Genomic characterization of L. amylovorus Slp:s
To initiate comparative studies on the role of L. amylovorus surface layer proteins in the probiotic interactions described above, the numbers and sequence similarities of the slp genes in the genomes of the strains were initially analysed. The genomic investigation of the eight strains revealed several slp genes in each strain. Genes with homology to L. acidophilus NCFM slpA and slpB[12] were identified, and the homologous L. amylovorus genes were named slpA and slpB, respectively. Furthermore, slp-like genes of a third type were detected in all of the eight genomes and these were designated as slpC. The slp sequences, along with the deduced amino acid sequences, are shown in Additional file 1. All the eight strains studied carried only one slpA-homologue, except for GRL 1117, which had two distinct slpA-like genes (slpA1 and slpA2). Only one slpB-homologue was identified in GRL 1112, 1114, 1115, 1116, 1117 and 1118 as well as in DSM 16698, whereas DSM 20531T carried two slpB-like genes (slpB1 and slpB2). The highest variation was found in the number of slpC-type genes: strains DSM 20531T and GRL 1115 carried one, DSM 16698 possessed three (slpC1, slpC2, slpC3), and the rest of the strains had two slpC-type genes (slpC1 and slpC2). Exceptionally, the gene slpC3 of DSM 16698 was found to be located on a plasmid. A phylogenetic tree was constructed based on the deduced amino acid sequences of the slpA, slpB and slpC gene products (Figure 6). The tree clearly shows that the SlpA-like sequences have diversified most during evolution, while the SlpB-type proteins have remained more similar to each other whereas the predicted SlpC-type proteins could be clustered into three distinct groups.
Figure 6  Phylogeny of L. amylovorus Slp:s. A neighbour-joining phylogenetic tree based on L. amylovorus Slp sequences was generated by creating a multiple amino acid sequence alignment of the predicted S-layer proteins with MUSCLE [39], by eliminating poorly aligned positions using GBLOCKS [40], and by generating phylogenies using the PhyML package [41]. Numbers 1–3 indicate the presence of several slp genes in the same strain. *, the corresponding gene is expressed.

E