Introduction

The genus Anoxybacillus, which belongs to the phylum Firmicutes, was initially proposed by Pikuta et al. in 2000 [1], with Anoxybacillus pushchinoensis as the type species. Its description was subsequently amended by Pikuta et al. in 2003 [2]. At the time of writing, there are 24 species within this genus with validly published names, along with three species that have not been validly published (http://www.bacterio.net). Anoxybacillus species are widely distributed and can be found in geothermally heated environments. The taxonomic classification of Anoxybacillus members has traditionally relied on 16S rRNA gene sequence analysis and DNA–DNA hybridization (DDH). However, it is known that the discriminatory power of 16S rRNA gene analysis is often limited when it comes to distinguishing closely related species, such as those within the Anoxybacillus genus. DDH is a time-consuming and labor-intensive method, and the establishment of a central database is impractical. Recently, phylogenetic approaches utilizing whole-genome sequence-based metrics, such as average nucleotide identity (ANI), digital DDH (dDDH), and average amino acid identity (AAI), have emerged as important tools for the classification of prokaryotic taxa. These methods have been employed in the reclassification of various bacterial taxa [3, 4].

Anoxybacillus rupiensis DSM 17127T was isolated from different springs in the area of Rupi basin by Derekova et al. in 2007 [5] and its validation was documented in the International Journal of Systematic and Evolutionary Microbiology (Validation List No. 119) [6]. Anoxybacillus geothermalis GSsed3T was isolated from deposits at the entrance filters of the geothermal research facility of Groß Schönebeck in northern Germany by Filippidou et al. in 2016 [7], as validly named species. In the original article, Filippidou et al. [7] proposed A. geothermalis GSsed3T as a new species within the genus Anoxybacillus, primarily based on the DNA–DNA hybridization (DDH) value between A. rupiensis DSM 17127T and A. geothermalis GSsed3T and based on genomic average nucleotide identity (ANI) value and pairwise digital DNA–DNA hybridization (dDDH) values between A. amylolyticus MR3CT and A. geothermalis GSsed3T. Due to the unavailability of the genome sequence of A. rupiensis DSM 17127T in any database, Filippidou et al. [7] were unable to determine the average nucleotide identity (ANI) value and pairwise digital DNA–DNA hybridization values between A. rupiensis DSM 17127T and A. geothermalis GSsed3T. During our genome-based analysis, we observed that A. rupiensis DSM 17127T and A. geothermalis GSsed3T shared similar features. As a result, we attempted to clarify the relationship between A. rupiensis DSM 17127T and A. geothermalis GSsed3T using genomics-based methods. The data presented in this study provide evidence that A. geothermalis GSsed3T is a later heterotypic synonym of A. rupiensis DSM 17127T.

Materials and Methods

A. rupiensis DSM 17127T and A. geothermalis GSsed3T were obtained from the German Collection of Microorganisms and Cell Cultures GmbH (DSMZ). Two type strains were cultivated on Nutrient Agar medium and incubated at a temperature of 55 °C for a duration of 24 h. In this study, the genome sequence of A. rupiensis DSM 17127T was determined, while the genome sequence of A. geothermalis GSsed3T (JYCG00000000) was downloaded from the GenBank database. For the complete genome sequencing, genomic DNA was isolated from A. geothermalis GSsed3T culture using the QIAamp DNA Mini Kit according to the manufacturer’s instructions. The whole-genome sequencing of A. geothermalis GSsed3T was performed using 2 × 250 bp paired-end reads on an Illumina HiSeq 2500 platform by MicrobesNG (University of Birmingham, United Kingdom). The reads were assembled using the complete SPAdes assembly strategy on the PATRIC web server (https://patricbrc.org/) [8]. Genome annotation was performed using Rapid Annotations using Subsystems Technology (RAST) server [9]. The draft genome sequences obtained in this study have been submitted to the National Center for Biotechnology Information (NCBI) database and can be accessed under the accession number JAQOTG010000000.

