Antimicrobial activity and preliminary mode of action of PlnEF expressed in Escherichia coli against Staphylococci

Strains belonging to the genus of Staphylococci, such as Staphylococcus aureus are common pathologic bacteria which may cause nosocomial cross infection and food contamination. Plantaricin EF (PlnEF), a two-peptide nonlantibiotic bacteriocin produced by Lactobacillus plantarum strains, shows great inhibitory effects against Gram-positive Staphylococci strains. To overproduce this two-peptide bacteriocin, plnE and plnF genes were cloned into pET32a (+) vector and heterologously expressed in Escherichia coli by fusion with His6-tag in this study. The purified fusion proteins were cleaved by enterokinase, then PlnE and PlnF peptides without extra amino acids were obtained by a two-step purification method, ultrafiltration centrifuge (10 kDa) followed by a reverse-phase HPLC. Purity of PlnE and PlnF, determined by analytical HPLC, is ∼98%, and their molecular mass confirmed by ESI-MS was 3545.14 Da and 3703.1 Da, respectively. It was found that the two peptides had significant antimicrobial activities against Staphylococcus citreus and Staphylococcus aureus strains and they functioned synergistically. PlnEF exerted its bactericidal activity against Staphylococci strains by permeabilizing the cell membrane, causing influx and efflux of various molecules across the transmembrane barrier, eventually leading to cell death.