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J. Biol. Chem., Vol. 278, Issue 15, 13173-13182, April 11, 2003

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Mutational Analysis of TraR

CORRELATING FUNCTION WITH MOLECULAR STRUCTURE OF A QUORUM-SENSING TRANSCRIPTIONAL ACTIVATOR^*

Zhao-Qing Luo^§, Audra J. Smyth, Ping Gao, Yinping Qin, and Stephen K. Farrand^¶

From the Departments of  Crop Sciences and ^¶ Microbiology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801

Received for publication, October 1, 2002, and in revised form, February 3, 2003

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

TraR, the quorum-sensing activator of the Agrobacterium tumefaciens Ti plasmid conjugation system, induces gene expression in response to its quormone, N-(3-oxooctanoyl)-L-homoserine lactone. Ligand binding results in dimerization of TraR and is required for its activity. Analysis of N- and C-terminal deletion mutants of TraR localized the quormone-binding domain to a region between residues 39 and 140 and the primary dimerization domain to a region between residues 119 and 156. The dominant-negative properties of these mutants predicted a second dimerization domain at the C terminus of the protein. Analysis of fusions of N-terminal fragments of TraR to cI' confirmed the dimerization activity of these two domains. Fifteen single amino acid substitution mutants of TraR defective in dimerization were isolated. According to the analysis of these mutants, Asp-70 and Gly-113 are essential for quormone binding, whereas Ala-38 and Ala-105 are important, but not essential. Additional residues located within the N-terminal half of TraR, including three located in -helix 9, contribute to dimerization, but are not required for ligand binding. These results and the recently reported crystal structure of TraR are consistent with and complement each other and together define some of the structural and functional relationships of this quorum-sensing activator.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Many bacteria use a cell-cell signaling system to control expression of specialized gene sets in response to their population size. Called quorum sensing, the bacteria measure their cell numbers by sensing the accumulation within the environment of a small diffusible signal molecule, the quormone, which they themselves produce (reviewed in Ref. 1). Gram-negative bacteria often use N-acylated homoserine lactones (acyl-HSL)¹ as their quormone, with signal specificity being determined by the length and degree of saturation of the acyl side chain as well as the nature of the chemical group at C-3. For example, Vibrio fischeri, which regulates the expression of the lux operon by quorum sensing (2), produces and responds to N-(3-oxohexanoyl)-L-homoserine lactone (3-oxo-C₆-HSL) (3), whereas Rhizobium leguminosarum regulates expression of a network of genes (4) in a quorum-dependent manner using N-(3-hydroxy-7-cis-tetradecanoyl)-L-homoserine lactone as the signal (5, 6).

In the acyl-HSL-mediated systems examined to date, expression of the target genes is controlled by a dedicated transcription factor, usually an activator (1). These proteins, of which LuxR is the prototype, require the quormone to activate transcription. Genetic evidence from studies of LuxR suggests that the activator binds the acyl-HSL ligand and, in doing so, converts from an inactive to an active form (7). LuxR (and by inference, all other members of the family) is believed to be composed of two functional regions, a C-terminal DNA recognition and interaction domain and an N-terminal quormone-binding domain (7).

Recent studies of TraR, the quorum-sensing transcriptional activator responsible for controlling conjugal transfer of the Agrobacterium tumefaciens Ti plasmids, are consistent with this model. TraR activates expression of the three operons of the Ti plasmid tra regulon from promoters that contain an 18-bp inverted repeat called the tra box (8). The acyl-HSL quormone N-(3-oxooctanoyl)-L-homoserine lactone (3-oxo-C₈-HSL) (9) is required for TraR to bind the tra box (10). Moreover, purified TraR binds the ligand tightly (11, 12), with such binding resulting in dimerization of the protein (11). No higher order multimers are formed at detectable levels (11), indicating that TraR is active only in the dimer form. These observations suggest a model in which, in the absence of the quormone, TraR fails to dimerize and cannot bind the tra box. In the presence of the signal, TraR binds the quormone, thereby forming stable homodimers. The dimer binds the tra boxes and, in interaction with RNA polymerase, activates transcription.

TraR is the only activator of the LuxR family that can be purified in its full-sized active form, making it an ideal candidate for molecular and biochemical analysis (12). Moreover, genetic assays and biochemical techniques are available to examine the DNA-binding, dimerization, and transcriptional activation properties of this regulator (10-12). Mutations in the C-terminal domain of TraR abolish DNA binding (10), consistent with the presence of a helix-turn-helix motif located within this region of the protein (13, 14). In addition, certain residues located at the far N terminus and in the middle of the protein are important for transcriptional activation, but not for DNA binding (10). Analysis of a series of stable N- and C-terminal deletion mutants identified a region of TraR located between residues 49 and 156 that is important for dimerization (11). These results suggest that, like LuxR, TraR is composed of an N-terminal dimerization domain and a C-terminal DNA-binding domain, a conclusion that is supported by the recent reports of the x-ray crystal structure of TraR bound to its DNA recognition element (15, 16). However, although the x-ray structure predicts regions and residues involved in ligand binding as well as in dimerization, the importance and contributions of these residues in the two processes have not been assessed by biochemical or genetic analyses.

