Sample collection and isolation of bacterium

Sediment samples were collected twice from a hot spring at Suryakund ((24°08′58″N, 85°38′44″E), Jharkhand, India. The surface temperature of the sediment sample at the site of collection was 80°C and the pH was 7.4. No specific permissions were required for the collection of samples from this hot spring. Moreover, this location did not involve endangered and protected species and it is not under regulatory body concerned with protection of wildlife. Samples collected in a sterile container were transported to the laboratory without temperature control. 2.0 g (wet weight) sediment sample was transferred into a 250 ml conical flask containing 50 ml trypticase soy broth (Difco) pH 7.2 and incubated on a shaker (ISF-I-V; Adolf Kuhner AG) at 55°C and 200 rpm After overnight incubation, the suspension was serially diluted and plated onto trypticase soy agar (Difco) plates pH 7.2 and incubated at 55°C for three days. Morphologically similar pale yellow, rough surface circular colony that appeared on trypticase soy agar plate were picked and purified by repeated streaking on the same medium. For short term preservation, isolates were streaked on trypticase soy agar and stored at 4°C. For long-term preservation, the culture was stored at -80°C in 15% (v/v) glycerol.

For comparative study, closely related type strains were procured from Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Germany, National Collection of Industrial, Marine and Food Bacteria (NCIMB), UK, Korean Collection for Type Cultures (KCTC), Korea and from BCCM/LMG Bacteria collection, Universiteit Gent, Belgium. Unless indicated otherwise, all strains were grown and maintained as mentioned previously.

Phenotypic, biochemical and physiological characterization

Cell morphology was examined by transmission electron microscopy (model FEI Morgagni 268D) as described by Jyoti et al. [18]. Gram character was determined using Gram staining kit (Becton-Dickinson, USA) following manufacturer’s instruction. The formation of spores was observed by growing culture for 48 h in half strength TSB supplemented with 5 mg/l MnSO₄, 4 H₂O. Spores were detected by using Schaeffer and Fulton Spore Stain Kit (Hi-Media) following manufacturer’s instruction. For phenotypic characterization, all isolates and reference type strains were tested under same laboratory conditions. Tests like oxidase, catalase indole, methyl red, Voges Proskauer, lysine decarboxylase, ornithine decarboxylase, citrate and malonate utilization, hydrolysis of starch, urea, ONPG and nitrate reduction was performed by previously described methods [20]. Utilization of different carbohydrates as sole source of carbon and energy with concomitant production of acid was tested in peptone water basal media (Hi-Media) supplemented with substrates at concentration of 5.0 g/l and phenol red of 0.18 g/l. Tests were considered positive, weakly positive and negative based on the media colour (yellow, light yellow or pink and red respectively). In addition to classical tests, oxidation of organic compounds as sole source of carbon was determined using API50CH, API20E (bioMérieux) and Hi25 identification kit (Hi-Media) following manufacturer’s instruction except incubation was performed at 55°C for 18 h. Yeast extract-peptone broth containing 0-5.0% (w/v) NaCl was inoculated and incubated at 55°C for 4 days to test for salt tolerance. Ethanol tolerance was determined by growing the cells in TSB medium containing ethanol at a final concentration of 0-15% (v/v). Anaerobic growth was tested using the BD GasPak EZ system (Becton-Dickinson). The temperature for growth in the range of 30-60°C was examined in TSB medium at pH 7.5 for 2 days. Similarly, the pH range for growth was examined in TSB medium prepared in buffered solutions [21] in the pH range 3.5-11.5 in steps of 0.5 pH unit at 55°C for 48 h.

DNA isolation, PCR amplification and sequencing of 16S rRNA

Isolation of chromosomal DNA, amplification and sequencing of 16S rRNA gene of the isolates were performed by the methods described earlier [22]. Primers used for the amplification of 16S rRNA were 5’-GAG TTT GAT CCT GGC TCA-3’ (forward primer) and 5’-AGA AAG GAG GTG ATC CAG CC-3’ (reverse primer) [23]. The nucleotide sequences thus obtained were assembled using the sequence alignment editor program Bioedit (http://www.mbio.ncsu.edu/Bio-Edit/bioedit.html) and was compared with those in GenBank after BLAST searches [24] and using the EzTaxon-e server [25]. 16S rRNA sequences of valid strains obtained from EzTaxon-e server were considered for secondary structure based phylogenetic analysis where E.coli 16S rRNA was used as reference template. All sequences were aligned using template based alignment algorithm CRWalign [26], edited and refined manually. The base positions were assigned according to E.coli numbering and helix numbering was adopted from ARB database [27]. The 16S rRNA based phylogenetic tree was constructed according to the Kimura two-parameter model using the MEGA 5 [28] software package (The Biodesign Institute, Arizona, USA). A phylogenetic tree was constructed using the neighbor joining method of Saitou & Nei [29] and with the maximum likelihood algorithms. The statistical significance of branch points was calculated by 1,000 bootstrap re-samplings of the data [30].

