INTRODUCTION

Top Abstract Introduction Materials and Methods Results and Discussion References

DmpR and XylR are highly homologous Pseudomonas-derived transcriptional regulators (12, 28) that share many mechanistic features. DmpR is the specific regulator of the Po promoter, which controls the (methyl)phenol catabolic dmp operon of pVI150 (reviewed in reference 26). XylR performs an analogous regulatory role at the Pu promoter controlling the upper pathway operon of the TOL plasmid pWW0, which encodes the enzymes for conversion of toluene and xylenes to carboxylic acids (reviewed in reference 23). Although the sequences of the Po and Pu promoter regions differ, the RNA polymerase and the regulator binding motifs are sufficiently similar to allow efficient cross regulation (7). DmpR and XylR belong to the prokaryotic family of enhancer binding proteins that regulate transcription via RNA polymerase utilizing the alternative sigma factor, ⁵⁴ (^N), that recognizes 24 GG and 12 GC promoters (14). Like other family members, DmpR and XylR have a distinct domain structure consisting of an N-terminal signal reception A-domain linked to a central activation C-domain by a short B-domain, and a C-terminal D-domain mediating DNA binding (Fig. 1) (15).

View larger version (39K): [in this window] [in a new window]   FIG. 1.   (A) Schematic representation of dmpR- (lines and open boxes) and xylR (shaded boxes)-derived DNAs in key constructs used in this study. The domain structure of DmpR and XylR, described in the text, is superimposed on the DNA restriction map. The hatched boxes represent the extent of the NTP Walker motif (32) found in this class of regulators (G--G-GKE--A---H--S [15]). The flags indicate the presence of an 8-amino-acid carboxy-terminal fusion with the Flag epitope tag. (B) Extents of PCR-generated fragments used in DNA-shuffling experiments described in the text. The lowercase letters refer to the primers used (see below), while the plasmid numbers refer to the template used in each case. The open arrowheads indicate the locations of the primers (b and c) used in the final step of the DNA-shuffling protocol and to identify derivatives of pVI546 harboring full-length hybrid A-domains, as described in Materials and Methods. The primer sequences, with NdeI and SnaBI sites underlined where applicable, are as follows: a, 5'-CGCCATATGCCGATCAAGTACAAG-3'; b, 5'-CGATCAGGTCCCCCGGGATCGCC-3'; c, 5'-CCGTCGATTGATCATTTGGTTG-3'; d, 5'-TCAGGCGGTTACGTAGGTTGGCAA-3'; e, 5'-GGTTGGTACGTAGCGACAACAGTTGCGAT-3'; f, 5'-GCCATATGTCGCTTACATACAAACC-3'; g, 5'-CAGATTTCCACCTCGAAGGAGTC-3'; h, 5'-CAGCCAGATCCGTTTCGTTGCCGCC-3'; i, 5'-CTACGATCGGGTCGCTTTTGAAGT-3'; j, 5'-CCGCAGAATCATTTTCCAGGAAACT-3'; k, 5'-CTCATTCATCAGGCCCAGCTCAGT-3'; l, 5'-GCCCATATGATCCACTTCCAGAGCATG-3'; m, 5'-CGCGGATCCGTTCTTGAAATACTGTTT-3'.

Most ⁵⁴-dependent activators are constitutively produced, but their activities are controlled in response to environmental signals (reviewed in reference 27). DmpR and XylR belong to a subgroup of ligand-responsive regulators. In each case, the activity of the regulator is controlled by interdomain repression where, in the absence of aromatic effectors, the regulatory A-domain inhibits the transcription-promoting activities of the C-domain (6, 16, 17, 21, 22, 30). DmpR and XylR are responsive to distinct, nonoverlapping profiles of aromatic compounds (1, 29, 30), which include some priority pollutants. In this respect, these transcriptional regulators serve as the "aromatic sensors" of the cell, which directly detect the presence of the substrates for the catabolic pathways they control. Utilization of these natural sensing systems and their mutant derivatives, which can respond to different aromatic effectors, provides a potentially inexpensive and simple way to monitor environmental contamination.

