Title: N-acyl homoserine lactone binding to the CarR receptor determines quorum-sensing specificity in Erwinia
Abstract: Article15 February 2000free access N-acyl homoserine lactone binding to the CarR receptor determines quorum-sensing specificity in Erwinia Martin Welch Martin Welch Department of Biochemistry, Tennis Court Road, Cambridge, CB2 1QW UK Search for more papers by this author Daniel E. Todd Daniel E. Todd Department of Biochemistry, Tennis Court Road, Cambridge, CB2 1QW UK Search for more papers by this author Neil A. Whitehead Neil A. Whitehead Department of Biochemistry, Tennis Court Road, Cambridge, CB2 1QW UK Search for more papers by this author Simon J. McGowan Simon J. McGowan Department of Biochemistry, Tennis Court Road, Cambridge, CB2 1QW UK Search for more papers by this author Barrie W. Bycroft Barrie W. Bycroft Department of Pharmaceutical Sciences, University of Nottingham, Nottingham, UK Search for more papers by this author George P.C. Salmond Corresponding Author George P.C. Salmond Department of Biochemistry, Tennis Court Road, Cambridge, CB2 1QW UK Search for more papers by this author Martin Welch Martin Welch Department of Biochemistry, Tennis Court Road, Cambridge, CB2 1QW UK Search for more papers by this author Daniel E. Todd Daniel E. Todd Department of Biochemistry, Tennis Court Road, Cambridge, CB2 1QW UK Search for more papers by this author Neil A. Whitehead Neil A. Whitehead Department of Biochemistry, Tennis Court Road, Cambridge, CB2 1QW UK Search for more papers by this author Simon J. McGowan Simon J. McGowan Department of Biochemistry, Tennis Court Road, Cambridge, CB2 1QW UK Search for more papers by this author Barrie W. Bycroft Barrie W. Bycroft Department of Pharmaceutical Sciences, University of Nottingham, Nottingham, UK Search for more papers by this author George P.C. Salmond Corresponding Author George P.C. Salmond Department of Biochemistry, Tennis Court Road, Cambridge, CB2 1QW UK Search for more papers by this author Author Information Martin Welch1, Daniel E. Todd1, Neil A. Whitehead1, Simon J. McGowan1, Barrie W. Bycroft2 and George P.C. Salmond 1 1Department of Biochemistry, Tennis Court Road, Cambridge, CB2 1QW UK 2Department of Pharmaceutical Sciences, University of Nottingham, Nottingham, UK *Corresponding author. E-mail: [email protected] The EMBO Journal (2000)19:631-641https://doi.org/10.1093/emboj/19.4.631 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Quorum sensing via an N-acyl homoserine lactone (HSL) pheromone controls the biosynthesis of a carbapenem antibiotic in Erwinia carotovora. Transcription of the carbapenem biosynthetic genes is dependent on the LuxR-type activator protein, CarR. Equilibrium binding of a range of HSL molecules, which are thought to activate CarR to bind to its DNA target sequence, was examined using fluorescence quenching, DNA bandshift analysis, limited proteolysis and reporter gene assays. CarR bound the most physiologically relevant ligand, N-(3-oxohexanoyl)-L-homoserine lactone, with a stoichiometry of two molecules of ligand per dimer of protein and a dissociation constant of 1.8 μM, in good agreement with the concentration of HSL required to activate carbapenem production in vivo. In the presence of HSL, CarR formed a very high molecular weight complex with its target DNA, indicating that the ligand causes the protein to multimerize. Chemical cross-linking analysis supported this interpretation. Our data show that the ability of a given HSL to facilitate CarR binding to its target DNA sequence is directly proportional to the affinity of the HSL for the protein. Introduction In the last decade, it has become increasingly clear that a plethora of physiological processes, including virulence, secondary metabolism and bioluminescence, are activated in response to changes in the cell density of a bacterial population (for a recent review, see Fuqua et al., 1996). This phenomenon is known as quorum sensing and involves intercellular communication via N-acyl homoserine lactones (HSLs). These signalling molecules are synthesized in the cytoplasm by enzymes homologous to the LuxI protein from the bioluminescent bacterium Vibrio fischeri (reviewed by Meighen, 