RP-HPLC separation profile of muropeptides obtained from <i>L. casei</i> BL23 PG.

<p>PG was digested by mutanolysin (A) or by mutanolysin and recombinant Lc-p75 (B). Numbers correspond to the main muropeptides which amounts changed between Panel A and B. Letters indicate new peaks present in Panel B and absent in Panel A. Complete annotation of the chromatograms is presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032301#pone.0032301.s004" target="_blank">Figure S3</a>.</p

Detection of Lc-p75 in culture supernatant.

<p>Analysis by SDS-PAGE (A, B) and zymogram assay (C, D) of the culture supernatant of wild type BL23 (A, C) and negative mutant (B, D) grown on AOAC medium. Lc-p75 is indicated by an arrow.</p

Indirect immunofluorescence localization of Strep-tagged Lc-p75 in <i>L. casei</i>.

<p>Strep-tagged Lc-p75 was localized in overexpressing strain (A) and in complemented negative mutant (B) with monoclonal antibody directed against Strep-tag as first antibody.</p

Main muropeptides from <i>L. casei</i> BL23 PG hydrolyzed by Lc-p75 and main products of digestion.

a<p>Peak numbers refer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032301#pone-0032301-g002" target="_blank">Figure 2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032301#pone.0032301.s004" target="_blank">Figure S3</a>. New peaks obtained after Lc-p75 digestion, are indicated by letters.</p>b<p>Di, disaccharide dipeptide (L-Ala-D-iGln); Tri, disaccharide tripeptide (L-Ala-D-iGln-L-Lys); Tetra, disaccharide tetrapeptide (L-Ala-D-iGln-L-Lys-D-Ala); Disaccharide, GlcNAc-MurNAc; Ac, acetylation on MurNAc, iGln, isoglutamine; N, D-Asn; A, D-Ala; K, L-Lys.</p>c<p>Sodiated molecular ions were the most abundant ones on MALDI-TOF mass spectra for all muropeptides.</p>d<p>Percentage of each peak was calculated as the ratio of the peak area over the sum of areas of all the peaks identified in the corresponding chromatogram (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032301#pone.0032301.s008" target="_blank">Table S3</a>).</p>e<p>ND, non detected.</p

Phenotype comparison between wild type <i>L. casei</i> BL23, Lc-p75-negative mutant and complemented Lc-p75 mutant.

<p>Pictures of wild type <i>L. casei</i> (A, C, F, H), Lc-p75-negative mutant (B, D, I) and complemented Lc-p75 mutant (E). Colony morphology (A, B), phase contrast microscopy (C, D, E) fluorescence microscopy with merged FM-4-64 (red) and DAPI (blue) staining (F, G) and transmission electron microscopy (H, I).</p

Lc-p75 activity on purified muropeptides selected as substrates.

a<p>Di, disaccharide dipeptide; Tri, disaccharide tripeptide (L-Ala-D-iGln-L-Lys); Tetra, disaccharide tetrapeptide (L-Ala-D-iGln-L-Lys-D-Ala); Disaccharide, GlcNAc-MurNAc; iGln, isoglutamine; N, Asn; Ac, acetylation on MurNAc.</p>b<p>Similar amounts of each muropeptide were used for each test.</p>c<p>Percentage of each peak was calculated as the ratio of the peak area over the sum of areas of all the peaks identified in the corresponding chromatogram.</p>d<p>Other forms of muropeptides resulting from partial digestion of the substrate.</p

Schematic structure of <i>L. casei</i> BL23 peptidoglycan and site of cleavage determined for Lc-p75.

<p>GlcNAc, N-acetylglucosamine; MurNAc, N-acetylmuramic acid. MurNAc may be O-acetylated (O-Ac) or not in PG.</p

Effect of expression of <i>min::yfp</i> fusions on cell morphology and subcellular localization.

<p>Effect of expression of <i>minC::yfp</i> (A, MinC-YFP) and <i>minD::yfp</i> (B, MinD-YFP) in <i>L. plantarum</i> wild-type (WT) and <i>oatA</i> mutant (OatA⁻) without nisin induction (0 ng/ml) and with 2.5 ng/ml of nisin. Micrographs were obtained in bright field (BF) microscopy and fluorescence microscopy (YFP). For MinD-YFP (nisin 2.5 ng/ml), minicells are indicated by arrows in WT and three selected branched cells (insets) are added for OatA⁻. Bar scale, 2.0 µm.</p

Subcellular localization of OatA^TM10-YFP and OatB^TM10-YFP fusions.

<p>A) Schematic representation of fusion proteins (YFP, yellow star) and corresponding micrographs in phase contrast (PC) microscopy and fluorescence microscopy (YFP). Upper panels, <i>oatA</i> mutant producing cytoplasmic YFP (OatA^−/YFP) used as control; middle panels, <i>oatA</i> mutant producing OatA^TM1–10-YFP (OatA^−/OatA^TM1–10-YFP); lower panels, <i>oatB</i> mutant producing OatB^TM1–10-YFP (OatB^−/OatB^TM1–10-YFP). Induction of expression was performed with 10 ng/ml of nisin. Bar scale, 2.0 µm. B) Fluorescence ratio (FR; AU, arbitrary unit) between the fluorescence measured at mid-cell position and pole. A^−/A™, <i>oatA</i> mutant producing OatA^TM1–10-YFP and B^−/B™, <i>oatB</i> mutant producing OatB^TM1–10-YFP. Lines represent the mean value (n = 20, 3 independent replicates).</p

Study of the uncoupling between elongation and septation phases by time-lapse experiments.

<p>A) Cell length (µm) of the mother cell during the division process for the wild type (solid line) and the <i>oatA</i> mutant (dashed line). Each line corresponds to one individual cell. Time (min) was arbitrarily fixed to zero at the last view before any detectable cell invagination in bright field. Correspondences between time and division state are drawn upside of the graph for the wild type. One representative time-lapse experiment of three independent experiments for each strain (n ≥3 for each). All examined cells of the wild type and the <i>oatA</i> mutant (n = 15 for each) display their respective phenotype. B) Cell length increase (µm) measured during the division process after 5 min (T10–T5; T5, initial invagination), 10 min (T15–T5), and 15 min (T20–T5; T20, final septation). Time intervals are reported in panel A. Symbols: WT, wild-type; A⁻, <i>oatA</i> mutant; A^−/A⁺<i>oatA</i> mutant complemented with <i>oatA</i>^WT; A^−/A^*<i>oatA</i> mutant complemented with <i>oatA</i>^D510A/S511A; A^−/A™, <i>oatA</i> mutant complemented with <i>oatA</i>^TM1–10<i>::yfp</i>. All the variants of <i>oatA</i> are expressed at low basal level in absence of the nisin inducer. Mean values of 5 cells in each time-lapse experiment. Significance based on <i>t</i>-test; **, p-value <0.01.</p