DESTABILIZATION OF PSEUDOLYSOGENY IN ERWINIA HORTILCOLA: ANOTHER PHAGE INDUCED
Minchuk Ye1 Zlatohurska M2 Faidiuk Yu1,2 Tovkach F.2
1Taras Shevchenko National University of Kyiv,
ESC “Institute of Biology and Medicine”
2D.K. Zabolotny Institute of Microbiology and Virology of the NAS of Ukraine,
Department of bacteriophage molecular genetics
A phenomenon of phage mediated phage induction was described earlier by Tovkach and Zlatohurska and was attributed to induction of phages maintained in a cell in a pseudolysogenic state upon adsorption of infecting non-homologous phage. It was proposed as a process that underlies the release of temperate siphophages 49 and 59 (and probably podophage E105) from the cells of Erwinia horticola 60-2n, a fire-blight like symptom causing bacteria, infecting beech trees, Fagus sylvatica L.
Here we present a report on another case of induction on a related Eho strain 43II, infecting a different host plant – apple tree (Malus domestica Borkh.). Plaques of temperate type appeared when a supernatant obtained after centrifugation of overnight Eho 60-2n culture was applied on the lawn of Eho 43II. One could speculate that the phage originates from Eho 60-2n, but the same phage plaques were obtained independently upon infection of Eho 43II with lytic podophage E105. Thus, the origin of the phage lies within Eho 43II cells. On top of that, PCR using primers, specific to 49 and 59 phages’ genes has revealed that Eho 43II contains related DNA sequences within its cell. Electron microscopy of the samples adsorbed directly on the induced phage plaque revealed it to be a siphovirus as well (B1 morphotype). It features the capsid with Dmax (distance between opposite vertices) 56 nm, D min (distance between the opposite edges) 50 nm, and CL (capsid length, distance between the central and portal vertices) 53 nm, and a tail of approximately 146 nm.
The induced lines could be propagated (though with low titer) in further passages on Eho 43II. Two independently induced phage lines, named S1 and S2, were checked for host range using bacteria of three species, Eho, Erwinia amylovora (Eam), and Pantoea agglomerans (Pag), in comparance with erwiniaphages 49 and 59, KEY (Siphoviridae), and FE44 (Podoviridae). On the contrary to broad-host-range phages KEY (infecting all tested strains) and FE44 (infecting all strains except Pag and Eho strains 43II and 43I), phage lines S1 and S2 were highly specific to Eho, similar to phages 49 and 59. However, while 49 and 59 infect several Eho strains: 60-2n, its derivative 60-n, and Eho 450, phage lines S1 and S2 propagate exclusively on the cells of Eho 43II.
In order to obtain preparative quantities of phage lines S1 and S2, confluent lysis method was used; phages were concentrated using ultracentrifugation under 23 00 rpm for 3 h and purified with gradient ultracentrifugation in cesium chloride (prepared on STM buffer and STM buffer with addition of ficoll) for 3 h under 30,000 rpm. Electron microscopy revealed the phages to be highly unstable, as all samples contained degraded particles appearing as phage ghosts. On top of that, infection of S1 and S2 on Eho 43II leads to the formation of a massive amount of large aberrant capsids (80-120 nm in diameter) that may or may not be attached to tails, pointing at the abortive phenotype of the infection.
Further uncovering of relatedness between the induced phage and erwiniaphages 49 and 59 may shed light on features of the mobilome of Erwinia horticola, a pathogen causing fire-blight like symptoms in trees, the pseudolysogeny as a phenomenon of host-phage interaction, and the reasons for its destabilization.