Tailocin Purification from P. syringae

Tailocin was prepared and quantified from supernatants of P. syringae pv. syringae B728a as previously described (34 (link), 56 (link)). Overnight cultures of B728a were diluted 1:100 in King’s B medium and grown for 3 h at 28°C, and tailocin production was induced by the addition of mitomycin C (MP Biomedicals LLC, Solon, OH) to a concentration of 0.5 μg ml⁻¹. After a 24-h induction, supernatants were collected by centrifugation. Residual live cells were killed by treating the supernatant with chloroform. The aqueous phase was collected by centrifugation and then amended with NaCl and polyethylene glycol 8000 (PEG 8000) to final concentrations of 1 M and 10%, wt/vol, respectively. After 1 h of incubation on ice, the supernatant mixture was centrifuged at 16,000 × g for 30 min at 4°C. The resulting tailocin pellet was dissolved in 10 mM Tris (pH 7.0) and 10 mM MgSO₄. Residual PEG 8000 was removed by two extractions with equal volumes of chloroform. The activity of prepared tailocin was evaluated by spotting 5-μl serial dilutions onto soft agar overlay plates seeded with Pph. Tailocin activity is expressed in activity units (AU) derived from the highest dilution factor resulting in a visible inhibition zone (57 ).