Bacterial strains were isolated from Tao Dam hot spring, Khluang Wangchao National Park, Thailand, which is located in a remote region of Kampangpetch province. The spring was discovered recently and contains mineral (sulfide-rich) water with a surface temperature range of 50–75 °C and pH 8.0. The sediment was 60 °C at the time of sampling. The bacteria were isolated using tenfold serial dilutions of sediment in phosphate buffer (0.1 M K₂HPO₄ and 0.1 M KH₂PO₄, pH 8). The dilutions were spread on R2A agar (Difco, NJ, USA), and the plates were incubated overnight at 55 °C.

Bacterial isolates and the type strain of A. pallidus DSM 3670 were grown on R2A agar which contained one percent of AZ rhamnogalacturonan (Megazyme, Ireland). The plates were incubated overnight at 55 °C. A clear halo zone should appear when pectate lyase is produced. Positive clones were inoculated in 100 mL of M9 broth (pH 8) plus 1 g sodium pectate (Sigma, Germany). The flask was incubated at 55 °C with shaking at 150 rpm overnight. Bacterial cells were removed by centrifugation at 8,000 rpm for 10 min, and the supernatant was collected for the photometric assay, in which 0.5 mL of supernatant was added to 2.5 mL of substrate (60 mM Tris-HCl, pH 8.0, 0.6 mM CaCl₂, and 0.24% (w/v) sodium pectate). The reaction was incubated at 55 °C for 2 h. Pectate lyase activity was measured on a Genesys 10 uv spectrophotometer (Thermo Fisher Scientific, Madison, WI, USA) at 232 nm as described by Collmer and colleagues [19]. Pectate lyase from Aspergillus sp. (Megazyme) was used as the positive control, and sterile water and substrate were used as the negative control.

The morphology was examined under a Zeiss Gemini DSM852 field emission scanning electron microscope (Zeiss, Oberkochen, Germany) and Zeiss transmission electron microscope TEM910 (ProScan, 1024 × 1024, Scheuring, Germany). To prepare the samples for field emission scanning electron microscopy, the samples were fixed with a solution containing 2% glutaraldehyde and 5% formaldehyde in cacodylate buffer (0.1 M cacodylate, 0.09 M sucrose, 0.01 M MgCl₂, and 0.01 M CaCl₂, pH 6.9) for 1 h on ice and then washed with TE buffer (20 mM Tris and 2 mM EDTA, pH 6.9). The fixed bacterial cells were placed onto poly-l-lysine-coated cover slips, left for 5 min, washed once in TE buffer, and fixed with 2% glutaraldehyde. After washing with TE buffer, the samples were dehydrated with a graded series of acetone (10%, 30%, 50%, 70%, 90%, 100%) for 10 min at each step on ice, except that the final 100% acetone step was performed at room temperature. Samples were critical-point dried with liquid CO₂ (CPD 030, Bal-Tec, Liechtenstein), mounted onto aluminum stubs, sputter coated with gold (SCD 500, Bal-Tec), and examined in the field emission scanning electron microscope at an acceleration voltage of 5 kV using an Everhart–Thornley SE-detector and in-lens SE-detector at a 50:50 ratio. Images were stored onto a 230 MB MO-disk.

To embed the bacteria for transmission electron microscopy, the samples were fixed in a fixation solution containing 5% formaldehyde and 2% glutaraldehyde in cacodylate buffer (0.1 M cacodylate, 0.01 M CaCl₂, 0.01 M MgCl₂, 0.09 M sucrose, pH 6.9), washed with cacodylate buffer, fixed with 1% aqueous osmium for 1 h at room temperature, and washed. The pellets were embedded in 2% water agar and cut into small cubes. The samples were dehydrated in a graded series of acetone (10%, 20%, 50%) for 30 min on ice followed by contrast staining with 2% uranyl acetate in 70% acetone overnight at 4 °C, and the samples were dehydrated further with 90% and 100% acetone. At the 100% acetone step, the samples were allowed to reach room temperature and were then infiltrated with the epoxy resin according to Spurr’s formula for a medium resin [20]: 1 part 100% acetone/1 part resin overnight; 1 part 100% acetone/2 parts resin for 8 h; and pure resin overnight and several changes in the following 2 d. The samples were then transferred to resin-filled gelatin capsules and polymerized for 8 h at 75 °C. Ultrathin sections were cut with a diamond knife, picked up onto Formvar-coated copper grids (300 mesh), and counterstained with 2% aqueous uranyl acetate and lead citrate. After air-drying, the samples were examined in a Zeiss TEM910 transmission electron microscope at an acceleration voltage of 80 kV. The images were recorded digitally with a Slow-Scan CCD-Camera (ProScan, 1024 × 1024, Scheuring, Germany) with ITEM-Software (Olympus Soft Imaging Solutions, Germany).

The temperatures for growth of the strain TD1 were examined from 30–70 °C on R2A medium (Difco, NJ, USA). The salt tolerance was also assessed on R2A medium with 0–10% (w/v) NaCl at 55 °C. The phenotype of the strain TD1 was investigated using API 20E and API 50 CHB tests (bioMérieux, Nürtingen, Germany) at 55 °C, according to the manufacturer’s instructions.

Analysis of cellular fatty acids was performed at the German Collection of Microorganisms and Cell Cultures (DSMZ) (Braunschweig, Germany) according to the Sherlock Microbial Identification System manual (version 4.5; Microbial ID, Newark, DE). The profiles of cellular fatty acids were compared using the TSBA40 library database version 4.10 (Microbial ID, Newark, DE, USA).

DNA was extracted using MasterPure™ Gram Positive DNA Purification Kit (Biozym Scientific, Germany) according to the manufacturer’s instructions. The genomic G+C ratio was examined using HPLC at DSMZ. Primers 27f (5′-AGAGTTTGATCMTGGCTCAG-3′) and 1492r (5′-TACGGYTACCTTGTTACGACTT-3′) [21] were used for bacterial 16S rDNA amplification. The 50 μL total volume of PCR reaction contained 10 ng of DNA, 20 pmol of each primer, 80 μM dNTP, 1 × Taq reaction buffer, and 1.25 U Taq DNA polymerase (EURx, Poland). The PCR was started by heating the reactions to 94 °C for 3 min, followed by 30 cycles of 30 s at 94 °C, 30 sec at 52 °C, and 1 min at 72 °C, and ending with a final step at 72 °C for 3 min. The PCR product was purified using a MinElute PCR Purification Kit (QIAGEN, Germany). DNA sequencing was performed directly on the purified PCR product. The sequencing primers were 27f and 1492r. Big Dye v. 1.1 reagents (Applied Biosystems, Inc.) were used for the sequencing, and the analysis was performed on an ABI Genetic Analyzer sequencer (AB3130XL). The sequence was compared with the nonredundant database of sequences deposited at GenBank using BLAST [22]. 16S rDNA of the strain TD1 was aligned with other bacterial strains in the genera Geobacillus and Aeribacillus using MUSCLE 3.7 [23]. The size of the 16S rDNA for the alignment was about 1,500 bp. The evolutionary model was calculated using Modeltest 3.8 [24]. Bayesian inference was performed using MrBayes 3.1.2 [25]. The Bayesian posterior probabilities were obtained by performing two separate runs with four Markov chains. Each run was conducted with 2 × 10⁶ generations and sampled every 100 generations. Convergence was checked by examining the generation plot visualized with TRACER 1.4 [26]. Potential scale reduction factor was computed by MrBayes 3.1.2. A consensus tree was calculated after discarding the first 25% of the iterations as burn-in.