Probing of fucose and mannose residues on <i>L</i>. <i>rhamnosus</i> GG pili using AFM.

<p>Fig 1A and 1C depict the adhesion forces and Fig 1B and 1D the rupture length histograms (n = 1024) obtained in buffer, from the interaction between <i>L</i>. <i>rhamnosus</i> GG wild type and fucose- and mannose-binding lectin probes (AAL and HHA resp.). In Fig 1E and 1F the force data for the interaction of a pili-deficient Δ<i>spaCBA</i>::Tc^R mutant (CMPG5357) with the two lectin probes are displayed. Insets show representative retraction force curves.</p

Immunogold labeling reveals colocalization of SpaA and fucose on SpaCBA pili.

<p>Immunoelectron microscopy double labeling of <i>L</i>. <i>rhamnosus</i> GG cells (A and B) and the Δ<i>spaCBA</i>::Tc^R mutant (CMPG5357) (C) with SpaA antiserum and the fucose-specific <i>Aleuria aurantia</i> lectin (AAL). Detection of SpaA and AAL was done using 5 nm (white arrows) and 10 nm gold particles (black arrows) respectively. The scale bar represents 500 nm. Original overall pictures are shown as insets of A and B.</p

SpaCBA pili are glycosylated with mannose and fucose.

<p><b>(A) SpaCBA pili are glycosylated on <i>L</i>. <i>rhamnosus</i> GG cells—</b>Cell wall-associated proteins of <i>L</i>. <i>rhamnosus</i> GG wild type (1), the pilus-deficient Δ<i>spaCBA</i>::Tc^R mutant (CMPG5357, 2) and the Δ<i>welE</i>::Tc^R mutant on which the pili are overexposed (CMPG5351, 3), were probed with mannose- and fucose-specific lectins (HHA and AAL resp.). Pili content was visualized by probing with SpaC antiserum (SpaC, black arrow and HMW: high molecular weight pili). Interference of the Msp1 glycoprotein was ruled out (open arrow). Blots and gels were performed in triplicate. (LK = Precision Plus Protein^™ Kaleidoscope^™ Standard, Bio-Rad) <b>(B) Purified pili are glycosylated—</b>SDS-PAGE separated pili (pool A) were stained with PAS glycostain and Sypro^® to visualize their protein content. Pili content was shown by probing of Western blotted samples with SpaC antiserum (HMW: high molecular weight pili). Purified Msp1 (open arrow) was used as a positive control. Representative gels are shown, experiment was carried out in triplicate. (LK = Precision Plus Protein^™ Kaleidoscope^™ Standard, Bio-Rad) <b>(C) SpaCBA pili bind mannose-specific lectins—</b>Purified pili fractions (PRM and pili pool B) were subjected to PAS glycostaining and both Sypro^® and Silver stain to visualize their protein content. Absence of 75 kDa signals on PAS and lectin blots rule out the interference Msp1 (cf. open arrow). Western blotted samples were probed with SpaC antiserum (HMW: high molecular weight pili) and the mannose-specific lectins HHA and GNA, visualizing the pili content of the samples and the presence of mannose, respectively. Representative gels and blots of in triplicate-repeated experiment. (LK = Precision Plus Protein^™ Kaleidoscope^™ Standard, Bio-Rad) <b>(D&E) Mannose- and fucose-specific lectins bind SpaCBA pili—</b>Binding of lectins to plate-coated pili was measured to ELISA. Wells coated with coating buffer served as a negative control. Mannan and Lewis X were coated as a positive control for the mannose-specific HHA (<i>Hippeastrum</i> hybrid, D) and fucose-specific AAL (<i>Aleuria aurantia</i>, E) lectins, respectively. Error bars represent standard deviations of three independent experiments (paired t-test, p < 0.05)</p

Analysis of genome diversity in <i>L. rhamnosus</i> by mapped SOLiD sequencing.

