Mutational Analysis of the sbo-alb Locus of Bacillus subtilis : Identification of Genes Required for Subtilosin Production and Immunity

Abstract

ABSTRACT The Bacillus subtilis 168 derivative JH642 produces a bacteriocin, subtilosin, which possesses activity against Listeria monocytogenes . Inspection of the amino acid sequence of the presubtilosin polypeptide encoded by the gene sboA and sequence data from analysis of mature subtilosin indicate that the precursor subtilosin peptide undergoes several unique and unusual chemical modifications during its maturation process. The genes of the sbo-alb operon are believed to function in the synthesis and maturation of subtilosin. Nonpolar mutations introduced into each of the alb genes resulted in loss or reduction of subtilosin production. sboA , albA , and albF mutants showed no antilisterial activity, indicating that the products of these genes are critical for the production of active subtilosin. Mutations in albB , - C , and - D resulted in reduction of antilisterial activity and decreased immunity to subtilosin, particularly under anaerobic conditions. A new gene, sboX , encoding another bacteriocin-like product was discovered residing in a sequence overlapping the coding region of sboA . Construction of an sboX-lacZ translational fusion and analysis of its expression indicate that sboX is induced in stationary phase of anaerobic cultures of JH642. An in-frame deletion of the sboX coding sequence did not affect the antilisterial activity or production of or immunity to subtilosin. The results of this investigation show that the sbo-alb genes are required for the mechanisms of subtilosin synthesis and immunity.