20 research outputs found

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

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    <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

    Lc-p75 activity on purified muropeptides selected as substrates.

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    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

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

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    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

    Detection of Lc-p75 in culture supernatant.

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    <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>.

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    <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

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

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    <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

    Decreased growth rate and lack of tunicamycin sensitivity of <i>ΔgbcO</i> mutant.

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    <p>(<b>A</b>) Growth curves of NEM316 WT (solid squares), Δ<i>gbcO</i> mutant (circles) and Δ<i>gbcO</i>pTCVΩ<i>gbcO</i> (empty squares) strains. Cultures were performed in TH medium without antibiotics at 37°C in 96 wells plates in triplicate. Optical densities were recorded at 600 nm in a Tecan M200 apparatus with 5 sec agitation before measure. Average values of a typical experiment are presented. (<b>B</b>) Effect of various concentrations of tunicamycin on the growth rate of WT (solid squares), <i>ΔgbcO</i> (black circles) and Δ<i>gbcO</i>pTCVΩ<i>gbcO</i> (empty squares) strains. Tunicamycin, a general inhibitor of UDP-GlcNAc:lipid phosphate carrier transferase activities, inhibits the growth of WT and complemented strains but not that of <i>ΔgbcO</i> mutant suggesting that GbcO carries this activity. Experiments were performed in triplicate and results are reported as a percentage of the growth rate in absence of tunicamycin. Error bars represent ± S.E. of triplicate experiments.</p

    Structure of GBC and proposed scheme of GBC synthesis.

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    <p>(<b>A</b>) The multiantennary GBC is shown linked to an N-acetyl muramic (NAM) moiety, a component of PG. (<b>B</b>) The figure depicts the first steps of GBC synthesis where GbcO is proposed to catalyze the transfer of UDP-GlcNAc to a lipid phosphate carrier.</p

    Electron microscopy imaging of NEM316 WT, <i>ΔgbcO</i> mutant, and complemented strains.

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    <p>Bacteria were harvested in mid-log phase (OD<sub>600 nm</sub> = 0.5), fixed, and prepared as described in Supporting <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002756#s3" target="_blank">Materials and Methods</a> (see <b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002756#ppat.1002756.s005" target="_blank">Text S1</a></b>) (<b>A</b>) Representative views of scanning electron microcopy analysis illustrating the morphological alterations (size, form, and cell division abnormalities) due to <i>gbcO</i> inactivation. (<b>B, C</b>) Transmission electron microscopy views of uranyl acetate stained thin cryosections at two magnifications (see scale bars). The presence of the pellicle (electron dense outer layer) at the surface of WT and complemented strains observed at the higher magnification is highlighted with black arrows. An open triangle depicts the equatorial ring (EqR), a zone of active peptidoglycan synthesis seen in almost all WT and complemented cells but absent in the <i>ΔgbcO</i> mutant cells.</p
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