<p>The 100 <i>L. rhamnosus</i> strains were clustered using hierarchical clustering <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003683#pgen.1003683-Sturn1" target="_blank">[78]</a> based on their relative shared gene content with <i>L. rhamnosus</i> GG. Strain names were colour-coded as follows: green for dairy isolates, purple for intestinal isolates, orange for oral isolates, magenta for vaginal isolates and blue for clinical/other isolates. Four main groups or clusters were highlighted and numbered. The <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003683#pgen-1003683-g001" target="_blank">Figure 1</a> also shows the 17 variable chromosomal regions identified in GG, as further detailed in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003683#pgen-1003683-t001" target="_blank">Table 1</a>. Each row corresponds to one strain, and each column shows the genes in these variable regions, colour-coded as follows: blue for present and yellow for absent.</p

Pilosotype distribution in our <i>L. rhamnosus</i> collection.

<p>The table describes the niches or isolation sources, the number of strains per group and their pilosotype, <i>i.e.</i> the presence of an intact and functional SpaCBA pili cluster as determined in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003683#pgen-1003683-g006" target="_blank">Figure 6</a>. Probiotic strains GG, VIFIT, IDOF, AK-RO and CO-RO were classified as intestinal isolates. The group ‘Others’ contained strains of unspecified origins (clinical specimens) or from minor isolation source (<i>n</i><2), <i>i.e.</i> hip punction or pus.</p

CRISPR spacer oligotyping and CRISPR-associated protein diversity in <i>L. rhamnosus</i> species.

<p>Panel (A) illustrates the genetic organization of the CRISPR system and its associated genes in <i>L. rhamnosus</i> GG. Panel (B) shows the conservation (blue), the partial conservation (grey) or the absence (yellow) of <i>L. rhamnosus</i> GG spacers. The presence (green) or the absence (red) of the <i>cas</i> genes is also indicated in Panel (C). Strains are organized according to their genetic relatedness defined in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003683#pgen-1003683-g001" target="_blank">Figure 1</a>.</p

API 50CH fermentative profile of <i>L. rhamnosus</i> strains.

<p>Fermentation ability is indicated in black for positive, grey for partially positive and white for negative. Strains are organized according to their genetic relatedness as defined in the hierarchical clustering and coloured according to their respective niche/origin (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003683#pgen-1003683-g001" target="_blank">Figure 1</a>). Carbohydrates of interest are marked by a red asterisk. Black arrows show fermentative profile shifts among <i>L. rhamnosus</i> strains.</p

Features of the variable chromosomal regions found in <i>L. rhamnosus</i>.

<p>Features of the variable chromosomal regions found in <i>L. rhamnosus</i>.</p

Anthropocentric view of the <i>L. rhamnosus</i> species.

<p>The interactions between <i>L. rhamnosus</i> and the human cavities are frequent and occur in various contexts, <i>i.e.</i> consumption of food products (common scenario) or development of bacteremia (rare event). For each niche or isolation source, the strains were grouped according to their geno-phenotype (radar plot). The geno-phenotype is based on the scoring of distinctive genetic and phenotypic traits measured in this study, <i>i.e.</i> gene-content, CRISPR oligotype, bile resistance, pilosotype, sugar group I (dulcitol, D-arabinose and L-fucose), sugar group II (D-saccharose, D-maltose, methyl-α-D-glucopyranoside and D-turanose) and sugar group III(L-rhamnose, L-sorbose, D-ribose and D-lactose). The distinction between the two main geno-phenotypes mostly relies on gene acquisition and loss, point mutations, genetic reorganization that possibly reflect strain adaptation to an ecological niche.</p

Mucus adhesion and SpaCBA pili gene diversity among <i>L. rhamnosus</i>.

<p>Panel (A) shows the genotype and phenotype of all strains. Based on our genomic analysis, pilin and sortase genes were assigned as present (green) or divergent (red). Sequences of corresponding genes were further analyzed using blastx. The sequence identity was shown by an upper triangle superposed to the SOLiD genomic data, where the colour gradient corresponds to the identity percentage to GG pili genes. We also indicated if the strains were tested by immunoblotting analysis (DB), electron microscopy (EM) or <i>in vitro</i> competitive binding assay (AB). Green is for pili positive and red for pili negative. Panel (B) shows the human mucus binding ability (%) of all <i>L. rhamnosus</i> isolates ranked from the lowest to the highest mucus binder.</p