Bacillus telluris sp. nov. Isolated from Greenhouse Soil in Beijing, China.

A novel Gram-stain-positive, rod-shaped, endospore-forming bacterium, which we designated as strain 03113T, was isolated from greenhouse soil in Beijing, China. Phylogenetic analysis based on 16S rRNA gene sequences showed strain 03113T is in the genus Bacillus and had the highest similarity to Bacillus solani CCTCC AB 2014277T (98.14%). The strain grew at 4 °C-50 °C (optimum 37 °C), with 0-10% (w/v) NaCl (optimum 5%), and in the range of pH 3.0-12.0 (optimum pH 8.0). Menaquinone was identified as MK-7, and the major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, and phosphatidylethanolamine. The main major cellular fatty acids detected were anteiso-C15:0 (51.35%) and iso-C15:0 (11.06%), which are the predominant cellular fatty acids found in all recognized members of the genus Bacillus. The 16S rRNA gene sequence and core-genome analysis, the average nucleotide identity (ANI), and in silico DNA-DNA hybridization (DDH) value between strain 03113T and the most closely related species were 70.5% and 22.6%, respectively, which supported our conclusion that 03113T represented a novel species in the genus Bacillus. We demonstrated that type strain 03113T (=ACCC 03113T=JCM 33017T) was a novel species in the genus Bacillus, and the name Bacillus telluris sp. nov. was proposed. Strain 03113T secreted auxin IAA and carried the nitrogenase iron protein (nifH) gene, which indicated that strain 03113T has the potential to fix nitrogen and promote plant growth. Bacillus telluris sp. nov. 03113T is a potential candidate for the biofertilizers of organic agriculture areas.

1. Introduction

The genus Bacillus was first described by Cohn in 1872, and it is a genus of ubiquitous soil microorganisms [1]. It is comprised of endospore-forming, rod-shaped bacteria that are members of the phylum Firmicutes [2]. At the time of writing, there were 379 species in the genus Bacillus recorded on LPSN (www.bacterio.net/bacillus.html; Nov 2019). Based on their genetic similarity, Bacillus species can be classified into several groups, which include Bacillus cereus–Bacillus anthracis–Bacillus thuringiensis, Bacillus clausii–Bacillus halodurans, Bacillus coahuilensis–Bacillus sp. NRRLB-14911, and Bacillus subtilis–Bacillus licheniformis–Bacillus pumilus [3]. In addition, species in the genus Bacillus have a wide range of physiological and biochemical characteristics from psychrophilic to thermophilic, acidophilic to alkaliphilic, and some are halophilic [4], which allow them to live in a wide range of extreme habitats, such as desert sands, hot springs, and Arctic soils. In addition, the genus Bacillus is an extremely diverse group of bacteria that includes both the causative agent of anthrax (B. anthracis) [5,6] and several species that synthesize important antibiotics. In addition to medical uses, bacillus spores, due to their extreme tolerance of both heat and disinfectants, are used to test heat sterilization techniques and chemical disinfectants. Bacilli are also used in the detergent manufacturing industry for their ability to synthesize important enzymes.

In this study, we report a novel bacterial strain, 03113T, which was isolated from the greenhouse soil of Wangsiying, Chaoyang District, Beijing, China. Based on the phenotypic characteristics and phylogenetic analysis, strain 03113T represents a novel species in the genus Bacillus.

2. Materials and Methods

2.1. Bacterial Strains, Growth Conditions, and Cultivation

Strain 03113T was isolated from greenhouse soil from Wangsiying, Chaoyang District, Beijing, China (40°09′N, 116°42′E). We preserved the sample in freeze-dried milk ampoules at 4 °C and 20% (v/v) glycerol at −80 °C [7]. The type strains of species closely related to strain 03113T were used as reference strains under the same conditions for comparative taxonomic analysis, which included B. solani CCTCC AB 2014277T, B. praedii CCTCC AB 2015208T, and B. dabaoshanensis CCTCC AB 2013260T. All strains were maintained and cultivated in TSA or TSB (DifcoTM) medium plates at 30 °C, unless otherwise stated.