1991). However, the newly synthesized HSLs are believed to diffuse readily across the cell envelope into the growth medium, where they accumulate. This accumulation continues until the cell density (and, therefore, the HSL concentration) exceeds a critical value, whereupon specific physiological changes occur. Most models suggest that HSLs cause these changes by binding to intracellular receptor proteins, thereby activating these proteins to elicit the desired cellular response. The putative receptor proteins, of which LuxR from V.fischeri was the first to be identified, are almost exclusively transcriptional activators that turn on the expression of target genes in the presence of HSL (Fuqua et al., 1996). In this way, a bacterial population can achieve highly concerted expression of a defined set of genes using a diffusible pheromone as a signal of population density status. A widely accepted model by which LuxR-type proteins are thought to activate gene expression is that ligand binding to the N-terminal domain of the protein causes a conformational change, which leads to exposure of the DNA-binding domain at the C-terminus of the protein (Da Re et al., 1994). A recent variation on this mechanism is that the binding of HSL causes the protein to dimerize or form some other higher order assemblage competent to bind DNA (Zhu and Winans, 1999). However, although these hypotheses are experimentally tractable, very little biochemical work has been carried out to test them. Most notably, although the central theme of both mechanisms is that the LuxR homologue binds HSLs, this interaction has yet to be quantitated experimentally. Indeed, all attempts at measuring HSL binding to LuxR homologues have been either (i) indirect, assaying the ability of HSLs to activate reporter gene expression in vivo (Eberhard et al., 1986; Zhu et al., 1998) or the ability of HSL analogues to inhibit the binding of radiolabelled autoinducer to Escherichia coli cells expressing luxR (Schaefer et al., 1996); or (ii) qualitative, assaying the ability of a fixed concentration of HSL to cause a DNA bandshift in the presence of a LuxR homologue (Zhu and Winans, 1999). Without a more quantitative understanding of the binding of HSLs to LuxR homologues, it will be impossible to address questions about molecular recognition issues affecting specificity in quorum-sensing systems, or the precise mechanism of action of LuxR-type proteins. Quorum-sensing systems have been identified in a wide range of bacterial genera, particularly in a variety of plant and animal pathogens. One interpretation of the logic underlying a quorum-sensing system strategy in virulence is that the bacteria use it to mount a sustained attack on the host only when their numbers are high enough to ensure that they have a reasonable chance of success. A well characterized example of this is found in the Gram-negative phytopathogen, Erwinia carotovora subsp. carotovora (Pérombelon and Kelman, 1980; Jones et al., 1993; Pirhonen et al., 1993). The bacterium kills its host by producing an arsenal of cell wall-degrading exoenzymes in a cell density-dependent fashion (Andro et al., 1984; Collmer and Keen, 1986; Kotoujansky, 1987). Some strains of E.carotovora also manufacture a broad spectrum β-lactam antibiotic (1-carbapen-2-em-3-carboxylic acid, carbapenem; Bainton et al., 1992; Holden et al., 1998) in concert with virulence factor elaboration, presumably to suppress the growth of competing opportunistic bacteria in the nutritional windfall released at the site of infection. In quorum sensing-mediated control of carbapenem production, the LuxI homologue, CarI, is responsible for synthesizing N-(3-oxohexanoyl)-L-homoserine lactone (3-oxo-C6-HSL) (Bainton et al., 1992; Jones et al., 1993; Swift et al., 1993). The 3-oxo-C6-HSL synthesized by CarI is thought to bind to a LuxR homologue, CarR, which then activates transcription of the car genes (McGowan et al., 1995, 1996). carR is located ∼150 bp upstream of the car biosynthetic cluster (carA–H) but is not part of the same transcriptional unit. Several lines of genetic evidence indicate that the intergenic region between carR and carA contains the CarR-binding site (McGowan et