Genetic and Physiological Characterization of the Antibacterial Activity of Bacillus subtilis subsp. inaquosorum Strain T1 Effective Against pirABVp-Bearing Vibrio parahaemolyticus

Abstract

Acute hepatopancreatic necrosis disease (AHPND) is caused by PirAB toxin-producing Vibrio parahaemolyticus and has devastated the global shrimp aquaculture industry. One approach for preventing growth of AHPND-producing Vibrio spp. is through the application of beneficial bacteria capable of inhibiting these pathogens. In this study we focus on the inhibitory activity of Bacillus subtilis subsp. inaquosorum strain T1, which hinders V. parahaemolyticus growth in co-culture experiments in a density-dependent manner; inhibition was also obtained using cell-free supernatants from T1 stationary phase cultures. Using a mariner-based transposon mutagenesis, 17 mutants were identified having complete or partial loss of inhibitory activity. Of those having total activity loss, 13 had insertions within a 42.6 kb DNA region comprising 15 genes whose deduced products were homologous to non-ribosomal polypeptide synthetases (NRPSs), polyketide synthases (PKSs) and related activities, which were mapped as one transcriptional unit. Mutants with partial activity contained insertions in spo0A and oppA, indicating stationary phase control. Expression of lacZ transcriptional fusions to NRPS and PKS genes was negligible during growth and at their highest during early stationary phase. Inactivation of sigH resulted in loss of inhibitor activity, indicating a role for {sigma}H in transcription. Disruption of abrB resulted in NRPS and PKS gene overexpression during growth as well as enhanced growth inhibition. This is the first study examining expression and control of an NRPS-PKS region unique to the inaquosorum subspecies of B. subtilis and an understanding of factors involved in T1 inhibitor production will enable its development for use as a potential tool against AHPND Vibrio pathogens in shrimp aquaculture. IMPORTANCEThe shrimp aquaculture industry has been impacted by the rise of acute hepatopancreatic necrosis disease (AHPND), resulting in significant financial losses annually. Caused by strains of the bacterial pathogen, Vibrio parahaemolyticus, treatment of AHPND involves the use of antibiotics, which leads to a rise in antibiotic resistant strains. An alternative approach is through the application of beneficial microorganisms having inhibitory activities against AHPND-generating pathogens. In this study, we examine the genetic basis for the ability of Bacillus subtilis strain T1 to inhibit growth of an AHPND Vibrio strain and show that activity is associated with genes having the potential for synthesizing antibacterial compounds. We found that expression of these genes is under stationary phase control and showed that inactivation of a global transition state regulator results in enhancement of inhibitory activity against the AHPND Vibrio. Our approach for understanding the factors involved in production B. subtilis strain T1 inhibitory activity may allow for development of this strain for use as a potential tool for the prevention of AHPND outbreaks.