Physical map of the fosmid p90H6 and sub-cloning of <i>qsdB</i> coding for NAHLase activity.

<p>In A, GC content (%) and orientation (+/−) of the 34 putative <i>orfs</i> of the fosmid p90H6, and physical map of the pME6000-derivatives pMTHindIII and pMTXhoI barboring <i>qsdB</i> (<i>orf1</i>). In B, residual level of C6HSL, OC8HSL or C8HSL measured in the presence of <i>E. coli</i> strain DH5α harboring the empty vector pME6000, the fosmid p90H6, and the constructed pMTHindIII and pMTXhoI (symbols of the three formers are superimposed in the graphs).Three replicates were done. In C, C6HSL analysis by HPLC/MS: mass, retention time, and quantification of C6HSL before (t = 0) and 24 hours after incubation in the presence of cell-free extracts of <i>E. coli</i> strains DH5α(pME6000) and DH5α (pMTXhoI).</p

The AS-family catalytic triad K-S-S is required for NAHLase activity of QsdB.

<p>In A, the amino acids K70, S147 and S171 of the AS-family catalytic triad are underlined in the QsdB sequence. In B, concentration of residual OC8HSL after 24 hr-incubations in the presence of the wild type protein QsdB and its constructed derivatives K70A, S147A and S171A, all at 0.1 mg/ml; the protein-free reaction buffer was used as a control.</p

GCL-induced changes in the bacterial <i>rrs</i>-diversity.

<p>At 42-day, <i>rrs</i>-diversity was evaluated by DGGE (A) and pyrosequencing (B, C). In A, for each of the numbered bands, one of the closest <i>rrs</i> sequences was indicated and characterized by GenBank number, the name and taxonomical position of the bacteria of origin (Firm, Firmicutes; α, α-Proteobacteria; β, β-Proteobacteria; γ, γ-Proteobacteria), and the similarity index (SI) calculated at Ribosomal Database Project (<a href="http://rdp.cme.msu.edu" target="_blank">http://rdp.cme.msu.edu/</a>). The <i>rrs</i>-pyrosequencing data were analyzed at the class level (B) and, within the class of alphaproteobacteria, at the genus level (C).</p

ORFs of the p90H6 DNA-insert.

<p>ORFs of the p90H6 DNA-insert.</p

Relative abundance of NAHLase-encoding genes in GCL-treated and untreated plant cultures.

<p>Relative abundance of the <i>qsdB</i> (A), <i>qsdA</i> (B) and <i>attM</i> (C) genes in GCL-treated and untreated batches at 42-day was measured by qPCR. The <i>attM</i> intensity under the untreated condition (C) was used as a normalized reference (arbitrary value = 1) for calculation of the relative abundance of all genes.</p

QsdB-mediated quorum-quenching in <i>P.</i><i>carotovorum.</i>

<p>In A, relative abundance of NAHLs (OC8HSL as the main QS-signal) in wild-type <i>P. carotovorum</i> CFBP6276 (Pca6276), and <i>P.carotovorum</i> harboring the empty vector pME6000 and the pMTXhoI plasmid expressing QsdB. In B, symptoms on potato tubers (n = 14) inoculated with Pca6276, Pca6276 (pME6000), Pca6276 (pMTXhoI) or NaCl 0.8% as a negative control were classified according to their importance (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065473#s2" target="_blank">material and methods</a>) and compared using Kruskal-Wallis test (P<0.05). Different letters indicate statistical differences between the compared plant pathogens.</p

NAHL-degrading community in GCL-treated plant cultures.

<p>(A) Potato plants were cultivated under untreated and GCL-treated conditions during 42 days; vertical arrows indicate the two applications of GCL at 0.4g/L. At 42-day, total cultured bacteria (B), and percentage of NAHL-degraders (C) and NAHL-producers (D) were enumerated and calculated under both the GCL-treated and untreated conditions. The mean of three replicates are shown. Statistically different values (Mann & Whitney, α = 0.05) are noted by different letters.</p

Synthesis of the 4 diastereoisomers of QSI-1248.

<p>Synthesis of the 4 diastereoisomers of QSI-1248.</p

Structures of the QSIs identified in the chemical library.

<p>Structures of the QSIs identified in the chemical library.</p

IC50, MIC and MBC values of the tested compounds.

a<p>values are in µg/ml.</p>b<p>In brackets, inhibition (%) at 100 µg/ml.</p