al., 1995, 1996), although this is not defined. Given that the transcriptional activator, CarR, and the pheromone, C6ox-HSL, are thought to be interacting receptor and ligand, respectively, we initiated a study to quantitate the binding of various HSLs to CarR, and to monitor the effect that these ligands have on the binding of CarR to the carR–carA intergenic region. Results Purification of His6-tagged CarR The carR gene product from wild-type E.carotovora subsp. carotovora (strain ATTn10) was His6 tagged by insertion into the expression vector pQE31. The resulting construct (pcarR31) was then modified by insertion of a kanamycin cassette into the bla gene of the vector, yielding pcarR31Kn. (This was done in order to abolish the encoded ampicillin resistance, which would otherwise interfere with the assay for carbapenem.) To examine whether the His6-tagged protein was functional, we tested whether pcarR31Kn could restore carbapenem production to a carR mutant (GB3). We found that pcarR31Kn was able to restore carbapenem production in this mutant to wild-type levels, indicating that incorporation of the additional residues (MRGSHHHHHHTDPIEGR) at the N-terminus of the protein did not affect its biological activity (data not shown). When expressed in E.coli at 37°C, His6-CarR partitioned between the insoluble (inclusion bodies) and soluble fractions. The His6-CarR in the soluble fraction constituted ∼20% of the total recombinant protein. We attempted to increase the proportion of soluble protein by (i) growing the cells at a lower temperature (30°C) and (ii) expressing His6-CarR in the presence of CarI in the hope that the HSL molecules produced by the latter might stabilize a more soluble conformation of the protein. Neither method was effective. An alternative approach was to purify His6-CarR from the insoluble fraction. This was done by solubilizing the inclusion bodies in guanidinium hydrochloride. The denatured protein was then purified (by Ni–NTA chromatography) and refolded by rapid dilution into a buffer without denaturant. The properties of the refolded protein were indistinguishable from those of His6-CarR isolated from the soluble fraction of the cells. When His6-CarR was renatured, solutions of the protein remained clear for a few minutes, but then gradually turned cloudy as a precipitate formed. The relatively slow formation of this precipitate, which was not reduced visibly by inclusion of 3-oxo-C6-HSL in the renaturation buffer, suggested that it consisted of aggregated native protein. We conclude that CarR has an intrinsic tendency to aggregate. In line with this, neither refolded His6-CarR nor His6-CarR from the soluble fraction of the cells could be concentrated to more than ∼5 μM without inducing precipitation. Presumably, this explains why all of our attempts to increase the amount of soluble protein failed. Biochemical properties of His6-CarR Solutions of His6-CarR were too dilute to characterize by standard hydrodynamic methods, e.g. gel filtration chromatography. We therefore used chemical cross-linking to determine the multimeric status of the protein. The cross-linking mixture used for most experiments was 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) in the presence of N-hydroxysuccinimide (NHS) (Blat and Eisenbach, 1996), although glutaraldehyde gave the same results. In the absence of cross-linker, His6-CarR migrated on SDS–PAGE with an apparent molecular mass of 30 kDa (Figure 1), which is the same as the molecular mass predicted from the sequence (29 949 Da). However, in the presence of EDC/NHS, a pair of diffuse bands migrating with apparent mol. wts of 58 and 62 kDa appeared, along with a small amount of very high molecular weight material. The 58 and 62 kDa bands correspond to dimeric His6-CarR. The dimer migrates as two bands due to the presence of intramolecular cross-links within one or both protomers, which alter their mobilities. Control experiments (not shown) indicate that these intramolecular cross-links form very readily, and block intermolecular cross–linking, which is why only a relatively small fraction of the total protein appeared as dimers and high molecular weight material. In the presence of 