Hunting for Holins in the Paenibacillus Larvae Phage Willow Genome

Undergraduate #106
Discipline: Biological Sciences
Subcategory: Microbiology/Immunology/Virology

Erin K. Cassin - University of Nevada, Las Vegas
Co-Author(s): Christy Strong, Philippos Tsourkas, and Penny Amy, UNLV, Las Vegas, NV

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Honeybees (Apis mellifera) are responsible for pollinating seventy percent of the fruits and vegetables in the United States. Paenibacillus larvae is the causative agent in the fatal honeybee larvae infection American Foulbrood Disease, a significant contributor to the worldwide decline in honeybee populations. Current treatments utilize antibiotics, which has resulted in antibiotic resistant P. larvae throughout the U.S. Targeting P. larvae with bacteriophages may be a practical alternative since ‘phages’ are highly specific to their hosts. Laboratory treatments using phage have proven effective in prophylactic applications and for active P. larvae infections. Further research is needed to characterize the genetic control of the lytic mechanism in P. larvae phages. The goal of our research is to determine the contribution of putative genes in the lytic pathway. We hypothesize the putative bacteriocin, transposase, and N-acetylmuramoyl-L-alanine amidase, based on their DNA sequences, are involved in lysis. We will test this hypothesis by performing gene knockouts.
We determined P. larvae phage Willow, a potential biocontrol agent, can enter lysogeny. Isolation and confirmation of stable lysogens is essential for our gene knockouts. An established lysogeny confirmation test, the patch assay, was performed on suspected lysogens. Clearances in the naive lawn surrounding inoculation spots indicate Willow prophages in the spotted inoculum had been induced from lysogeny to lysis. Control plates displayed growth of lysogen spots with no naive lawn. A sensitivity assay was also used to confirm lysogeny. Prophages can provide immunity for their hosts against genetically similar phages. Six P. larvae phages dispensed in drops on a naive P. larvae lawn or a lysogen lawn containing Willow prophage. Following incubation, phages similar to the Willow prophage displayed plating efficiencies of 0.30 or lower. Distantly related phages had plating efficiencies of 0.63 to 1.0. These results indicate Willow exists as a prophage in P. larvae lysogens and confers immunity against related phages.
Willow knockouts will be performed by generating loss-of-function mutations in stable lysogens with the Targetron (Sigma-Aldrich) system. Each putative Willow gene will be inactivated by the insertion of a sequence specific mobile class II introns. Knockouts will be identified by their inability to lyse P. larvae in plaque assays. Inducible rescue plasmids containing functional gene inserts will be transformed into knockout P. larvae cultures to restore the wildtype phenotype. The presence of phage plaques will confirm a successful rescue event. Future research applications include purification of holin and lysin gene products for bactericidal products.

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Funder Acknowledgement(s): National Science Foundation Grant DBI REU 1358896

Faculty Advisor: Christy Strong, christy.strong@unlv.nevada.edu

Role: I performed all the bench work for this research: patch assay, sensitivity assays.