Effector specificity mutants and domain-swapping experiments with DmpR and XylR have shown that the effector specificity is mediated via the A-domain (4, 20, 30). However, the magnitude of the aromatic-stimulated transcriptional response varies depending on the position and nature of substituents on the aromatic ring. Recently, the A-domain of DmpR (amino acid residues 1 to 210) has been shown to be both necessary and sufficient to bind its ligand, phenol. Analysis of the binding data revealed a single phenol-binding site per monomer of DmpR (17). Furthermore, competition experiments using DmpR and a panel of aromatic compounds have shown that all the effectors tested act through the same binding site and that the affinity of DmpR for the different effectors determines the concentration at which a response can be detected, but not the magnitude of the response (18). Given the functional homology, it appears likely that all the multiple aromatic effectors of XylR/DmpR family members act through a single binding site on each protein. DNA shuffling between genes that share homology but encode proteins with distinctive properties is a powerful technique, pioneered by Stemmer and coworkers (3, 31), for directed evolution of desirable properties of individual enzymes and whole-enzyme systems (reviewed in reference 8). Here we use DNA shuffling between the A-domains of DmpR and XylR to identify a subregion of the A-domain that is primarily responsible for determining the distinct effector profiles of the two regulators.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results and Discussion References

DNA manipulations. DNA isolation and manipulations were performed using standard techniques (25). The construction of T7 expression plasmids based on pET3a (24) encoding either the entire DmpR regulator or various regions of DmpR as NdeI-to-BamHI fragments, with the BamHI site in frame with the eight codons of a Flag tag (10), has been described previously (17, 30). Plasmids expressing Flag-tagged peptides spanning residues 1 to 190 (pVI540), 6 to 200 (pVI541), 11 to 200 (pVI542), 16 to 200 (pVI543), and 21 to 200 (pVI544) were constructed in an analogous manner by PCR amplification of the corresponding coding regions as NdeI-to-BamHI fragments. T7 expression plasmids for DNA-shuffling products (see below) were constructed by cloning NdeI-to-HindIII fragments, spanning the entire regulator and Flag tag, into pET3H (30). Plasmid pVI577 expresses DmpR/XylR-4 (derived from pVI571) and pVI578 expresses XDX-2 (derived from pVI574), while pVI579 expresses DXD-1 (derived from pVI575).

Luciferase assays. For plate test screening, colonies of Pseudomonas putida KT2440::Po-luxAB (30) harboring various plasmids were replica plated and grown overnight on Luria agar plates containing antibiotics selective for the resident plasmid and supplemented with 2 mM effector or exposed to effector vapor (toluene or m-xylene). Inverted plates were exposed to decanal vapor, and the light emission was recorded by placing film over the plates. For quantification of luciferase activity, cultures were grown to late exponential phase (A₆₅₀, 2.5); at that time, aliquots of the cells were left unsupplemented or were supplemented with 2 mM effector or vapor and were further incubated with rigorous shaking for 3 h. Light emission due to luciferase expression was assayed as previously described (20).

Expression, purification, and [¹⁴C]phenol binding. Flag-tagged proteins were expressed, affinity purified, and tested for the ability to bind universally labeled [¹⁴C]phenol (5.22 GBq/mmol; Amersham Pharmacia Biotech) as previously described (18). Experiments were performed at a final concentration of 16 µM radiolabeled phenol (the K_d of wild-type DmpR). Background levels bound to a A-NifA-Flag derivative were subtracted to determine the specific binding of phenol as previously described (17). Values are expressed as percentages of phenol binding mediated by wild-type DmpR-Flag.

Protein analysis. Crude extracts of cytosolic proteins, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transfer to nitrocellulose filters, and Western blot analysis with polyclonal rabbit anti-A1-DmpR serum were as previously described (30). Antibody-decorated bands were revealed by using chemiluminescence reagents as directed by the supplier (Amersham Pharmacia Biotech). Differences in expression levels were assessed by comparison of dilution series of the test samples with those of wild-type DmpR-Flag. For N-terminal sequence analysis, samples were transferred to a polyvinylidene difluoride nitrocellulose filter (Bio-Rad), briefly stained, destained, and extensively washed prior to excision of a strip of filter from the center of the band corresponding to the unknown 60,000-Da protein.

