) and the videotest-razmer 5.0 software package (http://www.videotest.ru). The biomass was estimated from the average cell volume and abundance. For each station, a sample series, taken along the vertical line (0, 5, 10, 15, 25 and 50 m), was counted as a weighed arithmetic mean for 0–25 and 0–50-m layers. For T4-phage detection,

the water samples (500 mL) from depths between 5 and 10 m were used. The samples were filtered sequentially. Most organisms and particles larger than viruses were removed by filtration through polycarbonate filters (Millipore) with pore diameters of 1.2, 0.45 and 0.22 μm. The filtered subsamples (100 mL) were then concentrated on 0.02-μm Anopore Inorganic Membranes (Whatman). DNA was extracted from 0.02-μm filters using a DNA-sorb kit (InterLabService,

Russia) according to the manufacturer’s protocol. Degenerate g23 primers, MZIA1bis and MZIA6, were used for PCR amplification (Filée et al., 2005). selleck screening library PCR was performed using Amplisens kit (InterLabService). Two microliters of DNA template GDC-0980 mw was added to 8 μL of PCR mixture containing 1.5 mM MgCl2, 0.20 mM concentration of each deoxyribonucleoside triphosphate, 20 pmol each of the primers and 1.0 U of Taq polymerase. PCRs were performed as described by Filée et al. (2005). Amplicons were initially visualized by 4% acrylamide gel electrophoresis, followed by silver staining. Bands of the appropriate molecular mass were excised from gels, rinsed in plenty of water and frozen with 50 μL water. Water extracts were used as the DNA template for PCR. All of the reaction mixtures and conditions were the same as those in the

very first amplification, except that the PCR reaction volume was 50 μL. Purification of DNA fragments was performed by 0.8% agarose gel electrophoresis in 0.5 × TAE buffer (20 mM Tris-acetate, 5 mM EDTA, pH 8.0). PCR products were extracted by freezing agarose plugs, which contained the band, followed by centrifugation. The amplified DNA fragments were cloned using the InsTAclone kit (Fermentas). The positive clones were sequenced by the CEQ 8800 sequencer (Beckman Coulter). Sequences were aligned and formatted using clustal w software bioedit (v7.0.5) (Hall, 1999) and corrected manually with the help of the maximum-parsimony software (mega 4) (Tamura et al., 2007). Translated sequences were analyzed for the closest relatives by a blast search on the NCBI web site. The alignment sequences were compared with g23 fragments of known T4 phages obtained from the T4-like genome database (http://phage.bioc.tulane.edu) and with g23 clones of uncultured viruses of different origins. Phylogenetic trees were reconstructed with the Bayesian inference method using mrbayes v3.1.2 (Huelsenbeck & Ronquist, 2001). An appropriate model of amino acid substitution was selected previously by the prottest v2.4 program (Abascal et al., 2005) using the Bayesian information criterion.