The pairwise alignment feature implemented on the EZBioCloud server (https://www.ezbiocloud.net/tools/pairAlign) was used to compare the 16S rRNA gene sequence identity between A. rupiensis DSM 17127T and A. geothermalis GSsed3T. The 16S rRNA gene sequences of closely related type strains were obtained from the EzBioCloud server at https://www.ezbiocloud.net/ [10] and subsequently edited using the BioEdit software [11]. The ClustalW program [12] was employed for multiple sequence alignment of the 16S rRNA gene sequences. The Kimura’s two-parameter model [13] was employed to calculate the evolutionary distances. The phylogenetic trees were constructed using Mega-X software, employing the neighbor-joining method [14], maximum-parsimony method (Kluge and Farris, 1969), and maximum-likelihood method [15]. The bootstrap values were determined based on 1000 replications to assess the robustness of the tree topologies.

The phylogenetic analysis of A. rupiensis DSM 17127T and A. geothermalis GSsed3T was conducted using the type strain genomes server pipeline (TYGS) developed by Meier-Kolthoff and Göker [16]. The digital DNA–DNA hybridization (dDDH) value between the draft genome sequences of A. rupiensis DSM 17127T and A. geothermalis GSsed3T was calculated using Formula 2 of the online Genome-to-Genome Distance Calculator (GGDC) tool provided at http://ggdc.dsmz.de/distcalc2.php, as proposed by Meier-Kolthoff et al. [17]. To assess the genetic relationship between A. rupiensis DSM 17127T and A. geothermalis GSsed3T, average nucleotide identity (ANI) values were determined using the orthoANIu algorithm and an online ANI calculator available at www.ezbiocloud.net/tools/ani, as described by Lee et al. [18] and Yoon et al. [19]. A phylogenetic tree based on whole-genome sequences was constructed using the TYGS web server developed by Meier-Kolthoff and Göker [16]. The amino acid identity (AAI) value was calculated using the CompareM software (https://github.com/dparks1134/CompareM).

For the phylogenetic and pangenome analyses, the genomes of A. rupiensis DSM 17127T and A. geothermalis GSsed3T and all other Anoxybacillus species registered in RefSeq were re-annotated using Prokka 1.14.5 with default settings to ensure consistent annotation [20]. Phylogenetic trees were constructed using the ‛insert genome into species tree app’ version 2.2.0, which utilizes the FastTree 2 algorithm developed by Price et al. [21]. The pangenome was constructed using the ‛build pangenome with OrthoMCL app’ version 2.0, available on the KBase platform (https://www.kbase.us/), as described by Arkin et al. [22]. Pangenome-based phylogenomic analysis was performed using the ‘phylogenetic pangenome accumulation app’ version 1.4.0 developed by Li et al. [23] and Arkin et al. [22].

The assessment of A. rupiensis DSM 17127T and A. geothermalis GSsed3T’s biochemical characteristics was conducted using the API 20E, API 50CH strips, and the Vitek2 Bacilli Identification Card (BCL) microtest systems from bioMérieux, adhering to the instructions provided by the manufacturer. The polar lipids of strain A. salavatliensis DSM 22626T and A. gonensis G2T were extracted from 100-mg freeze-dried cells using two-dimensional thin-layer chromatography (TLC) according to the method of Tindall [24, 25]. Two-dimensional TLC on silica gel facilitated the separation of polar lipids. The initial phase involved the use of chloroform:methanol:water (65:25:4, v/v), while the subsequent phase utilized chloroform:methanol:acetic acid:water (80:12:15:4, v/v). For the detection process, 5% ethanolic molybdophosphoric acid was employed for total lipids, molybdenum blue for phospholipids, ninhydrin for aminolipids, and α-naphthol for glycolipids, as per the methodology outlined by Tindall et al. [26]. Reference standards included diphosphatidylglycerol (DPG), phosphatidic acid (PA), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylserine (PS), and phosphatidylinositol (PI). Extracting and purifying isoprenoid quinones from freeze-dried cells involved adhering to Collins’ procedure [27] and subjected the samples to analysis using high-performance liquid chromatography (HPLC).

