CdiGMP G8 inhibits HIV RT activity.

<p>(<b>A</b>) A graph of primer extension <i>vs.</i> time is shown for reactions conducted in the absence of presence of the indicated cdiGMP forms. Assays were performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053689#s2" target="_blank">Materials and Methods</a>. A fixed concentration of 80 µM cdiGMP form was used. Assays were conducted in either K⁺ or Na⁺ containing buffer depending on which cation was used in the preparation of the cdiGMP form. The experiment was repeated with similar results. (<b>B</b>) The amount of primer extension (5 minute time point), relative to assays conducted without added cdiGMP derivatives, <i>vs.</i> the concentration of cdiGMP G8 is shown. The graph is from an average of 3 experiments and error bars represent standard deviations. The IC₅₀ value in the presence of cdiGMP G8 prepared in K⁺ was determined as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053689#s2" target="_blank">Materials and Methods</a>. *The concentration of extended primer in (<b>A</b>) and the relative level of primer extension in (<b>B</b>) were determined by exposure of dried denaturing acrylamide gels using a phosphoimager. Reactions contained a total of 50 nM primer.</p

The cdiGMP G8 does not inhibit diguanylate cyclase activity in WspR D70E.

<p>(<b>A</b>) WspR D70E (1.18 µM) was incubated with 4 nM α-³²P-GTP with and without 500 µM cdiGMP M/D. Reaction products were assayed by TLC as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053689#s2" target="_blank">Materials and Methods</a> and the amounts of ³²P-cdiGMP M/D formed at specified time points were plotted. (<b>B</b>) WspR D70E (1.18 µM) was incubated with varying amounts of cdiGMP G8 or cdiGMP M/D and the fraction ³²P-cdiGMP M/D at 3 hours was assayed by TLC and plotted.</p

CdiGMP G8 binds HIV-1 reverse transcriptase, a parallel G-tetrad binding protein, but not thrombin protease, an anti-parallel binding protein.

<p>(<b>A</b>) HIV-1 RT (960 nM) was incubated with cdiGMP heated in 4 nM cdiGMP (−) and 500 µM cdiGMP (+) in the salt indicated. Binding was analyzed via DRaCALA. (<b>B</b>) Thrombin protease (8 µM) was incubated with ³²P-cdiGMP G8 (lane 1), ³²P-cdiGMP M/D (lane 2), ³²P-TBA25 (lane 3) and binding was analyzed via DRaCALA. ³²P-cdiGMP G8 (lane 4) and ³²P-TBA25 (lane 5) were spotted without protein as negative controls.</p

Thin layer chromatography separates ³²P-cdiGMP G8 from ³²P-cdiGMP M/D.

<p>(<b>A</b>) Four mixtures of ³²P-cdiGMP were heated, cooled, spotted on PEI-cellulose TLC plates, and separated with a mobile phase of 0.9 M KH₂PO₄ and 40% (v/v) saturated NH₄PO₄. ³²P-cdiGMP was prepared in buffer containing K⁺ (Lane 1 and 2) or Na⁺ (Lane 3 and 4) with no additional cdiGMP (Lane 1 and 3) or 500 µM cdiGMP (Lane 2 and 4). The retention factor (R_f) is indicated on the left of the TLC. (<b>B</b>) Samples in lane 2 and 4 from Panel A were two-fold serially diluted in water and allowed to incubate at room temperature for ten minutes. Samples were then spotted and separated by TLC as in (A) and the total ³²P-cdiGMP G8 (R_f = 0.06) was quantified and plotted. The first sample is undiluted and the dilution factor for the other samples is indicated on the X-axis. (C) UV-vis spectra of 500 µM cdiGMP with (red) and without (black) heat in K⁺ buffer. Absorbance was determined using a pathlength of 1.0 mm.</p

Summary of UV-Vis absorbance measurements for the cdiGMP G8 and the cdiGMP M/D.

<p>Summary of UV-Vis absorbance measurements for the cdiGMP G8 and the cdiGMP M/D.</p

The cdiGMP G8 does not interact with known cdiGMP M/D binding proteins.

