Purification and characterization of pediocin from probiotic Pediococcus pentosaceus GS4, MTCC 12683.

Pediococcus pentosaceus GS4 (MTCC 12683), a probiotic lactic acid bacterium (LAB), was found to produce bacteriocin in spent culture. Antibacterial and antagonistic potential of this bacteriocin against reference strains of Staphylococcus aureus (ATCC 25923), Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 25619), and Listeria monocytogenes (ATCC 15313) was proven by double-layer and well diffusion methods wherein nisin and ampicillin were used as positive controls. Bacteriocin in supernatant was purified and analyzed by SDS-PAGE, RP-HPLC, and circular dichroism (CD). The physico-chemical properties of purified bacteriocin were characterized being treated at different temperatures (30 to 110 °C), pH (3.0 to 12.0), with different enzymes (α-amylase, pepsin, and lysozyme), and organic solvents (hexane, ethanol, methanol, and acetone) respectively. The molar mass of bacteriocin (named pediocin GS4) was determined as 9.57 kDa. The single peak appears at the retention time of 2.403 with area amounting to 25.02% with nisin as positive control in RP-HPLC. CD analysis reveals that the compound appears to have the helix ratio of 40.2% with no beta sheet. The antibacterial activity of pediocin GS4 was optimum at 50 °C and at pH 5.0 and 7.0. The pediocin GS4 was not denatured by the treatment of amylase and lysozyme but was not active in the presence of organic solvents. This novel bacteriocin thus m ay be useful in food and health care industry.

Controlled functional expression of the bacteriocins pediocin PA-1 and bactofencin A in Escherichia coli.

The bacteriocins bactofencin A (class IId) and pediocin PA-1 (class IIa) are encoded by operons with a similarly clustered gene organization including a structural peptide, an immunity protein, an ABC transporter and accessory bacteriocin transporter protein. Cloning of these operons in E. coli TunerTM (DE3) on a pETcoco-2 derived vector resulted in successful secretion of both bacteriocins. A corresponding approach, involving the construction of vectors containing different combinations of these genes, revealed that the structural and the transporter genes alone are sufficient to permit heterologous production and secretion in this host. Even though the accessory protein, usually associated with optimal disulfide bond formation, was not required for bacteriocin synthesis, its presence did result in greater pediocin PA-1 production. The simplicity of the system and the fact that the associated bacteriocins could be recovered from the extracellular medium provides an opportunity to facilitate protein engineering and the overproduction of biologically-active bacteriocins at industrial scale. Additionally, this system could enable the characterization of new bacteriocin operons where genetic tools are not available for the native producers.

Common Mechanism of Cross-Resistance Development in Pathogenic Bacteria Bacillus cereus Against Alamethicin and Pediocin Involves Alteration in Lipid Composition.

To understand the mechanism of development of cross-resistance in food pathogen Bacillus cereus against an antimicrobial peptide pediocin and antibiotic alamethicin, the present study was designed. Pediococcus pentosaceus was taken as a source of pediocin, and it was purified by ammonium sulphate precipitation followed by cation exchange chromatography with 14.01-fold purity and 14.4 % recovery. B. cereus strains alamethicin-resistant strains (IC50 3.23 µg/ml) were selected from sensitive population with IC50 2.37 µg/ml. The development of resistance in B. cereus against alamethicin was associated with decrease in alamethicin-membrane interaction observed by in vitro assay. Resistant strain of B. cereus was found to harbour one additional general lipid as compared to sensitive strain, one amino group lacking phospholipid and one amino group containing phospholipid (ACP). In addition, ACP content was increased in resistant mutant (29.7 %) as compared to sensitive strain (14.56 %). The alamethicin-resistant mutant B. cereus also showed increased IC50 (58.8 AU/ml) for pediocin as compared to sensitive strain (IC50 47.8 AU/ml). Cross-resistance to pediocin and alamethicin in resistant mutant of B. cereus suggested a common mechanism of resistance. Therefore, this understanding could result in the development of peptide which will be effective against the resistant strains that share same mechanism of resistance.