Role of Exopolysaccharide in Pantoea agglomerans Interactions with Bacteriophages

Abstract

Fire blight is the common name given to a disease caused by phytopathogen, Erwinia amylovora. This pathogen infects plants belonging to the rosaceous family, particularly the commercially grown apple and pear cultivars in North America. Agriculture and Agri-Food Canada Research team has focused on the use of bacteriophages as biological control agents for the control of fire blight. Bacteriophage persistence in the orchard environment can be affected by various environmental factors such as exposure to ultraviolet light and dry conditions. In order to improve phage viability during application, a carrier-phage system was developed that uses a non-pathogenic epiphytic bacterium, named “the carrier”, that protects the bacteriophages during processing and field applications. The bacterium used is Pantoea agglomerans, which delivers and propagates the phages to the open blossoms, prior to the arrival of E. amylovora. In addition, the carrier alone may act as a biological control agent on the blossom surface, competing for space and producing antibiotics targeting E. amylovora. The aim of this study was study the interaction between the phage and the carrier bacterium, P. agglomerans. The exopolysaccharide (EPS) layer, is the first cellular component which bacteriophages encounter during the infection of P. agglomerans. PCR was conducted and it confirmed that 6 genes involved in EPS biosynthesis were present within the four different isolates of P. agglomerans. A CRISPR/Cas9 knockout and lambda red recombineering protocol was used to construct EPS deficient P. agglomerans mutants. The deficient populations were confirmed using colony appearance, PCR and Sangar sequencing. A quantitative PCR was conducted to quantify and compare the different bacteriophage populations between the wild-type and deficient EPS mutants. The EPS mutants were found to have shown a significantly decreased or complete lack of growth in both Myoviridae and Podoviridae infections.