Extracellular protease activity of biofilm mutants.

<p>Protease activity in cell-free supernatants from cultures grown in TSB for 12 hours was measured with Azocoll reagent as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010146#s4" target="_blank">Materials and Methods</a>. Supernatant protease activity of the wild-type strain (SH1000) was set to 1 as a reference. The graph shows the mean of three samples; error bars show standard deviation. Blue bars indicate mutants that show consistently higher levels of protease activity.</p

Assessment of biofilm formation in transposon mutants.

<p>Microtiter biofilm assays were performed in the absence (A) or presence (B) of the protease inhibitor α₂-macroglobulin in triplicate, and the percentages of biofilm formation for each mutant relative to that of the wild type are shown. Error bars show standard deviation. Color coding is as follows: black bars indicate mutants with normal extracellular protease activity, blue bars indicate high protease activity and biofilm recovery with α₂-macroglobulin, and red bars indicate the <i>graS</i> (37 C9) and <i>imp</i> (60 G7, 64 G10) mutations that did not recover with α₂-macroglobulin.</p

Extracellular nuclease activity of selected biofilm defective mutants.

<p>Nuclease activity was measured as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010146#s4" target="_blank">Materials and Methods</a> and plotted relative to SH1000 levels. For the <i>imp</i> mutant, only the results of transposon mutant 60 G7 are shown. Results with <i>imp</i> mutant 64 G10 were indistinguishable (data not shown). Underneath the plotted nuclease activity results are representative images of colonies grown on methyl green DNase agar plates. Clearing zones around the colonies is indicative of increased nuclease activity. Error bars show standard deviation.</p

Biofilm defective mutants.

a<p>Order of mutants listed matches the ordering in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010146#pone-0010146-g001" target="_blank">Figures 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010146#pone-0010146-g002" target="_blank">2</a>.</p>b<p>ORF numbers, gene names, and descriptions are from the National Microbial Pathogen Data Resource web site (<a href="http://www.nmpdr.org" target="_blank">www.nmpdr.org</a>).</p>c<p>Percentages of biofilm formation for each mutant relative to wild type represent the means (+/− standard deviations) from four independent experiments each performed in triplicate.</p

Flow cell biofilm formation of <i>graS</i> and <i>imp</i> mutants in different strain backgrounds.

<p><i>S. aureus</i> strains SH1000 (A) and LAC* (B), and <i>graS</i> and <i>imp</i> mutations in each strain, were grown in a flow cell apparatus for two days. A <i>z</i> series of images was obtained with CLSM, reconstructed with Volocity software, and each side of a grid square is 20 µm in the image reconstruction. The addition of a complementing plasmid containing either the <i>graRS</i> or <i>imp</i> genes is shown in the last column.</p

Transcriptome Analysis of Acetyl-Homoserine Lactone-Based Quorum Sensing Regulation in <i>Yersinia pestis</i>

<div><p>The etiologic agent of bubonic plague, <i>Yersinia pestis,</i> senses self-produced, secreted chemical signals in a process named quorum sensing. Though the closely related enteric pathogen <i>Y. pseudotuberculosis</i> uses quorum sensing system to regulate motility, the role of quorum sensing in <i>Y. pestis</i> has been unclear. In this study we performed transcriptional profiling experiments to identify <i>Y. pestis</i> quorum sensing regulated functions. Our analysis revealed that acyl-homoserine lactone-based quorum sensing controls the expression of several metabolic functions. Maltose fermentation and the glyoxylate bypass are induced by acyl-homoserine lactone signaling. This effect was observed at 30°C, indicating a potential role for quorum sensing regulation of metabolism at temperatures below the normal mammalian temperature. It is proposed that utilization of alternative carbon sources may enhance growth and/or survival during prolonged periods in natural habitats with limited nutrient sources, contributing to maintenance of plague in nature.</p></div

AHLs upregulate the maltose operon and enhance growth on maltose.

<p>(A) Growth of wild-type (WT) (black), and AHL mutant (dashed) <i>Y. pestis</i> in minimal maltose medium (light grey) was monitored in Bioscreen C microplate reader incubating at 28°C with agitation. (B) Complementation of AHL mutant bacteria with p<i>ypeIR</i> (black, dashed) or p<i>yspIR</i> (grey, dashed) restores growth on maltose, whereas control plasmid (black, solid) does not.</p

Selected genes significantly up-regulated by QS.

*<p>FC = fold change.</p

Identification of the <i>Y. pestis</i> quorum-sensing molecules.

<p>(A) HPLC fractionation profiles of ¹⁴C-labeled AHL produced by R88 <i>Y. pestis</i> (solid circles) compared to R115 QS^-<i>Y. pestis</i> (empty circles). The peaks absent from organic extracts of R115 <i>Y. pestis</i> supernatants correspond to C8-, C6-, and oxo-C6-AHL. (B) AI-2 production during the growth of R88 <i>Y. pestis</i> (solid circles) and R115 (empty circles) was monitored by adding the cell-free supernatants at the indicated time points to a <i>V. harveyii</i> reporter strain that is bioluminescent (RLU) in response to AI-2. Data are representative of at least three independent studies. (C and D) The production of AI-2 (C) and AHL (D) signals as a function of growth.</p

AHL quorum sensing upregulates glyoxylate bypass and enhances growth on acetate.

<p>(A) Growth of R88 (black), and an R115 AHL null mutant strain (dashed) of <i>Y. pestis</i> in minimal acetate medium (light grey is medium only control) was monitored in a Bioscreen C microplate reader incubating at 28°C with agitation. (B) Complementation of AHL mutant bacteria with p<i>ypeIR</i> (black, dashed) or p<i>yspIR</i> (grey, dashed) restores growth on acetate, whereas control plasmid (black, solid) does not.</p