Abstract
Several putative class II bacteriocin-like genes were identified in Lactobacillus casei ATCC 334 and all of which might encode peptides with a double-glycine leader. Six peptides encoded by these genes were heterologously expressed in Escherichia coli and then partially purified in order to test their bacteriocin activities. The results revealed that the mature LSEI_2163 peptide was a class IId bacteriocin that exhibited antimicrobial activity against some lactobacilli and several Listeria species. Similarly, mature LSEI_2386 was a putative pheromone peptide that also had significant bacteriocin activity against several Listeria species. The activities of both peptides tolerated 121◦C for 30 min, but not treatment with proteinase K or trypsin. The two Cys residues located at positions 4 and 24 in the mature LSEI_2163 peptide were shown by mass spectrometry to form a disulfide bridge, which was required for optimal antibacterial activity. However, replacement of one or both Cys with Ser would cause significant reduction of the antibacterial activity, the reduction being greater when only one of the Cys residues (C4S) was replaced than when both (C4S/C24S) were replaced. Both synthetic m2163 and m2386 behave as a random coil in aqueous solution whereas anα-helix is formed by both in the membrane mimic environment. However, the synthetic m2163 can also behave as anα-helix in a sodium phosphate buffer which would allow it to permeate into the cell membrane of sensitive bacteria and hence a corresponding bactericidal activity is detected. The ITC results indicated that the interaction between m2163 and POPG was weak. Therefore, we suspected that m2163 might have a specific target on the surface of sensitive bacteria. On the contrary, due to the presence of random coil conformation in sodium phosphate buffer, the bactericidal activity of synthetic m2386 is absent and no permeattion into the bacterial membrane is assumed.