Thurincin H Is a Nonhemolytic Bacteriocin of Bacillus thuringiensis with Potential for Applied Use

Thurincin H, a bacteriocin produced by Bacillus thuringiensis, exhibits antibacterial activity against Gram-positive and Gram-negative bacteria. While much is known about its expression and antimicrobial spectrum, its hemolytic property has yet to be established. In this study, thurincin H was produced in a plasmid-free acrystalliferous strain of B. thuringiensis (Bt Cry^-B) that naturally lacked antimicrobial and hemolytic activities. When grown in Tryptic Soy Broth (TSB), the bacteriocin's maximal production in Bt Cry^-B harboring the thurincin H genetic cluster (Bt Cry^-B/pThur) was observed at 24 h. Thurincin H was purified as a sole peptide of ~5 kDa using three purification steps, i.e., salt precipitation, ultrafiltration, and gel filtration chromatography. The bacteriocin showed inhibitory activity against B. cereus (5631 U), Bt Cry^-B (8827 U), E. faecium wild type (11,197 U), and E. faecium ATCC 19,434 (6950 U), but not against Bt Cry^-B/pThurH and Bt Cry^-B/pThurHΔThnA. In addition, a minimum inhibitory concentration (MIC) of 5.0 μg/mL against B. cereus 183 was observed. In silico predictions suggested that thuricin H lacks hemolytic activity, which was validated in vitro using 4 × the MIC, i.e., 20 μg/ml. Our data lay a foundation for the potential safe use of thurincin H as an antibacterial peptide for medical use, in food products, and for expression in probiotic bacteria.