In this study, we combine a genetic analysis with biochemical tests to identify regions of TraR required for ligand binding and dimerization. We also report the isolation of a series of substitution mutants of TraR defective in these properties and have used these mutants to identify residues of the activator that are critical for binding the quormone and for forming dimers. Our results indicate that certain conserved residues are essential for binding acyl-HSL; but, although ligand binding is required for the process, no single residue is essential for dimerization per se. In addition, although two residues were identified as being essential for ligand binding, substitutions at several positions that hinder dimerization alter, but do not abolish, the ligand-binding properties of the protein.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Bacterial Strains, Media, and Chemicals-- A. tumefaciens strain NTL4 (17) and Escherichia coli strains DH5 (18) and S17-1 (19) were used for all constructions and most experiments. E. coli strain JH372, which is lysogenized with 202 containing a P_R::lacZ reporter fusion (20), was used in the genetic assay for dimerization. E. coli strains were grown in Luria broth (Invitrogen) at 37 °C, and A. tumefaciens strains were grown at 28 °C on nutrient agar (Difco), in MG/L medium (21), and in AB minimal medium (22) supplemented with 0.2% mannitol (ABM medium) as sole carbon source. When required, antibiotics were added to the media at the concentrations described previously (23). X-Gal (Sigma) was included in the media at 40 µg/ml to monitor expression of lacZ reporter genes. IPTG (Sigma) was added at 100 µM to induce expression of traR when necessary. Synthetic preparations of 3-oxo-C₆-HSL, 3-oxo-C₈-HSL, and octanoylhomoserine lactone (C₈-HSL) were the generous gifts of Dr. David Lynn (Emory University) and Dr. Anatol Eberhard (Ithaca College) or were synthesized as previously described (24).

Plasmids-- Plasmid pRKLH4I41 is a derivative of pRK415 (25) that contains traR and a traG::lacZ reporter fusion (10). pZLQ, a broad-range host vector in which expression of inserts fused to a vector ATG can be regulated from the trc promoter by LacI^q, was constructed as follows. The trc promoter, as two reannealed 80-residue oligonucleotides,² and the rrnBT1T2 transcriptional termination signal from pBAD22 were independently cloned into pBBR1MCS-2 (26) to produce pZLEx1. In this vector, the trc promoter is followed by a polylinker that includes an NdeI site. The lacI^q gene from pKK38-I (a generous gift of Dr. David Nunn) was cloned into the unique XcaI site of pZLEx1 to generate pZLQ. Plasmid pZLQR contains the traR gene of pTiC58 under the control of the trc promoter of pZLQ (10).

DNA Manipulations-- Recombinant DNA techniques were performed as described by Sambrook et al. (18). Plasmid DNA was isolated using a miniprep kit from QIAGEN Inc. according to the manufacturer's instructions. Digestions with restriction endonucleases were carried out as described by the manufacturers of the enzymes. Plasmid DNA was introduced into E. coli by calcium chloride-mediated transformation (18) and into A. tumefaciens by electroporation (21) or by biparental matings from E. coli S17-1 (27).

Deletion Mutagenesis of traR-- The isolation and some properties of the N-terminal deletion mutants of traR used in this study were described previously (11). C-terminal deletion mutants were generated by digestion with exonuclease III (Promega, Madison, WI) as follows. A derivative of traR in which the initiator ATG is part of an NdeI site contained on a 1.3-kb XbaI-EcoRI fragment was cloned into pDSK519 (25) to generate pGPO2. A 53-bp double-stranded oligonucleotide that contains sets of translational terminators in each reading frame just 3' to recognition sites for BamHI and KpnI was synthesized.² This fragment was cloned immediately downstream of the traR termination codon of pGPO2, generating pGPO3. A preparation of pGPO3 digested with BamHI and KpnI was incubated with exonuclease III. At increasing incubation times, samples were removed; the reactions were terminated; and any overhangs at the sites of the deletions were filled in using Klenow fragment. The plasmid pools from each time point were religated and recovered by transformation into E. coli DH5. The extent of the deletion in the traR gene in a sampling of plasmids from each time point was assessed by agarose gel electrophoresis following digestion with NdeI and EcoRI, the latter which cuts just downstream of the translational termination sites introduced into pGPO3; and a set of plasmids with incremental deletions was retained. The precise size of the 3'-deletion in each plasmid was determined by nucleotide sequence analysis of the entire traR open reading frame. The deletion derivatives retained (see Fig. 1) were recloned as NdeI-EcoRI fragments into pZLQ to produce the deletion set pZLQRCX, where X is the number of 3'-codons removed.

Chemical Mutagenesis of Plasmid DNA-- A preparation of pZLQRC1, a C-terminal deletion mutant, isolated as described above, that lacks the terminal isoleucine residue, was treated with hydroxylamine as described previously (10) and introduced into NTL4(pRKLH4I41). Electroporatants were plated onto ABM medium supplemented with 3-oxo-C₈-HSL (2 nM) and X-gal. Under these conditions, TraR expressed from pRKLH4I41 fully activates the reporter, but coexpressed TraRC1 is strongly dominant-negative and completely inhibits this activation. Blue colonies, harboring mutants of traRC1 that have lost dominant negativity, were retained for further analysis. In each case, the mutant allele was recloned into a fresh vector and retested. In all cases, the mutant phenotype transferred with the recloning.

Reconstruction of the C Terminus of TraRC1 Substitution Mutants-- Plasmids coding for each of the substitution mutants were digested with Eco47III, a restriction enzyme that recognizes two sites, one located in the end of traR (nucleotide 641) and the other in the vector distal to the 3'-end of the gene. The 3'-end of wild-type traR was isolated by digesting pZLQR with Eco47III, and the recovered fragment was ligated with each of the Eco47III-digested mutants of pZLQRC1. Clones with the wild-type 3'-end inserted in the correct orientation were identified by restriction analysis, and the reconstituted genes were confirmed by nucleotide sequence analysis.

