(Received for publication, October 26, 1994; and in revised form, December 22, 1994)
From the
The XylR protein positively controls expression from the Pseudomonas putida TOL plasmid -dependent
``upper'' pathway operon promoter (Pu) and the xylS
gene promoter (Ps), in response to the presence of aromatic effectors.
Two mutant XylR regulators able to stimulate transcription from Pu and
Ps in the absence of effectors were isolated. These mutants exhibited
single point mutations, namely Asp
Asn and
Pro
Ser. Both mutations are located in the amino
termini domain of XylR, which is thought to be responsible for
interactions with effectors. The effector profile of XylRP85S was
similar to that of wild-type XylR protein; however, XylRD135N exhibited
an altered pattern of effector recognition: with m-nitrotoluene it stimulated transcription from the Pu
promoter above the high basal level, whereas this nitroarene inhibited
the wild-type regulator. Previous work (Delgado, A., and Ramos, J.
L.(1994) J. Biol. Chem. 269, 8059-8062) showed that residue 172
was involved in effector interactions, as mutant XylRE172K also
recognized m-nitrotoluene. However, double mutant
XylR135N/E172K did not stimulate transcription in the absence of
effector, but retained the ability to stimulate transcription with m-nitrotoluene. Transcription mediated by XylRD135N and
XylRP85S from Pu::lacZ was analyzed in detail. Like the
wild-type regulator, XylRD135N and XylRP85S required
for full transcription activation, but in contrast with the
wild-type regulator, XylRD135N, but not XylRP85S, stimulated
transcription from Pu in the absence of the integration host factor
protein. XylRD135N, also in contrast with XylR and XylRP85S, mediated
transcription from a mutant Pu promoter that lacked one of the upstream
regulator binding sites (
UAS1), but not when both upstream
regulator binding sites were deleted. The level of autoregulation of
XylRD135N was at least 2-fold higher than that found with the wild-type
XylR regulator and the mutant XylRP85S.
Pseudomonas putida harboring the TOL catabolic plasmid grows on toluene and related hydrocarbons, and is able to oxidize these compounds to Krebs cycle intermediates via the ``upper'' and the meta-cleavage pathways(1) . The XylR protein needs to be activated by effectors to stimulate transcription from the upper pathway operon promoter (Pu), and from the xylS gene promoter (Ps). Increased xylS mRNA levels lead to overproduction of the XylS regulator, which stimulate transcription from Pm, the meta-pathway operon promoter(2, 3, 4, 5) .
The XylR
protein is 566 amino acid residues long, and belongs to the NtrC/NifA
family of regulators(5) . These regulators exhibit four
domains, three of which are highly conserved among members of the
family (Fig. 1). The carboxyl-terminal domain D contains an
-helix-turn-
-helix DNA-binding motif. The central domain C,
the best conserved region in the family, seems to be involved in
interactions with RNA-polymerase and ATP hydrolysis to allow the
formation of open transcriptional complexes(6) . The B domain
(Q linker) is a short hydrophilic region whose role is probably to
serve as a linker between the C and A domains. The nonhomologous
NH
-terminal domain A has been implicated in signal
reception, either via a sensory protein as in the NtrB/NtrC pair, or
via interaction with a chemical signal as in DmpR (7, 8) and XylR (9, 10, 11) . This has been genetically
confirmed with the latter two regulators, in which mutations in the
NH
-terminal region altered effector
recognition(11) . (
)
Figure 1:
Domains of the XylR regulator and
location of point mutations. The organization of the XylR domains is
according to Inouye et al.(2) . The mutations located
at the NH-terminal of this regulator are
shown.
