Detection and expression analysis of slp genes in L. amylovorus
The presence of S-layer proteins on the surface of the L. amylovorus intestinal isolates GRL 1112 – GRL 1118 has previously been described [28]. The putative slp encoding genes were identified in silico in the draft genomes of the L. amylovorus strains based on homology with the publicly available L. acidophilus slp gene sequences. The identification of the expressed slp genes was based on the observed molecular weights of the proteins, obtained by analyzing overnight cultures of the strains by standard SDS-PAGE in 12% gels, and on the amino-terminal and/or internal amino acid sequences of the Slp:s. The amino-terminal sequences were obtained by an Edman-degradation-based Procise 494 HT sequencer (Life Technologies, Carlsbad, CA), and internal peptide sequences through a peptide mapping analysis: the proteins were digested in-gel by trypsin followed by analysis with liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) carried out with an EASY-nLC liquid chromatograph (Thermo Fisher Scientific, Germany) connected to a Velos Pro-Orbitrap Elite hybrid mass spectrometer (Thermo Fisher Scientific, Germany) with a nano-electrospray ion source (Thermo Fisher Scientific, Germany). Both amino-terminal sequencing and peptide mapping were performed in the Institute of Biotechnology (University of Helsinki, Finland).