Effect of Amino Acid Substitutions on the Activity of Carnobacteriocin B2

Abstract

Carnobacteriocin B2, a 48-amino acid antimicrobial peptide containing a YGNGV motif that is produced by the lactic acid bacterium Carnobacterium piscicola LV17B, was overexpressed as fusion with maltose-binding protein in Escherichia coli. This fusion protein was cleaved with Factor Xa to allow isolation of the mature bacteriocin that was identical in all respects to that obtained from C. piscicola. Similar methodology permitted production of the precursor precarnobacteriocin B2 (CbnB2P), which has an 18-amino acid leader, as well as six mutants of the mature peptide: CbnF3 (Tyr 3 3 Phe), CbnS33 (Phe 33 3 Ser), CbnI34 (Val 34 3 Ile), CbnI37 (Val 37 3 Ile), CbnG46 (Arg 46 3 Gly), and Cbn28 (truncated frameshift mutation: (carnobacteriocin B2 1-28) ؉ ELTHL). Examination of these compounds for antimicrobial activity showed that although CbnI34, CbnI37, and CbnG46 were fully active, CbnB2P, CbnF3, CbnS33, Cbn28, and all of the fusion proteins had greatly reduced or no antimicrobial activity. Expression of the immunity protein that protects against the action of the parent carnobacteriocin B2 in a previously sensitive organism also protects against the active mutants. Because carnobacteriocin B2 also acts as an inducer of bacteriocin production in C. piscicola, the ability of the precursor CbnB2P and the mutants to exert this effect was examined. All were able to induce Bac ؊ cultures and reestablish the Bac ؉ phenotype except for the truncated Cbn28. The results demonstrate that very minor changes in the peptide sequence may drastically alter antimicrobial activity but that the induction of bacteriocin production is much more tolerant of structural modification, especially at the N terminus.

Plantaricin A Is an Amphiphilic α-Helical Bacteriocin-like Pheromone Which Exerts Antimicrobial and Pheromone Activities through Different Mechanisms

“…These peptides are usually basic and between 30 and 60 residues long. The production of bacteriocins is, at least in some strains, controlled by a quorum sensing mechanism that involves a secreted peptide pheromone with bacteriocin-like characteristics (3)(4)(5)(6)(7). Like bacteriocins, these peptide pheromones are cationic and they are exported from the cell with help of a bacteriocin-type leader peptide and secretion machinery.…”

Enhancement of the Enterocin CRL35 Activity by a Synthetic Peptide Derived from the NH 2 -Terminal Sequence

“…The main effect of peptide 2 would be the increase in bacteriocin killing rate. Because this domain would be involved in the initial unspecific binding with the membrane and would not be related to pore formation (5,17), a plausible explanation might be that the first interaction of peptide 2 with the membrane would facilitate later binding and correct insertion of enterocin CRL35.…”

Determination of Essential and Variable Residues in Pediocin PA-1 by NNK Scanning

“…Furthermore, studies of the C-terminal 15-mer region of pediocin PA-1 revealed that the fragment interfered with the inhibitory activity of the native peptide (15). More recent studies have investigated multiple mutants with mutations at different residues (30,33,36). Six mutants with mutations at 6 different residues of carnobacteriocin B2 (36), 10 with mutations at 8 different residues of mesentericin Y105 (33), and 17 with mutations at 14 different residues of pediocin PA-1 (30) were generated by random PCR mutagenesis.…”