(Received for publication, October 25, 1995; and in revised form, January 16, 1996)
From the
In the presence of m-xylene, the protein XylR encoded
by the TOL plasmid of Pseudomonas putida, activates the
-dependent promoter Pu. Early activation
stages involve the release of the intramolecular repression caused by
the signal reception N-terminal (A domain) of XylR on the central
module of the protein. A genetic approach has been followed to locate
the specific segment within A domain of XylR that is directly
responsible for its down-regulation in the absence of inducer, as
compared to that involved in effector (m-xylene) binding. For
this, a reporter Escherichia coli strain carrying a monocopy
transcriptional fusion of Pu to lacZ was transformed
with a collection of plasmids encoding equivalent truncated varieties
of XylR, consisting of nested and internal deletions throughout the
entire A domain. Examination of the resulting phenotypes allowed the
assignment of the A domain region near the central activation domain,
as the portion of the protein responsible for the specific repression
of XylR activity in the absence of m-xylene.
Strains of the genus Pseudomonas harboring the TOL
plasmid pWW0 can grow on toluene, m-xylene, and p-xylene as the only carbon source owing to the activity of a
complex catabolic pathway (summarized in Fig. 1) that proceeds
in two major biochemical steps (Nakazawa et al., 1990). These
are determined by two independent operons that become coordinately
transcribed when bacteria face pathway substrates such as m-xylene (see Marqués and Ramos(1993)
for a review). The main regulator of the system is the so-called XylR
protein, a member of the family of prokaryotic enhancer-binding
regulators that act in concert with the alternative
Figure 1:
The TOL system of plasmid
pWW0 and domain organization of XylR. The TOL system for degradation of
toluene and m-/p-xylene includes two gene clusters,
the upper-operon and the meta-operon, as well as two
regulatory genes, xylS and xylR, downstream of the meta-operon. The
Figure 2:
Effect of sequential N-terminal deletions
through the A domain in the transcriptional activity of XylR. The
activity of the N-terminal truncated derivatives of the A module of
XylR (211 amino acids, Fig. 1) indicated in the figure was
monitored as accumulation of
Figure 3:
Phenotypes endowed by internal truncations
of the A domain of XylR. The ability of xylR alleles carrying
the internal deletions within the A domain indicated in the figure was
examined as for the sequential N-terminal deletions (Fig. 2).
The strategy to generate the internally truncated proteins involved the
production by PCR of two restriction fragments that were sequentially
assembled in vector pPr (see legend to Fig. 2). The
``left'' restriction fragments (flanked by EcoRI-XbaI sites) were produced by amplifying the
desired part of the A domain sequence with a direct EcoRI
primer and a reverse XbaI primer (Fig. 1). The
``right'' restriction fragments were similarly produced with
a direct XbaI primer, targeted to the site of interest within
the sequence of the A domain, and a reverse BamHI primer at
the end of the xylR sequence (Fig. 1). Their assembly
downstream of the Pr promoter in pPr gave rise to the
truncated proteins under examination. Accumulation of
To explore the presence and the location of specific portions
within the A module of XylR that are accountable for the central domain
repression, we chose a reporter system in which the effect of changes
in the regulator could be related immediately to a distinct phenotype
in activation of Pu. Since all transcriptional control
elements can be faithfully reproduced in Escherichia coli,
this reporter system employs a derivative of E. coli YMC10
that had been lysogenized with an specialized To have a preliminary indication on the portion of the A
domain of XylR involved in intramolecular repression, we sought to
divide the A domain (211 amino acids, Inouye et al.(1988) and
Shingler et al.(1993); see Fig. 2) in 6 large segments,
that were progressively deleted from the N terminus (Fig. 2).
