Computational protocol: Newly Isolated Paenibacillus tyrfis sp. nov., from Malaysian Tropical Peat Swamp Soil with Broad Spectrum Antimicrobial Activity

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[…] Genomic DNA was extracted from a 3 day old culture of strain MSt1T on TSA using GF-1 DNA extraction kit (Vivantis, Malaysia) according to manufacturer's instructions. The almost complete 16S rRNA gene sequence of strain MSt1T (1492 bp; DDBJ/EMBL/GenBank accession number KT216503) was amplified according to Lee et al. () using the primer pairs of 27f and 1492r. Briefly, 16S rRNA gene sequence was amplified using universal primer pair 27f (5′ AGAGTTTGATCMTGGCTCAG 3′) and 1492r (5′ TACGGYTACCTTGTTACGACTT 3′). PCR reaction mixture contained 5 × MyTaq Red Buffer, 1.25 U MyTaq DNA polymerase (Bioline, United Kingdom), 0.5 μM of 27f forward primer, 0.5 μM of 1492r reverse primer and 3 μL of DNA template with the final volume of 50 μL. The thermal cycling profile of initial denaturation at 95°C for 5 min followed by 30 cycles of denaturation at 95°C for 1 min, annealing at 55°C for 1 min and elongation at 72°C for 1 min were used.The resulting 16S rRNA gene sequence was compared to the sequences in the EzTaxon database (http://eztaxon-e.ezbiocloud.net/; Kim et al., ). The 16S rRNA gene sequence of strain MSt1T was aligned with sequences of closely related type species that had been retrieved from the GenBank/EMBL/DDBJ databases using CLUSTAL-X software (Thompson et al., ). The alignment was manually verified and adjusted prior to the construction of the phylogenetic tree using the neighbor-joining algorithm (Saitou and Nei, ) with the MEGA version 6 software (Tamura et al., ). The EzTaxon-e server (http://eztaxon-e.ezbiocloud.net/; Kim et al., ) was used for calculations of sequence similarity level. The stability of the resultant tree topologies were evaluated by using the bootstrap resampling method (1000 resampling) of Felsenstein (). The evolutionary distances were computed using Kimura's two-parameter model (Kimura, ). The 16S rRNA gene sequence of strain MSt1T was deposited at DDBJ/EMBL/GenBank under the accession no. KT216503. [...] The draft genome sequence of strain MSt1T was obtained as described in Aw et al. (). The draft genome of strain MSt1T was deposited at DDBJ/EMBL/GenBank under the accession no. JNVM00000000.As the 16S rRNA gene sequence of strain MSt1T showed more than 97% similarity when compared with its closely related type species, analyses that compare between genomes of closely related species such as ANI and DDH using the draft genome sequence of strain MSt1T were performed. ANI determination was performed at EzGenome (http://www.ezbiocloud.net/ezgenome/ani) as described by Goris et al. () by comparing the draft genome of strain MSt1T to its closely related isolates as determined from the phylogenetic analysis of the 16S rRNA sequences. Estimation of in silico pairwise DDH was performed with genome to genome distance calculator (GGDC 2.0; ; Meier-Kolthoff et al., ). […]

Pipeline specifications

Software tools Clustal W, MEGA, GGDC
Databases DDBJ EzBioCloud
Applications Phylogenetics, WGS analysis
Organisms Homo sapiens, Bacteria, Escherichia coli, Staphylococcus aureus, Candida albicans
Chemicals Fatty Acids, Phosphatidylglycerols, Vitamin K 2