Acyl-HSL Retention-- Retention of acyl-HSLs by cells expressing traR and its mutant alleles was assessed as follows. Cultures of NTL4 cells harboring traR and its mutants cloned into pZLQ were grown in MG/L medium containing IPTG and the appropriate acyl-HSL to OD₆₀₀ ~ 1.2. The cells were collected by centrifugation and washed six times with equal culture volumes of ABM medium and three times with 1.5-ml volumes of Agrowash (50 mM Tris-HCl (pH 8.0) and 0.5 M NaCl) containing 1% Sarkosyl. This washing regime removes all detectable acyl-HSL from agrobacteria that do not express traR.³ The final cell pellets were resuspended in 200 µl of extraction buffer (0.2 M Tris-HCl (pH 8.0), 0.2 M Na₂EDTA, 10% glycerol, 0.2 mM dithiothreitol, 1% Tween 20, and 2 mg/ml lysozyme) and incubated on ice for 20 min. The cells were disrupted by sonication; unbroken cells and cell debris were removed by centrifugation; and the cleared lysates were extracted twice with equal volumes of ethyl acetate. The organic phases were pooled; water was removed by addition of a small amount of anhydrous magnesium sulfate; and the extracts were taken to dryness. Residues were redissolved in 50-µl volumes of ethyl acetate.

Acyl-HSL Detection-- Acyl-HSLs in culture supernatants or in cell extracts were chromatographed on C₁₈ reversed-phase thin-layer plates as previously described (24). Acyl-HSLs were visualized following chromatography by overlaying the plates with the bioindicator strains A. tumefaciens NTL4(pZLR4) (28) and Chromobacterium violaceum CV026blu (29).

Dimerization Assays-- Dimerization of TraR and its deletion derivatives was assessed genetically using the cI' assay system of Hu (30) as previously described (11). Fusions between cI' and the appropriate traR derivative were constructed in pJH391 (20) using PCR (11). In all cases, correct expression and stability of the chimeric fusion proteins of cI' and the TraR derivative were confirmed by Western analysis using antisera against TraR (10) and the cI head group (a generous gift of Jennifer Leeds, Harvard University). Cultures of E. coli JH372 cells (20) expressing the fusion constructs were grown in medium with and without 3-oxo-C₈-HSL.

The His-tagged co-affinity retention assay for dimerization was conducted as previously described (11). Each mutant of traR cloned into pZLQ was coexpressed with His₆-traR in NT1(pKKHTR9) grown in the presence of 3-oxo-C₈-HSL at concentrations of 2 and 10 nM depending on the experiment.

SDS-PAGE and Western Analysis-- Cell lysates were prepared and subjected to electrophoresis on SDS-polyacrylamide gels, and proteins were detected by Coomassie staining or by reaction with appropriate antibodies as previously described (11, 31).

Nucleotide Sequence Analysis-- Complete double-stranded nucleotide sequence was determined for all mutants and genetic constructs using automated sequencing and dye terminator chemistries at the University of Illinois Keck Center.

Quantitative Assays for Activation, Repression, and Dominant Negativity-- The activation properties of TraR and its mutants cloned into pZLQ were assessed using the reporter strain NTL4(pH4I41) as previously described (10). The ability of the mutant alleles to bind DNA in vivo was assessed using a repression assay with DH5(pPBL1). In this strain, wild-type TraR represses expression of a lacZ fusion encoded by pPBL1 (10). The dominant-negative properties of TraRC1 and its substitution mutants cloned into pZLQ were assessed using NTL4(pRKLH4I41). The plasmid in this strain expresses wild-type TraR from its native weak promoter and codes for a TraR-activable traG::lacZ fusion. In all cases strains, were cultured in ABM medium with or without 3-oxo-C₈-HSL.

Acyl-HSL Response Assays-- The response of strains harboring mutant alleles of traR to acyl-HSLs was assessed as follows. NTL4(pH4I41) cultures harboring pZLQ derivatives expressing wild-type traR or its mutant alleles were grown in ABM medium containing IPTG to OD₆₀₀ ~ 0.9. The cultures were split into a series of 2-ml subcultures, to each of which the acyl-HSL being tested was added at various concentrations. The subcultures were incubated for additional 4 h, and samples were assayed for -galactosidase activity.

-Galactosidase Assays-- Production of -galactosidase by A. tumefaciens strains was quantified using a modification of the Miller method (44) as previously described (23). Activity is expressed as Miller units (18) or units of enzyme/10⁹ colony-forming units. Samples were assayed in triplicate, and experiments were repeated at least twice. In the absence of error bars or values of variation, results from a single representative experiment are shown.

Structural Analyses-- The three-dimensional structure of TraR and its mutants was analyzed using PyMOL Version 10.2 on a Macintosh. Potentials for interactions and bond distances were assessed using the program O (Version 8.0) on a Silicon Graphics workstation. The coordinates for the structure of TraR from pTiR10 used in these analyses are available in the Protein Data Bank under code IL3L (15).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

TraR Contains Two Dimerization Domains-- We previously identified a region between residues 49 and 156 of the 234-residue TraR protein that is required for quormone-dependent dimerization (11). By assessing dominant negativity exerted against wild-type TraR using a series of stable C-terminal deletion mutants constructed as described under "Experimental Procedures," we re-examined this activator for dimerization properties. These mutants, lacking from 1 (233 residues remaining) to 127 (107 residues remaining) amino acids (Fig. 1), failed to activate transcription and did not bind the tra box as measured using a repression assay (10) (data not shown). Moreover, each mutant protein was stable as assessed by Western analysis (data not shown). Mutants with deletions of 1 and 14 C-terminal residues exerted strong dominant negativity, inhibiting the activity of coexpressed wild-type TraR almost completely (Fig. 1). However, mutants with deletions removing between 20 and 69 residues, while retaining dominant negativity, exerted significantly lower levels of inhibition (Fig. 1). Deletions removing from 78 to 115 residues strongly reduced, but did not abolish, the dominant-negative phenotype, whereas mutants with deletions removing more than 115 residues completely lost dominant negativity (Fig. 1). We conclude from these results that at least two regions of TraR, one from residues 214 to 220 and the second from residues 119 to 156, contribute to dimerization of the activator.