Results and Discussion

Whole-genome sequencing has greatly contributed to resolving the taxonomic inconsistencies within prokaryotic taxa, leading to the reclassification of several bacterial species [28]. In this study, we conducted a comprehensive reassessment of the taxonomic relationship between A. rupiensis DSM 17127T and A. geothermalis GSsed3T by employing whole-genome phylogenetic analysis. A. geothermalis GSsed3T was isolated from deposits at the entrance filters of the geothermal research facility of Groß Schönebeck in northern Germany, while A. rupiensis DSM 17127T was isolated from different springs in the area of Rupi basin.

In their original study, Filippidou et al. [7] reported that A. geothermalis GSsed3T exhibited a 16S rRNA gene sequence similarity of 99.8% with A. rupiensis DSM 17127T. However, despite this high similarity, they reported that the DNA–DNA hybridization value between A. rupiensis DSM 17127T and A. geothermalis GSsed3T was determined to be 16%, which is below the species delineation threshold (70%) as defined by Wayne et al. [29]. In this study, we conducted a comprehensive analysis to assess the phylogenetic relationship between A. rupiensis DSM 17127T and A. geothermalis GSsed3T, focusing on various genomic parameters. The pairwise nucleotide sequence alignment of their 16S rRNA gene sequences revealed a high similarity of 99.8%, with three nucleotide mismatches. Furthermore, phylogenetic tree reconstructions based on the 16S rRNA gene sequences consistently clustered A. rupiensis DSM 17127T and A. geothermalis GSsed3T together in the neighbor-joining algorithm, showing robust clustering with a high bootstrap resampling value of 96% (Fig. 1). Similar results were obtained when employing the maximum-likelihood and maximum-parsimony algorithms (Figs. S1, S2), further supporting their close phylogenetic relationship.

Fig. 1
figure 1

Neighbor-joining (NJ) tree constructed based on 16S rRNA gene sequences available from the GenBank database. Bootstrap values (expressed as percentages of 1000 replications) greater than 50% are shown at branch points. Bar, 0.01 represents substitutions per nucleotide position. Paenibacillus polymyxa DSM 36T was used as the outgroup

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In the phylogenomic tree (Fig. 2), A. rupiensis DSM 17127T and A. geothermalis GSsed3T formed a distinct and well-supported branch separate from other type strains within the same genus, with a high bootstrap resampling value of 100%. The average nucleotide identity (ANI) value between A. rupiensis DSM 17127T and A. geothermalis GSsed3T was determined to be 98.60%, surpassing the suggested threshold value (95–96%) for species demarcation [30]. This finding confirms their high phylogenetic relatedness at the genomic level. Moreover, the average amino acid identity (AAI) value between A. rupiensis DSM 17127T and A. geothermalis GSsed3T was calculated as 98.50%, significantly exceeding the recommended cut-off for species delineation (AAI > 95%) [31], further supporting their classification within the same species. Furthermore, digital DNA–DNA hybridization (dDDH) analyses yielded a dDDH value of 85.6% between A. rupiensis DSM 17127T and A. geothermalis GSsed3T, surpassing the established cut-off (70%) for assigning bacterial strains to the same species [29]. These results reinforce the notion that A. geothermalis GSsed3T and A. rupiensis DSM 17127T should be considered members of the same species. Table 1 presents the AAI, ANI, and dDDH values calculated between A. rupiensis DSM 17127T, A. geothermalis GSsed3T, and other closely related type strains.

Fig. 2
figure 2

Phylogenetic tree based on whole-genome sequences of A. rupiensis DSM 17127T and A. geothermalis GSsed3T and related reference strains. The tree was inferred with FastME 2.1.6.1 [33] from genome blast distance phylogeny (GBDP) distances calculated from genome sequences using the TYGS server (https://tygs.dsmz.de) [16]. The branch lengths are scaled in terms of GBDP distance formula d5. The numbers at branches are GBDP pseudo-bootstrap support values ≥ 64% from 100 replications with an average branch support of 97.7%. The tree was rooted at the midpoint [34] (Color figure online)

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Table 1 AAI, ANI and dDDH values between A. rupiensis DSM 17127T, A. geothermalis GSsed3T, and closely related type strains
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The pangenomic analysis of Anoxybacillus species, including A. rupiensis DSM 17127T and A. geothermalis GSsed3T, resulted in the identification of 9726 orthologous clusters that constituted the pangenome. The numbers of core genes, strain-specific genes (singleton), and accessory genes (partial) were 128, 5062, and 4536, respectively. According to the pangenome-based phylogenomic analysis, A. rupiensis DSM 17127T and A. geothermalis GSsed3T formed a monophyletic clade and shared 2421 core genes, indicating their close evolutionary relationship (Fig. 3).