These deletions were generated by amplifying with PCR the sequences of
interest with an adequate collection of primers, so that the resulting
products were flanked by EcoRI and BamHI sites as
specified in the legend to Fig. 1. Transfer of the resulting DNA
fragments to the specialized expression vector pPr
(Pérez-Martín and de
Lorenzo(1995); see legend to Fig. 2) provided a translation
initiation sequence and a leading ATG for expression of the six xylR deletion alleles shown in Fig. 2. These were
named, respectively, The results above confirmed the existence of a
specific region within the A domain of XylR directly involved in
intramolecular repression located between amino acid positions 150 and
210. To narrow down the location of the boundary, we made two
additional deletions in 10-amino acid increments called XylR To verify the location of the repressor segment of
the A domain suggested by the phenotypes caused by the N-terminal
deletions, we constructed additional xylR alleles bearing
sequential truncations of the A domain starting in position 210 (i.e. at the C-terminal end of the module) and spanning
increasingly larger segments toward the N-terminal end (see legend of Fig. 3for the procedure employed for their construction). As
before, production in vivo of the predicted mutant proteins
was verified through autorepression assays and Western blot of the
corresponding cell extracts (not shown). The phenotypes endowed by each
of the constructions listed in Fig. 3were examined as before
with the results summarized in Fig. 3. As expected, deletions
spanning large portions of the A domain ( The semiconstitutive phenotype of deletions Taken together, these results led to the
conclusion that a protein segment as short at 40-50 amino acids
fully accounts for the repression caused by the whole A domain on XylR
in the absence of m-xylene. Our data are consistent also with
the notion that only that portion of the A module maintains specific
interactions with the central domain of the protein. In addition, the
phenotypes originated by the internally truncated proteins (Fig. 3) suggest that the XylR portion involved in effector
recognition may exist as a separate subdomain within the N-terminal
module. Prediction of secondary structure within the region carried out
with the PHD profile network method (Burkhard and Sander, 1993)
indicated that amino acids 219 to 221 could be organized as a somewhat
long (21 residues) The A domain of XylR
is the key component of this activator that endows specificity in the
response to m-xylene (Delgado and Ramos, 1994; Shingler and
Moore, 1994), so that direct effector binding is translated into
release of intramolecular repression (Delgado et al., 1995;
Pérez-Martín and de
Lorenzo, 1995). Although the precise mechanism by which this happens
remains unsolved, the data presented in this work suggest that
different portions of the A domain have specific roles in the process.
It is possible that, similarly to what may happen to NtrC (Fiedler and
Weiss, 1995), derepression could involve the dimerization of the
N-terminal module in response to the reception of the activating
signal. The different predictions raised by this hypothesis and other
alternatives are currently under study in our laboratory. factor
(Morett and Segovia, 1993; North et al.,
1993). In the presence of xylenes, XylR bound to upstream sequences
activates the Pu promoter of the upper-TOL operon,
thus triggering expression of the corresponding catabolic genes (de
Lorenzo et al., 1991; Abril et al., 1991). The very
early chain of events that translates the presence of xylenes into
activation of Pu, involve effector-mediated conversion of XylR
into a transcriptionally competent form. For this, the inducer binds
directly to the N-terminal, signal reception module of XylR termed the
A domain (Delgado and Ramos, 1994; Fernández et al., 1995; see Fig. 1) and triggers the release of
the repression caused by this domain on the central portion of the
protein that is involved in the contact and activation of the
-containing RNA polymerase
(Pérez-Martín and de
Lorenzo, 1995). This notion is based on the observation that deleting
the entire A domain of XylR gives rise to a truncated protein that
activates constitutively Pu in the absence of aromatic
effectors, both in vivo (Fernández et al., 1995) and in vitro (Pérez-Martín
and de Lorenzo, 1996). It seems, therefore, that the A domain of XylR
has at least two functions: (a) recognition of the aromatic
inducers and (b) intramolecular repression. Since these two
are obviously connected, the question arises as whether discrete
subdomains within the N-terminal module can be assigned to each of
them.
-dependent promoters Pu and Ps (underlined) are activated by the
cognate activator XylR in the presence of m-xylene, while the Pm promoter is activated by XylS in the presence of benzoate
or toluates. Functional domains of XylR are shown expanded, with an
indication of the amino acid positions corresponding to the boundaries
of each domain (Inouye et al., 1988; Shingler, 1996). These
include a signal reception N-terminal module (A domain), the central
(C) activation domain, and the C-terminal segment (D domain) containing
a helix-turn-helix (HTH) motif for DNA binding. The lower part
of the figure sketches the strategy used to amplify with the polymerase
chain reaction (PCR) specific DNA segments corresponding to different
portions of the XylR protein for expression of truncated variants of
the A domain. These include N-terminal deleted or internally truncated
proteins (see legends to Fig. 2and Fig. 3).
