Development of Bacteriophage Plaque Assay for Liberibacter

Development of Bacteriophage Plaque Assay for Liberibacter

Report Date: 04/15/2016
Project: 726-Liberibacter   Year: 2016
Category: Horticultural & Management
Author: Carlos Gonzalez
Sponsor: Citrus Research and Development Foundation

Liberibacter crescens strain BT-1, has recently been cultured under laboratory conditions and is the model system for our studies. We had previously reported that we had initiated studies using the well-studied R2 tailocin to design fusions between N-terminal tail fiber region of the R2 tailocin and C-terminal portions of tail spike from BT-1 prophages. Tailocins are protein assemblages that function like the tails of phages, by adsorbing to the bacterial cell and then puncturing the cell envelope. Unlike phages, tailocins do not have a capsid and thus inject no DNA, instead relying on the membrane puncturing activity to kill the cell. Like phages, tailocins use tail fibers to recognize specific receptors on the target cell surface. Tailocins are thus potent and specific lethal nanoparticles. The assembly of active tailocin requires chaperones specific to the C-terminal and the availability of chaperone can limit tailocin production. In order to understand the protein folding necessary to assemble functional hybrid tailocins, we are fusing different N-terminal region-encoding portions of the tail fiber gene of the well-studied tailocin R2 to a series of C-terminal regions of the P2 tail fiber. This is being accomplished in- trans using a broad host vector with a tac promoter. This series of experiments will allow us to understand the fusion(s) points necessary to construct active tailocins. Using the developed overlay system that incorporates several modifications of medium BM7, we are testing broad host phages, tailocins and environmental samples to identify phages active against L. crescens.


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