The two promoters regulated
by XylR belong to the -12/-24 class, and are recognized by
the RpoN factor (also called
and
NtrA)(4, 13, 14, 15) . In the Pu and
Ps promoters XylR binds to UASs (
)located between -120
and -180. Fine deletions in Pu (10, 16) and in
Ps(17, 19) , and in vivo(10) and in vitro footprinting experiments in the Pu promoter
region(20) , revealed that XylR recognizes a 5` motif, which
appears twice, in inverted orientation. These upstream activator
sequences were identified around -160 (UAS1) and around
-130 (UAS2). Deletion of one or both of these motifs abolished
transcription activation by wild-type XylR
regulator(10, 16, 18) . The
-40/-70 region of both Pu and Ps is rich in As and Ts, and
shows good homology to the consensus IHF-binding motif(19) . In
an IHF
-deficient Escherichia coli background, Abril et al.(10) and de Lorenzo et al.(20) showed that stimulation of
effector-activated XylR-dependent transcription from Pu was only about
10-25% of that obtained in an IHF
background. In
contrast, expression from the Ps promoter in an IHF
background was virtually unchanged with respect to the activity
level in an IHF
background (19) .
The
mechanism of activation by the /E complex requires
that the regulator make contact with the
/E complex
bound at the promoter site(21) . It has been proposed that the
intervening DNA sequences loop out to allow
interactions(21, 22, 23) . The role of IHF in
the Pu promoter is probably to assist in loop formation; it may also
assist in the formation and stabilization of the complex (24) .
In this study we show that mutant XylR regulators exhibiting single
point mutations (D135N and P85
S) stimulate transcription
from the Pu and Ps promoters in the absence of effector. The XylD135N
mutant also exhibited an altered pattern of effector recognition.
Transcription activation from Pu and Ps by these mutant regulators is
dependent on
, but expression from Pu with XylD135N
became independent of IHF protein. Another finding of interest was that
deletion of UAS1 still allowed moderate (i.e. about 50% of
maximal) induction from Pu with XylRD135N, whereas induction was
markedly diminished with the wild-type XylR and XylRP85S.
Plasmids used were pTS174
(Cm, xylR, P15 replicon)(2) ;
pAD1(Cm
, xylR7, a mutant allele encoding
XylRE172K, P15 replicon)(11) ; pAD6 (Cm
, xylR6, a mutant allele encoding XylRP85S, P15 replicon) (this
study); pAD49 (Cm
, xylR49, a mutant allele
encoding XylRD135N, P15 replicon) (this study); pRD579 (Ap
,
Pu::lacZ, pR1 replicon)(13) ; pAH100 (Ap
,
Ps::lacZ, pR1 replicon) (19) ; pAH120 (Ap
,
Pr::lacZ, pR1 replicon)(19) ; pTZ19 (Ap
);
pERD401 (Ap
, Pu::lacZ, pBR replicon)(10) ;
pERD411 (Ap
,
UAS1
UAS2Pu::lacZ, pBR
replicon)(10) ; pERD412 (Ap
,
UAS1Pu::lacZ, pBR replicon(10) ; pERD415
(Ap
,
2 bp at
-64Pu::lacZ)(10) ; pERD416 (
8 bp at
-64Pu::lacZ)(10) ; pERD419 (Ap
,
6 bp at -144Pu::lacZ) (10) and pERD420
(Ap
,
10 bp at
-144Pu::lacZ)(10) . Bacteria were grown at 30
°C in LB broth supplemented, when required, with 100 µg/ml
ampicillin and 30 µg/ml chloramphenicol.
The double mutant XylRE172K/D135N was constructed in vitro by replacing the 300-bp internal NheI site fragment of pAD1 with the corresponding one in pAD49, so that the resulting mutant allele bore two point mutations.
In the presence of m-methylbenzyl alcohol and o-nitrotoluene the wild-type XylR regulator stimulated transcription from Pu and Ps between 5- and 10-fold (Table 1), while with m-nitrotoluene the basal level not only did not increase, but dropped to one-half. The high basal transcription level mediated by the XylRD135N regulator from the Pu promoter increased 2-fold in response to the addition of the three aromatics noted above. These results suggest that XylRD135N is not only able to stimulate transcription in the absence of effectors, but is also able to recognize them and produce an additional transcription stimulus. However, when similar assays were done with XylRD135N and the Ps::lacZ fusion, although the basal level of expression in the absence of effectors was high, this level did not increase in response to the presence of the effectors (Table 1).
