Phenotypic, Genomic and Transcriptomic Characterization of Erwinia amylovora Bacteriophage Insensitive Mutant
Nassereldin Ibrahim 1*, Janet Lin 2, Darlene Nesbitt 3, Antonet Svircev 3, Joel Weadge 4, Hany Anany 1
- GRDC, Agriculture and Agri-Food Canada (AAFC), Guelph, ON, Canada.
- National Microbiology Laboratory, Public Health Agency of Canada.
- Vineland Station, Agriculture and Agri-Food Canada (AAFC), Vineland, ON, Canada.
- Wilfrid Laurier University, Waterloo, ON, Canada.
Introduction: Fire blight, caused by Erwinia amylovora, is a prominent concern for the apple and pear farmers due to its drastic economic effects. Traditionally, streptomycin is used for fire blight management, however, health and environmental concerns urged the need for alternative control methods. Lytic phages are considered as promising alternative to antibiotics as pesticide. However, phage resistance development might hinder its wide application as a natural biocontrol agent. Prevention of phage resistance development in the plant pathogenic bacteria requires investigating first the phage resistance mechanism(s).
Purpose: Generating and characterizing E. amylovora bacteriophage-insensitive mutant (BIM) resistant to ΦEa46-1 phage to understand E. amylovora resistance mechanism against phages and its impact on virulence fitness.
Method: Introducing a novel modification to liquid subculture method to generate BIM by addition of fresh medium and phage on collected cells every week for 3-4s. Analysis of the generated BIM is carried out on phenotypic, genomic, and transcriptomic levels.
Results: The modification of the liquid subculture method able to successfully produce a stable BIM to ΦEa46-1 phage. Difference in the biofilm formation was found between the BIM and the parent strain. Furthermore, significant differences found with Biolog Phenotype MicroArray PM7 plate for Peptide and Nitrogen sources metabolism. Genomic analysis results showed mutations in the two-component Rcs phosphorelay (mutations in rcsB, rcsC, and rcsD genes) and Ion-translocating oxidoreductase complex (rsxC and rsxD genes) when compared to the phage-sensitive parent strain. Mutations lead to DL amino acids insertion, D to V inversion, and promotor regions change. Full analysis of the transcriptomic and phenotypic data still in progress, as well as virulence fitness experiment.
Significance: Two-component Rcs phosphorelay and/or Ion-translocating oxidoreductase complex are playing vital role in phage resistance of E. amylovora. Deeper data analysis is required to explore this role in the resistance mechanism and virulence fitness.