Computational protocol: A Nanoscale Study of Carbon and Nitrogen Fluxes in Mats of Purple Sulfur Bacteria: Implications for Carbon Cycling at the Surface of Coastal Sediments

Similar protocols

Protocol publication

[…] Biomass from three microbial mats was collected close to where the cores were sampled using sterile plastic syringes and transferred in Eppendorf tubes. Samples were immediately frozen in liquid nitrogen and further stored at -80°C until analysis. For DNA extraction, cells were thawed and suspended in DNA lysis buffer (0.75 M sucrose, 50 mM Tris-HCl, pH 8) and processed using the procedure described by . Total RNA was extracted using the RNeasy Mini kit (Qiagen) following the manufacturer’s instructions. Any trace of genomic DNA was removed using a Turbo DNA-free kit (Ambion). DNA removal in RNA samples was confirmed by control PCR amplifications without the reverse transcription step. No amplification was detected in these controls. ThermoScript RT-PCR system (Invitrogen) was used for the reverse transcription of mRNA from total RNA samples. The cDNA synthesis was performed at 50°C using random primers.Small-subunit (16S) rRNA genes were amplified by polymerase chain reaction (PCR) using universal reverse primer 1492R and Bacteria-specific forward primer 8F (). PCR amplification of pufM genes was performed using the primers 557F and 750R designed by . Reaction mixture (25 μL) contained the following components: 5X buffer (10 μl), 2 mM MgCl2, 10 pmoles of each deoxyribonucleotide triphosphate (dATP, dCTP, dGTP, dTTP; Eurogentec), 10 pmoles of each oligonucleotide primer, 2.5 U of GoTaq Flexi DNA polymerase (Promega) and 50 to 100 ng of template DNA. Amplifications were carried out in a GeneAmp PCR system 9700 (Applied Biosystems, Foster City, CA, United States) with the following parameters: 95°C for 5 min, followed by 35 cycles of 95°C for 30 s, annealing at 55°C, respectively, and extension at 72°C for 60 s and 30 s for 16S rRNA and pufM gene amplification, respectively, with a final extension step at 72°C for 10 min. Amplicons were cloned directly or after gel extraction using the TOPO4-TA cloning kit (Invitrogen) according to the manufacturer’s instructions. Clones were sequenced using an ABI 3130 POP7 sequencer (Applied Biosystems) at the Biogenouest Sequencing-Genotyping Platform (Roscoff site).The sequences were trimmed to remove any vector and primer sequences. Chimeras were removed using Uchime (v4.2.40; ). Gene sequences were compared to sequences in public databases with BLASTn (). The 16S rRNA gene sequences were aligned the SILVA SSUref N99 references (v.128) using ARB aligner. The pufM DNA sequences were translated into amino acid sequences and aligned using the MAFFT E-INS-I algorithm. The resulting protein alignment was back-translated to nucleotide acid sequences using pal2nal (). Conservative values of 97% and 94% nucleic acid sequence similarity for 16S rRNA and pufM sequences, respectively, were chosen for clustering sequences into Operational Taxonomic Units (OTUs) using MOTHUR (). Representative sequences (defined as the sequence with the minimum distance to all other sequences in the OTU) were obtained using MOTHUR.A pufM database and a consensus Bayesian tree were built as described in . Representative sequences of each OTU (245 pb) and short pufM environmental reference sequences were aligned as above and added to the backbone tree using the ADD_BY_PARSIMONY algorithm implemented in ARB software (). Likewise, representative ssu sequences of each OTU (927 pb) were added to the SILVA reference tree using ARB. Non-informative taxa were removed from both final tree. […]

Pipeline specifications

Software tools UCHIME, BLASTN, MAFFT, PAL2NAL, mothur, ARB
Applications 16S rRNA-seq analysis, Nucleotide sequence alignment
Chemicals Carbon, Nitrogen, Sulfur, Pyruvic Acid, Ammonium Compounds