View larger version (17K): [in this window] [in a new window]   Fig. 1.   Dominant-negative properties of C-terminal TraR deletion mutants. C-terminal deletion mutants of traR, isolated as described under "Experimental Procedures," were introduced into NTL4 cells harboring pRKLH4I41, which codes for the wild-type activator and a lacZ fusion that reports transcriptional activation by TraR. Each strain was grown for 6 h in ABM medium containing IPTG to induce expression of the traR deletion derivative and 3-oxo-C8-HSL at 25 nM. Cells were harvested by centrifugation and assayed for -galactosidase activity as described under "Experimental Procedures." A, bars represent levels of enzyme activity in NTL4(pRKLH4I41) coexpressing a second copy of wild-type TraR (wt) or one of the deletion derivatives. B, shown are representations of the extents of the C-terminal deletions in the mutants tested.

The N-terminal Region of TraR Is Sufficient for Quormone-dependent Dimerization-- We tested a selected subset of N-terminal fragments of TraR for dimerization using the cI repression assay as described under "Experimental Procedures." In each case, the fragment was fused translationally to cI', the N-terminal DNA-binding domain of the cI repressor. As judged by Western analysis using anti-TraR and anti-cI' antibodies, all tested fusions were stable in cells grown in the presence or absence of the acyl-HSL quormone (data not shown). As we previously described (11), cI' fusions with wild-type TraR dimerized as well as control fusions of cI' with LeuZip-GCN4, the positive control (30), but only in cells grown with the acyl-HSL (Table I). Fusions of cI' to derivatives with deletions of between 14 and 69 C-terminal residues also dimerized; and again, dimerization was dependent on growth with the quormone (Table I). However, levels of repression were about half those observed for the cI'::TraR fusion. The fusion to TraRC78 retained a small amount of quormone-dependent dimerization activity, whereas the fusion to TraRC127 did not show significant activity even in cells grown with high levels of the signal (Table I).

View this table: [in this window] [in a new window]   Table I Dimerization of fusions of cI' to TraR and its C-terminal deletion mutants

We also tested fusions of cI' to a series of far C-terminal fragments of TraR ranging from the last 100 residues (positions 135-234) to the last 20 residues (positions 215-234). All such fusion proteins were stable as assessed by Western analysis using anti-cI' antibody (data not shown). None of these chimeric proteins repressed the lacZ reporter at detectable levels even in cells grown with 3-oxo-C₈-HSL (data not shown).

The Ligand-binding Region of TraR Spans a Large Segment of the N-terminal Domain-- Failure to dimerize could result from the inability to bind the signal. Alternatively, these deletion mutants might bind the signal, but lack residues required for subsequent dimerization. We assessed the ability of sets of N- and C-terminal deletion mutants to bind 3-oxo-C₈-HSL using a cellular quormone retention assay as described under "Experimental Procedures." Cells expressing wild-type TraR or TraR with N-terminal deletions up to 39 residues retained the signal (Fig. 2A). However, the quormone readily washed out of cells expressing mutants with deletions of 49 or more N-terminal residues (Fig. 2A) (data not shown). Similarly, cells expressing mutant forms of TraR with C-terminal deletions up to Asp-140 (TraRC94) retained as much quormone as cells expressing wild-type TraR (Fig. 2B). However, cells expressing mutants of TraR lacking 104 or more C-terminal residues (130-residue oligopeptide) did not retain detectable amounts of the acyl-HSL. Thus, the region of TraR between residues 39 and 140 is essential for signal binding (Fig. 3).

View larger version (78K): [in this window] [in a new window]   Fig. 2.   Acyl-HSL retention by cells expressing mutant alleles of traR. NTL4 cells harboring derivatives of pZLQ coding for N-terminal deletion mutants (A), C-terminal deletion mutants (B), and amino acid substitution mutants (C) of TraR were grown in MG/L medium supplemented with IPTG and 3-oxo-C8-HSL, harvested, and processed by washing. Cell-free lysates were prepared and extracted with ethyl acetate, and the organic extracts were examined for the acyl-HSL by thin layer chromatography as described under "Experimental Procedures." Lanes contain extracts from cells expressing the following. A, pZLQ empty vector (lane 1), traRN2-4 (lane 2), traRN2-9 (lane 3), traRN2-19 (lane 4), traRN2-29 (lane 5), traRN2-39 (lane 6), traRN2-49 (lane 7), and wild-type traR (lane 8). Lane 9 contains authentic 3-oxo-C8-HSL. B, pZLQ empty vector (lane 1), wild-type traR (lane 2), traRC14 (lane 3), traRC27 (lane 4), traRC38 (lane 5), traRC50 (lane 6), traRC69 (lane 7), traRC78 (lane 8), traRC94 (lane 9), traRC104 (lane 10), and traRC115 (lane 11). Lane 12 contains authentic 3-oxo-C8-HSL. C, wild-type traR (lane 2), traR-H54Y (lane 3), traR-S112F (lane 4), traR-S160F (lane 5), traR-D70N (lane 6), traR-A105V (lane 7), traR-E211K (lane 8), traR-G113S (lane 9), and empty vector (lane 10). Lane 1 contains a sample of authentic 3-oxo-C8-HSL.

View larger version (10K): [in this window] [in a new window]   Fig. 3.   Genetic and structural map of TraR. The 234-residue TraR protein is depicted in linear form as composed of N- and C-terminal segments joined by a flexible 12-residue linker (15, 16). The hatched region in the C terminus denotes the recognition helix of the DNA-binding domain. The two -helical motifs involved in dimerization as determined from x-ray crystallography (15, 16) are indicated by brackets below the map. The brackets above the map indicate the quormone-binding and dimerization regions as delimited by analysis of N- and C-terminal deletion mutants. The sites of single amino acid changes that affect function are shown, with those above the map influencing activity and dimerization properties, but not quormone binding, and those below the map affecting quormone binding and subsequent biological activities. Changes in the residues shown in boldface reduced quormone binding below detectable levels as measured by the retention assay (see Fig. 2).

