Cyanobacterial growth
Synechococcus sp. strain WH8109 was grown in artificial seawater (ASW) medium61, with modifications as described previously62. Cultures were grown at 21 °C under a light intensity of 20 µmol photons per m2 per s, under a 14 h–10 h light–dark cycle. Pour-plates were obtained using ASW medium with low-melting-point agarose at a final concentration of 0.28% with an additional 1 mM of sodium sulfite. A heterotrophic helper strain, Alteromonas sp. EZ55, was added to the pour-plate mixture for isolating Synechococcus colonies63.
qPCR with reverse transcription
For RNA extraction, a 1 ml culture sample was collected by centrifugation at 4 °C, 15,000g for 15 min, and the pellets were flash-frozen in liquid nitrogen. Cell pellets were thawed and incubated with lysozyme L6876-5G (Sigma-Aldrich) at a final concentration of 30 mM and 200 U of RNase inhibitor (Murine BioLabs) for 60 min at 37 °C. An equal volume of lysis buffer was added and cell debris was centrifuged at 4 °C, 16,000g for 1 min. Nucleic acids in the supernatant were precipitated with an equal volume of 95% ethanol and centrifuged as described above. RNA wash buffer (500 µl) from Monarch Total RNA Miniprep Kit (NEB, T2010) was added, and the sample was centrifuged again for 30 s. The supernatant was discarded and the step was repeated. DNase I reaction buffer (5 µl) and 4 U of DNase I from the TURBO DNA-free kit were added to the 45-µl sample and incubated for 10 min at 4 °C. The samples were incubated with 0.5 µl of 0.5 M of EDTA, pH 8.0, for 10 min at 75 °C.
Total RNA was reverse transcribed into cDNA using the LunaScript RT SuperMix Kit (New England Biolabs, E3010). The reaction mixture was prepared in a total volume of 20 µl, 4 µl of LunaScript RT SuperMix containing random hexamers and 16 µl of nuclease-free water, with the RNA sample added to reach the final volume. As a control, a similar mixture was prepared, but without addition of the reverse transcriptase enzyme. The reaction was incubated at 25 °C for 2 min, 55 °C for 10 min and 95 °C for 1 min.
Quantitative PCR (qPCR) reactions were prepared using LightCycler 480 SYBR Green I Master mix from Roche, combined with 0.2 µM of each primer and the nblA/rnpB DNA template. The reactions were run on the LightCycler 480 Real-Time PCR System. Cycle threshold fluorescence values for each reaction were determined using LightCycler 480 software. To quantify DNA copy numbers, a standard curve was generated by running tenfold serial dilutions of the template and correlating cycle threshold values to known DNA concentrations (Supplementary Data 6).
The genes of which the expression levels were analysed using qPCR were as follows: host nblA2 (Syncc8109_1607, see explanation below; primers: 5′-GCGATCAAGCGGTCAATCAAC-3′ and 5′-CTCTCTGCCGCACGTAGAGG-3′), host rnpB (Syncc8109_0157; primers: 5′-CATCGGCGGTGTGTTTCT-3′ and 5′-CAGGCTTGCTGGGT-3′), S-TIP37 nblA (primers: 5′-TTCCCGAGGCAGACAAGAG-3′ and 5′-TAATGGGATGGTGACTCGGC-3′), S-TIP37 DNA polymerase (STIP37_17B; primers: 5′-TGAGCTACTACGCAACAGGC-3′ and 5′-AGCGCGATCATTCAGGGAAG-3′). The Synechococcus sp. strain WH8109 nblA gene chosen for qPCR is the one that we reported previously6, although a more refined remote homology search with hhsearch64 using a custom NblA profile reveals that the genome carries four additional nblA-like genes and an nblA pseudogene. To clarify which homologue has the highest structural similarity to previously characterized proteins and is therefore most likely to have a function similar to that of freshwater cyanobacterial NblAs, we obtained the structures for the corresponding monomers with ColabFold65 and searched them against the Protein Data Bank (PDB) using Foldseek66. The best match was indeed obtained for NblA2 (Syncc8109_1607), with the highest similarity to the NblA protein from Nostoc sp. strain PCC 7120 (ref. 10; PDB: 1OJH chain E; probability, 0.94; TM-score, 0.6621).
Cyanophage-infection experiments
Before infection, Synechococcus sp. strain WH8109 was grown in liquid medium to mid-log growth of around 1 × 108 cells per ml. Infection experiments were initiated by adding the S-TIP37 cyanophage strains at an MOI of 5. Infection dynamics were determined by collecting samples at hourly time intervals during the initial 6 h, followed by sampling every 2 h thereafter. The samples were filtered through a 0.22 µm syringe filter and the filtrate containing free cyanophages was plated to determine the number of infective cyanophages using the plaque assay67 in semi-solid pour-plates (see above). Statistical analysis of the infection course was performed using segmented linear regression by selecting near-linear ranges in the infection curves and obtaining linear fits for the relative cyanophage abundance (obtained by dividing the abundance in the extracellular medium by its maximum across all experiments) with the factors time after inoculation, cyanophage type (WT or mutant) and their interaction, and replicate as random effects using lme4 (v.1.1-31)68. The significance of the model terms was tested using the Anova function from the car package (v.3.1-1)69. The distributions of the residuals were checked using QQ plots.
Spectral measurements
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