Letter pubs.acs.org/journal/aidcbc
Pseudomonas aeruginosa LasR·DNA Binding Is Directly Inhibited by Quorum Sensing Antagonists Emma G. Suneby,† Leslie R. Herndon,‡ and Tanya L. Schneider*,† †
Department of Chemistry, Connecticut College, New London, Connecticut 06320, United States Department of Chemistry, Smith College, Northampton, Massachusetts 01063, United States
‡
S Supporting Information *
ABSTRACT: Inhibition of quorum sensing in Pseudomonas aeruginosa is of interest as a possible antivirulence strategy for this pathogenic bacterium. The LasR regulator protein is important in coordinating gene expression in response to quorum sensing signaling molecules. One predominant strategy for LasR inhibition is the development of small-molecule antagonists that mimic the native autoinducer, though the mechanism by which they inactivate LasR is not known. This work reveals that multiple antagonists function by binding to and stabilizing LasR in a conformation that renders it unable to bind DNA. Further analysis of purified LasR complexed with known antagonists indicates that DNA binding can be recovered with the addition of native autoinducer, providing insights into the reversibility of ligand binding for this transcription factor. This in vitro assay could be used to assess future promising antagonists and complements existing cell-based reporter assays. KEYWORDS: Pseudomonas aeruginosa, quorum sensing, LasR, transcription factor, antivirulence
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targets for inhibition, most work to date has focused on inhibiting the LasR transcription factor through the design of small-molecule antagonists.12 LasR dimerization and DNA binding are controlled by the binding of its native ligand, N-(3-oxododecanoyl)-L-homoserine lactone (3O-C12-HSL).13,14 It has been proposed that LasR complexed with its native ligand and bound to target DNA may additionally interact with RNA polymerase to promote gene expression.15 Numerous antagonists have been designed to disrupt this specific LasR−ligand interaction, ranging in degree of structural similarity to 3O-C12-HSL and functioning through both covalent 16,17 and non-covalent interactions with LasR.12,18,19 Antagonist efficacy has typically been measured in cell-based reporter assays, and recent work by Blackwell and co-workers describes a careful, systematic look at the properties of many known antagonists under consistent assay conditions.20 Additional studies have targeted increased understanding of the nature of LasR interactions with its autoinducer. The ligand binding domain (LBD) of LasR has been crystallized, providing structural details of the discriminating interactions between LasR and 3O-C12-HSL21 as well as several autoinducer mimics.22 Key polar interactions between LasR and varied ligands have been investigated by measuring the impact of mutagenesis on ligand-mediated LasR activation or inhibition, revealing residues of particular importance in the LasR binding pocket that may help to guide further agonist and antagonist design.23,24
acteria commonly employ a communication strategy termed quorum sensing (QS) to signal local population density through the biosynthesis and exchange of small molecules or peptides.1 QS contributes to the fitness of an organism, allowing the coordination of gene expression beneficial to the bacterial colony. Processes governed by QS include the timely production of virulence factors and biofilm development.2 Because QS is linked to virulence in pathogenic bacteria, there is interest in developing inhibitors of QS as a possible antimicrobial therapy.3 One microbe of concern is the Gram-negative bacterium Pseudomonas aeruginosa, an opportunistic human pathogen that establishes infections in immunocompromised individuals and is particularly problematic in cystic fibrosis patients.4,5 This multidrug-resistant pathogen requires new therapeutic strategies, and the possibility of QS inhibition in this case has received considerable attention.6,7 Since QS inhibition may reduce bacterial virulence without impacting bacterial growth, it has been proposed that selective pressures that lead to resistance might be lessened with this form of antibacterial therapy,8 and recent work suggests that the spread of resistance to QS inhibitors in P. aeruginosa is likely to be limited.9 QS in P. aeruginosa is primarily governed by N-acyl Lhomoserine lactone autoinducer molecules, though some signaling is mediated by 2-alkyl-4-quinolones, and the pathways are interconnected.10 Acylhomoserine lactone (AHL) signaling molecules are biosynthesized by the LuxI-type synthases LasI and RhlI, while detection of AHLs is mediated by the LuxRtype regulatory proteins LasR and RhlR, which link the binding of specific AHLs to downstream gene expression.11 Though the synthases and regulatory proteins both represent interesting © XXXX American Chemical Society
Received: September 15, 2016
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Table 1. Mass Spectrometric Confirmation of Soluble LasR−Ligand Complexation
