Master's Thesis Defense: Sarah Avery

Title: A Study of the Antagonistic Activity of Bacillus subtilis Strain T1 Against Shrimp Pathogen Vibrio parahaemolyticus Strain D4

Speaker: Sarah Avery, Master's Candidate

Abstract: Acute hepatopancreatic necrosis disease (AHPND), also known as early mortality syndrome (EMS), is caused by strains of Vibrio parahaemolyticus containing the PirAB toxin and has been devastating to shrimp aquaculture globally.  Current methods for prevention rely on the use of antibiotics that leads to the development of antibiotic-resistant bacteria.  In vitro, Bacillus subtilis strain T1 was found to possess antagonistic activity against EMS-causing V. parahaemolyticus strain D4 and is a candidate for use as a probiotic in the aquaculture industry.  Competitive growth experiments examined the effect of T1 on D4 growth.  Using qPCR to assess T1 and D4 growth, these studies showed that T1 was capable of inhibiting D4 growth in a density-dependent manner, with complete inhibition occurring when the T1 starting density was 104-fold higher than D4.  Using a mariner-based transposon system, T1 mutants were generated to identify genes involved in D4-inhibitory activity.  Of over 3,000 colonies screened using an overlay-based assay, 17 were identified as having either complete or partial loss of activity.  Eleven mutants contained insertions within an ~30-kb DNA cluster that included lipopeptide and polyketide biosynthesis genes.  One of these mutants, A3-41, which contained an insertion within a non-ribosomal peptide synthetase gene utilized for lipopeptide biosynthesis, was found to have lost the ability to inhibit D4 growth in co-culture experiments.  Two mutants were found to contain insertions within stationary phase regulators, spo0A (sporulation gene regulator) and oppA (first gene of the oligopeptide transporter system operon) suggesting that D4 inhibitory activity is associated with a stationary phase product.  Consistent with the mutagenesis results, supernatant fractions prepared from stationary phase cultures of T1 were found to inhibit D4 growth in a dose-dependent manner while culture supernatants prepared from mutant A3-41 were not inhibitory.  These results indicate that T1 produces and excretes a stationary phase metabolite(s) that has inhibitory activity against D4 and has implications for the use of T1 as a probiotic in shrimp aquaculture.