Molecular characterization
Genomic DNA extraction.
Genomic DNA extraction was carried out by colony polymerase chain reaction
(PCR) method. Colony from fresh bacterial culture was properly mixed in 20 ml tris(hydroxymethyl) aminomethane–ethylenediaminetetraacetic acid buffer and heated at 95C for 10 min in a PCR machine.
After PCR, centrifugation was carried out at
8000 r/min for 5 min, and the supernatant was taken as genomic DNA.
PCR amplification of 16S rRNA gene.
For 16S ribosomal RNA (rRNA) amplification of isolated bacterial strains, oligonucleotide primers, 9F (GAGTTTGAT CCTGGCTCAG) and 1510R (GGCTACCTTGTTACGA) were used as described by Ahmed et al. (2007). The DNA template without inoculation was used as the negative control. Thermal cycling was performed by initial denaturation at 94C for 2 min, 35 cycles of denaturation at 94C for 1 min, annealing at 50C for 1 min, and elongation at 72C for 1 min. Cycling was done at final elongation step at 72C for 10 min. The PCR amplified products were separated on 0.8% agar-
ose gel and observed under gel doc system.
Phylogenetic analysis.
The amplified PCR products of 16S rRNA gene of isolated bacterial strains were sequenced by primers 27F (50-AGAGTTTGATCMTGGCTCAG-30) and 1492R (50-ACCTTGTTAC-GACTT-30) using a commercial service of Macrogen Inc. (Korea; www.dna.macrogen.com)). The sequences obtained were compared with the sequences present in the DNA DataBank of Japan (DDBJ)/the European Molecular Biology Laboratory (EMBL)/GenBank databases by the BLAST program of the National Centre of Biotechnology Information. The sequences of strains were also retrieved for constructing the phylogenetic trees. Phylogenetic analysis was done by MEGA version 5. Phylogenetic tree was made from unambiguously arranged nucleotides using an algorithm (Saitouand Nei, 1987).