the LasR·DNA binding affinity. Our DNA binding assays provide additional evidence for the reversibility of LasR−ligand complexation25 and represent an in vitro assay for LasR activity that will prove useful in the development and testing of future potent antagonists. We began investigating the impact of quorum sensing antagonists on LasR·DNA binding by determining whether expression of LasR in the presence of non-native ligands yields soluble protein for further characterization. Full-length LasR was expressed in Escherichia coli grown in media supplemented with each of the ligands listed in Table 1. Some ligands (1−4) closely resemble native 3O-C12-HSL, varying only in the acyl chain length and presence of the 3-oxo group. Ligands 5−8 are molecules identified as some of the most potent LasR antagonists in cell-based LasR reporter assays and feature replacement of the acyl chain with aromatic rings.20,26,27 Most notably, ligand 7 was recently developed as a particularly stable
Though multiple potent LasR antagonists are now known, their biochemical mechanism of action has been challenging to discern. In general, they are believed to compete directly with the native ligand, possibly disrupting protein folding in order to yield insoluble or unstable LasR.21 It is also conceivable that antagonists impact the DNA binding affinity of LasR or change its interactions with RNA polymerase. Because of difficulties with protein solubility, however, in vitro DNA binding assays with LasR have been limited. Here we worked with purified LasR to investigate the biochemical mechanism by which a collection of known potent antagonists function. Expression of LasR in the presence of various ligands reveals that antagonists impact the yield of soluble LasR. For LasR that does remain soluble with antagonists bound, we have found that antagonists alter the protein secondary structure and disrupt LasR·DNA binding. Additional experiments indicate that even small changes in the ligand structure have a significant impact on B
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Figure 1. Solubility of LasR−ligand complexes. SDS-PAGE analysis of whole-cell (W) and soluble (S) fractions of E. coli expressing LasR in the presence of 50 μM 3O-C12-HSL (native ligand), 50 μM ligands 1−9, or no ligand (DMF alone) as a control. The expected molecular weight for fulllength LasR is 27.9 kDa.
non-native ligands. The interaction between LasR and one of its known target sequences, the lasB operator 1 (OP1) site,14 has been measured directly by EMSA for LasR expressed in the presence of 3O-C12-HSL, giving an apparent equilibrium dissociation constant of 400 nM.31 Here we found that LasR, when interacting with nearly all of the ligands tested, shows no ability to bind DNA even at 10 times the concentration required to give significant binding when associated with its native autoinducer (Figure 2a). It appears that most of these
LasR antagonist, as introduction of the 2-nitrophenyl substituent in place of the native homoserine lactone renders the molecule less susceptible to hydrolysis.28 Ligand 9 is another known LasR antagonist24 with enhanced hydrolytic stability due to its aniline headgroup, and ligand 10 is a synthetic derivative of a naturally occurring halogenated furanone and has been proposed to impact LasR-mediated quorum sensing activity in P. aeruginosa.29 Ligand 10 proved to inhibit E. coli growth significantly in our experiments, as has also been previously reported,20 and could not be analyzed further. An initial indication of LasR solubility in the presence of varied ligands was obtained through SDS-PAGE comparison of whole cells and soluble fractions from cell lysates (Figure 1). For reference, LasR expressed in the presence of native 3O-C12HSL and in the absence of any ligand were also included. While LasR was detected in all of the whole-cell samples, the solubility varied with the ligand added. As anticipated, soluble LasR was not observed in the absence of ligand, in accord with prior work with the isolated LasR LBD.21 Soluble LasR was noted in cultures prepared with the addition of ligands 1−4 that are structurally similar to 3O-C12-HSL. These somewhat conservative changes to the acyl chain length, ranging from nine to 14 carbons, diminish the amount of soluble LasR compared with 3O-C12-HSL through visual inspection. Some soluble LasR was also detected in cell lysates resulting from growths in the presence of ligands 5−9, though the relative amount of soluble protein was significantly reduced compared with LasR expressed with the native autoinducer. Since these ligands differ more considerably in structure from 3O-C12-HSL, it may not be surprising that they do not stimulate native LasR folding to the same degree. Therefore, one mechanism by which these antagonists may impact LasR activity is through interactions with the nascent protein that render it poorly folded and less soluble, thus