Comparative genome sequence analysis of several species in the genus Tepidimonas and the description of a novel species Tepidimonas charontis sp. nov.

We performed high-quality genome sequencing of eight strains of the species of the genus Tepidimonas and examined the genomes of closely related strains from the databases to understand why Tepidimonas taiwanensis is the only strain of this genus that utilizes glucose and fructose for growth. We found that the assimilation of these hexoses by T. taiwanensis was due to the presence of two transporters that are absent in all other genomes of strains of members of the genus Tepidimonas examined. Some strains lack genes coding for glucokinase, but the Embden–Meyerhof–Parnas pathway appears to be otherwise complete. The pentose phosphate pathway has a complete set of genes, but genes of the Entner–Doudoroff pathway were not identified in the genomes of any of the strains examined. Genome analysis using average nucleotide identity (ANIb), digital DNA–DNA hybridization (dDDH), average amino acid identity (AAI) and phylogenetic analysis of 400 conserved genes was performed to assess the taxonomic classification of the organisms. Two isolates of the genus Tepidimonas from the hot spring at São Pedro do Sul, Portugal, designated SPSP-6T and SPSPC-18 were also examined in this study. These organisms are mixotrophic, have an optimum growth temperature of about 50 ºC, utilize several organic acids and amino acids for growth but do not grow on sugars. Distinctive phenotypic, 16S rRNA gene sequence and genomic characteristics of strains SPSP-6T and SPSPC-18 lead us to propose a novel species based on strain SPSP-6T for which we recommend the name Tepidimonas charontis sp. nov. (=CECT 9683T=LMG 30884T).

With the exception of T. taiwanensis, none of the strains of this genus grow in a minimal medium with glucose and fructose as sole carbon and energy source. The type strains of all species of this genus assimilate individual organic acids and amino acids for growth. Moreover, the strains examined oxidize thiosulfate in the presence of an organic carbon source, indicating that the strains are mixotrophic.
Chemoorganotrophic and mixotrophic bacteria that do not utilize sugars for growth are not rare; these organisms may lack specific sugar transporters or enzymes involved in the Embden-Meyerhof-Parnas, the Entner-Doudoroff or the pentose phosphate pathways. With the objective of understanding the conundrum that only one type strain of this genus is capable of growing on hexoses, we performed highquality draft genome sequencing of eight type strains of T. ignava, T. aquatica, T. fonticaldi, T. taiwanensis, T. thermarum, T. sediminis, T. alkaliphilus and the type strain of one isolate of the genus Tepidimonas from the hot spring at São Pedro do Sul, Portugal, designated SPSP-6 T . We also scrutinized two genome sequences of strains closely related to the type strain of T. taiwanensis I1-1 T , namely strains VT154-175 and MB2 as well as a strain closely related to T. fonticaldi designated PL17 [8,9]. We also propose that strain SPSP-6 T represents a novel species for which we recommend the name Tepidimonas charontis sp. nov.
The growth temperature ranges of SPSP-6 T and SPSPC-18 were examined with 5 °C increments between 20 and 65 °C by measuring the turbidity at 610 nm of cultures in liquid Degryse medium as described previously. The pH range for growth was examined at 50 °C in the same medium by using 50 mM MES, HEPES, TAPS and CAPSO over a pH range of 6.0 to 10.0 with 0.5 unit increments, in a rotary water-bath shaker. Growth with added salt, 0.5 and 1 % (w/v) NaCl, was determined in liquid medium. Catalase and oxidase activities and nitrate reduction were examined as described previously [12].
Single-carbon source assimilation tests were performed in a minimal medium composed of Degryse medium 162 basal salts containing filter-sterilized single carbon sources (2.0 g l −1 ), ammonium sulfate (0.5 g l −1 ) and a vitamin and nucleotide solution at a final concentration of 40 µg l −1 [4]. Growth of the strains on single carbon sources was examined by measuring the turbidity of cultures (at 610 nm) in 20 ml screw-capped tubes containing 10 ml medium for up to 7 days.
