Parameters to fit to variable confinement size model

<p>Parameters to fit to variable confinement size model</p

Parameters to fit mixed diffusion mode model

<p>Parameters to fit mixed diffusion mode model</p

Confined Mobility of TonB and FepA in <i>Escherichia coli</i> Membranes - Fig 5

<p><b>Effect of (a) deletion of ExbB/D (triangles), (b) presence of monoclonal antibody against FepA (triangles), and (c) presence of MreB–disrupting drug A22 (triangles) on MSDs of TonB (circles).</b> Fitting suggests that the effect of deletion of ExbB/D and disruption of MreB is to reduce the size of confinement of TonB while the effect of anti-FepA is to increase the mobility of TonB which appears as a decrease in confinement size.</p

MSD of TonB and of FepA.

<p>a) MSD of TonB (solid circles) and of FepA (solid squares). b) Fit to confined diffusion model for TonB (data: solid circles, fit: open circles) which gives best fit parameters of a confinement radius of μm with short-time (microsecond timescale) diffusion coefficient in the range μm²/s. c) Fit to confined diffusion model for FepA (data: solid squares, fit: open squares) which gives best fit parameters of a confinement radius of μm with short-time (microsecond timescale) diffusion coefficient in of μm²/s.</p

Schematic of the interaction between the TonB-ExbB-ExbD complex in the inner membrane and its interaction with the Ferric enterobactin (FeEnt) TBDT FepA in the outer membrane of <i>E</i>. <i>coli</i>.

<p>In this study, either FepA was labeled extracellularly with Alexa-555 or TonB was labeled intracellularly with GFP. IM: inner membrane, PG: peptidoglycan layer, OM: outer membrane.</p

Colonization Competition between <i>E. coli</i> MG1655 and CAT0, CAT4 or CAT40.

<p>In each trial three streptomycin-treated mice were simultaneously fed with a mutant strain and wild type parent MG1655, which are both streptomycin resistant. If even a slight advantage exists between the two strains, the conditions of the large intestine select for the preferred strain, which dominates within a few days. If neither strain has an advantage, then the two strains co-colonize at almost equal levels. Fecal plate counts determined the relative colonizing abilities (the log difference in CFU/g of feces). Three-log differences or greater between the mutant and wild-type strain indicates in a major colonization defect. A 1.5 to 3 log difference shows a significant colonization defect, and a 1 to 1.5 log difference denotes a minor defect. Log differences less than 1 are not significant. In these experiments pairs of bacteria were orally inoculated into mice on day 0, and their presence in feces was monitored for 15 days. The plotted data represents the mean of two or more independent trials; error bars represent standard deviations of the means. <i>E. coli</i> MG1655 out-competed CAT0, CAT4 and CAT40 for colonization. Unexpectedly, CAT40 showed 1000-fold better persistence than CAT4, and maintained colonization at almost the same level as MG1655 for the first week.</p

SDS-PAGE of OM fractions.

<p>The prototypic <i>E. coli</i> strain MG1655 and its derivatives CAT4 (<i>Δfiu, ΔfepA, Δcir, ΔfecA::Cm</i>) and CAT40 (<i>Δfiu, ΔfepA, Δcir, ΔfecA, ΔentA::Cm</i>) were grown in LB broth, subcultured at 1% into iron-deficient MOPS minimal media at 37°C and grown to late log phase. The bacteria were collected by centrifugation, lysed in a French pressure cell and their OM fractions were purified, resolved by SDS-PAGE, and the gels were stained with coomassie blue R. Fiu, FepA and Cir are seen in MG1655 (Lane 1), but absent from CAT4 (lane 2) and CAT40 (lane 3). Molecular weight standards were included in lane 4. FecA, which is inducible by growth in the presence of citrate, is not visible in this experiment, but its absence was verified by PCR.</p

Enterobactin quantification in vivo.

<p>Mice were inoculated on day 0 and on day 12 caecal mucus from mice that were uncolonized or orally inoculated with <i>E. coli</i> strains MG1655, CAT4 or CAT40 was collected and diluted into 5 mM NaHPO₄, pH 6.9. Samples from five individual mice in each experimental group were consolidated, the solution was clarified by centrifugation, 100 uL of each supernatant was mixed with 10 µCi of ⁵⁹FeCl₃, the samples were incubated on ice for an hour and then chromatographed on Sephadex LH20 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050020#s4" target="_blank">Materials and Methods</a>). We chromatographed authentic FeEnt with the mucus as an internal marker, and determined and plotted the absorbances at 280 nm and 495 nm, and the radioactivity of each fraction. The black and red dashed lines show the absorbances of the eluted fractions at 280 nm and 495 nm, respectively: the blue line depicts their radioactivity.</p

<i>E. coli</i> strains and plasmids.

<p><i>E. coli</i> strains and plasmids.</p

Summary of enterobactin production in vivo.

<p>The amount of FeEnt produced by the colonizing bacteria was standardized in relation to the amount of protein in the mucosal samples (CPM/A₂₈₀ nm). The plotted data therefore depicts the relative amount of enterobactin production by each of the strains on day 12 after inoculation. This experiment was performed only once, but each data point represents the mean values from pooled extracts of 5 animals. Despite the fact that the cell numbers of <i>E. coli</i> CAT4 were 4-logs lower than those of MG1655 at this time, we found more ⁵⁹FeEnt in the former strain's gut mucus. This finding, that CAT4 hyperexcretes enterobactin in vivo, corroborated previous findings <i>in vitro </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050020#pone.0050020-Cox1" target="_blank">[31]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050020#pone.0050020-Young1" target="_blank">[32]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050020#pone.0050020-Young2" target="_blank">[60]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050020#pone.0050020-Seiffert1" target="_blank">[61]</a>. Even though they were present at 10,000-fold lower abundance (a finding that was substantiated by statistical analysis of three colonization experiments), CAT4 cells secreted as much or more enterobactin as wild type bacteria. This was validated by the background control, uncolonized mice treated with streptomycin, that established a baseline for the detection of FeEnt in the mice.</p