reducing the pool of effective LasR. It is also possible that these antagonists bind poorly to LasR and thus fail to stabilize as much soluble protein. For further investigation of LasR expressed with these alternate ligands, soluble LasR was purified successfully from cultures grown with all of the ligands except 5 and 10 as noted above. Since 5 has been shown to be less potent than 6 in cellbased assays, it is conceivable that 5 does not interact as well with LasR, with negligible soluble protein produced.20,30 For each successful case, purified LasR was ligand-bound, as determined by mass spectrometry following dialysis. A sample of each purified LasR−ligand complex was denatured, and analysis of the resulting supernatant revealed the anticipated ligand mass (Table 1). Using purified ligand-bound LasR, electrophoretic mobility shift assays (EMSAs) were performed to determine whether the DNA binding affinity is impacted by LasR complexation with
Figure 2. Impact of varied ligands on LasR·DNA binding. (a) Representative EMSA with target lasB OP1 alone (lane 1) or incubated with 0.5 μM LasR complexed with native 3O-C12-HSL (lane 2). LasR (5 μM) expressed in the presence of ligand 1 (lane 3), 2 (lane 4), 3 (lane 5), 4 (lane 6), 6 (lane 7), 7 (lane 8), 8 (lane 9), or 9 (lane 10) was also assayed. (b) Representative EMSA with target lasB OP1 alone (lane 1) or incubated with 0.5 μM LasR complexed with native 3O-C12-HSL (lane 2). LasR expressed in the presence of ligand 2 (C12HSL) was evaluated at 5 μM (lane 3), 3.75 μM (lane 4), 2.8 μM (lane 5), or 2.1 μM (lane 6). LasR expressed in the presence of ligand 1 (C14-HSL) was evaluated at 5 μM (lane 7), 3.75 μM (lane 8), 2.8 μM (lane 9), or 2.1 μM (lane 10).
ligands, and all of the known antagonists, interact with LasR in a way that does not foster the production of an active DNAbinding transcription factor. Their probable competitive binding to the LasR ligand binding domain likely alters the native fold of the ligand-bound LasR, perhaps impacting dimerization and certainly disrupting high-affinity LasR·DNA binding. In contrast, DNA binding was observed under our assay conditions for LasR complexed with two non-native ligands C
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non-native ligands. CD studies revealed that the secondary structure is impacted when LasR is bound to several known antagonists (Figure 3). This finding follows from earlier work
(Figure 2a). One of these exceptions is ligand 2, C12-HSL, which differs from native 3O-C12-HSL by the lack of the 3-oxo group. While EMSA reveals that this LasR−C12-HSL complex has much lower DNA binding affinity than the native LasR complex, it does suggest that minor structural changes to the native ligand can yield a ligand that retains some ability to promote LasR folding and DNA recognition. Prior mutagenesis studies also provide support for similar LasR binding to 3OC12-HSL and C12-HSL.23 Interestingly, ligand 1, C14-HSL, also seems to facilitate LasR·DNA binding even though its acyl chain is two carbons longer than the native acyl chain. Figure 2b shows the relative binding affinities of LasR complexed with these two ligands compared with the native LasR complex, revealing that even these small structural changes significantly impact the DNA binding affinity. Our findings fit well with previous reporter assays that demonstrated LasR activity in the presence of C12-HSL and C14-HSL, again with weaker agonism displayed by these ligands than by 3O-C12-HSL.30,32,33 C10-HSL has also been shown to be an agonist, but it is still less potent than C12-HSL, which likely explains why DNA binding was not detected in our in vitro assay. Our studies of ligands 1−4 serve to further delineate the importance of the acyl chain length in promoting active LasR. Previous work in our lab31 and others34 has highlighted LasR Cys79, which is proximal to the end of the acyl chain of 3OC12-HSL in the ligand binding pocket21 and seems related to the folding of active LasR. Mutagenesis of Cys79 significantly reduces DNA binding affinity31 and LasR QS function.34 It may be that ligands shorter than 3O-C12-HSL do not constrain LasR in the same way and do not drive folding of active, dimeric LasR. The EMSA results presented here and prior circular dichroism (CD) studies indicating that LasR LBD folding and stability trend with the acyl chain length31 support this conclusion. It is not surprising that such structural changes impact LasR activity, as this transcription factor is effective as a QS regulator if it is uniquely responsive to its autoinducer. Our results also suggest that LasR complexed with nonnative ligands appears to be inactivated beyond simple disordering of the LBD or loss of the DNA binding affinity enhancement conferred by a properly folded, dimeric LasR LBD. Here we show that DNA binding affinity is lower for LasR expressed in the presence of multiple antagonists than for truncated LasR with the LBD completely removed. We previously demonstrated