Cultures for fatty acid analysis were grown on R2A and Degryse medium 162 at 50 °C for 24 h. Fatty acid methyl esters (FAMEs) were obtained from fresh wet biomass, separated, identified and quantified with the standard MIS Library Generation Software, version 6.0, aerobe TSBA method (Microbial ID, MIDI) as described previously [16].  [17]. The purity of the DNA was verified by 1 % agarose gel electrophoresis. DNA quantity was measured by fluorescence in an Invitrogen Qubit 2.0 fluorometer (Thermo Fisher Scientific). The DNA was prepared for genome sequencing using the Nextera XT DNA Library Preparation Kit (Illumina). Bacterial genomes were sequenced on the MiSeq (Illumina) with paired-end (PE) 2×300 bp reads. The draft genomes of strains MB2 (GCF_001481285.1), VT154-175 (GCF_000807215.1) and PL17 (GCF_001675355.1) of members of the genus Tepidimonas were obtained from public databases.
Multiple sequence alignments were performed using muscle [27]. Phylogenetic trees were reconstructed with the neighborjoining (NJ) and maximum-likelihood (ML) algorithms using mega (version X) [28]. For the NJ and ML algorithms, genetic distances were calculated with the Jukes-Cantor model [29]. Bootstrap analysis based on 1000 replicates was used to evaluate the resulting tree topologies.
Pairwise average nucleotide identity based on blast (ANIb) was analyzed with JSpecies [30]. Digital DNA-DNA hybridization (dDDH) was determined with the Genome-to-Genome distance Calculator [31]. The amino acid identity (AAI) phylogenetic tree based on 400 universally conserved protein sequences was produced with PhyloPhlAn [32] to provide additional information on the relationships between members of the genus Tepidimonas.
The assembled genomes of the strains of members of the genus Tepidimonas ranged from 2465 kbp for T. alkaliphilus strain YIM 72238 T to 3009 kbp for strain T. fonticaldi AT-A2 T ( Table 1). The DNA G+C content of genomes ranged from 66.63 % for strain SPSP-6 T to 71.83 % for T. sediminis YIM 72259 T . The completeness of the genomes examined ranged from 98.91 % for the draft genomes of strains VT154-175 and AA-1 T to 100 % for the draft genome of T. fonticaldi AT-A2 T . The genomes of strains of members of the genus Tepidimonas had a variable number of rRNA genes ranging from three in T. alkaliphilus YIM 72238 T to twelve in the genome of strain VT154-175.
Genes coding for enzymes involved in the hydrolysis of starch, cyclodextrin and pullulan, namely alpha-amylase (EC 3.2.1.1), beta-amylase (EC 3.2.1.2), pullulanase (EC 3.2.1.41) and cyclomaltodextrinase (EC 2.4.1.19), were not identified in the genomes of any strains of members of the genus Tepidimonas. Therefore, it should not be possible for these organisms to obtain glucose or maltose from starch that could be taken up in pure culture. The type strain of T. taiwanensis has been reported to hydrolyze starch [3], while another study has reported that starch was not hydrolyzed by the same organism, corroborating the absence of starch hydrolyzing-enzymes from the genome analyses [4].
