Bacterial 16S rRNA Gene Sequencing Protocol

Genomic DNA was extracted from the bacterial strain according to the manufacturer’s instructions using an InstaGene Matrix kit (Bio-Rad, Hercules, CA, USA) for the polymerase chain reaction (PCR) amplification of the 16S rRNA gene. A pair of universal primers, 27F and 1492R, was used to amplify the 16S rRNA gene [25 (link)]. The PCR products were purified using a multiscreen filter plate (Millipore Corp, Bedford, MA, USA) and sequenced using the primers 518F (5′-CCA GCA GCC GCG GTA ATA CG-3′) and 800R (5′-TAC CAG GGT ATC TAA TCC-3′), with a PRISM BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA). The nearly full-length 16S rRNA sequence was assembled using SeqMan software (DNASTAR Inc., Madison, WI, USA). Sequence similarity was determined by comparison with existing sequences available in the GenBank database using the EZBioCloud server [26 (link)]. The MEGA 7 program was used to align sequences and reconstruct phylogenetic trees based on the BLAST results [25 (link),27 (link)].