Identification of phage receptors in the fire blight pathogen Erwinia amylovora

Erwinia amylovora is a member of the Erwiniaceae and the causative agent of fire blight, a severe disease of Rosaceae plants. E. amylovora usually infects host blossoms via the stigma and invades the ovary. It spreads through the xylem of an infected plant. In the xylem E. amylovora produces exopolysaccharides (EPS), e.g., a capsule, which leads to ooze formation and canker development. The capsule is composed of amylovoran, levan, and cellulose, respectively.

Transposon mutagenesis of E. amylovora revealed that adsorption of the T7-like phage L1 and the SP6-like phage S2 (both Autographiviridae) is dependent on the amylovoran synthesis (ams) operon. Accordingly, both phages exhibit a Depolymerase (Dpo) with 59 % amino acid identity. The enzyme is a structural component of the virion. DpoL1 binds specifically to amylovoran and cuts the galactose backbone. In addition, adsorption of phage S6 (Schitoviridae) and M7 (Myoviridae) depends on the bacterial cellulose synthesis (bcs) operon. Deletion of the bcs operon or associated genes (bcsA, bcsC, and bcsZ) verified the crucial role of bacterial cellulose for S6 and M7 infection. Application of the cellulose binding dye Congo Red blocked infection by both phages. In addition, we demonstrate that infective S6 virions degraded cellulose to glucose molecules and that Gp95, a phage encoded cellulase, is involved to catalyze the reaction. In vitro treatment of a growing E. amylovora culture with DpoL1 or GP95 did not inhibit growth. However, application of DpoL1 together with the capsule-independent phage Y2 (Chaseviridae) revealed a strong synergistic inhibitory effect and caused a 4 log reduction of viable cell counts. Y2 is highly specific to E. amylovora and solely relies on LPS for adsorption. Transposon mutagenesis revealed four so far unrecognized genes in E. amylovora, which play a central role in both, LPS and/or EPS synthesis. Deletion of each gene affected either LPS or EPS composition and resulted in highly reduced adsorption and efficacy of plating not only for Y2, but also for other phages that infect E. amylovora. Most of the mutants were highly attenuated in virulence on detached blossoms.