown at 45°C, in a medium consisting of the following constituents (l− 1): 1 g NaNO3, 0.5 g yeast extract, 0.5 g Casamino acids, 0.5 g disodium fumarate, 0.5 g disodium succinate, and 50 mg cyanocobalamin. Headspace of the culturing bottles was filled with N2 gas, and pH of the medium was adjusted to 7.0. Their cellular fatty acid profiles were obtained with the Sherlock Microbial Identification System (MIDI) version 6.0 (database; TSBA6). As for J5BT, cells grown in the S5 medium under oxic conditions were also subjected to the same analysis.
Effects of temperature on growth of strain M52T were examined by culturing at various temperatures (15, 18, 22, 25, 28, 30, 32, 35, 37, 40, 42, 45, 48, 50, 53, 55, 57 and 60°C), in the medium used for maintenance. The other tests for phenotypic characterization were all conducted at 45°C.
Chemolithoautotrophic growth under nitrate-reducing conditions was tested in the medium used for the maintenance, by replacing thiosulfate with one of electron donors listed below; elemental sulfur (0.5 g l− 1), sulfide (2 mM), tetrathionate (10 mM) and hydrogen gas (H2/N2/CO2 50:40:10 v/v/v; 200 kPa in total pressure). Utilization of organic substrates for heterotrophic growth was tested in 10 mM MOPS-NaOH buffer (pH 7.0), supplemented with NaNO3 (1 g l− 1), yeast extract (0.1 g l− 1), Casamino acids (0.1 g l− 1) and cyanocobalamin (50 mg l− 1). The medium was dispensed in closed culture bottles and the headspace was filled with N2 gas. One of the following organic substrates were added to the medium (mM); pyruvate (5), lactate (5), acetate (5), propionate (2.5), succinate (2.5), fumarate (2.5), malate (2.5), butyrate (2.5), benzoate (2.5), isobutyrate (2.5), methanol (5), ethanol (2.5), formate (5), citrate (5), glucose (2.5), xylose (2.5), phenol (2), o-cresol (1), m-cresol (1).
Effect of pH on growth of strain M52T was tested with pH-buffered media modified from the medium used for the fatty acid analysis. The media were buffered with 10 mM of MES, MOPS or Tricine, and adjusted to varying pH with NaOH. The tested pH were as follows: 5.4, 5.5, 5.6, 5.8, 5.9, 6.0, 6.1, 6.3, 6.5, 6.7, 6.9, 6.9 and 7.3 with MES; 6.4, 6.6, 6.8, 6.9, 7.1, 7.2, 7.3, 7.4, 7.6, 7.8 and 8.0 with MOPS; 7.0, 7.8, 8.0, 8.2, 8.4, 8.6, 8.8 and 9.0 with Tricine.
The complete genome sequences of strains J5BT and M52T were obtained in the previous studies (Watanabe et al, 2019; Ospino et al., 2019). As overall genome relatedness indices between them, values of average nucleotide identity (ANI) and digital DNA–DNA hybridization (dDDH) were calculated by using online tools as follows. The ANI was computed by ANI calculator available in EzBioCloud, based on the OrthoANIu algorithm (Yoon et al., 2017). The dDDH value was calculated using Genome-to-Genome Distance Calculator (GGDC) provided by DSMZ (Meier-Kolthoff et al., 2013), applying the formula 2.
The genome sequences were annotated with DFAST (Tanizawa et al., 2018), to identify protein coding sequences and RNA genes. Genes for sulfur oxidation were identified as described previously (Watanabe et al, 2019).
The full sequences of the 16S rRNA gene identified in the genomes were subjected to the blastn search at NCBI against the nucleotide collection (nr/nt) database. Phylogenetic analyses were conducted using the program MEGA version 11 (Tamura et al., 2021). The 16S rRNA gene sequences were aligned with reference sequences, using the MUSCLE algorithm. The references sequences were those of type strains of species with validly published names in the order Nitrosomonadales and the genus Rugosibacter. The best model for calculation of genetic distances was selected by using the model selection tool in MEGA, as the model with the lowest score of the Bayesian Information Criterion (BIC).