	RESULTS AND DISCUSSION

Top Abstract Introduction Materials and Methods Results and Discussion References

Deletions in the A-domain cause association with GroEL. Previous analysis of the [¹⁴C]phenol-binding capacity of Flag epitope-tagged peptides of DmpR has shown that amino acid residues 1 to 200 are sufficient for full phenol binding (compared to that of the wild-type protein) (17). As a first strategy to further define the region of DmpR responsible for aromatic-ligand binding, we constructed a series of derivatives that would express truncated derivatives of the A-domain as Flag epitope-tagged peptides. However, further deletions resulting in peptides spanning residues 1 to 190, 6 to 200, 11 to 200, 16 to 200, and 21 to 200 all gave rise to preparations in which the peptide copurified with an unknown protein with an M_r of 60,000 and were incapable of binding [¹⁴C]phenol (data not shown). N-terminal sequencing of the copurified protein revealed the amino acid sequence AAKDVKFGNDARV, which is identical to that of the protein-folding chaperone GroEL of Escherichia coli. These results suggests that the A-domain of DmpR, defined as residues 1 to 210 by sequence alignment with other members of the family (28), is a discrete domain and that truncation of the domain at either the N or C terminus results in unfolded peptides. This finding prompted us to use DNA shuffling to generate hybrid A-domains in the wild-type context of the remainder of DmpR as a strategy to identify the key region(s) involved in the specificity of ligand binding.

Construction of a dmpR-shuffling template. The domain structures of DmpR and XylR are schematically illustrated in Fig. 1. Previous manipulation to introduce a silent NdeI site overlapping the ATG initiation codons of dmpR and xylR, and introduction of a silent mutation generating a ScaI site in the coding region of xylR analogous to that in dmpR, allowed construction of a hybrid gene in which codons 1 to 233 of dmpR were exchanged for those of xylR (Fig. 1A, pVI406). The encoded protein, XylR/DmpR-ScaI, was tightly regulated in response to aromatic effectors of XylR but was unable to respond to effectors of DmpR, demonstrating that the A-domain of XylR is fully functional and capable of exerting its repressive property and effector response in the context of DmpR (see below) (29). Because of the presence of two other ScaI sites in the vector, this site was not useful for DNA-shuffling purposes. Therefore, a unique silent SnaBI was introduced into the B linker of dmpR so that replacement of the NdeI (codon 1)-to-SnaBI (overlapping codons 221 to 222) region would result in replacement of the entire A-domain (Fig. 1A, pVI545). To facilitate the identification of clones encoding full-length hybrid A-domains, two other manipulations were made. First, a diagnostic unique ClaI site was also introduced, and second, an internal deletion between the SacI site (overlapping codons 112 to 113) and the NdeI site (overlapping codons 188 to 190) was made to give the final template into which hybrid NdeI-to-SnaBI cassettes would be cloned (Fig. 1A, pVI546).

DNA shuffling round 1: generation of XylR/DmpR hybrids. The general strategy for all DNA-shuffling experiments followed the same basic steps, as detailed in Materials and Methods. First, PCR-generated fragments spanning the A-domains of dmpR and xylR (depicted in Fig. 1B) were subject to limited DNase I treatment, and the resulting random fragments of 30 to 300 bp were used to generate extended hybrid DNA fragments in a self-priming PCR. The resulting products were then used to generate DNA spanning from upstream of the NdeI site to downstream of the SnaBI site in primer-directed PCRs. Finally, purified NdeI-to-SnaBI fragments were cloned into the DNA-shuffling template pVI546 (Fig. 1A) to regenerate an entire regulator under control of the native promoter of dmpR.

View larger version (61K): [in this window] [in a new window]   FIG. 2.   A-domain DNA and amino acid sequence alignments of DmpR and XylR. The NdeI and SnaBI sites used in the DNA-shuffling protocol are shown in boldface. The 12 different junction points found in the hybrid regulators are marked by lines. Colons indicate identical bases, and dashes indicate identical amino acid residues.