that the isolated LasR DNA binding domain (DBD) is able to bind DNA, though with an equilibrium dissociation constant nearly 2 orders of magnitude greater than that for full-length LasR with native autoinducer.31 This difference in binding affinity is likely due to the loss of the organized LBD. In that study, we measured the DNA binding affinity of the isolated DBD under EMSA conditions similar to those reported here and found that 3.6 μM DBD gave about 40% target DNA bound. Since we did not see any DNA binding by 5 μM full-length LasR complexed with most of our non-native ligands, it seems likely that these antagonists promote an alternate conformation of LasR that is more disruptive than simple excision of the LBD. This result is similar to findings regarding antagonists for the LuxR-type regulator protein CviR from Chromobacterium violaceum, which also bind in place of the native autoinducer and disrupt DNA binding35 by promoting an inactive conformation of the CviR DNA binding domain.36 On the basis of the observed altered DNA binding affinity, it seemed likely that LasR folding is changed in the presence of
Figure 3. Wavelength-dependent CD spectra comparing the relative folding of 5 μM LasR expressed in the presence of native 3O-C12-HSL (blue), 6 (red), 7 (green), and 8 (purple).
that illustrated the altered folding and stability of the LasR LBD in the presence of AHLs with different acyl chain lengths.31 Here we also found that the thermal stability of LasR varies with the identity of the ligand bound. Not surprisingly, LasR appears to be most stable in the presence of native 3O-C12-HSL (Tm = 53.0 °C). LasR complexed with ligands 6 (Tm = 38.9 °C) and 8 (Tm = 39.6 °C) gave similar but reduced thermal stabilities. LasR with ligand 7, which showed the most difference in secondary structure from the native ligand, was unexpectedly stable (Tm = 50.7 °C), suggesting that LasR interactions with this antagonist promote a robust unnatural fold that does not permit DNA binding. Recent CD assessment of the LasR LBD with covalent LasR inhibitors also indicated a change in fold and reduction in thermal stability compared with the native ligand.17 Most studies indicate that LasR binds tightly, even with extensive dialysis, to 3O-C12-HSL,14,21 with one recent report suggesting that LasR·autoinducer binding may be reversible and thus allow for rapid quorum sensing response.25 Here we measured whether LasR·DNA binding could be recovered if purified LasR complexed with a non-native ligand were incubated with 3O-C12-HSL. Recovery of DNA binding was observed by EMSA for several LasR−antagonist complexes, suggesting that the addition of native 3O-C12-HSL leads to replacement of the antagonist and refolding of LasR into a DNA-binding-competent state (Figure 4). Recovery of binding for LasR−3 complexes was much more successful than for LasR complexed with ligands 6−8, suggesting that these known antagonists form a more stable complex with LasR and are not easily exchanged. Ligand 3, C10-HSL, also more closely resembles the native ligand and may induce a LasR fold more similar to that in the native complex. The reverse exchange of ligands is also possible, with DNA binding by LasR complexed with native 3O-C12-HSL disrupted by incubation with added antagonists 5−8 prior to the addition of DNA (Figure 5). The addition of 25 μM antagonist to 0.5 μM LasR complexed with native ligand led to reduced DNA binding, with nearly complete loss of DNA binding at 500 μM added antagonist (Figure 5c). One exception is the behavior of ligand 7, which does appear to reverse LasR·DNA binding fractionally, but this trend does not increase at higher D
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interaction between LasR and 7 is similarly unique. Our CD results also indicate that 7 has a distinct effect on the structure of LasR. Structural work would clarify the nature of the LasR−7 complex, including whether 7 occupies the same binding pocket as 3O-C12-HSL. Since the triphenyl agonists that served as part of the inspiration for the design of 7 do bind in the LasR ligand binding pocket even though they are structurally dissimilar to 3O-C12-HSL,22 it seems possible that 7 does as well. In summary, we have demonstrated conclusively that known antagonists disrupt LasR activity through some stabilization of a soluble LasR−antagonist complex that no longer binds DNA. Our work also provides additional evidence describing the reversibility of LasR−ligand complexes that may be relevant for the design of antagonists effective in a complex in vivo environment with competing ligands. While this study investigated only a subset of potent LasR antagonists, our methods should be applicable to future in vitro characterization and comparison of other LasR antagonists of interest. This approach will complement existing cell-based strategies for the evaluation of LasR antagonists, helping in the development of new antibacterial tools for the treatment of P. aeruginosa infections.