<p>(<b>A</b>) Representative DRaCALA spots for binding reactions of either ³²P-cdiGMP M/D or ³²P-cdiGMP G8 with the indicated protein. Binding was assayed by DRaCALA. (<b>B</b>) Quantification of fraction bound from DRaCALA of ³²P-cdiGMP G8 reactions (gray) and ³²P-cdiGMP M/D reactions (black).</p

Cartoon depicting cdiGMP structures in solution.

<p>(<b>A</b>) The <i>trans</i>-monomer has the two bases oriented away from each other. (<b>B</b>) The <i>cis</i>-monomer has the two bases overlapping and is a precursor for higher order cdiGMP polymorphs. (<b>C</b>) The <i>cis</i>-dimer forms when two <i>cis</i>-monomers intercalate. This structure is stabilized by both π-stacking and hydrogen bonds between phosphates and bases. (<b>D</b>) The G-quadruplex (G4) forms when the bases from four <i>cis</i>-monomers interact via Hoogsteen base pairing and π-stacking. Monovalent cations, such as potassium, and high concentrations of cdiGMP promote this structure. The cation is located either in the middle of the G-tetrad plane or between G-tetrads and has been excluded here for clarity. (<b>E</b>) The G-octaplex (G8) forms when two G4 structures sandwich each other. This structure is stabilized by hydrogen bonding and base stacking. When potassium and high concentrations are present, the G8 is the predominant higher order rotaform.</p

Comparative assembly using multiple genomes.

<p>The target genome is shown in the center, aligned to two related genomes, A and B. The DNA sequence of the target diverges from the reference genomes in distinct loci, labeled X, Y, and Z. The comparative assembly based on genome A contains a gap corresponding to region Y, while the assembly based on genome B contains two gaps, corresponding to X and Z. The merged assembly will cover all of the target genome with no gaps.</p

Major steps in the assembly of <i>P. aeruginosa</i> from 33 bp Solexa reads.

<p>The first column indicates the assembly strategies described in the text. Singletons refers to the number of reads that were not used to produce the contigs generated by each method.</p

High-Throughput Screening Using the Differential Radial Capillary Action of Ligand Assay Identifies Ebselen As an Inhibitor of Diguanylate Cyclases

The rise of bacterial resistance to traditional antibiotics has motivated recent efforts to identify new drug candidates that target virulence factors or their regulatory pathways. One such antivirulence target is the cyclic-di-GMP (cdiGMP) signaling pathway, which regulates biofilm formation, motility, and pathogenesis. <i>Pseudomonas aeruginosa</i> is an important opportunistic pathogen that utilizes cdiGMP-regulated polysaccharides, including alginate and pellicle polysaccharide (PEL), to mediate virulence and antibiotic resistance. CdiGMP activates PEL and alginate biosynthesis by binding to specific receptors including PelD and Alg44. Mutations that abrogate cdiGMP binding to these receptors prevent polysaccharide production. Identification of small molecules that can inhibit cdiGMP binding to the allosteric sites on these proteins could mimic binding defective mutants and potentially reduce biofilm formation or alginate secretion. Here, we report the development of a rapid and quantitative high-throughput screen for inhibitors of protein-cdiGMP interactions based on the differential radial capillary action of ligand assay (DRaCALA). Using this approach, we identified ebselen as an inhibitor of cdiGMP binding to receptors containing an RxxD domain including PelD and diguanylate cyclases (DGC). Ebselen reduces diguanylate cyclase activity by covalently modifying cysteine residues. Ebselen oxide, the selenone analogue of ebselen, also inhibits cdiGMP binding through the same covalent mechanism. Ebselen and ebselen oxide inhibit cdiGMP regulation of biofilm formation and flagella-mediated motility in <i>P. aeruginosa</i> through inhibition of diguanylate cyclases. The identification of ebselen provides a proof-of-principle that a DRaCALA high-throughput screening approach can be used to identify bioactive agents that reverse regulation of cdiGMP signaling by targeting cdiGMP-binding domains