Fig. 3
figure 3

Pangenome-based phylogenomic analysis of Anoxybacillus species. Orthologous gene sets within a pangenome are partitioned into three categories: core (blue), singleton (red), and partial pangenome (pink). Pangenome-based phylogenomic analysis was created by the OrthoMCL and phylogenetic pangenome accumulation (v1.4.0) app (Color figure online)

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Furthermore, the validation of this conclusion has been established through a comparison of phenotypic and chemotaxonomic characteristics between A. rupiensis DSM 17127T and A. geothermalis GSsed3T. In the API 20E, API 50CH, and Vitek2 BCL system, A. rupiensis DSM 17127T and A. geothermalis GSsed3T exhibited similar biochemical features with minor exceptions (Table 2). As an illustration, acid production from d-galactose, sucrose, and arginine dihydrolase were negative for A. rupiensis DSM 17127T, whereas A. geothermalis GSsed3T showed positive results. On the other hand, urease, ornithine decarboxylase, tryptophanase, and acid production from d-trehalose and d-mannose were positive for A. rupiensis DSM 17127T, whereas A. geothermalis GSsed3T yielded negative results. Both species exhibited positive results for lysine decarboxylase, tyrosine arylamidase, citrate utilization, catalase, phenylalanine arylamidase, Ala-Phe-Pro-Arylamidase, α-glucosidase acid production from d-xylose, d-mannitol, d-glucose, d-fructose, d-ribose, d-maltose, glycerol, esculin ferric citrate, d-saccharose, and starch. Also, A. rupiensis DSM 17127T and A. geothermalis GSsed3T exhibited negative results for nitrate reduction, leucine-arylamidase, tryptophan deaminase, glycine arylamidase, gelatin hydrolysis, Voges–Proskauer, β-galactosidase, polymyxin B resistance, l-proline arylamidase, β-N-acetyl-glucosaminidase, hydrogen sulfide production, l-lysine arylamidase, alanine arylamidase, pyruvate, β-xylosidase, l-aspartate arylamidase, l-pyrrolydonyl arylamidase, phosphoryl choline, cyclodextrine, methyl-d-xyloside, α-mannosidase, N-acetyl-glucosamine, kanamycin resistance, β-mannosidase, methyl-a-d-glucopyranoside, salicin, d-cellobiose, d-trehalose, inulin, d-melezitose, glycogen, d-turanose, growth in 6.5% NaCI, oleandomycin resistance, acid production from l-arabinose, d-sorbitol, d-melibiose, l-xylose, d-raffinose, d-tagatose, d-arabitol, arbutin, inositol, l-rhamnose, putrescine, erythritol, d-arabinose, methyl-d-xylopyranoside, l-sorbose, dulcitol, amygdalin, d-lactose, xylitol, gentiobiose, d-lyxose, d-fucose, l-fucose, l-arabitol, potassium gluconate, potassium 2-ketogluconate, and potassium 5-ketogluconate. The total count of phenotypic tests conducted utilizing the API 20E, API 50CH, and Vitek2 BCL system amounted to 91. It was determined that there was a difference between A. rupiensis DSM 17127T and A. geothermalis GSsed3T in only 8 tests and the difference value was 9%.

Table 2 The biochemical characteristics of A. geothermalis GSsed3T and A. rupiensis DSM 17127T
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In the original article, Derekova et al. [5] were not determined the polar lipids of A. rupiensis DSM 17127T. In the present study, the polar lipids found in A. rupiensis DSM 17127T were diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), unidentified amino phospholipid (APL), unidentified phospholipid-1 (PL1), unidentified phospholipid-2 (PL2), unidentified lipid-1 (L1), unidentified lipid-2 (L2), and unidentified lipid-3 (L3), whereas A. geothermalis GSsed3T consisted of DPG, PG, PE, APL, PL1, L1, and L3. Polar lipid composition showed very similar profile between two species (Fig. 4). The respiratory quinone of A. rupiensis DSM 17127T and A. geothermalis GSsed3T was menaquinone MK-7. Most of the chemotaxonomic and phenotypic features between A. rupiensis DSM 17127T and A. geothermalis GSsed3T were almost identical except for a few exceptions as is shown in Table 2 and Fig. 4. The disagreement for phenotypic and chemotaxonomic was probably due to their different ecological niches.