Figure 4. Reversibility of binding of LasR to unnatural ligands. (a) Representative EMSA with target lasB OP1 alone (lane 1) or incubated with 0.5 μM LasR complexed with native 3O-C12-HSL (lane 2). LasR (5 μM) expressed in the presence of ligand 3 (C10-HSL) was assayed with added 3O-C12-HSL [0 μM (lane 3), 5 μM (lane 4), 25 μM (lane 5), or 100 μM (lane 6)]. LasR (5 μM) expressed in the presence of ligand 8 was assayed with added 3O-C12-HSL [0 μM (lane 7), 5 μM (lane 8), 25 μM (lane 9), or 100 μM (lane 10)]. (b) Representative EMSA with target lasB OP1 alone (lane 1) or incubated with 0.5 μM LasR complexed with native 3O-C12-HSL (lane 2). LasR (5 μM) expressed in the presence of ligand 7 was assayed with added 3O-C12-HSL [0 μM (lane 3), 5 μM (lane 4), 25 μM (lane 5), or 100 μM (lane 6)]. LasR (5 μM) expressed in the presence of ligand 6 was assayed with added 3O-C12-HSL [0 μM (lane 7), 5 μM (lane 8), 25 μM (lane 9), or 100 μM (lane 10)].
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsinfecdis.6b00163. Experimental methods for preparation of materials and biochemical assays (PDF)
concentrations. Ligand 7 is the most structurally different among the antagonists tested, and these results suggest that the
Figure 5. Reversibility of binding of LasR to native 3O-C12-HSL. (a) Representative EMSA with target lasB OP1 alone (lane 1) or incubated with 0.5 μM LasR complexed with native 3O-C12-HSL and 1% THF as a control (lane 2) or with added ligand 6 (lanes 3−6) or 5 (lanes 7−10). The ligand was added at 25 μM, 100 μM, 500 μM, or 1 mM in each case. (b) Representative EMSA with target lasB OP1 alone (lane 1) or incubated with 0.5 μM LasR complexed with native 3O-C12-HSL and 1% THF as a control (lane 2) or with added ligand 7 (lanes 3−6) or 8 (lanes 7−10). The ligand was added at 25 μM, 100 μM, 500 μM, or 1 mM in each case. (c) Comparison of ligand exchange with LasR−3O-C12-HSL as measured by the relative fraction of target lasB OP1 bound with added 5 (○), 6 (●), 7 (□), or 8 (■). E
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(12) Welsh, M. A., and Blackwell, H. E. (2016) Chemical probes of quorum sensing: from compound development to biological discovery. FEMS Microbiol. Rev. 40, 774−794. (13) Kiratisin, P., Tucker, K. D., and Passador, L. (2002) LasR, a transcriptional activator of Pseudomonas aeruginosa virulence genes, functions as a multimer. J. Bacteriol. 184, 4912−4919. (14) Schuster, M., Urbanowski, M. L., and Greenberg, E. P. (2004) Promoter specificity in Pseudomonas aeruginosa quorum sensing revealed by DNA binding of purified LasR. Proc. Natl. Acad. Sci. U. S. A. 101, 15833−15839. (15) Gilbert, K. B., Kim, T. H., Gupta, R., Greenberg, E. P., and Schuster, M. (2009) Global position analysis of the Pseudomonas aeruginosa quorum-sensing transcription factor LasR. Mol. Microbiol. 73, 1072−1085. (16) O’Brien, K. T., Noto, J. G., Nichols-O’Neill, L., and Perez, L. J. (2015) Potent irreversible inhibitors of LasR quorum sensing in Pseudomonas aeruginosa. ACS Med. Chem. Lett. 6, 162−167. (17) Amara, N., Gregor, R., Rayo, J., Dandela, R., Daniel, E., Liubin, N., Willems, H. M., Ben-Zvi, A., Krom, B. P., and Meijler, M. M. (2016) Fine-tuning covalent inhibition of bacterial quorum sensing. ChemBioChem 17, 825−835. (18) Galloway, W. R. J. D., Hodgkinson, J. T., Bowden, S. D., Welch, M., and Spring, D. R. (2011) Quorum sensing in Gram-negative bacteria: small-molecule modulation of AHL and AI-2 quorum sensing pathways. Chem. Rev. 111, 28−67. (19) Mattmann, M. E., and Blackwell, H. E. (2010) Small molecules that modulate quorum sensing and