Overlap and PHN-Families

<div><p>By partitioning the network with the overlap procedure for increasing value of θ<i>,</i> we separated the PHN into regions of increasing compactness. The maximum value of the modularity measure <i>Q</i> (see <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.0020173#s4" target="_blank">Materials and Methods</a>) allowed us to identify the optimal cutoff value to partition the PHN into families of homologous proteins.</p><p>(Main Graph) <i>Q</i> is shown as a function of θ<i> </i>. The maximum value of <i>Q</i> = 0.723 is found for θ<i> </i> = 0.5.</p><p>(Inset Graph) The dark circles represent the compactness index <i>η</i> after the partitioning (see <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.0020173#s4" target="_blank">Materials and Methods</a>) as a function of θ<i> </i>. The white triangle is the value of <i>η</i> of the original PHN for <i>ɛ</i> = 10⁻⁵, which corresponds to the limiting value θ<i> </i> = 0.</p><p>doi:10.1371/journal.pcbi. 0020173.g005</p></div

PHN Topology

<div><p>The compactness index, <i>η,</i> and the clustering index, <i>C,</i> shown here as a function of the E-value cutoff <i>ɛ,</i> describe the global and local topology of the network, respectively. For growing values of <i>ɛ, η</i> rapidly decreases towards 0, while <i>C</i> always has values well above 0.8. These results indicate that the PHN is formed by compact regions that are loosely connected to form globally sparse connected components.</p><p>doi:10.1371/journal.pcbi. 0020173.g004</p></div

PHN Giant Component

<div><p>The fraction <i>n_G</i> of nodes included in the largest connected component of the PHN is shown as a function of the homology cutoff <i>ɛ</i>.</p><p>doi:10.1371/journal.pcbi. 0020173.g003</p></div

VirB3 PHN-Families Phylogeny

<div><p>The PHN-Families of nonconserved genes correlate with their molecular phylogeny. Shown here is the Maximum Likelihood tree of the 33 VIRB3 proteins classified in three PHN-Families (see <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.0020173#pcbi-0020173-st001" target="_blank">Table S1</a>). PHN-Families are enclosed in circles, color-coded as in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.0020173#pcbi-0020173-g008" target="_blank">Figure 8</a>, and coincide with monophyletic branches of the phylogenetic tree. Numbers are bootstrap values, and the ruler shows the number of point-accepted mutations.</p><p>doi:10.1371/journal.pcbi. 0020173.g007</p></div

SctJ PHN-Family: Network and Phylogeny

<div><p>In this example we show a representation of a single PHN-family, compared with a reconstruction of the evolutionary history of its components based on molecular phylogenetic data. The two subgroups clearly visible in the PHN representation coincide with monophyletic clades of the phylogenetic tree.</p><p>(A) Network representation of the SctJ PHN-family (see <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.0020173#pcbi-0020173-sd001" target="_blank">Protocol S1</a>). Spheres represent proteins; edges are homology relations, color-coded according to the homology level <i>ɛ_ij</i>. The two subgroups are YscJ (T3SS) and FliF (flagellar) proteins. For <i>ɛ</i> = 10⁻⁵, this portion of the PHN falls in the giant component, for the presence of false homology relations with seven outlier proteins (blue spheres, external links to the giant component not shown). After the overlap procedure with θ<i> </i> = 0.5, false links are removed, and all the members of the SctJ family fall in a single PHN-family, shown by the circle.</p><p>(B) Maximum likelihood phylogenetic tree of the SctJ family. Numbers are bootstrap values. The YscJ and FliF subgroups correspond to two distinct evolutionary clades. Organism and group names in the T3SS clade refer to the T3SS classification shown in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.0020173#pcbi-0020173-g008" target="_blank">Figure 8</a>.</p><p>doi:10.1371/journal.pcbi. 0020173.g006</p></div

Dose Dependent Binding of RrgA to Selected Extracellular Matrix (ECM) Components.

<p>Shown are the results of binding increasing concentrations of purified T4 pilus proteins HisTag-RrgA, -RrgB and -RrgC (A) and HMW pilus preparations (B) to fibronectin, collagen I and laminin. BSA and delta pilus mock preparation served as negative controls. Binding was quantified by ELISA at an absorbance of 405 nm. Points represent the means (error bars, standard errors of the means) of measurements made in triplicate.</p

Triple Immunoelectron-Microscopy (IEM) Analysis of the Pilus Subunits of <i>Streptococcus pneumoniae</i>.

<p>Isolated pili material (A) was incubated with antisera raised against His-tagged RrgA, RrgB and RrgC and conjugated respectively to 15 nm, 5 nm and 10 nm gold particles. The image shows the pilus backbone stained with gold-labelled antibodies raised against the main pneumococcal pilus component (RrgB). Clusters of RrgA ancillary proteins (open arrows) are present along the entire pilus. Single copies of the ancillary protein RrgC (arrows) were found alone or co-localized with the RrgA clusters. Scale bar, 100 nm. The same protocol for triple immunogold EM has been applied to bacteria preparation <i>of Streptococcus pneumoniae</i> T4 (B), showing a similar pattern of gold distribution (scale bar, 100 nm).</p

Micrograph of Negative Stained Whole Cell <i>Streptococcus pneumoniae</i> TIGR4.

<p>Sample stained with 1% buffered phosphotungstic acid (PTA). Open arrows indicate an individual single pilus; arrow indicates bundles of individual pili. Scale bar, 200 nm. (Philips TEM CM200 FEG microscope at 50000× magnification, working at low-dose conditions).</p

Model of a Pneumococcal Pilus.

<p>Model showing T4 pneumococcal pilus composed of at least two RrgB protofilaments (green) arranged in a coiled-coil superstructure with surface located ancillary proteins (RrgA and RrgC) is based on cryo-EM, freeze drying/metal shadowing EM, IEM and biochemical data. (R) and (L) illustrate a possible right and left handed orientation of the thin pilus respectively. Outlines are not drawn to scale.</p

Images of Gaussian Filtered Thin Individual Pili Show Their Structural Composition.

<p>(A) Images of original pilus taken at low dose conditions. (B) Gaussian filtered thin individual pili show their structural composition. Inset reveals an enlarged view of the thin pilus corresponding to the pilus crossover. Crossover position (arrow) for thin pilus is indicated. The protofilament diameter was measured as 3.5 nm. Crossover diameters (arrows) were calculated as 6.8 nm for thin pili (9.5 nm diameter). Scale bars, 10 nm.</p