Summary of pilus phenotypes observed in derivatives of <i>Lactococcus lactis</i> NZ9000, <i>Lactobacillus rhamnosus</i> GG and PB12.

<p>The AA replacement is shaded in light grey within the GYPSY motif (bold AA residues).</p

Western blotting analysis of <i>L</i>. <i>lactis</i> NZ9000 expressing different <i>spaA-srtC</i>1 gene variants, where the GYPSY motif has been altered by AA substitution.

<p><b>SpaA pilin proteins were detected using anti-SpaA polyclonal antibodies.</b> Legend: #, protein weight marker; HMWL, high molecular weight ladder; *, band corresponding to elongated pilus structures.</p

Electron microscopy observations of <i>Lb</i>. <i>rhamnosus</i> PB12 and its SrtC1-complemented derivatives.

<p>Bacterial cells were immunogold-labeled with anti-SpaA serum and gold particles (10 nm). Legend: A, <i>Lb</i>. <i>rhamnosus</i> GG; B, <i>Lb</i>. <i>rhamnosus</i> PB12; C, <i>Lb</i>. <i>rhamnosus</i> PB12 + SrtC1.</p

Western blotting analysis of <i>L</i>. <i>lactis</i> MG1363 PPiA KO mutant and its complemented mutant, where the SpaA-SrtC1 cassette was introduced.

<p>SpaA pilin proteins were detected using anti-SpaA polyclonal antibodies. Legend: #, protein weight marker; HMWL, high molecular weight ladder; *, band corresponding to elongated pilus structures.</p

Bacterial strains and plasmids used in the present study.

<p>Bacterial strains and plasmids used in the present study.</p

Electron microscopy observations of <i>L</i>. <i>lactis</i> NZ9000 producing various SpaA pilus structures.

<p>Bacterial cells were immunogold-labeled with anti-SpaA serum and gold particles (10 nm). Legend: A, NZ9000 + SpaA-SrtC1; B, NZ9000 + SpaA-SrtC1^{Y29G, P30G}; C, NZ9000 + SpaA-SrtC1^P30G; D, NZ9000 + SpaA-SrtC1^Y29G.</p

Modelling of the region containing the GYPSY motif of the <i>Lb</i>. <i>rhamnosus</i> GG SrtC1 with kinked proline residue.

<p>(A) Output prediction results using PSIPRED server [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0153373#pone.0153373.ref043" target="_blank">43</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0153373#pone.0153373.ref044" target="_blank">44</a>] indicated that the GYPSY motif introduces a breakage within an α-helix as opposed to some mutant variants; (B) and (C), <i>ab initio</i> model of the GYPSY motif. The kinked proline residue is shaded in fuchsia. The relevant AA modification(s) introduced in the present study are shown in vivid green.</p

Mucus binding assays of <i>Lb</i>. <i>rhamnosus</i> GG and PB12 expressing different <i>srtC</i>1 gene variants, where the GYPSY motif has been altered by AA substitution.

<p>The averages and standard deviations were obtained from a total of twelve technical replicates from three biological replicates.</p

Sequence alignment of the N-terminal domains of pilin-specific sortases from various piliated Gram-positive species and LOGO motif analysis.

<p>(A) The alignment was generated using MUSCLE [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0153373#pone.0153373.ref041" target="_blank">41</a>]. The so-called GYPSY motif is highlighted in yellow, with the tyrosine residue and the proline residue constituting the functional core of the motif. Residues with a partial degree of conservation within the GYPSY motif are shaded in light grey. The start methionine is highlighted in green. Protein sequences were obtained from public databases, as indicated in the text. In the table, the start of the three protein sequences marked with an asterisk (*) were re-analyzed in the present study. In addition, all sortase sequences excluding <i>E</i>. <i>faecium</i> SrtC4, <i>S</i>. <i>pneumoniae</i> TIGR4 SrtD and <i>S</i>. <i>agalactiae</i> 2603V/R sag0650 were analyzed using the MEME suite [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0153373#pone.0153373.ref042" target="_blank">42</a>], resulting in the LOGO motif depicted in (B). Legend: blue, hydrophobic residue; green, polar, non-charged residue; magenta, acidic residue and red, positively charged residue.</p

Electron microscopy observations of <i>Lb</i>. <i>rhamnosus</i> PB12 complemented with SrtC1 variants.

<p>Bacterial cells were immunogold-labeled with anti-SpaA serum and gold particles (10 nm). Legend: A, <i>Lb</i>. <i>rhamnosus</i> PB12 complemented with SrtC1 ^{Y29G, P30G}; B, <i>Lb</i>. <i>rhamnosus</i> PB12 complemented SrtC1 ^Y29G; C, <i>L</i>. <i>rhamnosus</i> PB12 complemented SrtC1 ^P30G.</p