control virulence in Pseudomonas aeruginosa. J. Org. Chem. 75, 6737−6746. (20) Moore, J. D., Rossi, F. M., Welsh, M. A., Nyffeler, K. E., and Blackwell, H. E. (2015) A comparative analysis of synthetic quorum sensing modulators in Pseudomonas aeruginosa: new insights into mechanism, active efflux susceptibility, phenotypic response, and nextgeneration ligand design. J. Am. Chem. Soc. 137, 14626−14639. (21) Bottomley, M. J., Muraglia, E., Bazzo, R., and Carfi, A. (2007) Molecular insights into quorum sensing in the human pathogen Pseudomonas aeruginosa from the structure of the virulence regulator LasR bound to its autoinducer. J. Biol. Chem. 282, 13592−13600. (22) Zou, Y., and Nair, S. K. (2009) Molecular basis for the recognition of structurally distinct autoinducer mimics by the Pseudomonas aeruginosa LasR quorum-sensing signaling receptor. Chem. Biol. 16, 961−970. (23) Gerdt, J. P., McInnis, C. E., Schell, T. L., and Blackwell, H. E. (2015) Unraveling the contributions of hydrogen-bonding interactions to the activity of native and non-native ligands in the quorum-sensing receptor LasR. Org. Biomol. Chem. 13, 1453−1462. (24) Gerdt, J. P., McInnis, C. E., Schell, T. L., Rossi, F. M., and Blackwell, H. E. (2014) Mutational analysis of the quorum-sensing receptor LasR reveals interactions that govern activation and inhibition by nonlactone ligands. Chem. Biol. 21, 1361−1369. (25) Sappington, K. J., Dandekar, A. A., Oinuma, K., and Greenberg, E. P. (2011) Reversible signal binding by the Pseudomonas aeruginosa quorum-sensing signal receptor LasR. mBio 2, e00011-11. (26) Geske, G. D., O’Neill, J. C., Miller, D. M., Mattmann, M. E., and Blackwell, H. E. (2007) Modulation of bacterial quorum sensing with synthetic ligands: systematic evaluation of N-acylated homoserine lactones in multiple species and new insights into their mechanisms of action. J. Am. Chem. Soc. 129, 13613−13625. (27) Geske, G. D., Wezeman, R. J., Siegel, A. P., and Blackwell, H. E. (2005) Small molecule inhibitors of bacterial quorum sensing and biofilm formation. J. Am. Chem. Soc. 127, 12762−12763. (28) O’Reilly, M. C., and Blackwell, H. E. (2016) Structure-based design and biological evaluation of triphenyl scaffold-based hybrid compounds as hydrolytically stable modulators of a LuxR-type quorum sensing receptor. ACS Infect. Dis. 2, 32−38. (29) Hentzer, M., Wu, H., Andersen, J. B., Riedel, K., Rasmussen, T. B., Bagge, N., Kumar, N., Schembri, M. A., Song, Z., Kristoffersen, P., Manefield, M., Costerton, J. W., Molin, S., Eberl, L., Steinberg, P., Kjelleberg, S., Høiby, N., and Givskov, M. (2003) Attenuation of
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Phone: (860) 439-2481. ORCID
Tanya L. Schneider: 0000-0002-0643-0170 Author Contributions
E.G.S and T.L.S. expressed and purified LasR. E.G.S. and L.R.H synthesized small-molecule ligands. E.G.S. and T.L.S. performed EMSA, mass spectrometry, and CD spectroscopy. T.L.S. wrote the manuscript with input and approval from all authors. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by a Cottrell College Science Award from the Research Corporation for Science Advancement. E.G.S. was supported by a summer fellowship from the Sherman Fairchild Foundation.
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ABBREVIATIONS QS, quorum sensing; 3O-C12-HSL, N-(3-oxododecanoyl)-Lhomoserine lactone; LBD, ligand binding domain; DBD, DNA binding domain; CD, circular dichroism; EMSA, electrophoretic mobility shift assay
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REFERENCES
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