Residues of TraR Required for Binding Quormone and for Dimerization-- Mutant TraRC1, which lacks the C-terminal isoleucine residue, did not activate transcription and exerted strong dominant negativity when coexpressed with the wild-type activator (Fig. 1) (data not shown). We infer from these properties that, like TraRC2 (10, 11), TraRC1 forms ligand-dependent stable but inactive heterodimers with wild-type TraR. We reasoned that stable mutants of TraRC1 unable to bind ligand or able to bind ligand but unable to dimerize should lose the dominant-negative character. We mutagenized a clone expressing traRC1 in vitro, introduced the treated DNA into NTL4(pRKLH4I41), and assessed electroporatants for loss of the dominant-negative phenotype as described under "Experimental Procedures." From 180 candidates, we identified 34 such mutants that produced normal levels of protein of the same size as TraRC1, as assessed by Western analysis (data not shown). Following sequence analysis, 15 mutants with unique substitutions were retained for further analysis (Table II). Several mutants, including A38V, L155F, and E211K, were isolated in more than one independent screen. Two Asp-70 mutants were isolated, one substituted with asparagine (D70N) and the other with glutamate (D70E). Most of the substitutions are located within the N-terminal region of TraR (Fig. 3 and Table II). However, two of the mutations, T190I and E211K, are located at the C-terminal end of the protein, with E211K mapping within the recognition helix of the helix-turn-helix domain (Fig. 3). When overexpressed in trans to wild-type TraR, four mutants, T51I, D70N, D70E, and G113S, exerted greatly reduced levels of inhibition of the activator (Table II). The remaining mutants retained significant levels of dominant negativity.

View this table: [in this window] [in a new window]   Table II Characteristics and properties of substitution mutants of TraR and TraRC1

We reconstructed the intact C terminus of each of the 15 mutants and tested these constructs for their ability to activate transcription in vivo. The four mutants exerting very weak dominant negativity failed to activate expression of the traG::lacZ reporter, even in cells grown with concentrations of 3-oxo-C₈-HSL well above that needed to saturate wild-type TraR (Table II) (data not shown). Ten of the 11 mutants that retained some dominant negativity activated the reporter to low levels in a signal-dependent manner (Table II). The T190I mutant did not significantly activate the reporter at the concentration of signal tested.

Asp-70 and Gly-113 Are Essential for Quormone Binding-- We assessed the capacity of each mutant to bind 3-oxo-C₈-HSL using our acyl-HSL retention assay. Cells expressing three of the mutants, D70E, D70N, and G113S, reproducibly failed to retain detectable levels of the quormone (Fig. 2C). The A105V mutant gave variable results; in some assays, the cells retained barely detectable amounts of ligand, whereas in others, the cells failed to retain the signal (Fig. 2C). A strain expressing the A38V mutant reproducibly retained a very small amount of signal (data not shown). The remaining 10 mutants retained amounts of signal indistinguishable from that retained by cells expressing wild-type TraR (Fig. 2C) (data not shown).

The Mutants Are Defective in Dimerization-- We examined each mutant for its capacity to dimerize using an assay in which heterodimers formed between the mutant and His-tagged wild-type forms of TraR can be recovered by nickel affinity chromatography of extracts from cells expressing both genes (11). In each case, the cells were grown with saturating amounts of 3-oxo-C₈-HSL, the cognate quormone. Western analysis using anti-TraR antibody indicated that, for all but two of the mutants, the cells produced approximately equal amounts of both forms of TraR (Fig. 4A). E88K and A38V were detectable, but were present at somewhat lower amounts compared with His₆-TraR. D70N, D70E, and G113S, the three mutants completely defective in signal binding, were not co-retained on the nickel column, suggesting that they failed to form detectable heterodimers with the His-tagged protein (Fig. 4B) (data not shown). A38V, which bound very small amounts of signal, and A105V, which gave variable results, displayed barely detectable levels of heterodimers (Fig. 4B) (data not shown). Each of the remaining nine mutants yielded detectable heterodimers, but the amounts of mutant proteins co-retained on the affinity columns were lower than that of coexpressed wild-type TraR in the control culture (Fig. 4B) (data not shown).

View larger version (103K): [in this window] [in a new window]   Fig. 4.   TraR mutants are defective in forming dimers. Wild-type TraR or its amino acid substitution mutants cloned into pZLQ were coexpressed with His6-TraR cloned into pKKHTR9 in A. tumefaciens NT1 cells grown in ABM medium with IPTG and 100 nM 3-oxo-C8-HSL. Cleared lysates were prepared; a sample of each was subjected to SDS-PAGE and Western analysis to gauge levels of expression of the two proteins; and the remainder were applied to a nickel affinity column. The protein samples eluted from the column were analyzed by SDS-PAGE and detected by Western blotting as described under "Experimental Procedures." A, Western analysis of His6-TraR (denoted by the arrow) and wild-type (WT) or mutant TraR proteins coexpressed in the same cells. B, Western analysis of TraR proteins recovered from nickel affinity columns. The first lane in each panel contains molecular mass markers (M).

Mutants Partially Defective in Dimerization Show Altered Responses to the Acyl-HSL Signals-- TraR responds with highest sensitivity to 3-oxo-C₈-HSL (9, 24, 32). The protein is activated by other acyl-HSLs, but activation requires much higher concentrations of the non-cognate signal (9, 10, 24, 28, 32). We examined a subset of the dimerization-defective C-terminally repaired substitution mutants of TraR that still bound the signal for their ability to activate a reporter in response to different concentrations of the cognate and two non-cognate acyl-HSL quormones. Wild-type TraR activated the traG::lacZ reporter to maximal levels when the assay strain was grown with 3-oxo-C₈-HSL at concentrations as low as 0.1 nM (Fig. 5). Under the conditions of the assay, the non-cognate but agonistic quormones 3-oxo-C₆-HSL and C₈-HSL (9, 24, 28, 32) yielded maximal activation at similar concentrations. We suspect that this similar sensitivity is due to the high level of expression of the traR gene in the reporter strain (32).