2.2. Phenotypic Characterization

Biochemical characteristics of strain 03113T were investigated. Growth at eight different temperatures (4, 15, 25, 30, 37, 40, 45, and 50 °C) was tested on TSA plates. The pH values (pH 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, and 12.0, with increments of 1.0 pH unit) were tested in LB medium. Growth at various NaCl concentrations was tested over the range 0%−12% (w/v) NaCl (at intervals of 1%) by incubating at 30 °C [8]. Gram staining was performed using the Gram-stain kit [9]. Cell morphology was observed by light microscopy (CX21; Olympus) and transmission electron microscopy. Endospores were examined according to the Schaeffer–Fulton staining method [10]. Motility was examined on motility agar [11]. Catalase activity was determined by investigating bubble production with 3% (v/v) H2O2, and oxidase activity was determined using 1% (v/v) tetramethyl-p-phenylenediamine. The basic biochemical characteristics were investigated on API-20NE, API 50CH (BioMérieux) [12], and BIOLOG GEN III MicroPlate (BIOLOG), according to the manufacturer’s instructions. The type strains of B. solani CCTCC AB 2014277T, B. praedii CCTCC AB 2015208T, and B. dabaoshanensis CCTCC AB 2013260T were used as reference strains.

2.3. Chemotaxonomic Analysis

For the measurement of chemotaxonomic characteristics, the menaquinone system was analyzed as described by Collins et al. [13] using reversed-phase HPLC [14]. The analysis of polar lipids by two-dimensional TLC was performed according to the method described by Minnikin et al. [15]. The cellular fatty acid is a useful and functional tool to identify species in the genus Bacillus and related genera. After 48 h of incubation at 30 °C on TSA, cellular fatty acids were extracted and analyzed using the method described by Sasser [16] and identified with the MIDI Sherlock Microbial Identification System (Library RTSA6 6.0, MIDI Sherlock Software Package, Version 6.0; Agilent 6890N).

2.4. Phylogenetic 16S rRNA Gene Analysis

Genomic DNA was extracted from a single colony of the novel strain grown on TSA plates at 30 °C for 2 d using Bacteria DNA Kit (Tiangen, Beijing, China), according to the manufacturer’s protocol. The 16S rRNA gene was amplified by PCR and sequenced using the universal primers 27F(5’-AGAGTTTGATCCTGGCTCAG-3’) and 1492R(5’-GGTTACCTTGTTACGACTT-3’) [17]. Pairwise 16S rRNA gene sequence similarities were calculated using the EzTaxon-e database (http://eztaxon-e.ezbiocloud.net/) [18]. The CLUSTAL_W algorithm was used for sequence alignments using the neighbour-joining [19,20] and maximum-likelihood [21] methods that were implemented with Mega 7.0 software for phylogenetic analysis. Evolutionary distances were computed by using the Kimura two-parameter model [22]. The robustness of the tree branches was estimated by bootstrap analysis with 1000 replications [23]. The GenBank/EMBL/DDBJ accession number of 16S rRNA sequence is MN907472.

2.5. Complete Genome Sequencing and Analysis

To confirm the results of the 16S rRNA gene sequence similarity analysis, the complete genome sequence of the novel species was performed. The genome was sequenced by Personal Biotechnology Co., Ltd (Shanghai, PR China). Genomes of the most closely related species chosen above were retrieved from the GenBank database in NCBI. Reads of each data set were filtered by using AdapterRemoval (ver. 2.1.7) [24], and high-quality paired-end reads were assembled using A5-MiSeq v20150522 [25]. The open reading frames (ORFs) were predicted by GeneMarkS (ver. 4.32 April 2015) [26]. The tRNA genes were predicted by tRNAscan-SE 94 (ver. 1.3.1) and the rRNA genes by Barrnap (0.9-dev) 95 (https://github.com/tseemann/barrnap) [27]. Calculations of average nucleotide identity (ANI) were performed using JSpecies software (http://www.imedea.uib.es/jspecies). In silico DNA—DNA hybridization (DDH) estimates were performed using Genome-to-Genome Distance Calculator (GGDC) with the BLAST+ (recommended) method [28]. The partial genome files were uploaded to the GGDC 2.0 web interface (http://ggdc.dsmz.de/ggdc.php#), and Formula 2 was used as recommended for the calculation of DDH values. As a further extension of genome-based phylogeny, the GGDC website was used to establish the phylogenomic tree of strain 03113T and other closely related Bacillus species.

