Biogeography and interactions among Bacterial, microeukaryotic and T4-like viral assemblages in lakes from both polar zones. Microbial assemblages in polar lakes

Samples were taken from freshwater bodies in the Arctic (Spitsbergen Island, Svalbard archipelago, Norway), and along the Antarctic Peninsula. Ninety liters from each water body were pre-filtered through a 30 μm nylon mesh. Subsequent filtration by 0.45μm tangential flow filtration (TFF) using a Centramate holder (Pall) separated the free viral community (defined as the <0.45μm fraction) from the cellular (and associated virus) fraction (defined as the fraction between 0.45 and 30 μm). Viral fractions were subsequently concentrated 100 times by 70-kDa TFF. All stocks were preserved at -20ºC/-80ºC prior to DNA extraction. Cellular fraction community DNA was extracted using PowerSoil DNA isolation kit. Viral DNA was obtained from frozen stocks thawed at 4ºC, and passed through a 25% sucrose cushion by centrifugation for 16 h at 60 000 g and 4ºC. The pellets were re-suspended in 10 mM Tris pH 8, 1 mM EDTA, and filtered using a 0.45 μm syringe filter. Viral concentrates were then treated with DNAse I (500 U.ml-1), Nuclease S7 (420 U.ml-1), RNAse A (100 μg.ml-1) and RNAse H (2 U per reaction) for 30 min at room temperature to remove free nucleic acids. Nuclease reactions were stopped with 12 mM EDTA/2 mM EGTA, and viral capsids and envelopes were then disrupted with SDS (0.5%) and proteinase K (200 μg.ml-1) treatment. Finally, viral DNA was extracted with phenol-chloroform and ethanol precipitated. Bacterial 16S rRNA marker genes were amplified from the cellular fractions using primers 8F15B and 515R14AM. All PCR reactions were carried out using 1μl of template DNA, 0.5 μl Phusion High-fidelity polymerase, 20 nmol dNTPs, 20 pmol of each primer, 1.5μl DMSO, 0.4 mM MgCl2, in a final volume of 50 μl. Reaction conditions included an initial denaturation step of 30s at 98ºC, followed by 25 cycles of 10s at 98ºC, 30s at 53ºC, 30s at 72ºC, and a final elongation step of 5 min at 72ºC. For the analysis of the microeukaryotic and T4-like viral assemblages we followed a two-step barcoding strategy that allows a universal set of bar-coded sequencing primers to be appended to an amplified PCR product without introducing discernible biases. In the first step, one of the target-specific primers is modified to include a linker sequence. After amplification, a second primer consisting of the bar code and linker is used to tag the amplicon. The eukaryotic community in the cellular fractions was analyzed using primers targeting the 18S rRNA gene modified from those described by Bates et al. (F515; 5′-GTGCCAGCMGCCGCGGTAA-3′ and R1119 5′-GGTGCCCTTCCGTCA-3′),. The T4-like virus communities in both cellular and free viral fractions were assessed using primers modified from those reported by Filée et al. targeting the g23 major capsid protein gene (MZIA1bis 5′-GATATTTGIGGIGTTCAGCCIATGA-3′ and MZIA6 5′-CGCGGTTGATTTCCAGCATGATTTC-3′). In this case, proper amplification for all samples proved difficult. Nevertheless, we were able in most cases to produce the desired products performing an initial amplification using the unmodified primers (45 cycles, melting temperature of 50ºC), followed by agarose gel extraction of DNA bands of appropriate size. The resulting products were re-amplified using the modified primers (10 cycles), at this point linking with the two-steps protocol. Unfortunately, we were unable to produce reliable g23 sequence data for the free virus fraction samples from Antarctic sites Domo, Cierva, and Refugio (in the latter, amplification from the cellular fraction also failed), and hence these sites were removed from some subsequent analyses. Final concentrations of PCR products were measured using a PicoGreen dsDNA Assay Kit, equal amounts for each sample pooled, agarose gel-extracted using the QIAquick Gel Extraction Kit, and sequenced using a Roche 454 FLX sequencer with titanium chemistry.