Isolation of Spontaneous POP72-Resistant Pcc27 Mutants

To isolate spontaneous POP72-resistant mutants, a high-titer overlay assay was performed as previously described (Kim and Ryu, 2011). Briefly, 5 mL of soft TSB agar (0.4% agar) inoculated with a mixture of serially diluted (10-fold) Pcc27 culture at stationary phase and 100 μL of POP72 suspension (~10⁹ PFU mL⁻¹) were poured on TSA plates. The solidified plates were incubated for 24 h at 28°C. Randomly selected single colonies from each plate were isolated by repeated subcultures on TSA plates. The phage resistance and the absence of phages from the isolates were verified using a spot assay.