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

Acute hepatopancreatic necrosis disease (AHPND) is caused by PirAB toxin-producing and has devastated the global shrimp aquaculture industry. One approach for preventing growth of AHPND-producing spp. is through the application of beneficial bacteria capable of inhibiting these pathogens. In this study we focus on the inhibitory activity of subsp. strain T1, which hinders 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 -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 and , indicating stationary phase control. Expression of transcriptional fusions to NRPS and PKS genes was negligible during growth and at their highest during early stationary phase. Inactivation of resulted in loss of inhibitor activity, indicating a role for σ in transcription. Disruption of 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 subspecies of and an understanding of factors involved in T1 inhibitor production will enable its development for use as a potential tool against AHPND pathogens in shrimp aquaculture.