2.6. Analysis of Core Orthologous Genes

To identify orthologous genes among the strains in Bacillus species, 13 Bacillus strains were selected for the core genome analysis based on their biological control properties. The 13 bacteria included B. solani CCTCC AB 2014277T, B. praedii CCTCC AB 2015208T, B. glycinifermentans GO-13, B. acidicola FJAT-2406, B. salacetis SKP7-4, B. shackletonii LMG 18435, B. circulans NBRC 13626, B. foraminis Bac44, B. persicus DSM 25386, B. oceanisediminis CGMCC 1.10115, B. firmus NCTC 10335, B. gottheilii FJAT-2394, and 03113T. The Bacterial Pan Genome Analysis (BPGA) pipeline [29] was used for the pan-genome analyses. The clustering tool USEARCH was used to cluster protein families. The OrthoFinder [30,31] was used to perform an all-versus-all BLAST search based on nucleotide gene sequences of strain 03113T and other related strains of the genus Bacillus to identify clusters of orthologous genes (OGs). Those OGs shared among all taxa and present in a single copy per genome were selected. They were aligned with Mafft [32] and subsequently concatenated. A phylogenetic tree based on orthologous proteins of the Bacillus genus was constructed by RA×ML version 8.2.12, based on the maximum-likelihood method.

2.7. Plant Growth-Promoting Characteristics

The performance of secreting plant growth hormone indoleacetic acid (IAA) of strain 03113T was measured by the PC Salkowski colorimetric method described by Glickmann and Dessaux [33]. The qualitative and quantitative analyses of siderophore production were conducted by the method described by Machuca and Milagres [34]. Phosphate solubilization was measured on inorganic and organic phosphate media [35]. All experimental analyses were performed in triplicate to ensure reproducibility. The results were expressed as the mean value of these determinations.

3. Results and Discussion

3.1. Phenotypic Characterization of 03113T

The colonies of strain 03113T were Gram-stain-positive and rod-shaped with a size range of 1−2 mm in diameter (Figure S1a). The size of the cells was observed by light microscopy. The cells produced ellipsoidal endospores that were positioned terminally (Figure S1b), and the cells were motile. Catalase and oxidase activity were positive. According to API 50CH tests, reactions of galactose, sorbose, rhamnose, dulcitol, α-metyl-D-glucoside, arbutin, esculin, melibiose, sucrose, trehalose, and D-turanose were positive but the other three reference strains were negative. With API 20NE, strain 03113T was positive for lysine, but the other three reference strains were negative. The phenotypic properties differentiating between strain 03113T and its closest phylogenetic neighbors are shown in Table 1.

Table 1

Differential phenotypic characteristics of strain 03113T and closely related strains in the genus Bacillus.

Characteristic	1	2	3	4
Optimal growth conditions
Temperature for growth (°C)	37	30–37	35	30
pH for growth	8.0	7.0	9.0	9.0
NaCl concentration for growth (%, w/v)	5	1	0	4
The Acid produced from (API 50CH)
L-arabinose	+	+	+	−
Esculin	+	+	−	−
API 20NE
β-galactosidase	+	+	−	−
Lysine	+	−	−	−
Lohn gelatin	+	+	+	−
Utilization (Biolog GEN III)
dextrin	+	+	−	+
d-maltos, d-trehalose, sucrose, d-turanose, d-raffinose	+	−	−	−
N-acetyl-d-glucosamine	−	+	+	−
N-acetyl-β-d-mannosamine, acetic acid	−	+	−	−
Stachyose, d-mannose	w	−	−	−
N-acetyl-d-galactosamine	−	w	−	−
α-d-glucose	+	−	w	−
d-fructose	+	+	w	−
Inosine, d-serine, glycerol, d-glucose-6-PO4, d-fructose-6-PO4, nalidixic acid, lithium chloride, aztreonam, lincomycin	−	+	+	+
Troleandomycin	−	−	+	−
L-aspartic acid, L-glutamic acid, L-histidine, L-pyroglutamic acid, L-serine, L-lactic acid, sodium butyrate	-	+	+	−

