Isolation of a Variant of Subtilosin A with Hemolytic Activity

Bacillus subtilis produces an anionic bacteriocin called subtilosin A that possesses antibacterial activity against certain gram-positive bacteria. In this study, we uncovered a hemolytic mutant ofB. subtilis that produces an altered form of subtilosin A. The mutant bacteriocin, named subtilosin A1, has a replacement of threonine at position 6 with isoleucine. In addition to the hemolytic activity, subtilosin A1 was found to exhibit enhanced antimicrobial activity against specific bacterial strains. TheB. subtilis albB mutant that does not produce a putative immunity peptide was more sensitive to both subtilosin A and subtilosin A1. A spontaneous suppressor mutation ofalbB that restored resistance to subtilosin A and subtilosin A1 was obtained. Thesbr (s ub tilosinr esistance) mutation conferring the resistance is not linked to thesboA-alb locus. Thesbr mutation does not increase the resistance ofB. subtilis to other cell envelope-targeted antimicrobial agents, indicating that the mutation specifically confers the resistance to subtilosins. The findings suggest possible bioengineering approaches for obtaining anionic bacteriocins with enhanced and/or altered bactericidal activity. Furthermore, future identification of the subtilosin-resistant mutation could provide insights into the mechanism of subtilosin A activity.