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 P85S) 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.

EXPERIMENTAL PROCEDURES

Bacterial Strains, Plasmids, and Growth Conditions

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, UAS1UAS2Pu::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.

XylR Mutants

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.

-Galactosidase Assays

DNA Techniques

RESULTS

XylR Mutants That Stimulate Transcription in the Absence of Effectors: The Sequence Change of the xylR Mutant Alleles

Expression from the Pu and Ps Promoters Mediated by the Mutant XylR Regulators and Their Effector Profile

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.

Role of and IHF in Transcription Stimulation from the Wild-type Pu Promoter by XylRD135N and XylRP85S

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 (Pu8bp::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 Pu8bp::lacZ and Pu2bp::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.

Role of the UASs in the Pu Promoter in Transcription Regulation Mediated by the XylRD135N and XylRP85S Regulator

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).

XylR, XylRD135N, and XylRP85S Regulate Their Own Synthesis

DISCUSSION

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 D135N change was abolished by the E172K 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 P85S 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.

FOOTNOTES

*

This work was supported by Comisión Interministerial de Ciencia y Tecnología Grants BI0 091/0659 and AMB94-1038-(02-01) and Commission of the European Communities Grant BT 092-284. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§

Present address: University of Granada, Dept. of Biochemistry and Molecular Biology, School of Pharmacy, E-18014 Granada, Spain.

¶

To whom all correspondence should be addressed: Estación Experimental del Zaidin, Apdo 419, E-18008 Granada, Spain. Tel.: 34-58-121011; Fax: 34-58-129600.

()

Pavel, H., Forsman, M., and Shingler, V.(1994) J. Bacteriol.176, 7550-7557.

()

The abbreviations used are: UAS, upstream activator sequences; IHF, integration host factor; /E complex, RNA-polymerase holoenzyme containing ; bp, base pair.

()

S. Fernández, J. Pérez-Martin, and V. de Lorenzo, submitted for publication.

()

A. Delgado and V. Shingler, unpublished data.

ACKNOWLEDGEMENTS

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