Strains: 1, 03113T; 2, B. dabaoshanensis CCTCC AB 2013260T; 3, B. solani CCTCC AB 2014277T; 4, B. praedii CCTCC AB 2015208T. All strains were negative for sodium thiosulfate, tryptophan, d-cellobiose, gentiobiose, α-d-lactose, d-melibiose, β-methyl-d-glucoside, d-salicin, N-acetyl neuraminic acid, d-galactose, 3-methyl glucose, L-fucose, L-rhamnose, fusidic acid, d-sorbitol, d-mannitol, myo-inositol, d-aspartic acid, minocycline, pectin, d-galacturonic acid, glucuronamide, mucic acid, quinic acid, p-hydroxy-phenylacetic acid, citric acid, α-keto-glutaric acid, d-malic acid, γ-amino-butryric acid, α-hydroxy-butyric acid, β-hydroxy-D, L-butyric acid, α-keto-butyric acid, propionic acid, and formic acid. All strains were positive for sodium lactate and potassium tellurite. All data were from the present study. +, positive; w, weakly positive; −, negative.

3.2. Analysis of Isoprenoid Quinones, Polar Lipids, and Cellular Fatty Acids

The main isoprenoid quinone of strain 03113T was identified as MK-7. The polar lipids detected were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylserine, three unknown aminophospholipids, and one unknown phospholipid, which was consistent with the predominant component of B. solani CCTCC AB 2014277T [7]. The major fatty acids of strain 03113T were anteiso-C15:0 (51.35%), iso-C15:0 (11.06%), and iso-C14:0 (7.13%), which were similar to those of the reference strains (Table 2). Iso- and anteiso- branched fatty acids of the 14-17 carbon series are typical for the genus Bacillus [36], which indicated that strain 03113T is a member of this genus. However, the proportions of the novel strain were different from B. solani CCTCC AB 2014277T, B. praedii CCTCC AB 2015208T, and B. dabaoshanensis CCTCC AB 2013260T. For instance, the content of anteiso-C15:0 in strain 03113T was much higher than in the reference strains, but the concentration of iso-C15:0 was much lower than in the related reference strains.

Table 2

The cellular fatty acid content of strain 03113T and representative strains of closely related species of the genus Bacillus. Strains: 1, 03113T; 2, B. dabaoshanensis CCTCC AB 2013260T; 3, B. solani CCTCC AB 2014277T; 4, B. praedii CCTCC AB 2015208T. All data were obtained in this study. Partial values lower than 1% are not shown in the table. ND, Not detected.

Fatty acid	1	2a	3	4
C14:0	1.96	1.1	1.77	1.68
C16:0	7.50	2.60	1.98	2.20
iso-C14:0	7.13	ND	6.61	5.13
iso-C15:0	11.06	42.9	45.43	54.12
iso-C16:0	8.73	6.7	6.07	5.61
anteiso-C15:0	51.35	24.1	27.16	20.15
anteiso-C17:0	6.71	6.2	3.88	3.13
C16:1ω7c alcohol	ND	ND	2.56	2.36
C16:1ωw11c	ND	ND	1.21	1.12
Summed Feature 3 *	<1	2.5	ND	ND
Summed Feature 8 †	<1	1.5	ND	ND

* Summed feature 3 comprises C16: 1ω6c and/or C16: 1ω7c. † Summed feature 8 comprises C18: 1ω6c and/or C18: 1ω7c.a Data were obtained from: Cui et al. [37].

3.3. Phylogenetic Analysis of 16S rRNA

The complete 16S rRNA gene sequence (1347 bp) was discovered from the draft genome of the novel strain. Pairwise comparisons showed that strain 03113T was related most closely to B. solani CCTCC AB 2014277T (98.14% similarity), followed by B. praedii CCTCC AB 2015208T (98.07%), and B. dabaoshanensis CCTCC AB 2013260T (98.0%). Phylogenetic trees were reconstructed using the maximum-likelihood, neighbour-joining, and minimum-evolution methods. All three treeing methods yielded a similar phylogeny. Strain 03113T was located within the genus Bacillus and had a separated clade based on the phylogenetic trees of 16S rRNA genes (Figure 1, Figure S2, Figure S3), indicating that 03113T was a novel species of genus Bacillus.

Figure 1

Neighbour-joining phylogenetic tree based on the 16S rRNA gene sequence of strain 03113T and other closely related Bacillus species. The significance of each branch is indicated by a bootstrap value (%) calculated for 1000 subsets. Genbank accession numbers are given in parentheses. Bar, 0.005 substitutions per nucleotide position. Isolating source label has been annotated in the back.

