Computational protocol: Roles of thermophilic thiosulfate-reducing bacteria and methanogenic archaea in the biocorrosion of oil pipelines

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Protocol publication

[…] The highest dilution MPN tubes in which all three replicates were scored positive for sulfide production and the resulting enrichments that showed electron acceptor loss and sulfide production above controls were chosen for DNA analyses. Five milliliters of each culture was centrifuged for 15 min at 6000 × g. The cell pellet was washed twice in 1 ml of a sterile phosphate-buffered saline (PBS) followed by centrifugation for 3 min at 8000 × g. The final pellet was resuspended in 500 μl of sterile PBS solution and used immediately for DNA extraction or frozen at −20°C.DNA was extracted from 250-μl samples using the PowerSoil DNA Isolation Kit (MOBIO Laboratories). Due to low cell biomass, two sets of tubes per sample were pooled onto one spin filter in order to achieve a higher DNA concentration. The DNA was then extracted from the spin filter into 75 μl sterile molecular biology-grade water (Eppendorf, Hamburg, Germany) and stored at −20°C until needed.The bacterial 16S rRNA gene was amplified using the forward bacterial primer GM5F (5′-CCTACGGGAGGCAGCAG-3′) with a GC clamp (Muyzer et al., ) and the universal reverse primer D907R (5′-CCGTCAATTCCTTTRAGTTT-3′) (Amann et al., ). Archaeal amplification was conducted using the forward primer Arc333 with GC clamp and the reverse primer Arc958R (Struchtemeyer et al., ). Thermal cycling of all PCR reactions was performed with a “touchdown” PCR method (Muyzer et al., ). Denaturing gradient gel electrophoresis (DGGE) was then performed using the PCR-amplified bacterial and archaeal 16S rRNA gene products. The protocol described by Muyzer et al. () was modified by using a 6% polyacrylamide gel and a 40–80% denaturing gradient, and running the gel in 1X Tris-acetate-EDTA (TAE) buffer solution for 16 h at 65 V at a constant 60°C temperature (Muyzer et al., ). The DGGE gels were stained with SYBR Safe and photographed under UV light. Bands of interest were excised and suspended in 30 μl of PCR-grade water; 5 μl of the excised DNA solution was used as template DNA for PCR reactions as described above. The 16S rRNA gene PCR products amplified from the excised DGGE bands were then sequenced at the Oklahoma Medical Research Foundation (Oklahoma City, OK) on an ABI 3730 capillary sequencer using the primers mentioned above.Bacterial 16S rRNA gene clone libraries were prepared from DNA extracted using the PowerSoil DNA Isolation Kit from thiosulfate-using and methanogenic enrichments that had been transferred at least four times. Fragments of nearly full length 16S rRNA gene were generated by PCR amplification using the primers 8F and 1492R (Stackebrandt and Goodfellow, ) and cloned into Escherichia coli using the TOPO TA Cloning Kit for Sequencing (Invitrogen Corp., Carlsbad, CA). Transformed colonies were picked and transferred to 96-well microtiter plates containing Luria-Bertani broth with 25% (v/v) glycerol and 50 μ g·ml−1 kanamycin, and grown overnight at 37°C. Plasmid DNA was purified from the transformed cells and sequenced using the M13F and M13R vector flanking regions as priming sites on an ABI 3730 capillary sequencer (Microgen: Laboratory for Genomics and Bioinformatics, Oklahoma City, OK).The consensus bacterial 16S rRNA gene sequences were obtained using Sequencher version 5.1 (Gene Codes, Ann Arbor, MI). The software package mothur v.1.30.2 (Schloss et al., ) was used to remove vector and chimera sequences, and define operational taxonomic units (OTUs) at a 97% identity level. A sequence representing each OTU was chosen and compared to previously reported 16S rRNA gene sequences using the BLASTN version 2.2.21+ (Zhang et al., ). A naïve Bayesian rRNA classifier (Wang et al., ) based on a 80% confidence threshold available through the RDP was used to assign phylogenetically consistent taxonomy to each representative sequence. Each representative sequence was rescreened to exclude chimeras using Pintail (Ashelford et al., ). Sequences that most closely matched the representative OTU sequence and selected outgroup sequences were aligned with CLUSTALX (version 1.81) (Thompson et al., ). Tree construction was performed using the Neighbor-Joining method (Saitou and Nei, ) with bootstrap values of 70% or greater used to determine each clade based on 1000 replicates (Felsenstein, ). Each of the four bacterial 16S rRNA gene sequence libraries contained between 16 and 21 sequences, for a total of 74 near full-length sequences (approximately 1480 bp). […]

Pipeline specifications

Software tools Sequencher, mothur, BLASTN, Clustal W
Databases MicroGen
Application 16S rRNA-seq analysis
Organisms Sus scrofa, Saccharomyces cerevisiae, Bacteria, Homo sapiens
Chemicals Carbon, Iron, Sulfur