Genetic and Physiological Characterization of the Antibacterial Activity ofBacillus subtilissubsp.inaquosorumStrain T1 Effective AgainstpirAB^Vp-Bearing Vibrioparahaemolyticus

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 pathogen inhibition. 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 NRPS and PKS lacZ transcriptional fusions were negligible during growth and highest during early stationary phase. Inactivation of sigH resulted in loss of inhibitor activity, indicating a role for σH 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 the 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.