The isolation and characterization of a temperate phage, Y46/(E2), from Erwinia herbicola Y46

Abstract

A temperate phage was induced from exponential phase cells of Erwinia herbicola Y46 by treatment with mitomycin C. The phage was purified by single plaque isolation, and produced in bulk by successive cultivation in young cultures of E. herbicola Y178. Phages were concentrated from culture filtrates by rate zonal centrifugation and resuspension in 0.02 M Tris buffer, pH 7.2, twice, yielding suspensions of about 5 × 1011 PFU/ml. Purification was achieved by centrifugation in buffered sucrose solutions. The band at the 30/40% sucrose interface yielded intact particles having regular hexagonal heads and long contractile tails, with base plates. Fibers were not seen. The mean dimensions were head, 51 nm; neck length, 11 nm; overall tail length, extended, 98 nm and contracted, 75 nm; diameter of tail sheath, 24 nm. The phage was stable from pH 4.0 to 11.0, but unstable at pH 3.0, the response being independent of the suspending medium used. At pH 3.0, a survival curve having a biphasic appearance was observed, which was not due to a mixed population of phages. Stability to heat was good up to 45 °C, above which a logarithmic decline with temperature increase occurred. The average inactivation rate constant at 50 °C and pH 6.8 was 0.15 min−1. Adsorption to E. herbicola Y178 cells exhibited first-order kinetics, the adsorption rate constant being 2.5 × 10−10 ml/min. One-step growth-curve experiments indicated a burst size of 35–40, and a minimum latent period of 80 min. Probit analysis gave a mean latent period of 140 min (SD 25). The phage caused lysis of only E. herbicola strains Y178 and Y186.