Confirmation of transposon insertion events into <i>B.</i><i>breve</i> UCC2003 and <i>B. breve</i> NCFB2258 genome by Southern hybridization.

<p>Blots of twenty-six randomly selected mutants of <i>B. breve</i> UCC2003 (Panel I, two blots) and sixteen randomly selected mutants of <i>B. breve</i> NCFB2258 (Panel II, single blot) are shown. Lanes labeled from A to R correspond to mutants whose insertion site was subsequently sequenced as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064699#pone-0064699-t003" target="_blank">Table 3</a>.</p

Genomic position and surrounding regions of the insertion site of a number of transposon insertion mutants that were isolated based on their inability to grow on one or more carbohydrates.

<p>The diagram was drawn to scale using <i>B. breve</i> UCC2003 genome sequence information. White open arrowheads represent the location of the transposon with the specific location indicated as the genome coordinate based on accession number CP000303. Red arrows represent transposon-disrupted open reading frames. Grey arrows represent flanking open reading frames. Lollipops indicate transcriptional terminators predicted by ARNold Web server (<a href="http://rna.igmors.u-psud.fr/toolbox/arnold/" target="_blank">http://rna.igmors.u-psud.fr/toolbox/arnold/</a>).</p

Strains, plasmids and primers used.

<p>Restriction sites are underlined and a rho-transcriptional terminator sequence is highlighted in bold. NCFB, National Collection of Food Bacteria.</p

Growth profiles of <i>B.</i><i>breve</i> UCC2003 and derived transposon mutant strains, 101C6 (I), 181D10 (II) on lactose, galactose and ribose; and 164B7 (III) on lactose, pullulan and ribose.

<p>Presented data are average of duplicate independent growth experiments.</p

Transformation efficiencies and transposition frequencies.

*<p>Transformation efficiencies and transposition frequencies were determined following electroporation of a replicative vector, namely pAM5, or a tetracycline-resistant TN5 transposome, respectively, into <i>B. breve</i> UCC2003 competent cells grown in different carbohydrates. Transposition events are average of independent duplicates.</p

List of <i>Bifidobacterium</i> strains, with additional information on the source of isolation.

<p>For each strain, the relative levels of AI-2 production (means +/- SEM) in the diluted supernatant are given compared to AI-2 levels produced by the biosensor itself ( = 100%). Data presented are mean +/- SEM from triplicate experiments. In addition, the results for the PCR assay with primers directed against <i>luxS</i> are shown. (* +, PCR positive result, -, PCR negative result; ** T, type strain of the species).</p

Luminescence signal of the <i>V. harveyi</i> BB170 biosensor strain in the presence of sterile and neutralized supernatant of <i>B. breve</i> UCC2003, the insertion mutant <i>B. breve</i> UCC2003-luxS and the complemented strain <i>B. breve</i> UCC2003-luxS [pBC1.2luxS].

<p>Data obtained with <i>E. coli</i> DH5α (a strain not producing AI-2) and a medium-only control are included as reference. Data shown are means ± SEM. (*, luminescence is significantly lower than than produced with supernatant of <i>B. breve</i> UCC2003, p<0.05, compared to WT; n = 3).</p

Comparison of the <i>lux</i>S genetic loci of <i>B. breve</i> UCC2003 with corresponding <i>lux</i>S loci from other sequenced bifidobacteria.

<p>Each solid arrow indicates an open reading frame. The lengths of the arrows are proportional to the length of the predicted open reading frame. The colour coding which is indicative of putative function, is indicated within the arrow. Orthologs are marked with the same colour while the amino acid identity of each predicted protein is indicated as a percentage relative to its equivalent protein encoded by <i>B. breve</i> UCC2003.</p

Murine colonization trial.

<p>CFU g⁻¹ feces of <i>B. breve</i> UCC2003 (dark blue) and <i>B. breve</i> UCC2003-luxS (red) administered individually, or simultaneously {a mixture of equal numbers of <i>B. breve</i> UCC2003 (pale blue) and <i>B. breve</i> UCC2003-luxS (pink)}. Administration started at day 0 and was continued for 3 consecutive days. Data shown are mean ± SEM. (n = 7).</p

Bacterial strains and plasmids used in this study.

<p>Bacterial strains and plasmids used in this study.</p