DNA extraction, PCR amplification for bacterial isolates
The DNA extraction from pure colonies of bacterial isolates was carried by using GenElute™ Bacterial Genomic DNA Kit Protocol, Sigma, following the manufacturer’s protocol. Eluted DNA samples were checked in agarose gel. 16S rRNA gene was amplified in an Eppendorf Mastercycler using primer pair 27f (5’ -AGAGTTTGATYMTGGCTCAG) (24) and 1401r (5’ - CGGTGTGTACAAGACCC) (25). In brief, PCR amplification was performed in a 25 µl reaction mixture containing 1x PCR buffer, 1.5 mM MgCl2, 200 µM dNTPs, 10 pmol of each primer, 1 U of Taq DNA polymerase, and 30–50 ng of gDNA. The cycling conditions were as follows: initial denaturation at 95 °C for 5 min. This was followed by 35 cycles of 95 °C for 30 s, 55 °C for 45 s, 72 °C for 60 s, and a final extension at 72 °C for 10 min. The amplicons were resolved on 1.5 % (w/v) agarose gel containing 0.05 % ethidium bromide along with a 100 bp ladder for size and mass calculation of PCR products and visualized under Chemi-Doc System (BioRad). The amplified PCR products were purified using the Gen-elute PCR cleanup kit (Sigma). The purified PCR products of bacterial gene fragments were sequenced at the 1st Base sequencing company, Singapore using an automated DNA sequencer. Contigs were assembled based on their phred scores (>15) and identified by aligning in the NCBI reference rRNA database using the blastn algorithm.