3.4. Whole-Genome Analysis

A total of 5,033,596 reads were obtained from draft genome sequencing of strain 03113T, which yielded a genome of 4,856,532 reads in length. N50 value was 190,698 bp, and the largest contig was 198,446 bp. The genome was predicted to contain a total of 4288 genes, which included 4241 protein-coding genes, 2 rRNA genes, and 45 tRNA genes. The genomic DNA G+C content of strain 03113T was 36.08 mol%. The phylogenomic tree based on the GGDC web also revealed the distinct phylogeny of strain 03113T and its close relationship with B. solani CCTCC AB 2014277T, B. praedii CCTCC AB 2015208T, and B. dabaoshanensis CCTCC AB 2013260T (Figure 2). ANIb and ANIm values of strain 03113T with the type strain of the most closely related species, B. solani CCTCC AB 2014277T, were 70.5% and 85.9%, respectively. All ANI values were much lower than the 96.0% cut-off value that was proposed previously for the genus Bacillus [38,39]. The DDH value of strain 03113T and B. solani CCTCC AB 2014277T was 22.6%, which was much lower than 70%. The ANI and DDH between strain 03113T and the other reference species B. praedii CCTCC AB 2015208T were 70.5% and 20.9%, respectively. This genome sequence, which was deposited in the GenBank/EMBL/DDBJ database under accession number VATK00000000, was used for further analysis. Thus, complete genome analysis combined with 16S rRNA phylogenetic, physiological, and biochemical properties all supported the conclusion that strain 03113T should be considered a novel species in the genus Bacillus.

Figure 2

Phylogenomic tree generated with Genome-to-Genome Distance Calculator (GGDC), showing the phylogenomic position of strain 03113T and the type strains of related species of Bacillus. The numbers at the nodes indicate the gene support index. Bar, 0.02 substitutions per position.

3.5. Phylogenomic Comparative Analysis of Bacillus species

Based on the above database, we conducted a preliminary analysis of the pan-genome, which showed that 840 shared orthologous coding sequences were clustered into the core genome of Bacillus, 32,926 were represented in the accessory genome, and 22,024 were identified as strain-unique genes (Figure S4a). Therefore, a highly reliable mathematical extrapolation of the pan and core genome was constructed (Figure S4b). The total genes increase in the pan-genome of Bacillus with the rise in the analyzed genome number, suggesting that the pan-genome was open. The previous reports showed that the genes’ number of core genomes was highly conserved, while many strain-unique genomes and accessory genomes are thought to contribute to species diversity [40], which indicated that species in the genus Bacillus were also multifarious. A phylogenetic tree reconstructed based on the concatenated alignment of these 840 core orthologous proteins (Figure S4c) showed that strain 03113T clustered closely with known species, indicating that it was a member of the genus Bacillus. This is consistent with the previous results.

3.6. Plant Growth-Promoting Characteristics of Isolates

The qualitative determination indicated that strain 03113T secreted auxin IAA, and the colour reaction was pink, at a concentration of 175.94 μg/mL (Figure S5). Strain 03113T did not generate a color ring on the CAS flat plate, and no clear zone was observed around each of the colonies of strain 03113T on inorganic or organic phosphate media, which indicated that it did not produce siderophores or dissolve phosphate.

Strain 03113T carried the nitrogenase iron protein (nifH) gene, based on the genome annotation, which is a key enzyme for fixing nitrogen in bacteria. The nifH gene of 03113T had a very low similarity with published sequences based on Blast in Genbank (https://blast.ncbi.nlm.nih.gov/Blast.cgi). It was mostly related to B. alkalidiazotrophicus MS6 and B. arsenniciselenatis E1H with a similarity of 80%, which indicated that strain 03113T has the potential to fix nitrogen.

4. Conclusions

From the phenotypic and chemotaxonomic properties of strain 03113T, 16S rRNA gene sequence comparisons, and DNA–DNA hybridization, we concluded that strain 03113T (=ACCC 03113T=JCM 33017T) was distinguished from the known species in the genus Bacillus. Based on the present polyphasic analysis, strain 03113T is considered to represent a novel species within the genus Bacillus, for which we propose the name Bacillus telluris sp. nov. The description of Bacillus telluris sp. nov. is summarized in Appendix A.