S-layer from Lactobacillus kefiri as nanotechnological tool: looking for the missing gene.

MALAMUD M; CARASI P; BRONSOMS S; TREJO SA; SERRADELL MA

Mar del Plata

Congreso; X Congreso de SAMIGE; 2014

Surface (S) layers are (glyco)-proteinaceous cell envelope structures ubiquitously found in bacterial species and in Archaea. The subunits are held together and attached to the underlying cell surface by non-covalent interactions and have an intrinsic tendency to self-assembly. Lactobacillus kefiri is a potentially probiotic microorganism carrying a highly glycosylated S-layer protein which showed to have interesting functional properties. Previous MALDI-TOF-MS analysis showed a great structural heterogeneity among S-layer proteins from different L. kefiri strains, but no more information about their sequences were obtained until now. The aim of this work is to show the most recent results obtained by MALDI-TOF and LC-MS/MS analysis of the S-layer protein from the autoaggregative strain L. kefiri CIDCA 8348, and the way that information was employed to gain insight into the primary sequence of this glycoprotein.A SDS-PAGE band corresponding to the S-layer protein from L. kefiri CIDCA 8348 was excised, then it was digested with trypsin, and finally a peptide mass fingerprint (PMF) was carried out in a MALDI-TOF mass spectrometer. Searching was performed against NCBInr/SwissProt database using MASCOT software. As observed few years ago, no significant matching was obtained at global protein sequence level between S-layer protein from CIDCA 8348 and other S-layer proteins available in databases. Interestingly, PMF analysis showed that our protein shares seven different fragments with the S-layer proteins from L. buchneri CD034 or L. parafarraginis F0439, two phylogenetically related strains. A 100% homology was observed with fragments from L. buchneri CD034 or L. parafarraginis F0439, using MS/MS analysis and a de novo sequencing method. Each fragment contains from 9 to 25 amino acid residues, showing a 20 % of total homology with proteins of the same family. Based on the genetic code reported for L. buchneri CD034, primers were designed and used for PCR reaction. A fragment of 320 bp length was amplified from DNA of L. kefiri CIDCA 8348, and the sequence showed a 99% homology with L. buchneri ´s fragment. Since our goal is to sequence the S-layer gene of L. kefiri, this information as well as new primers designed in the flanking area of L. buchneri S-layer gene, will be used as a starting point for sequencing. Amplification from DNA of other aggregative and non-aggregative L. kefiri strains will be also performed.