View larger version (28K): [in this window] [in a new window]   Fig. 5.   Response of amino acid substitution mutants of TraR to 3-oxo-C8-HSL and its acyl-HSL analogs. Subcultures of A. tumefaciens NTL4 cells harboring the TraR-dependent reporter plasmid pH4I41 and pZLQ derivatives coding for wild-type TraR or one of its C-terminally repaired substitution mutants were grown for 4 h in ABM medium containing IPTG and increasing concentrations of 3-oxo-C8-HSL (), 3-oxo-C6-HSL (), or C8-HSL (). Cells were harvested and assayed for -galactosidase activity as described under "Experimental Procedures." -Galactosidase activity is expressed as units/109 colony-forming units. Concentrations of the acyl-HSLs (AI) are in nM. The experiment was repeated twice for each mutant, and the results of a representative set of assays are presented.

Three of the mutants, A38V, H54Y, and L155F, responded to 3-oxo-C₈-HSL at concentrations similar to that of wild-type TraR (Fig. 5). However, these three mutants activated expression of the reporter to levels only one-fifth to one-half that attained by cells expressing wild-type TraR, even in cultures grown with saturating levels of signal. The H54Y and L155F mutants responded to 3-oxo-C₆-HSL and C₈-HSL at concentrations similar to that of wild-type TraR, whereas the A38V mutant was substantially less sensitive to the two non-cognate signals. When grown with 3-oxo-C₈-HSL, the A105V mutant activated the reporter to levels as high as those observed with wild-type TraR (Fig. 5). However, compared with the parent, full activation by this mutant required ~10-fold higher levels of 3-oxo-C₈-HSL and ~1000-fold higher levels of the two non-cognate signals. Compared with wild-type TraR and other mutants, the T51I mutant gave a pattern of reporter activation that spanned a much broader range of signal concentrations and required the cognate quormone at concentrations above 20 nM for its maximal activity (Fig. 5). Moreover, like the A38V and A105V mutants, this mutant was much less responsive to the non-cognate signals. The T190I mutant did not detectably activate the reporter at any concentration of signal tested (Fig. 5).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

We reported previously that purified active TraR is a homodimer and that formation of stable dimers depends upon binding the acyl-HSL quormone (11). Recently reported crystallographic analyses confirm this prediction and identify sets of residues, all in the N-terminal half of TraR, that contribute to ligand binding and to dimerization (15, 16). Our studies of dimerization-defective mutants of TraR generally support these assignments. However, we show that substitutions at residues predicted to be involved in quormone binding exert different effects, ranging from abolishing binding outright to modifying the nature of the binding, resulting in altered responses to cognate and non-cognate signals.

Of the 15 substitution mutants we isolated, alterations at only two residues, Asp-70 and Gly-113, completely abolished quormone binding. Not surprisingly, both mutants failed to dimerize and lacked detectable biological activity. The two amino acids are among five residues that are conserved in all acyl-HSL-dependent members of the LuxR family (1). Moreover, the corresponding residues of LuxR (Asp-79) and LasR (Asp-73) also are important for transcriptional activation (33, 34) and, in the case of LasR, for dimerization (34). That a mutation at Asp-70 affects binding is predictable; in the crystal structure, the side chain carboxyl group of this residue forms a hydrogen bond with the imino group of the quormone (Fig. 6A) (15, 16). Our analyses show that Asp-70 and its contributed hydrogen bond are essential for binding signal.

View larger version (88K): [in this window] [in a new window]   Fig. 6.   Locations within the three-dimensional structure of TraR of the residues in which substitution mutations were isolated. Each panel shows a dimer of TraR complexed with its ligand (arrows) and its DNA-binding site. Shown are locations of substitution mutations in regions of TraR involved in ligand binding (A), dimerization (B), DNA binding (C), and unknown functions (D).

The two replacements at Asp-70, D70N and D70E, provide an informative contrast. Whereas C-terminally repaired D70N was completely inactive, the D70E mutant was very weakly active in cells grown with high levels of quormone (Table II). Asparagine differs from the native Asp predominantly in charge, whereas the glutamate retains the acidic function, but the side chain is larger by one methylene group. We conclude that, although the bulkiness of the side group at position 70 is important, this residue must contain an acidic function, a prediction that is consistent with the structural studies (15, 16).

The G113S mutation exerted a profound effect on TraR activity; and clearly, the glycine at this position is essential for quormone binding and subsequent dimerization. The corresponding residues in LuxR (Gly-121) and LasR (Gly-113) also are required for biological activity (2, 33, 34) and, in LasR, for dimerization (34). The structural analysis did not predict a direct interaction between Gly-113, located in -sheet 4, and the acyl-HSL (Figs. 3 and 6A) (15, 16), but the glycine may position Trp-85, located in -sheet 3, which could interact with the quormone (Fig. 7A) (15). Zhang et al. (15) concluded that Trp-85 interacts with the acyl side chain, whereas Vannini et al. (16) predicted that this residue interacts with the lactone moiety. Our examination of the crystal structure is consistent with the latter interpretation (Fig. 7A). We suggest that the bulkier side group of the serine substitution in the G113S mutant perturbs the correct positioning of Trp-85 (Fig. 7B), thereby inhibiting quormone binding.

View larger version (40K): [in this window] [in a new window]   Fig. 7.   Specific interactions between residues of TraR and between residues of TraR and the acyl-HSL ligand. A, possible interactions of Trp-85 with the homoserine lactone moiety of 3-oxo-C8-HSL and with Gly-113. B, the serine substitution in the G113S mutant with its altered side group in white. C, possible main chain hydrogen bonding between Thr-190 and the amide of Glu-193 located in -helix 11, the scaffold helix of the helix-turn-helix motif. D, the isoleucine substitution at Thr-190. Groups with oxygen atoms are shown in red, and those with nitrogen atoms are shown in blue. Distances are in Angstroms.