Determination of DNA base ratio and DNA-DNA homology study

For determination of DNA G+C content, DNA was degraded enzymatically into nucleosides as described by Mesbah et al. [31]. The obtained nucleoside mixture was then separated by HPLC (Shimadzu) using an analytical column (Vydac 201 SP54, C18, 5μm; 250 x 4.6 mm) equipped with a guard column (201 GD54H; Vydac). Non methylated lambda phage DNA (Sigma) was used as the calibration reference.

DNA-DNA re-association study is considered as standard method for bacterial species delineation [32]. Hence, DNA-DNA re-association study was performed among the isolates and between strain JS1^T and other closely related type species showing 16S rRNA sequence similarity more than 97% following the method described by Ezaki et al. [33]. DNA from E. coli strain HB101 was taken as an unrelated negative control. Hybridization was performed following the methods described earlier [20].

ERIC and REP-PCR

ERIC-PCR (enterobacterial repetitive intergenic consensus sequence PCR) and REP-PCR (repetitive extragenic palindromic PCR) was performed with the isolated strains by method as described previously [34]. For ERIC PCR, two oligonucleotide primers were 5ʹ-ATG TAA GCT CCT GGG GAT TCA C-3ʹ and 5ʹ-AAG TAA GTG ACT GGG GTG AGC G-3ʹ. For Rep PCR two (18-mer) oligonucleotide primers were REP 1R-1 (5ʹ-III ICG ICG ICA TCI GGC-3’) and REP-21 (5ʹ-ICG ICT TAT CIG GCC TAC-3ʹ) [35]. The fingerprints were analyzed by using Bionumerics software (Applied Maths, Bio-Rad).

Chemotaxonomic analysis

For the analysis of polar lipids, and nature of diaminoacids in the peptidoglycan layer, cells were grown in TSB medium to mid log phase in a rotary shaker at 55°C. Polar lipids were determined by 2D-TLC following the method of Bligh & Dyer [36]. Cellular fatty acids were extracted from stationary phase cells grown on TSA at 55°C for 3 days and analyzed by previously described method [19]. Diaminopimelic acid (DAP) and cell wall sugar was detected following the method of Staneck & Roberts [37] by using the TLC on cellulose plates (Merck, cat no. 1.05577.0001, Germany).

Nucleotide sequence accession number

The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene of strain JS1^T, JS5, JS11 and JS15 are KC958552, KF772607, KF772610 and KF772608 respectively.

Description of Anoxybacillus suryakundensis sp. nov

Anoxybacillus suryakundensis (sur.ya.kund.en’sis. N.L. masc. adj. suryakundensis pertaining to Suryakund in Jharkhand, India, the geographical origin of isolation of the type strain).

Cells are non motile, straight rod of length 1-4 µm and diameter 0.3-0.8 µm. Occurs single or in pair. Facultative anaerobe. Gram-positive. Endospores are spherical, located centrally or terminally and slightly swell sporangia. Aerobically grown colonies on TSA are pale yellow, rough surface, round with an uneven edge of diameter 1-2 mm. Moderately thermophilic and alkalitolerant. Grows at 40-60°C and pH 5.5-11.5 and can tolerate 3.5% NaCl. Optimum growth was observed at 55°C and pH 7.5. Catalase and oxidase positive. Positive for methyl red test and hydrolysis of gelatin, starch, esculin and DNA. Negative for reduction of nitrate, lysine decarboxylase, arginine dihydrolase, ornithine decarboxylase, phenylalanie deaminase, H₂S production, indole, Voges Proskauer and hydrolysis of ONPG and urea. Acid is produced from oxidation of glucose, sucrose, fructose, trehalose, mannose, mannitol, lactose, maltose and starch. Negative for utilization of raffinose, galactose, xylose, rhamnose, malonate, citrate and arabinose. Cell wall contains glucose as sole sugar and DAP is of meso type. The major fatty acids are C_16:0iso, C_15:0iso, C_{17:1anteisow9c} and C_17:0iso. Polar lipids include a phosphatidylgylycerol (PG), a diphosphatidylglycerol (DPG), a phosphatidylethnolamine (PE), a phosphatidylcholine (PC), a phosphatidyl monomethylethanolamine (PMME) and four unidentified lipids (L1-4). The G+C content of DNA is 42.1 mol%. Anoxybacillus suryakundensis strain JS1^T, was isolated from a hot spring at Suryakund, Jharkhand, India.