Analysis by In Vitro Site-Directed Mutagenesis of the PlnE Component of the Two-Peptide Bacteriocin Plantaricin EF

Abstract

Bacteriocins are ribosomally synthesized antimicrobial peptides (AMPs) and proteins produced by bacteria. The two-peptide (class-IIb) bacteriocins consist of two different peptides and optimal antimicrobial activity requires the presence of both peptides in about equimolar amounts. They exert their antimicrobial activity by rendering the target-cell membrane permeable for various small molecules. The two-peptide bacteriocin plantaricin EF consists of the two helical peptides PlnE and PlnF and is produced by some strains of lactic acid bacteria (LAB). Both PlnE and PlnF contain GxxxG motifs which might be involved in helix-helix interactions between the two peptides. Both peptides also contain aromatic tryptophan and/or tyrosine residues, which are often located in the membrane interface of membrane proteins.

In this study, variants of the PlnE peptide, which are mutated in its two GxxxG motifs (G5xxxG9 and G20xxxG24) and in its tyrosine residue (Y6), were constructed, since such constructs might reveal how the PlnE and PlnF peptides interact with each other and with target-cell membranes. A total of 27 mutants were constructed by in vitro site-directed mutagenesis and 11 of the constructed peptide variants, all involving individual residue replacements of the two glycine residues in the G5xxxG9 motif, were subsequently expressed, purified and assayed in complementation with the wild type PlnF peptide. The results revealed that the glycine residue at position 9 (G9) seems to be in a sterically restricted environment, since substituting G9 with large hydrophobic and large hydrophilic residues resulted in at least a 100 fold reduction in activity. It also seems that this glycine residue is in a hydrophobic environment, since replacement with a small hydrophobic residue (Ala) was less detrimental (about 10 fold reduction in activity) than replacement with a small hydrophilic residue (Ser; 60 to 70 fold reduction in activity). In contrast to G9, the glycine residue at position 5 (G5) seems not to be restricted by spatial constraints, since substituting with large hydrophobic residues (Leu/Ile) resulted in only a 2 to 6 fold reduction in antimicrobial activity. Similarly to G9, G5 also appears to be in a hydrophobic environment, since replacement with a large hydrophilic residue was detrimental (about 100 fold reduction in activity). The fact that G5 was not spatially restricted indicates that the G5xxxG9 motif is not a helix-helix interacting motif. G9, being in a sterically restricted environment, may nevertheless be involved in interactions with PlnF and/or a docking protein.