Since the mutant XylRE172K had been described before (11) as able to activate transcription from Pu with m-nitrotoluene, a hybrid protein XylRD135N/E172K was constructed in vitro, and its ability to stimulate transcription in vivo from Pu and Ps fused to lacZ was tested. The double mutant protein XylRD135N/E172K was not able to mediate transcription from the Pu and Ps promoters in the absence of effectors (Table 1), however, it was able to stimulate transcription from the Pu and the Ps promoters in the presence of o- and m-nitrotoluene (Table 1). The XylRD135N/E172K mutant recognized only weakly m-methylbenzyl alcohol.
The XylRP85S mutant showed an effector profile similar to
the wild-type protein. The high basal transcription levels mediated by
this regulator from Pu and Ps increased approximately 2-fold in
response to the addition of m-methylbenzyl alcohol and o-nitrotoluene (Table 1). Interestingly,
-galactosidase activity expressed from Pu and Ps and mediated by
XylRP85S in the presence of m-nitrotoluene was about one-half
of the
-galactosidase activity measured in the absence of aromatic
compounds (Table 1), a fact also observed with the wild-type
regulator. Therefore, m-nitrotoluene seems to act as an
inhibitor of transcription stimulation mediated by XylR and XylRP85S,
but apparently behaves as a positive effector for XylRD135N, XylRE172K,
and the double mutant XylRD135N/E172K.
The stimulation of transcription from Pu by XylRD135N and XylRP85S in the absence of effectors was further confirmed when the transcription initiation point of the Pu promoter was determined by primer extension. The transcript expressed from Pu was observed both in the absence and presence of m-methylbenzyl alcohol when the cell bore the xylR allele encoding XylRD135N or XylRP85S, whereas with the wild-type regulator it was only seen in the presence of the aromatic compound. The transcription initiation point in all cases was always the same, and matched that determined by Inouye et al.(29) . The results obtained with the wild-type regulator and mutant XylRD135N are presented in Fig. 2.
Figure 2:
Induction of mRNA synthesis from Pu by the
wild-type XylR and mutant XylRD135N. E. coli pERD401(Pu::lacZ) bearing pTS174 (XylR) or pAD49
(XylRD135N) were grown overnight on LB medium supplemented with
appropriate antibiotics. Bacterial cells were diluted 1/100 in the same
fresh medium, and after 1 h of cell growth, two aliquots were taken;
one sample was supplemented with 5 mMm-methylbenzyl
alcohol. After 30 min, samples were withdrawn for mRNA analyses. Primer
extension analysis was done by hybridizing 20 µg of total RNA to a
5`-P-labeled oligonucleotide complementary to the
Pu-derived transcript(3) . The extended products (indicated by
an arrow) were 134 nucleotides and were separated in
polyacrylamide gel electrophoresis. mRNA was prepared from the
following cultures: Lanes 1 and 2, E. coli (pERD401, pTS174); in the absence (lane 1) and in the
presence (lane 2) of the effector. Lanes 3 and 4, E. coli (pERD401, pAD49) in the absence (lane
3) and presence (lane 4) of the
effector.
Figure 3:
Activation of Pu and mutant Pu in
different IHF isogenic backgrounds. E. coli S90C
(IHF) and E. coli DBP101
(IHF
) were transformed with the Ap
plasmid bearing the wild-type or mutant Pu::lacZ fusion
indicated below, together with the Cm
plasmids pTS174 or
pAD49, which encode for XylR and XylRD135N, respectively. Bacteria were
grown in the presence (+) and absence(-) of 1 mMm-methylbenzyl alcohol. Other details are given under
``Experimental Procedures'' and in the legend of Table 1.
Stimulation of transcription by XylRD135N, XylRP85S, and by
wild-type XylR from the Pu and Ps promoters was not observed in the
-deficient background provided by ET8045, either in
the presence or absence of m-methylbenzyl alcohol. In fact, in
all assays
-galactosidase activity was below 15 Miller units. This
contrasted with the full transcriptional stimulation in the isogenic
ET8000 background with the wild-type
regulator activated by m-methylbenzyl alcohol and the mutant
regulators, both in the presence and absence of this aromatic (see Table 1). These results confirm the dependence on
of transcription activation from Pu.