A mutation at Ala-38 also affected signal retention. However, although the mutant was defective, unlike the Asp-70 and G-113 mutants, activity was restored by addition of excess quormone (Fig. 5). Ala-38 is located in the ligand-binding site (Fig. 6A) and, along with Thr-129, is predicted to contribute to a water bridge with the 3-oxo group of the quormone (15). The A38V mutant still bound a small amount of signal, suggesting that the valine substitution can participate, although poorly, in the coordination of the water molecule. Alternatively, the decreased binding may reflect the contribution of Thr-129 alone. Consistent with its limited signal binding, the A38V mutant formed heterodimers, albeit at low levels, with His-tagged TraR (Fig. 4B). Moreover, although functional, the C-terminally repaired mutant activated expression of the reporter to levels only half those of the wild-type activator (Fig. 5). Furthermore, the mutant was as sensitive as wild-type TraR to the cognate acyl-HSL, but was considerably less sensitive to the non-cognate signals (Fig. 5). We suggest that the bulkier valine substitution interferes with the water bridge, resulting in a decreased affinity for the quormone. However, this defect can be partially overcome by addition of excess ligand. Given the disproportionately decreased sensitivity to 3-oxo-C₆-HSL, we speculate that, in the absence of a water bridge, the van der Waals interactions with the acyl side chain take on added importance for signal binding. Ala-38 is not highly conserved among sequenced homologs of LuxR (1). Among those members that recognize acyl-HSLs with oxo or hydroxy substitutions at C-3 in which a water bridge might be important, the analogous positions can be occupied by alanine, serine, and the considerably bulkier residues threonine and leucine, but none contain valine (1).

Like the A38V mutant, cells expressing the A105V mutant retained only small amounts of quormone (Fig. 2C). Moreover, the two mutants formed very weak heterodimers with His₆-TraR (Fig. 4) and also exhibited reduced sensitivity to the non-cognate signals (Fig. 5). However, whereas A38V activated the reporter to only about half its maximal level, the A105V mutant induced the same reporter to wild-type levels. Ala-105 contributes to quormone binding through a van der Waals interaction with the homoserine lactone moiety (Fig. 6A) (16). Moreover, the residue is conserved in most members of the LuxR family (1), and an A105V mutation in LasR has similar effects on the activity of the Pseudomonas aeruginosa activator (34). We suggest that the bulkier valine substitution at this position affects the capacity of TraR to initially bind the quormone. However, once bound, the protein forms stable dimers, and the ligand is retained strongly. This conclusion is consistent with the wild-type levels of activity exhibited by the mutant when exposed to higher levels of quormone (Fig. 5). Moreover, when tested at high levels of signal, cells expressing the A105V mutant retained as much signal as wild-type TraR (data not shown).

Strains expressing the T51I mutant retained levels of ligand indistinguishable from those expressing wild-type TraR. Thr-51 is predicted to form a van der Waals contact with the distal region of the acyl chain of the quormone (Fig. 6A) (15). That strains expressing the T51I mutant retained ligand indicates that the isoleucine substitution has little effect on signal binding. However, the mutant lost most of the dominant negativity of the parent (Table II) and did not form strong heterodimers with His₆-TraR. Moreover, the C-terminally repaired T51I mutant was activable by excess quormone, but to levels only one-fourth that of wild-type TraR (Fig. 5). These observations suggest that the isoleucine substitution, although having little effect on signal binding, interferes strongly with the formation or stability of the dimers. It is conceivable that replacing the hydrophilic threonine residue with the bulkier hydrophobic isoleucine introduces a steric perturbation that inhibits dimerization.

Of the remaining nine dimerization-defective mutants, three, A149V, L155F, and S160F, map to -helix 9 (Ref. 15; -helix 6 in Ref. 16), the motif predicted to constitute the primary dimer interface of TraR (Figs. 3 and 6B) (15, 16). All three mutants bound quormone as well as wild-type TraR (Fig. 2) (data not shown). Moreover, all three retained some dominant negativity and, in their C-terminally repaired forms, activated the traG reporter, albeit to low levels, suggesting that they still form dimers (Table II). These properties are consistent with the analysis of the C-terminal deletion mutants; TraRC115, from which all of -helix 9 was removed, retained some dominant negativity (Fig. 1), suggesting that it can form weak heterodimers with wild-type TraR. The response of the L155F mutant to cognate and non-cognate quormones emphasizes the importance of the -helix 9 region; the mutant activated the reporter, but only to very low levels even at saturating amounts of signal (Fig. 5). Moreover, the mutant responded to the non-cognate quormones in a manner similar to that of the cognate acyl-HSL, consistent with our conclusion that the phenylalanine substitution alters dimerization of the protein, but not its signal-binding properties.

Four mutants, L25F, H54Y, E88K, and S112F, map to the N-terminal region of TraR (Fig. 3), but are not predicted to contribute to dimerization or signal binding (15, 16). All four mutant proteins bound quormone as well as wild-type TraR (Fig. 2) (data not shown), and all retained some biological activity (Table II). However, the mutants did not interact with His₆-TraR (Fig. 4B) (data not shown). Leu-25 and Glu-88 lie within -helices 2 and 6, respectively, and Ser-112 is located within -sheet 4 (Fig. 6D) (15). His-54 is located within a group of residues involved in quormone binding (15); but, in the three-dimensional structure, this amino acid is distant from the binding site (Fig. 6D). All four residues cluster in the same general region (Fig. 6D), and it is conceivable that steric effects induced by substitutions at these sites adversely alter the dimer properties of the protein.

