Identification of arsenic tolerant NR5
Genomic DNA of NR5 was extracted using Nucleopore bacteria DNA extraction kit (Genetix Biotech, India), and isolated DNA was visualized in 0.8% agarose gel to ensure the proper extraction and purification. Yield and purity of extracted DNA was also checked by Nabi-UV/Visible Nano Spectrophotometer (MicroDigital, Korea) by measuring absorption ratio of A260/A230, and A260/A280. Subsequently, 16S rRNA gene was amplified in extracted genomic DNA through universal primers 27F (5’ AGAGTTTGATCCTGGCTCAG 3’) and 1542R (5’ AAGGAGGTGATCCAGCCGCA 3’). A 25 µL reaction mixture was prepared using 1.0 µL of 100pM concertation of forward and reverse primers by Integrated DNA Technologies (IDT, Coralville, IA), dNTPs (200nM), and 2 µl of 100 ng template. In MyCycler (BioRad thermal cycler). PCR reaction was setup with the conditions - initial denaturation 95°C for 5 minutes, 35 cycles of denaturation at 95°C for 30 seconds, 52°C for 30 seconds and 72°C for 1 minute and final extension at 72 °C for 5 minutes. The amplified product was visualized on 1.5% agarose gel, and the bands obtained were compared to standard 1Kb DNA ladder (genetic biotech, India). The amplified band was further purified through Nucleopore PCR clean up kit (NP-36105, Genetix Biotech, India) as per protocol provided by the manufacturer. Sequencing PCR was performed with universal primers as mentioned above and Big Dye Terminator v3.1 cycle sequencing kit (Applied Biosystems, USA) in a 3130×l Genetic Analyzer (Applied Biosystems, USA). The amplicon sequence obtained from ABI sequencer were analyzed for quality check and contig was prepared manually using Finch TV software (Geospiza, Inc). Sequence similarity was carried out by EZBioCloud database and also analyzed by NCBI-BLASTn algorithm (Yoon et al. 2017). Maximum percent identity score of first 20 sequence were selected and used for multiple sequence alignment using EZEditor2 (Jeon et al. 2014). Subsequently phylogenetic tree was constructed by Neighbor-Joining method with 1000 bootstrap value in MEGA X (Kumar et al. 2018). 16S rRNA gene sequence of the bacterium was submitted to NCBI GenBank, and well-characterized strain was submitted to ICAR-National Agriculturally Important Microorganisms Culture Collection (NAIMCC), Mau, India.