The ability of the type strain of T. taiwanensis to grow on glucose and fructose has been reproduced in laboratories that examined these phenotypic characteristics [4,6]. Likewise, the inability of the other species of this genus to grow on hexoses is also well attested. The genome sequences of the organisms used in this study clarified the likely reasons why the type strain of T. taiwanensis is able to use glucose and fructose while the other strains are not (Table S1, available in the online version of this article). The genome analysis indicated that glucose and fructose transporters only occur in the type strain of T. taiwanensis, as well as strains MB2 and VT154-175, where putative ABC glucose/mannose (gtsABCD) and   Although the type strain of T. taiwanensis grows on glucose and fructose, this strain, like all strains of this genus, does not grow on any other carbohydrates examined, such as mannose, galactose, trehalose, maltose, sucrose, ribose, l-arabinose, xylose or polyol (Table S1). We were unable to identify genes in any strains of members of the genus Tepidimonas that could channel these carbohydrates to the Emden-MeyerhofParnas or the pentose phosphate pathways. Experimental nitrate reduction to nitrite has been observed for the type strains of T. aquatica, T. fonticaldi, T. thermarum and T. taiwanensis but not or the type strain of T. ignava and strain SPSP-6 T [5]. The strains of the members of the genus Tepidimonas have variable genes involved in nitrogen metabolism (Table S2). For example, the type strain of T. fonticaldi possesses the most complete set of genes of the species of this genus, being predicted to be capable of reducing nitrate to nitrous oxide via the products of norB (nitric oxide reductase, large subunit) and norC (nitric oxide reductase, small subunit). The other strains, including the closely related strain PL17, appear to lack genes norB and norC. Genes coding for nitrate/nitrite transporters nasA/narK and the nitrate reductase complex narGHIJ were identified in the genomes of T. aquatica CLN-1 T , T. fonticaldi AT-A2 T , strain PL17, T. taiwanensis I1-1 T , strain VT154-175 and T. thermarum AA-1 T , but not in T. alkaliphilus YIM 72238 T , strain SPSP-6 T and strain MB2. T. ignava SPS-1037 T and T. sediminis YIM 72259 T have genes coding for the nitrate/nitrite transporters nasA/narK but the nitrate reductase complex narGHIJ was not identified in the genome sequences. Nitrate did not appear to be reduced by T. thermarum AA-1 T experimentally in one study but his strain has been reported to reduce nitrate in another study [4,5]. However, the genome predicts that nitrate should be reduced to nitrite because this organism possesses narGHIJ. The only gene involved in the reduction of nitrate identified in SPSP-6 T and T. alkaliphilus YIM 72238 T was NirB (nitrite reductase, NADH-dependent large subunit) (Tables S1 and S2).
With the exception of strains PL17, MB2 and VT154-175 whose phenotypic characteristics are not available, and the type strains of T. sediminis and T. alkaliphilus where thiosulfate oxidation was not examined, all other strains of members of the genus Tepidimonas oxidize thiosulfate to sulfate experimentally. However, all genomes predict that thiosulfate is oxidized to sulfate via the sox pathway, namely the soxXABCDYZ genes.
The three subunit orthologs of the tripartite ATP-independent periplasmic transporter (TRAP) that transport the C 4dicarboxylates malate/fumarate (DctM, DctP and DctQ) were identified in T. aquatica CLN-1 T , T. ignava SPS-1037 T , T. alkaliphilus YIM 72238 T and T. taiwanensis I1-1 T . These organisms use malate and fumarate as single carbon sources [33]. The other type strains appear to have only the genes for the DctM and DctP components or the DctM component alone. The type strain of T. thermarum and strain SPSP-6 T do not grow on malate or fumarate. The type strain of T. fonticaldi does not grow on malate but growth on fumarate was not tested, while the type strains of T. sediminis and T. alkaliphilus were not tested for the utilization of malate or fumarate (Table S1).
We only identified the genes for the tripartate tricarboxylate ABC system transporter for citrate composed of three subunits (TctA, TctB and TctC) in the genome of the type strain of T. taiwanensis and strain MB2. Strain VT154-175 has two components (TctB and TctC), while the other strains of members of the genus Tepidimonas appear to have only one component [34]. The type strain of T. taiwanensis is the only organism, among those examined that uses citrate for growth (Table S1).
