Quantification of TEM images (Figs. 2–4).

<p>For each HSL isoform 600 particles were analyzed with regard to size of the two structural domains in the monomer and for presence of a head-to-head arrangement of the dimer. Large and small domain refer to the situation in HSL_adi and HSL_beta, where the labeling pattern shows that the larger domain corresponds to the C-terminal catalytic domain and the small domain to the N-terminal non-catalytic domain.</p

Negative stain electron microscopy analysis of HSL_tes.

<p>Electron microscopy images depicting HSL_tes either native <b>(A–C)</b> or with Au-labelled anti-His-Fab fragments only <b>(D and E)</b> or in combination with Au-labelled anti-exon T- Fab fragments <b>(F and G)</b>. Partials <b>(B–G)</b> show either monomers <b>(B, D and F)</b> or dimers <b>(C, E and G)</b> in different orientations. As a guide to the TEM images, cartoons are included below each subpanel, showing the domain corresponding to the large domain in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011193#pone-0011193-g002" target="_blank">Figs. 2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011193#pone-0011193-g003" target="_blank">3</a>in red, the other domain in green and the gold-labelled antibodies in black. Scale bars are 50 nm <b>(A)</b> and 25 nm <b>(B–G)</b>. Au particles have a diameter of 3 nm (Anti-His₆) or 5 nm (Anti-exon T).</p

Purity and identity of recombinant HSL isoforms.

<p>Purified recombinant proteins were analysed by SDS-PAGE (A) and western blot (B–D). <b>(A)</b> Lane 1: MWM, lane 2: purified HSL_tes, lane 3: purified HSL_beta, lane 4: purified HSL_adi. The identity of the respective purified isoforms was verified by western blot analysis using polyclonal antibodies generated against full-length HSL_adi<b>(B)</b>, polyclonal antibodies directed against amino acids encoded by exon A <b>(C)</b>, or polyclonal antibodies recognizing the amino acid sequence encoded by exon T <b>(D)</b>.</p

Negative stain electron microscopy analysis of HSL_beta.

<p>Electron microscopy images depicting HSL_beta either native <b>(A–C)</b> or with Au-labelled anti-exon A Fab fragments <b>(D–E)</b>. Partials <b>(B–E)</b> show either monomers <b>(B and D)</b> or dimers <b>(C and E)</b> in different orientations. As a guide to the TEM images, cartoons are included below each subpanel, showing the large domain in red, the small domain in blue and the gold-labelled Fab fragment in black. Scale bars are 50 nm <b>(A)</b> and 25 nm <b>(B–E)</b>. Au particles have a diameter of 3 nm.</p

Cold adaptation of HSL isoforms.

<p>Esterase activity towards the <i>p</i>-nitrophenolbutyrate (pNPB) substrate of the three HSL isoforms and bovine lipoprotein lipase (LPL) at different assay temperatures. Results shown are mean ± SEM of seven or eight experiments (HSL isoforms) or two experiments (LPL) in triplicate.</p

Negative stain electron microscopy analysis of HSL_adi.

<p>Electron microscopy images depicting HSL_adi either native <b>(A–C)</b> or with Au-labelled anti-his Fab framents <b>(D–E)</b>. Partials <b>(B–E)</b> show either monomers <b>(B and D)</b> or dimers <b>(C and E)</b> in different orientations. As a guide to the TEM images, cartoons are included below each subpanel, showing the large domain in red, the small domain in blue and the gold-labelled Fab fragment in black. Scale bars are 50 nm <b>(A)</b> and 25 nm <b>(B–E)</b>. Au particles have a diameter of 3 nm.</p

Phosphorylation and dephosphorylation of HSL isoforms purified from Sf9 cells.

<p>HSL_adi, HSL_beta and HSL_tes were phosphorylated by PKA in the presence of ³²P-labeled ATP. Reactions were subjected to SDS-PAGE and the amount of incorporated phosphate was determined by autoradiography and scintillation counting of gel bands followed by calculation from standards made from the original reactions <b>(A)</b>. HSL_adi, HSL_beta and HSL_tes were dephosphorylated by calf intestinal phosphatase and assayed using triolein as substrate. Data represents mean ± SEM of six assays <b>(B)</b>. HSL_adi, HSL_beta and HSL_tes were phosphorylated by PKA and assayed using triolein as substrate. Data represents mean ± SEM of triplicate assays. *P<0.05; *** P<0.0005, unpaired t-test <b>(C)</b>.</p

Bacteria and plasmids used in this study and the presence of <i>slr</i> gene in <i>S. pyogenes</i> strains.

1<p>Presence of the <i>slr</i> gene was determined using PCR as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020345#s2" target="_blank">Material and Methods</a>.</p>2<p>WHO Collaborating Center for Reference and Research on Streptococci, Institute of Hygiene and Epidemiology, Prague, Czech Republic.</p>3<p>National Center for Disease Control reference codes.</p

Binding of Slr to immobilized collagen I.

<p>A: Collagen I denatured with 4 M guanidine hydrochloride (+) and non-denatured collagen I (−) were applied to a PVDF membrane and incubated with radiolabeled Slr. The amount of collagen is indicated to the right. B: Binding of gold labeled Slr (the dots) and collagen I using electron microscopy. The dots are bound to C- and N terminal as well as 70 respectively 100 nm further in on the monomeric collagen I (panel I). The short span between light strands represents the overlap region, whiles the dark span represents the gap region on a collagen I fibril. Binding can be observed in both regions, but is mostly concentrated to the overlap region (panel II). Scale bar  = 100 nm.</p

Characteristics of the Slr protein.

<p>A: Schematic comparison of the InlA from <i>L. monocytogenes</i> and Slr. The identity and similarity between the two LRR regions are stated. The cell wall anchoring motif for InlA and the signal peptide II recognition motif for Slr are indicated as well as the histidine triad motifs. B: Sequence alignments of the two groups of LRRs (A and B repeats) in Slr and comparison of the LRR consensus sequences of Slr and InlA.</p