3-oxo-C6-HSL, the 62 kDa band became better defined, although the total amount of dimer was almost unchanged. However, even low concentrations of 3-oxo-C6-HSL significantly increased the amount of high molecular weight cross-linked products. Taken together, these data indicate that His6-CarR exists as a preformed dimer, and that the main effect of 3-oxo-C6-HSL binding is to increase the tendency of these dimers to form multimers. Figure 1.Chemical cross-linking of CarR. Reaction mixtures contained His6-CarR (1.37 μM dimer), EDC (5 mM), NHS (10 mM) and 3-oxo-C6-HSL. After 30 min incubation, the reactions were stopped and the samples were resolved on a 10–20% polyacrylamide–SDS gradient gel. Lane 1: no cross-linker. The concentrations of HSL in the remaining reactions were: 0 μM (lane 2), 1 μM (lane 3), 5 μM (lane 4), 10 μM (lane 5), 20 μM (lane 6), 50 μM (lane 7), 100 μM (lane 8) and 200 μM (lane 9). Lane 10: molecular weight markers (in kDa). Download figure Download PowerPoint To examine further how 3-oxo-C6-HSL affects the properties of His6-CarR, we performed a limited tryptic digestion on the protein in the absence and presence of HSL. The results were striking. In the absence of the HSL, His6-CarR was digested extensively by the protease after just 50 min (Figure 2A). However, in the presence of 3-oxo-C6-HSL (47 μM), His6-CarR was much more resistant to digestion with substantial amounts of the protein, remaining essentially intact even after 200 min of digestion. [The major (∼28 kDa) band present in trypsin-treated His6-CarR corresponds to the intact protein cleaved at the factor Xa site.] We conclude that HSL binding to His6-CarR alters the accessibility of scissile bonds to tryptic cleavage. Interestingly, although the rate of proteolysis was affected by the presence of the HSL, the overall pattern of digestion was not. This suggests that the HSL does not cause a major conformational change in the protein (thereby exposing new tryptic cleavage sites). Instead, and in line with the cross-linking data above, HSL binding to His6-CarR probably leads to a change in the aggregation state of the protein. It is likely that this change in aggregation state accounts for the altered kinetics of trypsinolysis. Addition of pSMG101 (which carries the CarR-binding site) to the reaction mixtures had no effect on the rate or extent of trypsinolysis (data not shown). Figure 2.Limited trypsinolysis of His6-CarR. (A) Time course of limited trypsinolysis. Reaction mixtures containing His6-CarR (1.5 μM dimer) with or without 3-oxo-C6-HSL (47 μM) were incubated for the indicated periods of time with trypsin (0.3 μg/ml) and then resolved on a 12–22% polyacrylamide–SDS gradient gel. The positions of the molecular weight markers (in kDa) are shown. The contents of each lane and the reaction times (in min) are as indicated. (B) Effect of different HSLs on the extent of trypsinolysis. Reaction mixtures contained His6-CarR (2.25 μM dimer), unsubstituted HSL (25 μM) and trypsin (3 μg/ml). The acyl chain length of the HSL used in each reaction and the positions of selected molecular weight markers are indicated. In the lane labelled 'N', no HSL was present. Download figure Download PowerPoint The result above prompted us to test whether other HSLs might also protect His6-CarR from digestion by trypsin. We therefore performed a limited trypsinolysis over a 5 h period in the presence of a homologous series of unsubstituted HSLs (all 25 μM concentration), each with an acyl chain containing a different number of carbon atoms (from C4 to C11). The result is shown in Figure 2B. A clear decrease in tryptic sensitivity is observed with those HSLs carrying acyl chains of seven, eight and nine carbon atoms. We conclude that these HSLs bind to His6-CarR better than the others we tested. In line with this, when the same experiment was carried out using a homologous series of 3-oxo-substituted HSLs, optimum protection from trypsin was afforded by 3-oxo-C8-HSL (data not shown). Ligand binding by His6-CarR CarR contains three tryptophan residues, W20, W44 and W72 (Holden et al., 1998). Of these residues, W44 and W72 are highly conserved in most LuxR homologues. All three tryptophan residues are located