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 Y 178. 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 times 10(11) 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 lonb 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 biphasic appearance was observed, which was not due to a mixed population of phages. Stability to heat was good up to 45 degrees C, above which a logarithmic decline with temperature increase occurred. The average inactivation rate constant at 50 degrees C and pH 6.8 was 0.15 min-1. Adsorption to E. herbicola Y 178 cells exhibited first-order kinetics, the adsorption rate constant being 2.5 times 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.

Absence of lysogeny in wild populations of Erwinia amylovora and Pantoea agglomerans

“…Prophages were also not observed in a genomic screening of 82 P. agglomerans isolates from rosaceous hosts in southern Ontario, Canada even though lysogeny has been previously reported in this species (Erskine, ; Harrison and Gibbins, ). Further testing of a subgroup of E. amylovora and P. agglomerans did not show any signs of release of phage particles spontaneously or after lytic cycle induction attempts with mitomycin C. These results indicate that lysogeny in E amylovora and P. agglomerans , although possible, is extremely rare and/or absent.…”

“…phages are known to be able to cross the species barrier and infect the orchard epiphyte P. agglomerans (Erskine, ; Lehman, ; Svircev et al ., 2010; 2011) . Independently, Erskine () and Harrison and Gibbins () reported that phages that could infect E. amylovora were capable of a lysogeny in P. agglomerans . In the present study, no evidence of lysogeny was apparent in P. agglomerans , which suggested that this bacterium does not provide a reservoir for Erwinia spp.…”

“…azadirachtae (Borkar, 1997), Erwinia carotovora subsp. carotovora (Faltus and Kishko, 1980;Ruban et al, 1981;Toth et al, 1993;Tovkach, 2002a,b) and P. agglomerans (previously known as Erwinia herbicola) (Harrison and Gibbins, 1975). Furthermore, the genomes of all fully sequenced E. amylovora phages lack the necessary genes for prophage integration (Lehman et al, 2009;Born et al, 2011;Nagy et al, 2012).…”