Functioning of a hybrid BRP‐β‐lactamase protein in the release of cloacin DF13 and lysis of Escherichia coli cells

The gene encoding a hybrid BRP‐Bla protein consisting of the pCloDF13 encoded BRP signal sequence, 25 of the 28 amino acid residues of the mature bacteriocin release protein (BRP) and the mature portion of β‐lactamese (Bla) was subcloned in the expression vector pEB112. A similar construct was made using a mutant gene encoding a BRP‐Bla protein in which the cysteine residue at the +1 position was changed into a glycine residue. The expression, processing, functioning and subcellular localization of the ‘wild‐type’ and mutant hybrid protein at high‐level expression conditions were studied. The ‘wild‐type’ BRP‐Bla protein was mainly found in the outer membranes and possessed all the activities of the BRP itseld; the protein was able to bring about the release of cloacin DF13 and caused apparent cell‐lycis after high‐level synthesis. The mutant hybrid protein was predominantly located in the inner membranes, was inactive in the release of cloacin DF13, but caused apparent cell‐lysis only after strong induction.