in the N-terminal one-third of the molecule, which in the case of LuxR is thought to contain the HSL-binding domain (Shadel et al., 1990; Slock et al., 1990; Hanzelka and Greenberg, 1995). Since tryptophan is strongly fluorescent, and since one or more of these tryptophan residues might be located close to the ligand-binding site (or be perturbed in some way by ligand binding), we used the intrinsic tryptophan fluorescence of CarR as a probe to monitor HSL binding. HSLs were titrated against His6-CarR and the fluorescence emission spectra were recorded after each addition. The addition of HSLs caused significant fluorescence quenching (by up to 50%) without any change in λmax (335 nm; data not shown). The extent of quenching produced by each HSL was dependent on its concentration and its structure (Figure 3). The pattern of quenching fell into three distinct groups. The first group incorporated those HSLs carrying unsubstituted aliphatic carbon chains. These HSLs caused <10% quenching at any of the concentrations tested, irrespective of the chain length. Of the remaining HSLs, all of which carried a 3-oxo substitution in the hydrocarbon chain, one group (composed of 3-oxo-C8-HSL and 3-oxo-C6-HSL) strongly quenched the fluorescence of the protein at approximately stoichiometric quantities, while the second group (composed of 3-oxo-C4-HSL, 3-oxo-C10-HSL and 3-oxo-C12-HSL) caused significant quenching only when added in excess quantities. The simplest interpretation of these data is that the affinity of the HSLs for CarR follows the order 3-oxo-C8-HSL>3-oxo-C6-HSL>[3-oxo-C4-HSL, 3-oxo-C10-HSL, 3-oxo-C12-HSL]>unsubstituted. Figure 3.HSL binding by His6-CarR. HSLs were titrated against His6-CarR (0.8 μM dimer) and the extent of fluorescence quenching was measured. For clarity, linefits are only drawn for selected HSLs. Key: 3-oxo-C4-HSL (▴), 3-oxo-C6-HSL (●), 3-oxo-C8-HSL (▪), 3-oxo-C10-HSL (♦), 3-oxo-C12-HSL (⊙), C4-HSL (▵), C6-HSL (○), C8-HSL (□), C10-HSL (⋄) and C12-HSL (+). Download figure Download PowerPoint Ligand-dependent transcription of carA in vivo To measure the effect of the different HSLs on the transcription of carA in vivo, we monitored β-galactosidase production by a carI mutant (GB7I) containing a lacZ transcriptional fusion in carA. The activity of the reporter gene product was assayed through the growth curve. None of the HSLs affected the growth rate of the cells and, for those ligands that induced significant β-galactosidase activity, induction was almost immediate (data not shown). In these cases, the level of induction rose and then levelled off during the stationary phase, and no peaks of β-galactosidase activity were observed. In view of this, we decided that the most informative way of plotting the data was as the cumulative reporter gene activity measured through the growth curve for each HSL (Figure 4). For the non-substituted HSLs, only C6-HSL and C7-HSL induced significant activity. In the case of the 3-oxo-substituted ligands, the overall potency of the HSLs was 1–2 orders of magnitude greater than that of the non-substituted analogues. Furthermore, although the optimal β-galactosidase activity was centred on 3-oxo-C6-HSL, both 3-oxo-C8-HSL and, to a much lesser extent, 3-oxo-C4-HSL also induced a substantial response. Figure 4.HSL-dependent carA transcription in vivo. Cumulative β-galactosidase activity [expressed as the sum of the ΔA420/min/ml (OD unit) measurements made for each HSL through the growth curve] of GB7I in the presence of the HSLs indicated (all 1 μg/ml concentration). (○) 3-oxo-substituted HSLs; (●) unsubstituted HSLs. For comparison, the cumulative β-galactosidase activity in the absence of HSL was 0.587 units. Download figure Download PowerPoint HSL binding to His6-CarR changes its interaction with DNA Using gel mobility shifts as an assay, we examined whether there was any correlation between the binding of HSL to His6-CarR and the binding of the protein to its target DNA. The target DNA used was a 370 bp HindIII fragment encompassing the entire carR–carA intergenic region (hereafter termed the IGR) or, as a control, a 340 bp DraI fragment of vector DNA. In each reaction, the concentration of labelled DNA used