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 inaquosorum strain T1, which hinders V. parahaemolyticus growth in co-culture experiments in a density-dependent manner; inhibition was also observed using cell-free supernatants from T1 stationary phase cultures. Using mariner-based transposon mutagenesis, 17 mutants were identified having complete or partial loss of inhibitory activity. Of those displaying total loss of activity, 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. Our characterization of the expression and control of an NRPS-PKS gene cluster in B. inaquosorum T1 provides an understanding of factors involved in inhibitor production, enabling this strain's development for use as a tool against AHPND Vibrio pathogens in shrimp aquaculture.