SBIR Phase I: Using enzymes secreted by soil amoebae to fight bacterial biofilms in agriculture

The broader impact/commercial potential of this Small Business Innovation Research (SBIR) Phase I project is development of a novel technology platform for the treatment of bacterial pathogens that pose a persistent threat to plant health both in the field and during storage of the harvest. Many bacteria kill their host by forming biofilms on the plant surface. Central to this proposal are findings that biofilms confer tolerance of bacteria to antimicrobial agents, whether these antimicrobial agents are antibiotics, disinfectants, or germicides. Dispersing these biofilms is necessary to remove contaminating bacteria from the plant surface. This proposal is addressing these challenges by developing safe and natural biofilm degrading enzymes from the collection of over 3,000 unique microorganisms called Dictyostelids isolated from five continents and several island nations. The focus of this proof-of-principle project is discovery of enzymes for treatment of bacterial potato soft rot. This disease is socially and commercially impacting our nation as potatoes are the most consumed vegetable in the U.S. with a total market value exceeding $3.5 B. The proposed project advances the use of Dictyostelid strains capable of dispersing biofilms formed by Dickeya and Pectobacterium and feeding on planktonic bacteria liberated from the corresponding bacterial biofilms. To be able to destroy those biofilms they must be able to break down biofilm integrity. This project uses proprietary technology licensed to the firm for a world-wide use in the agricultural markets. Suspected members of Glycoside Hydrolase Family of enzymes (GHF) have been already identified by bioinformatics and in crude secreted products. This project has two objectives: (1) Using polynucleotide chain reaction it will amplify Dictyostelids DNA encoding GHF and clone it into an Escherichia coli compatible vector with an in-frame Histidine-Tag sequence for purification using a specialized affinity column, and (2) Affinity purified enzymes will be used to disperse Dickeya and Pectobacterium biofilms established on 96-well polystyrene plates. The best performing enzymes will be further examined for their ability to disperse biofilms on a potato surface and prevent soft rot. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.