Fig. 4
figure 4

Two-dimensional thin-layer chromatogram of polar lipids of A A. rupiensis DSM 17127T and B A. geothermalis GSsed3T. DPG diphosphatidylglycerol, PG phosphatidylglycerol, PE phosphatidylethanolamine, APL unidentified aminophospolipid, PL unidentified phospholipid, L unidentified lipid (Color figure online)

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Collectively, the findings of this study indicate that A. rupiensis DSM 17127T and A. geothermalis GSsed3T should be regarded as the same species. Therefore, based on the phylogenetic analysis using whole-genome sequences and following rule 42 of the Bacteriological Code [32], we propose that A. geothermalis GSsed3T, originally described by Filippidou et al. in 2016 [7], be reclassified as a later heterotypic synonym of A. rupiensis DSM 17127T, as initially described by Derekova et al. in 2007. The type strain for A. rupiensis is DSM 17127T (= R270T = NBIMCC 8387T), and GSsed3 (= CCOS808 = ATCC BAA2555) represents an additional strain of A. rupiensis.

Emended Species Description of Anoxybacillus rupiensis Derekova et al. 2007

Anoxybacillus rupiensis (N.L. masc. adj. rupiensis, originating from Rupi Basin, referring to the place of isolation of the type strain).

The description is the same as given by Derekova et al. (2007) with the following modification.

The respiratory quinone is menaquinone MK-7. Major polar lipids include diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), unidentified amino phospholipid (APL), unidentified phospholipid-1 (PL1), unidentified phospholipid-2 (PL2), unidentified lipid-1 (L1), unidentified lipid-2 (L2), and unidentified lipid-3 (L3). In API 50CH, API 20E, and Vitek2 BCL system, the following activities were positive for lysine decarboxylase, tyrosine arylamidase, citrate utilization, catalase, phenylalanine arylamidase, Ala-Phe-Pro-Arylamidase, α-glucosidase, urease, ornithine decarboxylase, tryptophanase and acid production from d-trehalose, d-mannose, d-xylose, d-mannitol, d-glucose, d-fructose, d-ribose, d-maltose, glycerol, esculin ferric citrate, d-saccharose, and starch. Negative for nitrate reduction, leucine arylamidase, tryptophan deaminase, glycine arylamidase, gelatin hydrolysis, Voges–Proskauer, β-galactosidase, polymyxin B resistance, l-proline arylamidase, β-N-acetyl-glucosaminidase, hydrogen sulfide production, l-lysine arylamidase, alanine arylamidase, pyruvate, β-xylosidase, l-aspartate arylamidase, l-pyrrolydonyl arylamidase, phosphoryl choline, cyclodextrine, methyl-d-xyloside, α-mannosidase, N-acetyl-glucosamine, kanamycin resistance, β-mannosidase, methyl-a-d-glucopyranoside, salicin, d-cellobiose, d-trehalose, inulin, d-melezitose, glycogen, d-turanose, growth in 6.5% NaCI, oleandomycin resistance, acid production from l-arabinose, d-sorbitol, d-melibiose, l-xylose, d-raffinose, d-tagatose, d-arabitol, arbutin, inositol, l-rhamnose, putrescine, erythritol, d-arabinose, methyl-d-xylopyranoside, l-sorbose, dulcitol, amygdalin, d-lactose, xylitol, gentiobiose, d-lyxose, d-fucose, l-fucose, l-arabitol, potassium gluconate, potassium 2-ketogluconate, and potassium 5-ketogluconate. The DNA G + C content of the type strain 17127T (= R270T = NBIMCC 8387T) is 42.27 mol% (genome-based).