was ∼3 nM, which is approximately the concentration of a single regulatory DNA element in the cell. First, we measured whether His6-CarR had any intrinsic affinity for the IGR in the absence of HSL (Figure 5A). At low His6-CarR concentrations, a well defined band representing the minimal His6-CarR–DNA complex appeared just above the free DNA band. This slightly retarded species apparently acts as a scaffold onto which further His6-CarR molecules bind, because at higher His6-CarR concentrations it was gradually replaced by a band showing an increasingly greater degree of gel retardation. [The observation that His6-CarR forms a minimal protein–DNA complex in the absence of HSL supports our earlier suggestion (cf. Figure 1) that CarR exists as a preformed dimer. This is because the CarR-binding site in the IGR is likely to be a region of dyad symmetry (McGowan et al., 1995). Such dyads usually bind dimeric proteins, with each protomer in the dimer binding separate arms of the dyad.] Figure 5.The binding of His6-CarR to the carR–carA intergenic region. (A) Binding of His6-CarR to the IGR in the absence of HSL. Gel retardation assays were performed as described in Materials and methods. Each reaction contained 3 nM DNA. The concentration of dimeric His6-CarR in the reactions was: 0 nM (lane 1), 31 nM (lane 2), 62 nM (lane 3), 93 nM (lane 4), 125 nM (lane 5), 187 nM (lane 6), 312 nM (lane 7), 437 nM (lane 8) and 624 nM (lane 9). (B) Binding of His6-CarR to the IGR in the presence of 3-oxo-C6-HSL. Each reaction contained 3 nM DNA. Lane 1: no His6-CarR. The remaining lanes all contained 345 nM dimeric His6-CarR and the following 3-oxo-C6-HSL concentrations: 0 μM (lane 2), 0.46 μM (lane 3), 1.1 μM (lane 4), 1.8 μM (lane 5), 4.6 μM (lane 6), 7.3 μM (lane 7), 18.3 μM (lane 8) and 73.3 μM (lane 9). Lane 10 contained 73.3 μM 3-oxo-C6-HSL and a 66 bp unlabelled synthetic oligonucleotide (2 μM) spanning a region of inverse and direct repeats in the IGR. The sequence of the central (54 bp) portion of this oligo is shown (the remaining 12 bp of the oligo that are not shown constitute BamHI and HindIII restriction sites at each end of the molecule). Download figure Download PowerPoint Next, we titrated 3-oxo-C6-HSL against a fixed concentration of DNA and His6-CarR (Figure 5B). At the concentration of His6-CarR we chose to use (∼0.35 μM dimer), in the absence of 3-oxo-C6-HSL, most of the DNA was sequestered into the minimal protein–DNA complex (Figure 5B, lane 2). However, as the 3-oxo-C6-HSL concentration increased, the intensity of this band diminished and a very highly retarded species that only just entered the gel appeared (Figure 5B, lanes 3–9). The formation of this highly retarded species was abolished in the presence of an unlabelled synthetic 66 bp oligonucleotide (Figure 5B, lane 10) encompassing a region of the IGR containing dyad symmetry (McGowan et al., 1995). In control experiments, His6-CarR did not bind to the labelled 340 bp fragment of DraI vector DNA (data not shown). Based on these results, we estimate that the apparent Kd of 3-oxo-C6-HSL binding to His6-CarR is ∼1.8 μM. For comparison, when the same titration was carried out with 3-oxo-C8-HSL, the apparent Kd for binding was ∼0.5 μM (data not shown). From the low mobility of the protein–DNA complex formed in the presence of 3-oxo-C6-HSL, we conclude that it has a very high molecular weight. The effect of varying the acyl chain length of the HSLs on the binding of His6-CarR to the IGR was measured. As shown in Figure 6, in the presence of a fixed concentration of each ligand (25 μM), HSL with acyl chains containing 5–9 carbon atoms caused a significant gel shift, with the peak of activity being centred on C7-HSL and C8-HSL. However, in all cases, only a relatively small amount of the total DNA probe formed a ternary complex with the protein and the ligand. This suggests that although these ligands bind to His6-CarR, they do so with a much lower affinity than 3-oxo-C6-HSL. [We verified that this conclusion holds over a range of ligand concentrations by titrating selected unsubstituted HSLs against His6-CarR and monitoring the resulting gel shifts (data not shown).] Figure 6.The effect of HSL acyl chain