21 research outputs found

    Immunolocalization of the YhgE backbone pilin by TEM.

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    <p>Negative staining of <i>L. lactis</i> strains was performed using phosphotungstic acid. Strains were immobilized on Formvar-carbon-coated nickel grids and pilins were detected using as primary antibodies, guinea-pig anti-YhgE and rabbit anti-YhgD polyclonal antibodies. The preparations were treated with secondary antibodies consisting of anti-guinea-pig conjugated to 5 nm gold beads for YhgE and anti-rabbit conjugated to 15 nm gold beads for YhgD. Red arrowheads, YhgE pilin subunits present in the pilus fibers or in the cell wall; blue arrows, YhgD pilin subunits. Control refers to <i>L. lactis</i> IL1403 strain harboring pIL253 plasmid and IL pPil to <i>L. lactis</i> IL1403 strain in which the <i>pil</i> operon is overexpressed (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050989#pone-0050989-t001" target="_blank">Table 1</a>). (Scale bars, 200 nm).</p

    Bacterial strains and plasmids used in this study.

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    *<p>ColE1 and pAMβ1 refer to the replicon; Tet<sup>r</sup>, tetracycline resistance; Em<sup>r</sup>, erythromycin resistance; Kan<sup>r</sup>, kanamycin resistance; <i>srtC</i>*, mutated <i>srtC</i> gene encoding an inactive sortase C; plasmid and strain designations used in the text are indicated in parentheses.</p>$<p>Christine Delorme, INRA, Micalis-UMR1319, F78350-Jouy-en-Josas.</p

    Maximum height of biofilms obtained with <i>L. lactis</i> strains.

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    <p>Strains that yielded biofilms whose maximum height measured at 4 and 15 h of growth was significantly different (<i>P</i><0.05) to that of control <i>L. lactis</i> IL1403 are marked by asterisks. Standard error is indicated. For strain designation, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050989#pone-0050989-t001" target="_blank">Table 1</a>. Indicated values are the mean of 3 determinations per experiment.</p

    Auto-aggregation phenotype of <i>L. lactis</i> cultures.

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    <p>Strains over-expressing all or parts of the <i>pil</i> operon as indicated above the pictures were grown overnight under static conditions. Control refers to <i>L. lactis</i> IL1403 strain harboring the pIL253 plasmid. For strain designation, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050989#pone-0050989-t001" target="_blank">Table 1</a>.</p

    Western blot analysis of cell wall-anchored proteins of <i>L. lactis</i> strains using anti-YhgE antibodies.

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    <p>Equivalent protein amounts from <i>L. lactis</i> control strain and from derivatives expressing all or parts of the <i>pil</i> operon were separated on 3–8% gradient Tris-acetate Criterion XT SDS-PAGE gel and were detected by immunoblotting. Control refers to <i>L. lactis</i> IL1403 strain harboring pIL253 plasmid. For strain designation, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050989#pone-0050989-t001" target="_blank">Table 1</a>. The positions of molecular mass standards (in kilodaltons) are indicated and the YhgE monomer is shown by a black arrowhead.</p

    Immunolocalization of the YhhB basal pilin.

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    <p>Negative staining of <i>L. lactis</i> strains was performed using phosphotungstic acid. Strains were immobilized on Formvar-carbon-coated nickel grids and pilins were detected using as primary antibodies, guinea-pig anti-YhhB polyclonal antibodies. The preparations were treated with secondary antibodies consisting of anti-guinea-pig conjugated to 15 nm gold beads. The negatively stained pili are indicated by black arrows and the YhhB pilin is indicated with purple arrowheads. Control refers to <i>L. lactis</i> IL1403 strain harboring pIL253 plasmid and IL pPil to <i>L. lactis</i> IL1403 strain in which the <i>pil</i> operon is overexpressed (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050989#pone-0050989-t001" target="_blank">Table 1</a>). (Scale bars, 200 nm).</p

    Experimental configuration of single-molecule force spectroscopy assays.

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    <p>Scheme illustrating the measurement configuration that was used during the experiments with no force applied (A) and under force (B) when the stage was moving. C is a close-up of B.). The pilus attached to the trapped bead is disproportionally long for reasons of depiction. The 10.5-ÎĽm mounting bead (MB) was immobilized on the coverslip while the 1-ÎĽm probe bead (PB) was trapped by optical tweezers (OT). A piliated bacterium was non-specifically attached to the MB and a pilus to the PB. When the coverslip was moved, and the trap kept in a fixed position, a force was directly exerted on the pilus. (C) Assuming adhesion to be non-specific, the most likely situation is that a portion of the pilus was attached (white subunits) and not solely the adhesion pilin (red subunit). Only a part of the pilus (black subunits) was thus subjected to the applied force.</p

    Immunolocalization of the YhgE shaft pilin and of the YhgD cap pilin by SEM.

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    <p>Fixed bacteria were applied to glass cover slips and stained with primary antibodies consisting of guinea-pig anti-YhgE and rabbit anti-YhgD polyclonal antibodies. Preparations were further incubated with colloidal-gold-conjugated secondary antibodies anti-guinea-pig-15 nm gold beads and anti-rabbit-25 nm gold beads. The backbone (YhgE) and cap (YhgD) pilin subunits are indicated with small pink and large green arrowheads, respectively. Control refers to <i>L. lactis</i> IL1403 strain harboring pIL253 plasmid and IL pPil to <i>L. lactis</i> IL1403 strain in which the <i>pil</i> operon is over-expressed (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050989#pone-0050989-t001" target="_blank">Table 1</a>). (Scale bars, 500 nm).</p
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