Pili display and related auto-aggregation and biofilm phenotypes in <i>L. lactis</i> IL pPil and derivatives.

*<p>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

Immunolocalization of the YhhB basal pilin.

<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

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

<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

Bacterial strains and plasmids used in this study.

*<p>ColE1 and pAMβ1 refer to the replicon; Tet^r, tetracycline resistance; Em^r, erythromycin resistance; Kan^r, 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

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

<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

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

<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.

<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 YhgE backbone pilin by TEM.

<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

Distribution of YghE in cell wall and supernatant fractions of <i>L. lactis</i> strains.

<p>Equivalent protein amounts from control strain and of 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. A, Analysis of cell wall-anchored proteins; B, Analysis of both cell wall-anchored proteins and of supernatant-released proteins. 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>. The positions of molecular mass standards (in kilodaltons) are indicated and the YhgE monomer is indicated by a black arrow.</p

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

<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. ^a, Theoretical molecular weight corresponding to mature proteins (precursor proteins devoid of both signal sequence and CWA domain); ^b, the putative lysine residue (K) that is essential in pilin oligomerization is marked in bold; ^c, the putative glutamic acid residue (E) of the E-box is marked in bold.</p