-galactosidase in the E. coli strain
RSPu, that carried a chromosomal Pu-lacZ fusion. For construction and expression of each of the truncated
proteins, the following strategy was pursued. A DNA fragment containing
the wild-type xylR sequence was subjected to a PCR reaction
using as direct primers (Fig. 1) various oligonucleotides
(33-mers) that attached an EcoRI site to the left of the site
of deletion desired. For the reverse priming of the reaction, the same
oligonucleotide was used in all cases, that generated a BamHI
site following the STOP codon of the xylR sequence, TAG. The
amplified products were cloned as EcoRI-BamHI
fragments of different sizes at the same sites of vector pPr. This is a
derivative of pCG1 (Myers et al., 1987) in which the native Pr promoter of xylR within the TOL plasmid (Fig. 1) has been engineered in front of the same polylinker as
pTrc99A (Amman et al., 1988), that is led by an NcoI
site overlapping a first structural ATG. This allowed all truncated
proteins to be expressed through the very same native promoter and
translation initiation regions as the wild-type xylR. The use
of the direct EcoRI primers in the PCR reaction introduced in
all cases amino acid residues EF (corresponding to 5`-GAA TTC-3`), next
to the leading methionine of the truncated products. Replacement of the
second (Ser) and third (Leu) amino acid residue of the wild-type XylR
protein by EF had no effect on protein activity (not shown). For the
experiment of the figure, each of the E. coli
RSPu transformants were grown in LB medium (Miller, 1972) at 30 °C
up to an A
= 0.5, after which they were
exposed, as indicated, to saturating vapors of m-xylene.
Accumulation of
-galactosidase (Miller, 1972) was then measured
after 5 h of induction. The figures for the reporter product are
indicated with respect to the site of the deletions corresponding to
each truncated regulator. The values shown are the average of 3
independent experiments carried out with duplicate samples. The
approximate location of the repression subdomain suggested by the
results is indicated on top.
-galactosidase by each of the reporter strains, transformed with
the corresponding plasmids, was examined as described in the legend to Fig. 2. The hatched portion of the A module shows the
position of the repression subdomain.
phage containing a
transcriptional Pu-lacZ fusion
(Pérez-Martín and de
Lorenzo, 1995). This strain (E. coli
RSPu) was
transformed independently with each one of a collection of plasmids
bearing truncated xylR alleles that differed only in A domain
sequences (Fig. 1). These were generated with a polymerase chain
reaction-based strategy (PCR) (
)described in the legends of Fig. 2and Fig. 3. The activity of the truncated products
expressed in trans was measured as the accumulation in
vivo of
-galactosidase in the presence or absence of the XylR
effector m-xylene. Simultaneously to each activity assay, we
examined the level of expression of each XylR-derived protein through
Western blot assays (Fernández et al.,
1995) to ensure that the proteins were produced at similar levels (not
shown).
30,
120,
150,
180,
210,
and
226. The corresponding proteins were produced in vivo at levels comparable to those of the wild-type XylR with the same
expression system, as assessed by Western blot, and they were equally
able to shut down expression of the Pr promoter of the xylR gene in an in vivo autorepression assay
(Fernández et al. (1995); not shown).
These experiments verified that the proteins were expressed at similar
levels and that they were efficiently able to bind DNA. Intermediate
deletions between positions 30 and 120 neither produced any detectable
protein nor gave positive in an autorepression assay, and, thus, they
were not further considered (not shown). The six productive deletions
displayed very distinct phenotypes when the corresponding plasmids were
transformed in the Pu-lacZ E. coli reporter strain (Fig. 2). While truncation of the 30 leading amino acids
(XylR
30) gave rise to a protein with only a residual
responsiveness to the XylR inducer, m-xylene (Fig. 2),
subsequent removal of the protein segment up to position 120
(XylR
120) and beyond (XylR
150) gave rise to proteins unable
to activate the Pu promoter to any significant extent.
However, deletion of 30 additional amino acids (XylR
180) partially
restored the ability of the protein to promote transcription (Fig. 2), although responsiveness to m-xylene was lost
and the activity became constitutive. Deletion of 30 additional amino
acids (XylR
210), which removed the A domain entirely, increased
-galactosidase accumulation to its highest level. Further
deletions entering the Q-linker (XylR
226) did not result in an
additional increase of promoter activity, while deletions beyond
position 233, entering into the central C domain (Fig. 1),
abolished any transcriptional activity of the resulting truncated
proteins (not shown).
