Advances in the characterization of putative s-layer protein of Lactobacillus kefir

DELFEDERICO, LUCRECIA; HOLLMANN, AXEL; GARROTE, GRACIELA; ABRAHAM, ANALIA; DE ANTONI, GRACIELA; SEMORILE, LILIANA

TUCUMAN, ARGENTINA

Simposio; II Simposio Argentino-Italiano de Bacterias Lácticas; 2004

Crystalline bacterial cell surface layers (S-layers) represent one of the most observed cell envelope components of prokaryotic organisms (bacteria and archaea). S-layers are isoporous structures composed of a single protein or glycoprotein species endowed with the ability to assemble into a closed lattice during all stages of cell growth and cell division. Isolated S-layer subunits of a great variety of prokaryotic organisms are able to assemble into monomolecular arrays either in suspension, at liquid-surface interfaces, on lipid films, on liposomes and on solid supports. Due to these unique features, S-layers have broad application potential in molecular nanotechnology, nanobiotechnology and biomimetic. In a previous work, we have shown the presence of a preponderant band of 66-71 kDa in the SDS-PAGE of Lactobacillus kefir whole-cell proteins. Thin sections of these bacteria, analysed by transmission electron microscopy (TEM), showed an outermost layer over the bacterial cell wall, which was lost after the treatment of cells with chaotropic agents. The aims of this work were to demonstrate, by TEM,  the presence of S-layer as a two-dimensional macromolecular paracrystalline arrangement in L. kefir JCM 5818 and to initiate S-layer protein glycosilation studies by high performance anion-exchange chromatography (HPAEC). Whole bacterial cells and a dialyzed extract of surface proteins, obtained by guanidine hydrochloride treatment, were negatively stained with ammonium molybdate and analysed by TEM. Micrographs showed a regular two-dimensional arrangement of the protein subunits, characteristic of the S-layer proteins. To determine the ability of L. kefir S-layer to self-assemble on lipid surfaces, liposomes of soybean lecithin, cholesterol and stearilamine were prepared and incubated with monomers and oligomers of S-layer protein. Zeta potentials were determined to obtain information about lipid-protein interactions. Values of Z potential of S-layer coated liposomes decreased with the increase of the S-layer protein recrystallization on the liposome. On the other hand, S-layer was treated with TFA 2N and the glycosidic residues were recovered and analysed by HPAEC coupled with pulsed amperometric detection (HPAEC-PAD). The chromatogram showed peaks that evidence the presence of galactose residues, among others, in the L. kefir JCM 5818 S-layer protein. The data obtained confirm the identity of the extracted protein as a typical S-layer protein with galactose residues. Z-potential measures of S-layer coated liposomes showed the existence of interactions protein-lipid, which are dependent of S-layer concentration and incubation time.