Strains used in this study.

<p>*Formerly denoted as Newman <i>emp</i><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002434#ppat.1002434-Johnson1" target="_blank">[47]</a>. Sequencing analysis revealed that assumedly by phage transduction not only the <i>emp</i> gene but also the neighboring functional <i>vWbp</i> gene of strain Newman has been replaced by a truncated version of <i>vWbp</i> from strain RN4220. This was further confirmed by the fact that vWbp could be detected in the supernatants of 3 h Newman wildtype cultures but not of this <i>emp</i> mutant strain (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002434#ppat.1002434.s001" target="_blank">Fig. S1</a>).</p

Proposed model for the differential actions of Coa and vWbp.

<p><i>S. aureus</i> Newman forms two concentric barriers in the presence of fibrinogen in the growth medium: the outer MAM and the inner pseudocapsule; both contain fibrin. The MAM is dependent on vWbp and inhibits neutrophil immigration into the vicinity of the microcolony. The pseudocapsule is partially dependent on Coa and acts as a second barrier preventing neutrophil invasion of the microcolony.</p

Microcolony and MAM diameter of <i>S. aureus</i> Newman strains after growth in 3D-CoG/Fib.

<p>The average diameter of microcolonies and MAM was determined after 16 h of growth in 3D-CoG/Fib (A). Wildtype (wt), <i>coa</i> mutant and <i>eap</i> mutant formed microcolonies and MAM of comparable size. The <i>vWbp emp</i> double mutant did not form any MAM, despite being unaffected in pseudocapsule formation. A <i>sae</i> mutant (Newman-29) neither formed pseudocapsules nor MAM but grew in clusters significantly larger than wt colonies. USA300 microcolonies were comparable in size to wt, pseudocapsule formation was present, but the diameter of the MAM was significantly smaller compared to wt. Data are averaged from at least three independent experiments. Addition of plasmin (8 µg/ml) led to rapid degradation of both pseudocapsule and MAM surrounding <i>S. aureus</i> Newman colonies grown in 3D-CoG/Fib for 17h (B1–B4). The time stamp in the single panels is relative to plasmin addition, scale bar 25 µm.</p

Presence of pseudocapsule and MAM structures in various clinical isolates.

<p>Clinical <i>S. aureus</i> isolates were cultivated as described for strain Newman and evaluated after 16 h of growth. MSSA strain MP9-11 (A). MSSA strain MP3-11 (B). MSSA strain MP6-11 (C). MRSA strain MP10-11 (D). CA-MRSA strain USA300 (E). MSSA strain MP2-11 (F). MRSA strain ST239-CC8 (G). MSSA strain MP1-11 (H). Pseudocapsules (yellow arrowheads) and MAM-like structures (blue arrowheads) are indicated. A–C: scale bar 20 µm. D–H: scale bar 75 µm.</p

Localization of Coa and Emp to <i>S. aureus</i> pseudocapsules.

<p>Coa and Emp were detected in pseudocapsules by immunocytochemistry using rabbit antibodies specific for coagulase (anti-Coa, A) or Emp (anti-Emp, B and C). The pseudocapsule of the <i>coa</i> mutant is more irregularly shaped than its wildtype counterpart, illustrated by anti-Emp staining (C). Staphylococci were grown in 3D-CoG/Fib for 16 h and then fixed with 4% paraformaldehyde. Primary antibodies were detected by Alexa Fluor 555 anti-rabbit antibodies. Staphylococci were detected by staining DNA with DAPI (A1, B1, C1) and N-acetyl-glucosamine with FITC-Lectin (<i>T. vulgaris</i>) (A2, B2, B3). Staining of Coa and Emp was specific as the <i>coa</i> and <i>vwbp emp</i> mutant were not stained with the respective antisera (data not shown). Scale bar 7.5 µm. The images are representative of three independent experiments.</p

The thrombin inhibitor argatroban antagonizes staphylococcal barrier activity.

<p><i>S. aureus</i> Newman was grown in 3D-CoG/Fib in the presence of different argatroban concentrations. After 16 h of growth, the growth phenotypes were analyzed: at 10 nM argatroban, the MAM was diminished and at higher concentrations also pseudocapsule formation was absent (A). Challenging the system with neutrophils after 16–17 h revealed loss of both barrier functions in a concentration-dependent manner (B). Scale bar 25 µm. The images are representative of three independent experiments. Data are averaged from three independent experiments.</p

Plasmids used in this study.

<p>Plasmids used in this study.</p

Differential effect of SycO overproduction on cytosolic Yop levels in the presence of calcium ions

<p><b>Copyright information:</b></p><p>Taken from "The type three secretion chaperone SycO is integrated into the Yop regulatory network and binds to the Yop secretion protein YscM1"</p><p>http://www.biomedcentral.com/1471-2180/7/67</p><p>BMC Microbiology 2007;7():67-67.</p><p>Published online 5 Jul 2007</p><p>PMCID:PMC1933539.</p><p></p> Overproduction of SycO was analyzed in parental strain WA-314 and the isogenic deletion mutants Δand Δcultured at 37°C in the presence of 5 mM CaCl. After 2 h 1 mM IPTG was added and cultivation was continued for another 2 h. Pellet fractions were analyzed by immunoblotting with sera raised aginst YopO, SycO and YopH

Repression of Yop secretion by SycO overproduction is neither mediated by YscM1 nor by YscM2

<p><b>Copyright information:</b></p><p>Taken from "The type three secretion chaperone SycO is integrated into the Yop regulatory network and binds to the Yop secretion protein YscM1"</p><p>http://www.biomedcentral.com/1471-2180/7/67</p><p>BMC Microbiology 2007;7():67-67.</p><p>Published online 5 Jul 2007</p><p>PMCID:PMC1933539.</p><p></p> Overproduction of SycO was analyzed in parental strain WA-314 and the isogenic deletion mutants Δand Δas described in legend to Fig. 2. Analysis of the culture supernatant is represented in (A) and the cell lysates are shown in (B)

The role of TcsC in the stress-induced developmental program leading to a fluffy growth phenotype.

<p>Drop dilution assays were performed on AMM plates (supplemented with ammonium). Panel A: 1% oxygen; B: 2% oxygen; C: 2 mM farnesol; D: 200 µM farnesol; E: 2 mM farnesol; F: 100 mM MgSO₄; G: 100 mM CaCl₂; H: 100 mM MgSO₄; I: 50 mM CaCl₂; J: 500 mM CaCl₂. Side views of colonies from C and D are shown in panels E and F. The depicted colonies were photographed after 48 h at 37°C. AfS35 (top/left); Δ<i>tcsC</i> (middle); complemented strain (bottom/right).</p