160
and XylR
170. As shown in Fig. 2, while
160 remained
inactive,
170 gave a substantial, but not full, constitutive
activation of Pu, as compared to the maximum promoter
activation caused by the deletion of the entire A domain (XylR
210,
XylR
226). These results located the leftward limit of the
repressor subdomain at around amino acid position 170, and, therefore,
the whole functional module may span no more than 40 residues (or 60,
if we also include some amino acids present at the hinge B domain, Fig. 1).
60/210,
120/210, and
150/210) originated truncated proteins that were fully
constitutive. It was most remarkable, however, that deletions
160/210,
180/210, and
190/210 (that are totally or
partially deleted of the repressor subdomain), although capable of
activating transcription in the absence of inducer, maintained a
significant degree of responsiveness to m-xylene. Such
responsiveness was lost when the leading N terminus was deleted also
(truncation
30-
190/210). This suggested that such a leading
region is involved in effector recognition, lifting of intramolecular
repression, or both. Since deletions
160/210,
180/210, and
190/210 are predicted to offset considerably the relative
positioning of the remaining A sequence with respect to the central
domain of the protein, the regulation by m-xylene retained by
these proteins indicated that effector binding and associated changes
in protein structure do not involve per se specific
interdomain interactions. In addition, simultaneous deletion of the
segment 190-210 and the leading 30 amino acids gave rise to a
constitutive low activity regulator (Fig. 3), that may reflect
the need of an intact N terminus for any responsiveness to the
effector. Specific repression seems, therefore, to be due exclusively
to the portion of the A domain encompassing positions 160 to 226.
Furthermore, the fact that the internal deletion
160/185 (that
lacks the leftmost boundary of the repressor subdomain with the rest of
the protein) is virtually inactive (Fig. 3) suggested that the
protein segments determining the response to effector recognition may
be connected to each other close to positions 160-170. In fact,
deletion of the zone around 170 seems to originate a protein that is
inhibited unspecifically by the remainder of the A domain (see below).
Interestingly, Delgado and Ramos(1994) found that a mutation in residue
172 made the protein to respond to a new effector (m-nitrotoluene), thus indicating that some direct or indirect
determinants of ligand specificity may lie also around position 170.
160/210,
180/210, and
190/210 (Fig. 3) indicated also that
elimination of the repressor subdomain still affords a degree of
down-regulation of XylR by the remainder of the A module. This
phenomenon can be easily understood in light of the observations
reported elsewhere (Fernández et al.,
1995) on the inactivation of XylR through substitution of its A domain
by a bulky heterologous protein module. On this basis, it is very
likely that although deletions
160/210,
180/210, and
190/210 have lost the specific region involved in intramolecular
repression, they still retain a portion of the protein that inhibits
its full activity in the absence of inducer by a mere physical
hindrance of an activation surface and not because of an specific
interdomain interaction. The same argument explains the phenotype of
the deletion
160/185 (Fig. 3), that is virtually inactive in vivo in spite of having lost a protein segment that enters
a region involved in specific intramolecular repression (see above). In
this case, the lower activity of
160/185 (Fig. 3) as
compared to
180 (Fig. 2) is the likely result of the steric
hindrance caused by the remainder of the A domain present in the
truncated protein.
-helix. Interestingly, when compared to the
library of protein crystal coordinates of the Brookhaven data bank,
part of that
-helix and a few preceding residues (portion
204-222 of the A domain) bear significant resemblance to the
region of the eukaryotic protein c-Fos (residues 168 to 186) that is
involved in protein-protein interactions within a c-Fos/c-Jun/DNA
co-crystal (Brookhaven ID code: 1FOS). This structural resemblance
might be related to the specific interactions between the A domain and
the central domain of XylR proposed to control the activity of the
regulator
(Pérez-Martín and de
Lorenzo, 1995). On the contrary, no portion of the 160-220 region
of XylR showed any structural similarity to the N-terminal domain of
DctD, another member of the family of
-dependent
regulators whose activity is regulated through intramolecular
repression as well (Gu et al., 1994).