Intracellular DNA recovery for molecular analysis

The prokaryotic cells contained in the 10-μm pre-filtered source-seawater were concentrated onto 0.05 µm pore size polycarbonate filters (Sterlitech). The corresponding aerosol samples generated during the bubble-bursting experiment were obtained by pooling all quartz filters of the different pore sizes (0.05–10 μm). The DNA contained in the prokaryotic cells collected on the filters was extracted and purified using the QIAamp DNA Micro Kit (QIAGEN). This kit was preferred based on preliminary tests of DNA extractions from quartz filters, indicating higher DNA yields than with the MoBio UltraClean Microbial DNA isolation kit (Supplementary Figure ⁴). Before DNA extraction, the seawater and aerosol samples were treated with DNAse I (5U ml⁻¹) to remove any possible extracellular DNA contamination⁷¹. The purified extracts of intracellular DNA were then analysed by molecular fingerprinting (through ARISA - Automated Ribosomal Intergenic Spacer Analysis)⁷² to provide information on the bacterial diversity in seawater and in the corresponding produced aerosols.

For ARISA, the purified intracellular DNA was amplified using universal bacterial primers 16S-1392F (5′-GYACACACCGCCCGT-3′) and 23S-125R (5′-GGGTTBCCCCATTCRG-3′). This allowed the amplification of the ITS1 region in the rRNA operon plus ~282 bases of the 16 S and 23 S rRNA⁷². Primer 23S-125R was fluorescently labelled with the fluorochrome HEX (MWGspa BIOTECH). PCR reactions were performed in 50 µl volumes in a thermalcycler (Biometra) using the MasterTaq^® kit (Eppendorf), which reduces the effects of PCR-inhibiting contaminants. We used 30 PCR-cycles, consisting of 94 °C for 1 minute, 55 °C for 1 minute and 72 °C for 2 minute, preceded by 3 minutes of denaturation at 94 °C and followed by a final extension of 10 minutes at 72 °C. To check for eventual contamination of the filters, consumables and PCR reagents, negative controls of MoBio extracts from blank filters (i.e., control filters with no seawater or aerosol sample) containing the PCR-reaction mixture were run during each PCR reaction. All the negative controls produced no ARISA amplicons, confirming the lack of contamination and the high confidence of the analytical approach. Positive controls containing genomic DNA of E. coli were used and PCR amplicons were checked on agarose-TBE gel (1%), containing ethidium bromide for DNA staining and visualization. Four different reactions were run for each sample and then combined to form two duplicates, subsequently utilised for independent ARISA reactions. The two resulting PCR combined products were purified using the Wizard PCR clean-up system (Promega, Madison, Wis), resuspended in 50 µl of MilliQ water supplied with the clean-up system and then quantified spectrofluorimetrically as described above. For each ARISA run, 5 ng of purified amplicons were mixed with 14 µl of internal size standard (GS2500-ROX; Applied Biosystems, Foster City, Calif.) in deionised formamide, then denatured at 94 °C for 2 minutes and immediately chilled in ice. Automated detection of ARISA fragments were carried out using ABI Prism 3100 Genetic Analyzer (Applied Biosystems). ARISA fragments were determined using Peakscanner analytical software (ABI) and the results analysed using standardization of fluorescence among samples, elimination of “shoulder” and non-replicated peaks, and cut-off criterion⁷³. Bacterial genotype richness was expressed as the total number of peaks within each electropherogram, while the evenness (Pielou index, J’) was calculated in order to assess the relative importance of each taxon within the entire assemblage.