Every 15 min over a 5.75-h period of time, 2-mL aliquots were withdrawn from each sample and centrifuged for 15 min at 15 000 g at 4 °C. The cell-free supernatant was then titred for phage.

In addition, the light dependence of adsorption of the eight other cyanophages (S-BnM1, S-BP3, S-MM1, S-MM4, S-MM5, S-PWM1, S-PWM3 and S-BM3) to WH7803 and of S-PM2 to Synechococcus strain BL161 was also investigated. In these cases, phage adsorption was assayed at a single time point after 45 min of incubation. In order to determine the influence of light PF-02341066 manufacturer wavelength on cyanophage adsorption, cyanophage S-PM2 was added to samples of cells from cultures of Synechococcus sp. WH7803 (OD750 nm of 0.35–0.40) at an MOI of 0.02. Samples were incubated at 25 °C and illuminated with white light (peak wavelength, 470 nm), blue light (peak wavelength, 420 nm), green light (peak wavelength, 525 nm), yellow light (peak wavelength, 540 nm) or red light (peak wavelength 670 nm). The different light wavelengths

were generated using a Schott KL 1500 LCD Cold Light source (Schott-Fostec, LLC, Auburn, NY) at the same intensity of 15 μE m−2 s−1. Phage adsorption was assayed at 0, 20, 40 and 90 min of incubation as described above. DCMU and CCCP were each dissolved in 50 mL ethanol to a final concentration of 2 × 10−2 M to prepare stock solutions. Working solutions (1 × 10−4 M) were then prepared selleck kinase inhibitor by dilution with L-gulonolactone oxidase ASW. DCMU or CCCP was added to two samples of cells from Synechococcus sp. WH7803 cultures (OD750 nm of 0.35–0.40) to a final concentration of 1 × 10−5 M. These cultures were incubated for 1 h at 25 °C at a light intensity of 15 μE m−2 s−1. S-PM2 was then added to an MOI of 0.02. Phage adsorption was assayed at 0, 30, 60, 120 and 180 min of incubation as described above.

One litre of Synechococcus sp. WH7803 (OD750 nm=0.042) was incubated under a continuously modulated 12–12-h LD cycle at 25 °C for 10 days. When the culture reached OD750 nm=0.5, sampling began and six aliquots were collected at 0.25, 6, 11.75, 12.25, 18.25 and 23.75 h after the light period started (time 0). Cyanophage S-PM2 was then added at an MOI of 0.02. The ‘dark samples’ was wrapped in an aluminium foil to block all light and both samples were incubated at 25 °C with a light intensity of 15 μE m−2 s−1. Phage adsorption was assayed at 0 and 45 min of incubation as described above. The primers used for detecting the presence of the psbA regions in cyanophage genomes were based on known psbA gene sequences, which include 23 cyanobacterial psbA gene sequences and 16 cyanophage psbA gene sequences (see Supporting Information, Appendix S1). The following primers were designed manually: psbAF, 5′-CTTCTATCCNA TYTGGGAAG-3′; psbAR, 5′-TNAGGTTGAANGCCATN GTR-3′. Cyanophages were purified using caesium chloride gradients as described previously (Sambrook & Russell, 2001).