length on the binding of His6-CarR to DNA. Each reaction contained 3 nM DNA. Except for the lanes labelled 'P' (probe DNA alone) and 'N' (probe DNA and His6-CarR in the absence of HSL), the remaining lanes each contained 187 nM dimeric His6-CarR and 25 μM of the HSL indicated. Download figure Download PowerPoint Ligand binding to a soluble fragment of His6-CarR To investigate further the binding of HSLs to CarR, we expressed and purified a highly soluble portion of the protein (His6-CarR1–167; numbering based on untagged CarR) encompassing the ligand-binding domain. Those HSLs carrying a 3-oxo substituent on the acyl chain caused quenching of the fluorescence of His6-CarR1–167. However, whereas HSLs carrying unsubstituted acyl chains caused minor quenching of the fluorescence of the full-length protein, in the case of His6-CarR1–167 these HSLs enhanced its fluorescence. Although the extent of this enhancement was maximally only ∼30% of the total signal, the relative signal could be increased using quench-enhanced fluorescence (QEF). This approach is based upon the suppression of bulk protein fluorescence intensity using an extrinsic quencher (acrylamide) that does not affect the magnitude of the ligand-induced enhancement of fluorescence. Results from the two types of titrations performed (monitoring QEF in the case of the unsubstituted HSLs and intrinsic tryptophan quenching in the case of the 3-oxo-HSLs) are shown in Figure 7A and B, respectively. As in the case of the intact protein, the pattern of fluorescence quenching by the 3-oxo-substituted HSLs followed the order 3-oxo-C8>3-oxo-C6>[3-oxo-C4, 3-oxo-C10, 3-oxo-C12]. For the non-substituted HSLs, the pattern of fluorescence enhancement followed the order C8>C7>[C6, C9]>[C4, C5, C10]. The C11 and C12 unsubstituted HSLs did not significantly affect the fluorescence of the protein at any concentration tested. Figure 7.The binding of different HSLs to His6-CarR1–167. (A) Binding of unsubstituted HSLs to His6-CarR1–167 measured by quench-enhanced fluorescence. The concentration of dimeric His6-CarR1–167 was 4.4 μM. Key: C4-HSL (▵), C5-HSL, C6-HSL (○), C7-HSL, C8-HSL (□), C9-HSL (□;), C10-HSL (⋄), C11-HSL and C12-HSL (+). For clarity, solid lines have only been fitted through selected titrations. (B) Binding of 3-oxo-substituted HSLs to His6-CarR1–167 measured by fluorescence quenching. The concentration of dimeric His6-CarR1–167 was 2.2 μM. Key: 3-oxo-C4-HSL (▴), 3-oxo-C6-HSL (●), 3-oxo-C8-HSL (▪), 3-oxo-C10-HSL (♦) and 3-oxo-C12-HSL. Download figure Download PowerPoint Figure 8 summarizes the combined data for HSL binding to full-length His6-CarR and His6-CarR1–167. In the figure, the relative affinities are defined as the ligand concentrations required to yield the half-maximal change in fluorescence in Figures 3 and 7. The data clearly show three things. First, the ligand-binding spectrum of full-length His6-CarR is the same as that of His6-CarR1–167, indicating that the soluble fragment of CarR has the same binding specificity as the full-length protein. Secondly, the presence of a 3-oxo group on the ligand increases its affinity for CarR by at least an order of magnitude. Thirdly, and in line with the results in Figures 2B and 6, there is an optimum acyl chain length for binding of 7–8 carbon atoms. Figure 8.The optimal chain length for HSL binding to His6-CarR. The apparent Kd values of the HSL–His6-CarR and HSL–His6-CarR1–167 complexes are plotted against the number of carbon atoms in the acyl chain of each ligand. (⋄) Binding of 3-oxo-substituted HSLs to His6-CarR; (●) binding of 3-oxo-substituted HSLs to His6-CarR1–167; (○) binding of unsubstituted HSLs to His6-CarR1–167. Download figure Download PowerPoint Quantitative analysis of 3-oxo-C6-HSL binding to His6-CarR1–167 An intrinsic limitation of fluorescence titrations is that they do not readily yield to quantitative analysis. This is because the only information available to the experimenter is a signal for each of the ligand concentrations tested, i.e. the 'free' and 'bound' concentrations of the ligand are not known. However, these parameters can be calculated if specific binding models are applied (Hu and Eftink, 1993). Given that the protein i