Genetic and Physiological Characterization of the Antibacterial Activity ofBacillus subtilissubsp.inaquosorumStrain T1 Effective AgainstpirABVp-Bearing Vibrioparahaemolyticus
Authors: Avery Sarah E. , Ruzbarsky Susannah P. , Hise Amanda M. , Schreier Harold J. 
Published: August 2020

ABSTRACTAcute hepatopancreatic necrosis disease (AHPND) is caused by PirAB toxin-producingVibrio parahaemolyticusand has devastated the global shrimp aquaculture industry. One approach for preventing growth of AHPND-producingVibriospp. is through the application of beneficial bacteria capable of inhibiting these pathogens. In this study we focus on the inhibitory activity ofBacillus subtilissubsp.inaquosorumstrain T1, which hindersV. parahaemolyticusgrowth in co-culture experiments in a density-dependent manner; inhibition was also obtained using cell-free supernatants from T1 stationary phase cultures. Using amariner-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 inspo0AandoppA, indicating stationary phase control. Expression oflacZtranscriptional fusions to NRPS and PKS genes was negligible during growth and at their highest during early stationary phase. Inactivation ofsigHresulted in loss of inhibitor activity, indicating a role for σHin transcription. Disruption ofabrBresulted 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 theinaquosorumsubspecies ofB. subtilisand an understanding of factors involved in T1 inhibitor production will enable its development for use as a potential tool against AHPNDVibriopathogens 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 ofBacillus subtilisstrain T1 to inhibit growth of an AHPNDVibriostrain 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 AHPNDVibrio. Our approach for understanding the factors involved in productionB. subtilisstrain T1 inhibitory activity may allow for development of this strain for use as a potential tool for the prevention of AHPND outbreaks.