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emi412062-sup-0001-procedures.doc90K

Supplementary procedures.

emi412062-sup-0002-f1-5.pdf3845K

Fig. S1. Location of Lake Erhai in China and sampling locations in this study. Lake Erhai is located in the south-western Yungui Plateau, China (25°35′–25°58′N, 100°5′–100°17′E; water area = 249.8 km2; length = 42 km; average depth = 10.5 m; capacity = 2.88 × 109 m3). Surface water was sampled at 12 locations covering the whole lake from May to December in 2010. The field plankton biomass used for microcystin analysis was collected by pouring 50–100 l of lake water through plankton net (mesh size 45 μm). Besides, 0.5 l water was filtered through a 0.45 μm filter from which DNA was extracted subsequently. All the field samples were numbered according to sampling locations and months as depicted in Table 1. Microcystis strains isolated from this lake were grown in liquid CT medium (Ichimura, 1979) under a 12 h/12 h light/dark cycle with constant white light intensity of 30 μmol photons m−2 s−1 at 25°C. Microcystis cells of each strain were harvested by centrifugation (12 000 g). Before further processing, all the samples were preserved at −80°C.

Fig. S2. Rarefaction curves based on number of OTUs and Shannon index against the number of sequences for individual samples at the 0.01 (A and C) and 0.02 (B and D) distances respectively.

Fig. S3. Inter-sample rarefaction curves based on number of OTUs against the number of samples at the 0.01 and 0.02 distances respectively.

Fig. S4. Alignment of McyBA1 and McyCA sequences. PCC7806, M. aeruginosa PCC7806. Dots, amino acids identical to PCC7806 McyBA1; dashes, gaps introduced into the alignment; between arrows, probable recombination region; bold lines, short identical sequences.

Fig. S5. Restriction profiles of mcyBA1 fragments from field samples of four locations (N3, M1, M5 and S1). The M5/Aug. sample was lacked. M = DNA marker. mcy = undigested mcyBA1 fragment. EH1, EH9 and EH13 were reference strains belonging to Group IA, Group IB and Group II respectively (Table 3). A primer pair mcyBf1 (5′-TCCCTTCAATCTAACGACTCC-3′)/mcyBr1 (5′-CAAAGGCAGAAGGSACCATATA-3′) was designed targeting inner sequence of the mcyBA1 region. PCR amplification procedures were similar to the descriptions in the main text except an annealing temperature of 58°C. DNA samples from four locations (N3, M1, M5 and S1) and three Microcystis strains were used as templates. The purified products were digested by restriction endonuclease, SmoI (Fermentas, USA), according to the manufacturer's manual. Briefly, about 100–300 ng of purified PCR products were combined with 2 μl of 10× buffer and 5U of enzyme in a 20 μl digest. Reaction mixtures were incubated at 55°C for 16 h and complete digestion was confirmed by the results of reference Microcystis strains using 300 ng of purified PCR products. Thereafter, restriction digests were analysed by electrophoresis on an ethidium-bromide stained agarose gel (1%) in TAE buffer. Consequently, three characteristic fragments, 829 bp (or 853 bp), 494 bp and 666 bp were produced after digestion of the mcyBA1 fragments and representing the three groups (IA, IB and II) of McyBA1 respectively.

emi412062-sup-0003-ts1-4.doc195K

Table S1. Binding pocket residues of McyBA1 domain. Binding pocket residues of McyBA1 domain were identified by alignment with GrsA sequence (Stachelhaus et al., 1999).

Table S2. Paired comparison of samples from different months in the same location and from different locations in the same month.

Table S3. AMOVA analysis for geographic and seasonal changes of McyBA1 diversity.

Table S4. Concentrations of intracellular microcystins in lake water.

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