Computational protocol: Occurrence of Horizontal Gene Transfer of PIB-type ATPase Genes among Bacteria Isolated from the Uranium Rich Deposit of Domiasiat in North East India

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Protocol publication

[…] Amplicons of PIB-type ATPase genes (zntA/cadA pbrA-like) of the representative 16 isolates from different phyla were purified using the QIAquick Gel Extraction Spin Kit (QIAGEN, Germany) and sequenced using the Big Dye Terminator cycle sequencing kit v.3.1 (Applied Biosystems, USA) deploying the standard protocol and an automated Genetic Analyzer ABI 3130XL (Applied Biosystems, USA). The Basic Local Alignment Search Tool (BLAST, sub-program BLASTX) was used to determine the phylogenetic neighbours of PIB-type ATPase genes against the GenBank database (National Center for Biotechnology Information, Bethesda, USA). Molecular Evolutionary Genetics Analysis software (MEGA v4) was used for phylogenetic analyses . The sequences of identified phylogenetic neighbours were aligned with the sequences using ClustalW of MEGA4. The Escherichia coli kdpB gene was used as outlier. For phylogenetic tree construction we used the neighbor joining method with 1000 bootstrap replications for nodal support. Phylogenetic analyses based on the maximum likelihood and maximum parsimony of PIB-type ATPase amino acid sequences were in agreement with the data generated by the above described neighbour joining method. On average, 600 nucleotides were included in the phylogenetic analyses of the PIB-type ATPase sequences. The 16S rRNA gene sequences of the representative bacteria were amplified and sequenced as previously described . Phylogenetic neighbours were obtained against the database of type strains with validly published prokaryotic names (available online http://eztaxon-e.ezbiocloud.net/) . Phylogenetic analyses by MEGA4 were performed using an average of 1200 nucleotides of 16S rRNA encoding DNA sequence . The 16S rRNA gene sequence of Deinococcus radiodurans M21413 was taken as an outlier. The phylogenetic tree was constructed using the scale bars of 0.05 change per nucleotide position for the 16S rRNA gene and 0.1 change per amino acid position for PIB-type ATPase.The G+C content of each zntA/cadA/pbrA-like amplicon was calculated by using Oligo Calculator available at http://mcb.berkeley.edu/labs and were compared to the G+C contents of all other organisms belonging to the same genus. […]

Pipeline specifications

Software tools BLASTN, BLASTX, MEGA, MEGA-V, Clustal W
Applications Phylogenetics, GWAS
Chemicals Uranium