Abril et al.(10) and de Lorenzo et al.(20) showed
that in an IHF background, the level of transcription
stimulation from Pu by effector-activated XylR was about 10-25%
of the level in an IHF
background. These results were
confirmed in the present study: the level of effector-activated
wild-type XylR-dependent expression from Pu in an IHF
background was about 10% of that determined in the IHF
background (Fig. 3). Similar results were obtained when
the XylRP85S regulator was used instead of the wild-type regulator (not
shown). In contrast, in the IHF
background provided
by E. coli DPB101, expression from Pu::lacZ with
XylRD135N in the absence of effector was as high as 65% of the maximal
level in an IHF
background (Fig. 3).
Furthermore, in the presence of effector, the level of expression from
Pu in an IHF
background was as high as 83% of the
maximal level of expression from Pu in an IHF
background. Therefore, it seems that XylRD135N is not only able
to stimulate transcription from the Pu promoter in the absence of
effectors, but is also able to overcome the IHF requirement.
To
further confirm this finding, we determined activation from mutant Pu
promoters exhibiting an insertion of 2 or 8 bp within the IHF binding
site. The mutant promoters were fused to a promoterless lacZ
to yield plasmid pERD415 (Pu2bp::lacZ) and plasmid
pERD416 (Pu
8bp::lacZ) and these plasmids were
transformed in IHF
and IHF
backgrounds with XylR or XylRD135N.
-Galactosidase activity
was then determined in the absence and presence of m-methylbenzyl alcohol (Fig. 3). As expected, in the
IHF
and IHF
backgrounds, only low
levels of transcription from Pu
8bp::lacZ and
Pu
2bp::lacZ were found with wild-type XylR, regardless
of the presence of effectors. In contrast, the level of
-galactosidase expressed from these mutant promoters in
IHF
and IHF
backgrounds with
XylRD135N was between 27 and 47% of the maximal level of expression
determined for the wild-type regulator and the level of
-galactosidase was always higher in the presence of effectors.
These results confirm that transcription from Pu mediated by XylRD135N
abolishes the need for accessory DNA-binding IHF protein.
Figure 4: Activation of the wild-type and mutant Pu promoters by XylR and XylRD135N in the presence and absence of m-methylbenzyl alcohol. ET8000 bearing the wild-type or mutant Pu promoter with pTS174 (XylR) or pAD49 (XylRD135N) were grown for 5 h with vigorous shaking in the absence or presence of 1 mMm-methylbenzyl alcohol. Other experimental details are given under ``Experimental Procedures'' and in the legend of Table 1.
In agreement
with previous observations, the deletion of UAS1 and UAS1+UAS2 in
the Pu promoter eliminated the transcriptional response to
effector-activated XylR regulator (10, 16; see Fig. 4). Similar
results were obtained when XylRP85S was used instead of XylR (not
shown). In contrast, the XylRD135N regulator was still able to activate
transcription from Pu lacking UAS1, although only at 40-50%
of maximal transcription activation. However, the removal of both UASs
led to the loss of activation from
Pu (Fig. 4). These
results suggest that UAS2, the closest motif to the
binding site, at least was needed to activate transcription from
Pu.
To further confirm this, mutant Pu promoters in which the two
UASs were separated by one-half or a full helix turn were used. These
two mutant promoters did not respond to activated XylR regulator;
however, XylRD135N was still able to stimulate transcription from them.
In fact, transcription from PuUAS16bp UAS2 (plasmid pERD419)
approached 100% of the level determined for the wild-type promoter,
although when the two motives were separated by a full helix turn (as
in plasmid pERD420), the
-galactosidase level was about 40% of the
maximum (Fig. 4).