The pairwise 16S rRNA gene sequence similarity determined between strains SPSP-6 T and SPSC-18 was 100 %. These isolates shared pairwise 16S rRNA gene sequence similarities of 98.07, 98.38 and 98.44 % with the type strains of T. ignava, T. aquatica and T. taiwanensis. (Fig. 1, Table S3). The sequence similarity between T. fonticaldi AT-A2 T and strain PL17 was 99.86 %, indicating an extremely close relationship between the two organisms. A close relationship of T. fonticaldi AT-A2 T with 'T. arfidensis' of 99.58 % was also noted. The 16S rRNA gene sequence analysis also indicated T. taiwanensis I1-1 T to be closely related to strains MB2 and VT154-175 with sequence similarities of 99.93 and 99.58 %, respectively (Table S3). The phylogenetic results of 16S rRNA gene analysis indicate that SPSP-6 T is located within a cluster comprising the type strains of T. ignava, T. aquatica, T. taiwanensis, T. alkaliphilus and T. sediminis. However, SPSP-6 T is most closely related to the type strains of T. ignava, T. aquatica and T. taiwanensis (Figs 1 and  S1). The phylogenetic results based on 400 conserved genes were consistent with the 16S rRNA gene sequence results (Fig. 2), corroborating the phylogenetic relations observed for SPSP-6 T within the genus Tepidimonas. The phylogenetic analysis of 400 conserved genes also showed T. taiwanensis I1-1 T , strains MB2 and VT154-175 to be very closely related to each other indicating that the three strains represent one species. Strain PL17 and T. fonticaldi AT-A2 T should also probably be regarded as representing one species.
Additionally, the results of the genome comparisons, namely ANIb, AAI and dDDH corroborated the results of the 16S rRNA sequence analysis regarding the distinct species nature of the lineages (Tables 2, S4 and S5). Based on a threshold value of 95-96 % for species delineation [35,36], the ANIb values indicate that SPSP-6 T , with ANIb values of about 80 % with other species constitutes a separate species of the genus Tepidimonas. The ANIb value for T. fonticaldi AT-A2 T and strain PL17 of 97.46 % indicates a very close relationship between these strains. The same is true of T. taiwanensis I1-1 T with strains MB2 and VT154-175 that share ANIb values of 97.53 to 98.48 %. In addition to the genomic values, these are the only currently known strains that have genes for the transport of glucose and fructose, and utilize these hexoses for growth. On the basis of the ANIb values, the type strains of T. aquatica and T. ignava are closely related (93.22 %). Nevertheless, this value is below the threshold value for delineation of species ( Table 2).
The AAI values, generally taken to have a cut off value of around 70 % to delineate genera [37], indicate that the strains of members of the genus Tepidimonas, including strain SPSP-6 T , belong to one genus because of higher AAI values (Table  S4). The genome of Tepidicella xavieri has high AAI values with the species of the genus Tepidimonas (67.59 to 70.85 %) indicating that the organisms of the two genera are related, but within a transitional zone of AAI values, making it difficult to have an opinion on the classification of the sole strain of Tepidicella xavieri from the genomic analysis. However, the phylogeny obtained for the 16S rRNA gene and for 400 conserved genes sequence analysis indicates that Tepidicella xavieri is not closely related to the species of the genus Tepidimonas [38,39].
The genomic-based dDDH estimates have values of 79.8 to 87.20 % between T. taiwanensis strains I1-1 T , strains MB2 and VT154-175 (Table S5). These results are above the reference dDDH value of about 70 % to delineate separate species proposed by Stackebrandt et al. [35] leading us to the opinion that the three strains represent members of the species T. taiwanensis. Moreover, these three strains possess an ABC glucose/mannose transporter that all other strains seem to lack. The high dDDH value of 80.1 % between T. fonticaldi AT-A2 T and strain PL17 also supports the view that these two strains represent the same species. The low dDDH values between other organisms of the genus Tepidimonas, notably strain SPSP-6 T sharing no more than 25.1 %, support the view that the organisms examined represent distinct species of the genus Tepidimonas (Table S5). A small number of phenotypic characteristics of the type strains of the species of the genus Tepidimonas distinguish the strains from each other (Table S1). The fatty acid compositions of the strains were obtained after the organisms were grown in R2A and Degryse medium 162 agar plates for 24 h at 50 °C. These results indicated that the medium influenced the fatty acid composition to a large extent (Tables S6 and S7). For example, C 17 : 0 cyclo was not detected in T. ignava SPS-1037 T and T. sediminis YIM 72259 T grown on Degryse medium 162 but reached levels of 6.7 and 7.3 %, respectively, when they were grown on R2A agar. Nevertheless, the major fatty acids of all strains were C 16 : 0 , summed feature 3 (probably C 16 : 1 ω6c and/or C 16 : 1 ω7c) and in some cases, C 17 : 0 cyclo and summed feature 8 (C 18 : 1 ω6c and/or C 18 : 1 ω6c). However, there were differences in the concentrations of these fatty acids among the type strains. For example, the combination of C 17 : 0 cyclo and C 17 : 0 , after growth of the organisms on R2A and Degryse medium 162, can distinguish strains SPSP-6 T and SPSPC-18 from the other strains of species of this genus.
