Lactobacilli isolated from kefir grains : evidence of the presence of S-layer proteins Graciela Liliana Garrote 1 , Lucrecia Delfederico 2 , Rodrigo Bibiloni 1 , Analia Graciela Abraham 1 , Pablo Fernando Pe ´rez 1 , Liliana Semorile 2 and Graciela Liliana De Antoni 1 * 1 Centro de Investigacio ´ n y Desarrollo en Criotecnologı ´a de Alimentos (CIDCA), 47 y 116 (1900), Facultad de Ciencias Exactas, Universidad Nacional de La Plata, La Plata, Argentina 2 Laboratorio de Microbiologı ´a Molecular, Departamento de Ciencia y Tecnologı ´a, Universidad Nacional de Quilmes, Bernal, Argentina Received 30 September 2002 and accepted for publication 16 October 2003 In the present study we report for the first time the presence of S-layer proteins in Lactobacillus kefir and Lactobacillus parakefir isolated from kefir grains. Soluble whole-cell protein profile obtained either by mechanical disruption (X-press) or by a combined treatment with lysozyme and SDS on whole cells, showed a significant band of apparent molecular mass of 66–71 kDa as measured by SDS–PAGE. The intensity of this band was considerably reduced when cells were treated with 5 M-LiCl. The above mentioned proteins were recovered in the LiCl extracts. After dialysis and concentration, the proteins extracted were able to reassemble in a regular array. Negative staining of these protein preparations were analysed by transmission electron microscopy and a paracrystalline arrangement was seen. Thin sections of bacteria analysed by transmission electron micrographs showed an outermost layer over the bacterial cell wall, that was lost after the LiCl treatment. The production of this surface structure under different culture conditions was also evaluated. Finally, the relationship between the presence of S-layer proteins and surface properties (e.g. adhesion to Caco-2 cells, autoaggregation, and hemagglutination) was investigated. Keywords : Kefir, lactobacilli, S-layer, surface properties. Over the past three decades, it has been documented that S-layers comprise one of the most common surface struc- tures present in both domains Archaea and Bacteria. These structures are macromolecular paracrystalline arrays that completely cover the bacterial cell surface (Sleytr, 1978 ; Sa ´ra & Sleytr, 2000). S-layers are attached to the under- lying cell wall by non-covalent bonds and usually may be dissociated and solubilized into protein monomers by chaotropic agents such as guanidine hydrochloride and dissociating agents such as 5 M-LiCl (Sleytr & Beveridge, 1999). On the basis that S-layer-carrying organisms are ubiquitous in the biosphere, and that S-layer proteins rep- resent the most abundant cellular proteins within a certain microorganism, it is now evident that these metabolically expensive products must provide the organisms with a selectivity advantage in very dissimilar habitats (Sa ´ra & Sleytr, 2000). The widespread occurrence of this external structure in prokaryotic organisms has not always been appreciated, since S-layers are often lost during prolonged cultivation under laboratory conditions (Sa ´ra & Sleytr, 2000). Chemical analysis and genetic studies on a variety of S-layers showed that it consist of subunits of a single pro- tein or glycoprotein species with molecular masses ranging from 40 to 170 kDa in size. S-layer subunits are generally assembled in lattices with oblique, square or hexagonal symmetry (Sleytr & Beveridge, 1999) and show the intrin- sic tendency to reassemble into two-dimensional arrays after removal of the disrupting agent used in the extraction procedure (Sa ´ra & Sleytr, 2000). Several specific functions have been reported for S-layers, such as protective coats, molecular sieves, molecule and ion traps, structures in- volved in cell adhesion and surface recognition, and viru- lence factors (Sleytr & Beveridge, 1999). However, no general function found in all S-layers has been recognized (Sleytr & Beveridge, 1999). With regard to GRAS microorganisms (Generally Rec- ognized As Safe) such as lactic acid bacteria, S-layer proteins seem to be a typical surface structure in several *For correspondence ; e-mail : gdantoni@biol.unlp.edu.ar Journal of Dairy Research (2004) 71 222–230. f Proprietors of Journal of Dairy Research 2004 222 DOI: 10.1017/S0022029904000160 Printed in the United Kingdom