Computational protocol: Novel Methanotrophs of the Family Methylococcaceae from Different Geographical Regions and Habitats

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Protocol publication

[…] Genomic DNA was isolated using the GenElute Bacterial Genomic DNA kit (Sigma-Aldrich). The 16S rRNA genes were amplified as described previously [,] using a Veriti 96 well thermal cycler (Applied Biosystems, Carlsbad, CA, USA). The PCR products were purified and sequenced using the BigDye kit for automated DNA sequencers (ABI 3700 PE; Applied Biosystems). The genes encoding particulate methane monooxygenase (pmoA), soluble methane monooxygenase (mmoX), methanol dehydrogenase (mxaF) and dinitrogenase reductase H (nifH), as well as the ribulose-1,5-bisphosphate carboxylase/oxygenase (cbbL) were also PCR amplified from genomic DNA using the primers listed in . The PCR was performed using Dynazyme™ High-Fidelity DNA Polymerase (Finnzymes, Finland) and the following PCR program: initial denaturation at 95 °C for 5 min, 95 °C for 1 min, annealing at 55 °C for 1 min, extension at 72 °C for 1 min (30 cycles) and a final extension at 72 °C for 7 min. The reactions were performed in 50µL mixtures in 0.3mL microtubes. Methylococcus capsulatus strain Bath DNA and MilliQ water were used as the positive control and the negative control, respectively. After sequencing analysis, the 16S rRNA gene and the deduced PmoA amino acid sequences [] were compared to available sequences in the GenBank database using the NCBI tools, BLASTn and BLASTp. 16S rRNA gene sequences and deduced amino acid sequences of PmoA were aligned using the CLUSTAL W algorithm as implemented in the MEGA6 software package []. To confirm phylogenetic affiliation, trees of the 16S rRNA gene sequences were constructed using the neighbour joining, maximum likelihood, minimum evolution and maximum parsimony methods, and evolutionary distances were computed using Kimura 2-parameter model, Jones–Taylor/Thornton method or the Dayhoff model, which are also implemented in MEGA6 software. The tree topologies were decided by 1000 bootstrap replications. […]

Pipeline specifications

Software tools BLASTN, BLASTP, Clustal W, MEGA
Application Phylogenetics
Organisms Oryza sativa, Bovine orthopneumovirus
Chemicals Methane