CHARACTERIZATION OF THE PROTEIN CONFERRING IMMUNITY TO THE ANTIMICROBIAL PEPTIDE CARNOBACTERIOCIN B2 AND EXPRESSION OF CARNOBACTERIOCINS B2AND BM1

Cloning of a 16-kb DNA fragment from the 61-kb plasmid of Carnobacteri um piscicola LV17B into plasmidless C. piscicola LV17C restores the pr oduction of the plasmid-encoded carnobacteriocin B2 and the chromosoma lly-encoded carnobacteriocin BM1 and restores the immune phenotype. Th is fragment also has sufficient genetic information to allow the expre ssion of carnobacteriocin B2 and its immunity in a heterologous host. The gene locus (cbiB2) responsible for immunity to carnobacteriocin B2 is located downstream of the structural gene for carnobacteriocin B2 and encodes a protein of 111 amino acids (CbiB2). CbiB2 was expressed in Escherichia coil as a fusion of the maltose-binding protein and Cbi B2. The fusion protein was purified on an amylose column and cleaved w ith factor Xa, and pure CbiB2 was isolated by high-performance liquid chromatography. The N-terminal amino acid sequence and mass spectromet ry (molecular weight [mean +/- standard error], 12,662.2 +/- 3.4) of t he purified protein agree with the information deduced from the nucleo tide sequence of cbiB2. Western blot (immunoblot) analysis indicates t hat the majority of the intracellular pool of this immunity protein is in the cytoplasm and that a smaller proportion is associated with the membrane. CbiB2 confers immunity to carnobacteriocin B2, but not to c arnobacteriocin BM1, when it is expressed in homologous or heterologou s hosts. No protective effect is observed for sensitive cells growing in the presence of the bacteriocin when the immunity protein is added to the medium. The purified immunity protein does not show significant binding to microtiter plates coated with carnobacteriocin B2 and is n ot able to inactivate the bacteriocin in solution.