Abril et al.(10) substituted As for Gs -131, -139 in UAS2
and for Gs -160 and -169 in UAS1. These point mutations had
little effect on transcription from Pu in the presence of effectors,
which was further confirmed in this work (not shown). With the
XylRD135N mutant regulator, the basal level of expression in the
absence of effector with three of the point mutations (Gs -131,
-160, and -169), was as high as 65-95% of the level
with the wild-type Pu promoter, whereas with the G A -139
mutant, the level of expression was only about 35%. With all four
mutant Pu promoters the level of activity with XylRD135N in the
presence of m-methylbenzyl alcohol ranged from 80 to 100% of
that found for the wild-type Pu promoter (not shown).
The XylR protein belongs to the NtrC family of prokaryotic
enhancer-like positive regulators. These regulators become activated
either by phosphorylation of an aspartyl
residue(31, 32) , as in the case of NtrC, or through
the binding of an effector, as in the case of XylR and
DmpR(7, 8, 9, 10, 11) . The domain involved in signal reception in this family is the
nonconserved NH
-terminal domain, which is about
120-200 amino acids long (33, 34) (Fig. 1). This was deduced from the
following facts: (i) the Asp
residue is the phosphorylated
residue in NtrC(32, 33, 34, 35) ,
(ii) substitution of Lys for Glu
in XylR (11) and
Lys for Asp
in DmpR
resulted in mutant
regulators with altered effector specificity, and (iii) when the
sensing module of DmpR was replaced with that of XylR, the chimeric
protein responded to XylR effectors(8) .
NtrC possesses
ATPase activity, which is phosphorylation-dependent and strongly
stimulated by site-specific binding to DNA(36, 37) .
Isomerization and open complex formation by this family of regulators
require ATP hydrolysis(36) . Several laboratories have isolated
a mutant form of NtrC (Ser
Phe) in which ATPase
activity is independent of phosphorylation, and in which transcription
can be activated in the absence of phosphorylation. This mutant
stimulates transcription constitutively; therefore it seems that the
Ser
Phe change mimics the phosphorylation
state(35, 36, 38, 39) .
In this
study, we searched for XylR mutants that activated transcription in the
absence of effectors. These mutations would be expected to mimic the
conformational state of the regulator bound to their effectors. The
XylR mutants that activated transcription from Pu and Ps in the absence
of effectors exhibited a point mutation, which resulted in substitution
of Asn for Asp, and of Pro for Ser
. These
mutations are located at the NH
-terminal domain of the
regulator, the domain believed to be involved in effector binding. The
mechanism by which the wild-type XylR regulator became activated by
effector binding is still unknown, but the NH
-terminal
domain of this regulator may exert inhibitory effects on DNA binding
and ATPase activity of the COOH-terminal and central domains. This
effect could then be eliminated by the binding of effectors or through
mutations in the NH
-terminal domain as those found in this
study. This hypothesis is supported by the fact that removal of the
NH
-terminal domain of XylR resulted in a truncated protein
that activated transcription constitutively(
); this effect
is similar to that described with the DctD regulator of Rhizobium, a member of the NtrC family of
regulators(40) .
The high basal levels from Pu mediated by XylRD135N and XylRP85S increased further in the presence of o- and p-nitrotoluene. However, this did not occur in Ps. This difference in the pattern of stimulation of transcription from the Pu and the Ps promoters have previously been observed with the wild-type XylR regulator (3, 9) and with XylR7(11) .
XylRP85S exhibited an effector profile similar to that of the
wild-type regulator; however, the XylRD135N mutant was able to
recognize m-nitrotoluene as an effector (Table 1), in
contrast with the wild-type protein, for which this nitroarene behaved
as an inhibitor. Furthermore, XylRD135N is able to recognize cresols,
compounds not recognized by the wild-type XylR regulator. ()Delgado and Ramos (11) previously showed that the
Glu
Lys mutation in XylR also resulted in a mutant
regulator with altered effector specificity, which allowed this
regulator to recognize m-nitrotoluene as an effector. The
constitutive character conferred to XylR by the D135
N change was
abolished by the E172
K mutation, since the XylRD135N/E172K mutant
lost the ability to stimulate transcription in the absence of
effectors. However, the double mutant was able to stimulate
transcription from both the Pu and Ps promoters with o- and m-nitrotoluene. This result supports the notion that
interaction of the XylR regulator with its effectors leads to
conformational changes, which in turn resulted in altered patterns of
induction from cognate promoters.