Many of the validly described prokaryotic species are only based on a few distinctive phenotypic characteristics that could represent interspecific diversity, since these novel organisms are proposed on the basis of the description of one strain alone. The species of the genus Tepidimonas are an example of these considerations since strains SPSP-6 T
The novel species of the genus Tepidimonas represented by strain SPSP-6 T has very few phenotypic and chemotaxonomic characteristics that distinguish this strain from the type strains of the other species. The single carbon source assimilations, with the exception of T. taiwanensis, sulfur oxidation and the fatty acid composition are similar in all type strains of species of the genus Tepidimonas (Tables S1, S6 and S7). However, some phenotypic and chemotaxonomic characteristics indicate that the organism represents a novel species; the fatty acid composition indicated that SPSP-6 T and SPSPC-18 can be distinguished from other strains of members of the genus Tepidimonas by combining the relative proportions of C 17 : 0 cyclo and C 17 : 0 . Except for strains SPSP-6 T and T. alkaliphilus YIM 72238 T , which possess only homologues for nirB, all other strains possess genes coding for proteins involved in the reduction of nitrate to nitrite. Considering the phenotypic, genomic and the phylogenetic analysis based on 16S rRNA gene sequence and on 400 conserved genes sequences clearly confirms that strain SPSP-6 T represents a species level taxon, leading us to propose the name Tepidimonas charontis sp. nov.

DESCRIPTION Of Tepidimonas charonTis SP. NOv.
Tepidimonas charontis ( cha. ron'tis. L. gen. n. charontis of Charon, the boatman who required payment to ferry the ancient dead Greeks across the Rivers Styx and Acheron to Hades.
Forms short rod-shaped cells 0.5-0.8 µm in width and 1.0-2.0 µm in length. Endospores are not formed. The cells Gram-stain-negative and are motile by means of one polar flagellum. Colonies on Degryse medium 162 are not pigmented and are 1 to 2 mm in diameter after 48 h of growth. The optimum growth temperature is about 50 °C; growth occurs in the range of 25-60 °C. The optimum pH is between 7.5 and 9.0; the pH range for growth is pH 6.5-9.5. Mixotrophic. Aerobic. Nitrate is not reduced to nitrite. Oxidaseand catalase-positive. The major fatty acids are C 16 : 0 and C 16 : 1 ω6c and/or C 16 : 1 ω7c. Yeast extract or growth factors are required for growth. Thiosulfate is oxidized to sulfate. Several organic acids and amino acids are utilized for growth, namely succinate, lactate, pyruvate, acetate, glutamate, aspartate, l-alanine, l-asparagine, l-lysine, l-glutamine, l-isoleucine and l-ornithine, but the strains do not utilize carbohydrates or polyols.
The type strain SPSP-6 T (=CECT 9683=LMG 30884) was isolated from a hot spring at São Pedro do Sul in Central Portugal. The genomic DNA G+C content of the type strain is 66.63 % (determined by genome sequencing). Strain SPSPC-18 (=CECT 9684=LMG 30885) is an additional strain of this species.
GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequence of strains SPSP-6 T and SPSPC-18 are MH590702 and MH590703, respectively. The draft genome sequence of SPSP-6 T (VJON00000000) has been deposited in GenBank/EMBL/DDBJ.