DNA shuffling round 2: generation of DmpR/XylR hybrid A-domains. The results discussed above suggested that the strategy used in round 1 introduced a bias in the shuffling in favor of hybrids with 5' ends derived from xylR DNA. Therefore, to generate derivatives with 5' ends derived from dmpR DNA, we performed a second round of DNA shuffling in which the locations of the primers used to generate the starting DNA for the shuffling were reversed (Fig. 1B). Of 25 full-length derivatives tested as described above, 5 were found to be responsive to one or more of the test aromatic compounds. DNA sequence analysis of these five derivatives showed that this round of DNA shuffling, as expected, resulted in genes starting with dmpR sequences. On the basis of the junction point, the shuffling derivatives could be grouped into five classes (Table 1). The derivative with the most distal junction point at codon 221 regenerated full-length dmpR sequence. However, since it harbored a mutation that results in a single-amino-acid substitution (F132L) close to a previously defined effector specificity mutant (E135K [20]), this derivative was further analyzed as described below.

Effector responses of hybrid DmpR/XylR regulators. One representative from each class of hybrid regulators (Table 1) was quantitatively assayed in the luciferase reporter strain described above for the ability to respond to two sets of aromatic compounds. The responses are compared to those of wild-type DmpR and the XylR/DmpR-ScaI hybrid in Fig. 3. The first sets of aromatic compounds chosen (Fig. 3) are all effectors of DmpR and include phenol; 2-, 3-, and 4-methylphenol; and 3,4-dimethylphenol. The second set of aromatic compounds (Fig. 3) includes three effectors of XylR (toluene, m-xylene, and 3-methylbenzyl alcohol) and two structurally closely related compounds (2- and 4-methylbenzyl alcohol). The expression of the regulator in each case was also monitored by Western analysis using polyclonal rabbit antiserum directed against the C- to D-domains of DmpR, i.e., the region common to all the regulators tested (Fig. 3Q).

View larger version (41K): [in this window] [in a new window]   FIG. 3.   In vivo transcriptional response of Po mediated by DmpR and XylR hybrids. (A to P) Luciferase transcriptional response of P. putida KT2440::Po-luxAB harboring plasmids expressing the indicated derivatives (for plasmid designations, see Table 1) was measured in the absence of any effector (bars 1) or in the presence of two sets of aromatic effectors. The DmpR effectors (open bars) were phenol (bars 2), 2-methylphenol (bars 3), 3-methylphenol (bars 4), 4-methylphenol (bars 5), and 3,4-dimethylphenol (bars 6). The XylR and XylR-like effectors (shaded bars) were toluene (bars 7), m-xylene (bars 8), 2-methylbenzyl alcohol (bars 9), 3-methylbenzyl alcohol (bars 10), and 4-methylbenzyl alcohol (bars 11). The data are the averages of triplicate determinations in each of two independent experiments. The level of protein expression, estimated from panel Q (as described in Materials and Methods), is expressed as a percentage of that of DmpR-wt (wild type) for derivatives with significantly decreased expression levels (I, J, and K). (Q) Expression levels of the regulators. Western analysis of 30 µg of crude extract derived from cells exposed to 2-methylphenol shown in panels A to P, separated by SDS-11% PAGE, and probed with anti-DmpR serum as described in Materials and Methods is shown. LU, luciferase units. The error bars indicate standard deviations.

View larger version (50K): [in this window] [in a new window]   FIG. 4.   Schematic illustration of the relative contributions of DmpR (open boxes) and XylR (shaded boxes) amino acid sequences to the A-domains of the derivatives tested for aromatic responsiveness (Fig. 3). The derivatives are classified into four response profile groups as described in the text. The arrowheads mark the locations of single-amino-acid substitutions that give rise to novel effector response abilities of DmpR (open arrowheads) and XylR (shaded arrowheads) as described in the text. wt, wild type. Narrow ends indicate the extent of -domain linker sequences (Fig. 1), while stippled boxes indicate the junction points defined in Table 1.