Transcription from the Pu promoter
by wild-type XylR and the mutant XylR regulators isolated in this study
was analyzed in detail. Abril and Ramos (30) showed that three
binding sites are required for full transcriptional activation from Pu
with wild-type XylR, namely the site at
-12/-24, the IHF site at -40/-70, and the UASs
at -120/-180. The
factor is required
for transcriptional activation of Pu by mutants XylRD135N and XylRP85S.
Our previous results (10) and those of
Pérez-Martín et
al.(41) showed that IHF plays a mechanical role in the Pu
promoter, facilitating contacts between the distally located XylR and
/E complex.
The XylRD135N regulator, but
not XylRP85S, seems to bypass the barriers imposed by the lack of IHF,
since in an IHF-deficient background this mutant stimulated high levels
of transcription from Pu. Furthermore, when the IHF site in the Pu
promoter was destroyed by the insertion of 2 or 8 bp, the XylRD135N was
still able to stimulate (at about 40% of the maximal level)
transcription from the mutant promoters, whereas the wild-type
regulator was unable to stimulate transcription from these mutant
promoters (see Fig. 3). Therefore, the effector-independent
activation of transcription mediated by XylR mutants is not related
with the role of IHF in the Pu promoter. This is in agreement with the
fact that the XylR mutants were also able to stimulate transcription in
the absence of effectors from the Ps promoter, which does not require
IHF(18, 19) .
Our previous results (10) suggested that XylR dimers interact cooperatively to
stimulate transcription from Pu in the presence of effectors. This
required UAS1 and UAS2 located at a specific distance, a requirement
that also applies to XylRP85S. However, XylRD135N required only one
UAS, as XylRD135N was able to activate transcription from several
mutant Pu promoters that conserved the UAS2 motif and either lacked the
UAS1 motif or exhibited this motif displaced by a half or a full helix
turn. It therefore follows that the proper placement of the XylR
regulator or mutant regulators in the UASs is a sine qua non for transcriptional control of Pu. Wild-type XylR protein
regulates its own synthesis (2, 3, 9) by
controlling the expression from the XylR promoters (Pr), a process in
which is involved through an unknown
mechanism(17) . The XylR mutants isolated in the present study
conserved this property. The level of expression from Pr promoters was
reduced by half in the presence of wild-type XylR or mutant regulators
that required both UAS and IHF to mediate transcription from Pu,
namely, XylRP85S, XylRE172K, and XylRD135N/E172K (11) (Table 2). In contrast, XylRD135N, which bypassed
the IHF requirement and mediated transcription from mutant Pu
exhibiting only one UAS, mediated a stricter autoregulation, the level
of autorepression approaching 80%. Given the complex pattern of
interactions between the XylR regulator at the Pu and the Pr/Ps
promoter regions, we suggest that XylRD135N exhibits either increased
affinity for target DNA sequences or increased affinity for
, or both.
In summary, our results suggest that
the D135N and P85
S mutations in XylR allow transcription
stimulation from cognate promoters in the absence of effectors;
therefore these changes mimic the activation of the XylR regulator by
effectors. The XylRD135N mutant also exhibited an altered pattern of
effector recognition, as it stimulated transcription with m-nitrotoluene. Mutation E172K, which led to an altered
pattern of effector recognition, suppressed the ability of the mutant
XylD135N to activate transcription in the absence of effectors;
however, the double mutant XylRD135/E172K retained the ability to be
activated by effectors. On the basis of these findings, we suggest that
residues 85, 135, and 172 in XylR are either part of the recognition
pocket for the effector, or are involved in signal transmission from
the sensing domain of this regulator to the COOH-terminal domain
involved in DNA binding and central domains involved in ATP hydrolysis.
Some combination of these two functions may also be possible.