10 research outputs found

    Morphological aberrations induced by the overproduction of different variants of OatA in the <i>oatA</i> mutant.

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    <p>Induction was performed with 20 ng/ml of nisin. A) Selection of cells showing curvature (labeled C), asymmetrical septation (labeled A), dual septation (labeled D) observed in bright field (BF) or phase contrast (PC) microscopy and fluorescent microscopy (FM4–64, membrane staining). A<sup>−/</sup>A<sup>+++</sup>, <i>oatA</i> mutant overexpressing <i>oatA</i><sup>WT</sup>; A<sup>−/</sup>A<sup>* +++</sup>, <i>oatA</i> mutant overexpressing <i>oatA</i><sup>D510A/S511A</sup>; A<sup>−/</sup>A™<sup> +++</sup>, <i>oatA</i> mutant overexpressing <i>oatA</i><sup>TM1–10</sup><i>::yfp</i>. Bar scale, 2.0 µm. B) Selection of two tripartite cells (I and II) showing a central mini-cell (labeled MC) resulting from a dual septation event observed in bright field (BF) microscopy and fluorescent microscopy (YFP, OatA<sup>TM1–10</sup>-YFP fluorescence; DAPI, DNA staining).</p

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

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    <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<sup>−</sup>) 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<sup>−</sup>. Bar scale, 2.0 µm.</p

    Subcellular localization of OatA<sup>TM10</sup>-YFP and OatB<sup>TM10</sup>-YFP fusions.

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    <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<sup>−/</sup>YFP) used as control; middle panels, <i>oatA</i> mutant producing OatA<sup>TM1–10</sup>-YFP (OatA<sup>−/</sup>OatA<sup>TM1–10</sup>-YFP); lower panels, <i>oatB</i> mutant producing OatB<sup>TM1–10</sup>-YFP (OatB<sup>−/</sup>OatB<sup>TM1–10</sup>-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<sup>−/</sup>A™, <i>oatA</i> mutant producing OatA<sup>TM1–10</sup>-YFP and B<sup>−/</sup>B™, <i>oatB</i> mutant producing OatB<sup>TM1–10</sup>-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.

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    <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<sup>−</sup>, <i>oatA</i> mutant; A<sup>−/</sup>A<sup>+</sup><i>oatA</i> mutant complemented with <i>oatA</i><sup>WT</sup>; A<sup>−/</sup>A<sup>*</sup><i>oatA</i> mutant complemented with <i>oatA</i><sup>D510A/S511A</sup>; A<sup>−/</sup>A™, <i>oatA</i> mutant complemented with <i>oatA</i><sup>TM1–10</sup><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

    Temporal localization of OatA<sup>TM1–10</sup>-YFP during the cell cycle.

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    <p>Upper panels, selection of cells at different division stages observed in bright field (BF) microscopy and fluorescent microscopy (FM4–64, membrane staining; YFP, OatA<sup>TM1–10</sup>-YFP fluorescence). Induction of <i>oatA<sup>TM1–10</sup>::yfp</i> expression was performed with 10 ng/ml of nisin. I, septal localization of the YFP fusion prior to membrane invagination; II, co-localization of the YFP fusion and the septal membrane; III, polar localization at the end of septation; IV, reinitiation of septal localization in daughter cells. Bar scale, 1.0 µm. Lower panel, schematic representation of the cell cycle. Colors and numbers refer to above micrographs. Yellow represent a merge between FM4–64 and YFP fluorescences.</p

    Indirect immunofluorescence microscopy (A,B,C&D).

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    <p>Anti-Msp1 (A&B) and anti-Msp2 (C&D) rabbit antisera were used on wild-type (A&C) en <i>msp1</i> mutant cells (B&D). Anti-rabbit IgG antibodies conjugated with Alexa Fluor 488 (Invitrogen) were used to visualize the Msp1 and Msp2 localization on the cells.</p

    Main muropeptides from LGG PG digested with or without Msp1.

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    a<p>Peak numbers refer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031588#pone-0031588-g005" target="_blank">Figure 5</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031588#pone.0031588.s001" target="_blank">Figure S1</a>.</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, 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.0031588#pone-0031588-g005" target="_blank">Figure 5</a>).</p>e<p>ND, non detected.</p

    RP-HPLC separation profile of muropeptides from LGG digested with mutanolysin (A) and digested with mutanolysin and Msp1 (B).

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    <p>Peak numbers refer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031588#pone-0031588-t001" target="_blank">Table 1</a>. DS-di, disaccharide dipeptide (GlcNAc-MurNAc-L-Ala-D-Gln). The schematic structure of LGG peptidoglycan and site of cleavage of Msp1 is also represented. GlcNAc, N-acetylglucosamine; MurNAc, N-acetylmuramic acid.</p
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