Certain species of lactic acid bacteria produce and secrete bacteriocins, which are ribosomally synthesized antimicrobial peptides. These peptides recognize and kill target cells by rendering their membrane permeable for various small molecules. There has been an increased interest in lactic acid bacteria bacteriocins because of their potential use as food additives and pharmaceuticals. Plantaricin EF is a two-peptide bacteriocin produced by the lactic acid bacteria Lactobacillus plantarum C11. The two peptides constituting this bacteriocin are called Plantaricin E (PlnE) and Plantaricin F (PlnF). For optimal antimicrobial effect, the two peptides have to be present in equal molar amounts. Circular dichroism studies suggest that the peptides interact physically with each other upon contact with target membranes. The inter-peptide interactions between PlnE and PlnF are thought to be mediated by GxxxG motifs, which are located in their amphiphilic α-helical region. GxxxG motifs are known to confer helix-helix interactions between membrane-inserted polypeptides. In addition, tyrosine and tryptophan residues tend to be prominent in trans-membrane proteins, especially in the parts of proteins exposed to the interface region of the membrane. It is believed that these aromatic residues enhance stability because of interactions with membrane-lipids in the interface region.
To study the importance of GxxxG motifs in helix-helix interactions between PlnE and PlnF, the glycine-residues in PlnF have been altered by in vitro site-directed mutagenesis. The tyrosine and tryptophan residues in PlnF were also altered by in vitro site-directed mutagenesis, in order to investigate how PlnF will orient itself in target cell membranes. In order to do this, the gene encoding PlnF, plnF, and the gene encoding its cognate immunity protein, plnI, have been connected to the sakacin P leader-sequence, and cloned into the shuttle-vector pLPV111. The vector was transformed into the lactic acid bacteria Lactobacillus sake Lb790 containing the plasmid pSAK20. pLPV111 and pSAK20 are part of a heterologous expression system designed for expression of the many different bacteriocin. By using this expression system, PlnF is expressed separate from PlnE. This makes it easier to isolate and purify PlnF, as well as constructing and purifying the mutant version of PlnF, for subsequent structure and function analysis.