The remaining two mutations are located C-terminal to the linker joining the N- and C-terminal domains (Fig. 3). E211K is altered in a residue located in the recognition helix of the DNA-binding domain (Fig. 6C). Zhang et al. (15) predicted a van der Waals interaction between Glu-211 and T-8. In agreement with this assignment, the mutation substantially inhibited the dominant-negative properties of TraRC1 (Table II). This defect is consistent with the observation that E211K formed weak heterodimers with His₆-TraR (data not shown). Moreover, the C-terminally repaired mutant showed only weak activator activity (Table II). In contrast to these observations, an alanine substitution at Asn-220, the corresponding residue of LuxR, has little to no effect on transcriptional activation by this protein (35, 36).

The T190I mutant is unique among our isolates. Strains expressing this mutant retained normal levels of quormone, and the protein only weakly dimerized with His₆-TraR. However, like the ligand binding-defective mutants, in its C-terminally reconstituted form, T190I failed to detectably activate the lacZ reporter even at high quormone concentrations (Fig. 5). Although Thr-190 is not predicted to participate in signal binding or dimerization (15, 16), Vannini et al. (16) proposed that this residue makes hydrophobic contact with the phosphate group of cytosine 11 of the tra box, a conclusion consistent with its location near the helix-turn-helix domain (Fig. 6C). However, if this interaction is correct, it is difficult to understand how a substitution at this residue results in such a drastic loss of activity. Our analysis of the crystal structure predicts that the side group hydroxyl of Thr-190 forms a main chain hydrogen bond with the amide of Glu-193, which is located in -helix 11, the scaffold component of the helix-turn-helix motif (Fig. 7C). An isoleucine substitution at position 190 would be expected to disrupt this interaction (Fig. 7D). Given the extraordinary effect of the T190I mutation on the activity of the protein, we favor the latter structure and suggest that Thr-190 acts to initiate -helix 11 and thereby is critical for maintaining the integrity of the DNA-binding domain. Thr-190 is conserved in all functional forms of TraR from Agrobacterium and also in 75% of the other members of the LuxR family (1), strongly suggesting that this residue is important for binding DNA, but not for recognition sequence specificity. However, an alanine substitution at Ser-199, the corresponding residue of LuxR, only marginally affects the activity of this activator (35), suggesting that corresponding residues in this region of TraR and LuxR play different roles in conferring structural or functional properties to these two activators.

Our analysis of N- and C-terminal deletion derivatives of TraR predicts that the region between residues 119 and 156 contributes to dimerization, whereas the region between residues 39 and 140 is responsible for quormone binding (Fig. 3). Significantly, substitution mutations affecting these properties map to residues located within these boundaries (Fig. 6, A and B). In this regard, TraR, LuxR, and LasR share similar properties; LuxR (11) and LasR (34) form stable homomultimers, probably dimers, but only in cells grown with their cognate acyl-HSL. Deletion analyses localized the dimerization and acyl-HSL interaction regions to the N-terminal portion of these activators (34, 37-39). Coupled with the crystal structure of TraR (15, 16), these results demonstrate the functional two-domain structure for this activator. Moreover, given the similarities in amino acid sequence and the congruence of the genetic data (1, 11, 35, 37, 38, 40-43), many of these structural features are likely extendable to other members of the LuxR family.

Although the N-terminal half of TraR contains the primary dimerization domain, the dominant-negative properties of a series of deletion mutants (Fig. 1) suggest that the C-terminal end of each protomer exhibits dimerization properties. This conclusion is consistent with the crystal-based prediction that the regions encompassing -helix 13 (Ref. 15; -helix 10 of Ref. 16; see Fig. 3) at the far C terminus of each protomer interact with each other. This domain must confer only weak dimerization activity; we failed to isolate any mutants with substitutions in this region. Moreover, none of the cI'::C-terminal fusion constructs tested repressed the reporter. However, because cI' fusions with deletion derivatives lacking this C-terminal region were affected in their interaction properties (Table I), it is likely that this region of the protein is dimerized even in the absence of DNA, a conclusion consistent with that of Vannini et al. (16). We conclude that the N-terminal region is responsible for forming and maintaining the overall dimer structure of the activator, whereas the far C terminus stabilizes through weak dimerization interactions, a key structural feature associated with the DNA-binding domain of the protein. This conclusion is consistent with our observations that the intact C terminus of TraR is critical for DNA binding; mutants with deletions of as few as two C-terminal residues do not bind DNA as measured by our repressor assay (10). Given the molecular structure, the extensive collection of mutants, and the availability of in vivo and biochemical assays for the activity of TraR, studies of this quorum-sensing activator continue to expand our understanding of how this group of transcription factors recognizes its signal, binds its DNA recognition element, and activates expression of target genes.

	ACKNOWLEDGEMENT

We acknowledge Dr. Raven Huang (Department of Biochemistry, University of Illinois at Urbana-Champaign) for expertise and patience in introducing us to the mysteries of protein structure.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grant R01 GM52465 (to S. K. F.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^§ Present address: Dept. of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, MA 02148.

To whom correspondence should be addressed: Dept. of Crop Sciences, University of Illinois at Urbana-Champaign, 240 ERML, 1201 West Gregory Dr., Urbana, IL 61801. Tel.: 217-333-1524; Fax: 217-244-7830; E-mail: stephenf@uiuc.edu.

Published, JBC Papers in Press, February 4, 2003, DOI 10.1074/jbc.M210035200

² Sequence available on request.

³ Z.-Q. Luo, S. Su, and S. K. Farrand, submitted for publication.

	ABBREVIATIONS

The abbreviations used are: acyl-HSL, N-acylated homoserine lactone; 3-oxo-C₆-HSL, N-(3-oxohexanoyl)-L-homoserine lactone; 3-oxo-C₈-HSL, N-(3-oxooctanoyl)-L-homoserine lactone; C₈-HSL, N-octanoylhomoserine lactone; X-gal, 5-bromo-4-chloro-3-indolyl--D-galactopyranoside; IPTG, isopropyl--D-thiogalactopyranoside.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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