Computational protocol: Survival of Desulfotomaculum spores from estuarine sediments after serial autoclaving and high-temperature exposure

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Protocol publication

[…] Genomic DNA was extracted from a Desulfotomaculum sp. C1A60 cell pellet, obtained by centrifugation of a 50-ml culture, using the FastDNA Spin Kit for soil (MP Biomedicals, Santa Ana, CA, USA) and eluted in 100 μl molecular biology grade water (Severn Biotech Ltd, Kiddeminster, UK). The 16S rRNA and dissimilatory sulphite reductase (dsrAB) genes were amplified by PCR with primers 27F/1492R () and DSR1F/DSR4R (), respectively, using GoTaq Flexi DNA polymerase (Promega Corporation, Southampton, UK) and conditions previously described (; ). The dsrAB PCR product was then purified (QIAquick PCR Purification Kit, Qiagen, Manchester, UK), and sequenced directly with primers DSR1F, DSR4R and DSR3R () by Eurofins Genomics (Wolverhampton, UK). However, the 16S rRNA gene could not be sequenced directly because of multiple gene copies. Therefore, triplicate PCR reactions were pooled, purified and concentrated (Amicon Ultra Centrifugal filters; Millipore Ltd, Darmstadt, Germany) before being cloned (pGEM-T Easy Vector System I; Promega Corporation) and transformed into Escherichia coli JM109 competent cells according to the manufacturer's instructions. Colony PCR with transformed cell biomass was then used directly with M13 primers () and individual clone 16S rRNA gene products were sequenced (Eurofins Genomics).All sequence chromatograms were viewed and edited using Chromas Lite software version 2.1.1 (http://www.technelysium.com.au), and 16S rRNA and dsrAB gene consensus sequences were produced from overlapping sequences using BioEdit Sequence Alignment Editor version 7.2.0 (). Closest sequence matches were identified using the nucleotide blast (blastn) suite at the National Center for Biotechnology Information (NCBI; http://www.ncbi.nlm.nih.gov). [...] DNA was extracted from Tyne Estuary sediment slurries using the MoBio PowerSoil DNA Isolation Kit (Cambio Ltd., Cambridge, UK). Extracted DNA was amplified using primers 27F/1492R as above and PCR products were cloned using TOPO TA Cloning Kit (Life Technologies Ltd., Paisley, UK) according to the manufacturer's instructions. Clone inserts were amplified with vector primers pUCF (5′-GTTTTCCCAGTCACGAC-3′) and M13R and sequenced (Genevision, Newcastle Upon Tyne, UK). The 16S rRNA gene sequence from the Tyne Estuary sediment slurries, Desulfotomaculum sp. C1A60 clones and other representative Desulfotomaculum spp., including those used in this study, were aligned using ClustalX2 (), trimmed in BioEdit and phylogenetic trees were constructed using Minimum Evolution and LogDet distance method in MEGA version 6 (). Congruent trees were also obtained using other methods including neighbour-joining with Jukes–Cantor algorithm, maximum likelihood with the Tamura–Nei model and Maximum Parsimony. […]

Pipeline specifications

Software tools Chromas, BioEdit, BLASTN, Clustal W, MEGA
Applications Phylogenetics, Sanger sequencing
Organisms Desulfotomaculum kuznetsovii, Ceraesignum maximum