5 research outputs found

    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

    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

    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