Computational protocol: Naphthalene biodegradation under oxygen‐limiting conditions: community dynamics and the relevance of biofilm‐forming capacity

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Protocol publication

[…] DNA extraction for the initial environmental sample was performed as previously described (Martirani‐Von Abercron et al., ). For the enrichment cultures and isolated strains, genomic DNA was purified using the Wizard Genomic DNA Purification Kit as recommended by the manufacturer (Promega, Madison, WI, USA). Nucleic acid quantity and purity were determined with a NanoDrop ND‐1000 Spectrophotometer (NanoDrop Technologies Llc, Wilmington, Delaware, USA). A multiplex pyrosequencing amplicon approach was used for the characterization of the bacterial communities. The PCR amplifications of the hypervariable V1–V3 region of the 16S rRNA gene were carried out using the bacterial universal primers 6F and 532R containing 5′ tags with multiplex identifier and sequencing adapters (Table ). PCR amplifications were performed in 50 μl reactions containing 1× PCR Buffer (Bio‐Rad, Hercules, California), 200 μM dNTPs (Roche, Basel, Switzerland), 0.5 μM of each primer, 1 U of iProof™ High‐Fidelity DNA Polymerase (Bio‐Rad) and 20 ng of target DNA. The PCR program consisted of an initial denaturation step at 98°C for 30 s, followed by 25 cycles at 98°C for 10 s, 50°C for 20 s and 72°C for 30 s, with a final extension at 72°C for 5 min. PCRs were cleaned up using the Qiaquick MiniElute columns (Qiagen, Hilden, Germany) and checked in 1.5% agarose gel. Amplicon products were quantified using Qubit™ fluorometer (Invitrogen, Carlsbad, California), pooled at an equimolar ratio and sequenced using a 454 titanium amplicon sequencing kit and a Genome Sequencer FLX 454 at either Citius (University of Seville) or Macrogen (Korea). Analysis of the 16S 454 pyrosequencing data was performed following the Quantitative Insights Into Microbial Ecology (qiime v. 1.9.0) pipeline (Caporaso et al., ). Demultiplexing, primer removal, quality‐filtering, chimeras and singletons removal, and alpha‐ and beta‐diversity studies were carried out as defined in Martirani‐Von Abercron et al. (). [...] Gene fragments of bssA, ncr, nmsA, nahA, dbd and nod were amplified by PCR using available primer sets and the cycling conditions originally described (Table ). Genomic DNA from NaphS2 and Thauera aromatica K172 strains was used as a positive control for nmsA and ncrA, and bssA amplification, respectively. All PCRs were performed in 50 μl containing 1× PCR Buffer (Bio‐Rad), 200 μM dNTPs (Roche), 0.5 μM of each primer (Sigma‐Aldrich, St. Louis, Missouri, USA), 1 U of iProof™ High‐Fidelity DNA Polymerase (Bio‐Rad), and 10–20 ng of target DNA. The appropriately sized amplicons were purified and cloned in pCR2.1 (TA Cloning Kit; Invitrogen), and 16 positive clones for each gene were Sanger‐sequenced (IPBLN, CSIC, Granada, Spain). In addition to the available primers, six naphthalene dioxygenase (ndo) degenerated primers (Table ) were designed using the CODEHOP (COnsensus‐DEgenerate Hybrid Oligonucleotide Primers) software (; Rose et al., ) based on the alignment 29 sequences of dioxygenase proteins of different species (Table ). A gradient PCR was performed with annealing temperatures ranging from 50°C to 63°C for all primer combinations to determine optimal annealing temperatures. Touchdown PCR was used to avoid amplifying non‐specific sequences: after 1 min of denaturation at 98°C, the first 10 thermal cycles were 10 s at 98°C, 15 s at 63°C to 53°C (the annealing temperature was reduced 1°C per cycle from 63°C to 53°C) and 20 s at 72°C. The remaining 20 cycles were 98°C for 10 s, 55°C for 15 s and 72°C for 20 s, with the last cycle followed by a 5‐min extension at 72°C. Genomic DNA from P. putida KT2440 (NAH7; Fernández et al., ) was used as positive control. […]

Pipeline specifications

Software tools QIIME, CODEHOP
Applications Amplicon sequencing analysis, qPCR
Organisms Bacteria