Bacteriocin mesentericin Y105

Mesentericin Y105 (Mes-Y105) is a bacteriocin secreted by Leuconostoc mesenteroides which is particularly active on Listeria. It is constituted by 37 residues and reticulated by one disulfide bridge. It has two W residues, W18 and W37, which can be studied by fluorescence. Two single substituted W/F analogues were synthesized (Mes-Y105/W18 and Mes-Y105/W37) to differentiate the local environment around each W and to study their changes in the presence of lipid vesicles. Fluorescence experiments show that, for the pure Trp-analogues, W18 and W37 are fully exposed to solvent whatever pH and buffer conditions. In the presence of lipid vesicles, both became buried. Lipid affinities were estimated: they are weak for zwitterionic phospholipids but an order of magnitude higher for negatively charged phosphatidylserine (PS) and phosphatidylglycerol (PG) lipids. On negatively charged PG lipids, Mes-Y105 and Mes-Y105/W37 display comparable lipid affinities. A decrease in lipid affinity is observed for Mes-Y105/W18 compared to Mes-Y105, which means that W37 would seem to be required for increased lipid selectivity. In the lipid-bound state W18 is strongly dehydrated, probably embedded into the acyl chains, while W37 stands more at the interface. Mes-Y105 was also studied by polarization modulation infrared reflection absorption spectroscopy (PMIRRAS), alone and in various phospholipid environments, to obtain structural information and to assess lipid perturbations. At nanomolar concentrations close to those required for anti-Listeria activity, Mes-Y105 forms films at the air/water interface and inserts into negatively charged lipid monolayers. In situ infrared data show that Mes-Y105 binding only affects the polar head group vibrations while the lipid order of the acyl chains remains unaffected. The PMIRRAS show that Mes-Y105 folds into an N-terminal antiparallel beta-sheet followed by an alpha-helix, both structures being tilted (40 degrees) compared to the normal at the interface, which is in agreement with the thickness estimated by Brewster angle microscopy (BAM). All these data support the proposal of a new model for Mes-Y105 at the membrane interface.

A Leuconostoc mesenteroides ssp. mesenteroides was isolated from goat's milk on the basis of its ability to inhibit the growth of Listeria monocytogenes. The antimicrobial effect was due to the presence in the culture medium of a compound, named mesentericin Y105, excreted by the Leuconostoc mesenteroides Y105. The compound displayed known features of bacteriocins from lactic acid bacteria. It appeared as a proteinaceous molecule exhibiting a narrow inhibitory spectrum limited to genus Listeria. The apparent relative molecular mass, as indicated by activity detection after SDS-PAGE, was 2.5-3.0 kDa. The bacteriocin was purified to homogeneity by a simple three-step procedure: a crude supernatant obtained from an early-stationary-phase culture in a defined medium was subjected to affinity chromatography on a blue agarose column, followed by ultrafiltration through a 5 kDa cut-off membrane, and finally by reverse-phase HPLC on a C4 column. Microsequencing of the pure bacteriocin and of tryptic fragments showed that mesentericin Y105 is a 36 amino acid polypeptide whose primary structure is close to that of leucocin A-UAL 187, which contains an extra residue at the C-terminus and displays only two differences in the overlapping sequence. However, unlike leucocin A-UAL 187, mesentericin Y105 displayed a bactericidal mode of action.

Two different N-terminal extensions have been identified within class II bacteriocin precursors. The first one is a two-glycine-type leader peptide associated with a dedicated ATP-binding cassette transporter. The second is a signal peptide which directs the bacteriocin precursor to the general secretion machinery. Mesentericin Y105 is a class II anti-Listeria bacteriocin produced by Leuconostoc mesenteroides Y105 via a dedicated transport system (DTS). To investigate heterologous expression systems capable of producing mesentericin Y105 in various hosts, two different secretion vectors were constructed. One of them, containing the mesentericin Y105 structural gene fused to the segment encoding the divergicin A signal peptide, was introduced into Escherichia coli, Leuconostoc subsp. and Lactococcus subsp. In E. coli, mesentericin Y105 production was linked to a putative periplasmic toxicity. To take advantage of this secretion system, the mesentericin Y105 precursor was also produced in E. coli. It was demonstrated that this pre-bacteriocin exhibited some antagonistic activity against Listeria. To allow for a comparison between the two different transport systems, mesentericin Y105 production using the vector containing the mesentericin Y105 structural gene and its DTS transporter operon was examined. The production of mesentericin Y105 was monitored by a new fast purification method followed by MS analysis. It was shown that, in Leuconostoc, the production of mesentericin Y105 is enhanced via the DTS compared to the general secretion pathway.