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
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ABSTRACT |
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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 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.
Bacterial Strains, Media, and Chemicals--
A.
tumefaciens strain NTL4 (17) and Escherichia coli
strains DH5 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
LacIq, 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 lacIq 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 Chemical Mutagenesis of Plasmid DNA--
A preparation of
pZLQR Reconstruction of the C Terminus of TraR 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
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 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
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).
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.
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
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 (TraR Residues of TraR Required for Binding Quormone and for
Dimerization--
Mutant TraR
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).
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).
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).
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.
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
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(18) and S17-1 (19) were used for all constructions and
most experiments. E. coli strain JH372, which is lysogenized with
202 containing a PR::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).
. 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 pZLQR
CX, where X is the number of
3'-codons removed.
C1, 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 TraR
C1 is strongly
dominant-negative and completely inhibits this activation. Blue
colonies, harboring mutants of traR
C1 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.
C1 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 pZLQR
C1. 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.
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.
(pPBL1). In this strain, wild-type TraR
represses expression of a lacZ fusion encoded by
pPBL1 (10). The dominant-negative properties of TraR
C1 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.
-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.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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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.
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
TraR
C78 retained a small amount of quormone-dependent
dimerization activity, whereas the fusion to TraR
C127 did not show
significant activity even in cells grown with high levels of the signal
(Table I).
Dimerization of fusions of cI' to TraR and its C-terminal deletion
mutants
C94) 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).
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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),
traR N2-4 (lane 2), traR
N2-9
(lane 3), traR
N2-19 (lane 4),
traR
N2-29 (lane 5), traR
N2-39
(lane 6), traR
N2-49 (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), traR
C14 (lane 3),
traR
C27 (lane 4), traR
C38
(lane 5), traR
C50 (lane 6),
traR
C69 (lane 7), traR
C78
(lane 8), traR
C94 (lane 9),
traR
C104 (lane 10), and traR
C115
(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.
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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).
C1, 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 TraR
C2 (10, 11), TraR
C1 forms ligand-dependent stable
but inactive heterodimers with wild-type TraR. We reasoned that stable mutants of TraR
C1 unable to bind ligand or able to bind ligand but
unable to dimerize should lose the dominant-negative character. We
mutagenized a clone expressing traR
C1 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 TraR
C1, 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.
Characteristics and properties of substitution mutants of TraR and
TraRC1
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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).
View larger version (28K):
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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.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (88K):
[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.
|
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; TraR
C115, 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.
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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.
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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.
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