21 research outputs found

    Prophage excision and replication.

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    <p>Agarose gel analysis of prophage excision and circularization products corresponding to <i>attB</i> and <i>attP</i> regions, respectively, probed by PCR. (A) Experimental approach: two sets of primers were used to detect prophage excision from the chromosome. The first set in red targets the excision site on the chromosome (<i>attB</i>) and the second set in green targets prophage circular forms (<i>attP</i>). (B) Prophage excision products corresponding to <i>attB</i> region were probed by PCR in WT cultures induced with 2 µg/ml of mitomycin C (M), or ciprofloxacin (C) or uninduced (−) at 28, 37 and 42°C. Ch corresponds to amplification of a strain specific chromosomal gene. (C) Prophage excision products in strain <i>pp3<sup>−</sup> pp5<sup>−</sup></i> at 37°C. (D–F) Excision and circularization products probed by semi-quantitative PCR on 100, 10 and 1 pg of total bacterial DNA prepared from cultures of WT (D) and strains <i>pp3<sup>−</sup> pp5<sup>−</sup></i> (E) <i>pp4<sup>+</sup></i> (F) induced for 2 h with 2 µg/ml of ciprofloxacin at 37°C except for detection of pp4 products in the WT strain that were obtained from DNA prepared after induction at 42°C. Twenty and 32 PCR cycles were used to amplify products of pp1 and pp7 and products of pp3, pp4 and pp5, respectively. These results are representative of three independent experiments.</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

    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

    The <i>srtC</i> genomic locus in <i>L. lactis</i> IL1403.

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    <p>Gene names are indicated. Putative promoters and transcriptional terminators are schemed. Open triangles represent oligonucleotides that allowed cDNA amplification and RT-PCR experiments (see text). The gene products of <i>yhgD</i>, <i>yhgE</i>, <i>yhhB</i> (filled in light gray) all encode LPxTG motif (represented as black boxes)-containing proteins suggesting that they are substrates of SrtC (filled in darker gray). Some also harbor pilin motif (Pm) and E-box (Eb) motif (white boxes) indicated in the central table. The <i>yhgC</i> gene (filled in black) encodes a protein whose C-terminus shows homology with Rgg/GadR/MutR-type transcription regulators <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050989#pone.0050989-Sulavik1" target="_blank">[92]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050989#pone.0050989-Sanders1" target="_blank">[108]</a>. Note that the <i>yhhB</i> sequence contained a G743T substitution compared to the sequence deposited at the NCBI. <sup>a</sup>, Theoretical molecular weight corresponding to mature proteins (precursor proteins devoid of both signal sequence and CWA domain); <sup>b</sup>, the putative lysine residue (K) that is essential in pilin oligomerization is marked in bold; <sup>c</sup>, the putative glutamic acid residue (E) of the E-box is marked in bold.</p