Many hybrid DmpR/XylR regulators give low-level responses to aromatic effectors. The scale for the magnitude of the transcriptional response in the presence of different effectors in Fig. 3 is the same for all the derivatives. It is notable that some of the derivatives are poor at promoting transcription in the presence of their effectors (Fig. 3E to F and I to L). All derivatives are expressed from the same cis-acting regulatory element, but it is possible that the different hybrid proteins exhibit different stabilities. From the Western blot analysis (Fig. 3Q), it appears that the protein level of the regulator can account for the poor responses observed in some but not all cases. Reduced protein levels could partially or completely account for the reduced response levels of the derivatives shown in Fig. 3I to K. However, no major difference in protein levels could be observed for the derivatives shown in Fig. 3E, F, or L. This suggests that these derivatives are defective in their binding or response to aromatic effectors. The A-domain represses the essential activities of the C-domain required for transcriptional activation, namely, ATPase activity and interaction with the transcriptional apparatus, until aromatic effectors are bound. Mechanistically, the A-domain-mediated control of these two processes can be uncoupled, since some aromatic compounds that can be bound by DmpR elicit an ATPase activity but not a transcriptional response (18). Therefore, the defect in the abilities of the hybrid regulators to promote a transcriptional response can be predicted to lie on any of three levels: (i) at the level of correct folding of the hybrid A-domain, (ii) at the level of effector binding affinity, or (iii) at the level of the ability of the effector-bound form to allow interaction with the transcriptional apparatus. To distinguish the basis for the low levels of response observed, we analyzed the [¹⁴C]phenol-binding ability of DmpR/XylR-4, which exhibits a low-level response to phenol despite approximately wild-type protein levels. The ability of this derivative (Fig. 3E) to bind [¹⁴C]phenol is compared to that of wild-type DmpR in Fig. 5. The results show that DmpR/XylR-4 has approximately twofold-reduced ability to bind 16 µM phenol. In terms of phenol binding, this decrease is reminiscent of the effector specificity mutant DmpR-E135K, which has two- to fourfold-reduced affinity for phenol. However, DmpR-E135K-Flag is capable of a full in vivo transcriptional response at the 2 mM effector concentration used in the transcriptional reporter assays here (18). Thus, it appears likely that, despite the reduced ability of DmpR/XylR-4 to bind phenol, the main defect in this derivative lies at the level of transmission of the binding signal to generate an effector-bound form that allows productive interaction with the transcriptional apparatus.

View larger version (25K): [in this window] [in a new window]   FIG. 5.   Comparison of [14C]phenol binding of DmpR-Flag and DNA-shuffling derivatives. The data are the averages (+ standard deviations) of triplicate determinations performed at the Kd for wild-type DmpR-Flag (16 µM) as described in Materials and Methods. Binding by wild-type DmpR-Flag was set at 100%. The inset shows a Coomassie blue stain of 3 µg of proteins released from 3 µl of the beads and separated by SDS-11% PAGE.

Mosaic DmpR/XylR A-domains are predominantly nonresponsive to aromatic effectors. An initially surprising finding from the DNA shuffling rounds 1 and 2, was the complete absence of isolation of derivatives with mosaic A-domains in which more than two blocks of dmpR and xylR DNA had been shuffled. In these experiments, a total of 12 different junction points were found (Fig. 2), but no more than one junction was found per hybrid. This suggests that more complex shuffling of A-domain sequences may result in predominantly nonresponsive derivatives. To test this idea, we did three more rounds of shuffling with DNA, depicted in Fig. 1B (rounds 3 to 5). The design of the different DNA fragments used in these shuffling experiments served two purposes. First, it forced at least two junctions to generate a full-length A-domain. Second, it did not allow the possibility of a junction at the hybrid hot spot between codons 160 and 167 found in round 1, which results in the multiple effector responder XylR/DmpR-5 (Fig. 3H). From a total of 213 derivatives that had full-length A-domains derived from DNA shuffling rounds 3 to 5, no derivative capable of significant response to the three test effectors (2-methylphenol, toluene, and 3-methylbenzyl alcohol) was found. Therefore, to address the question of the aromatic responsiveness of mosaic A-domains, and as an independent test of whether residues 107 to 186 determine the specificity of activation, we artificially constructed four mosaic A-domains. These constructs were designed on the basis of the productive dmpR/xylR junction points found in rounds 1 and 2 and were generated as described in Materials and Methods. The extents of the contributions of DmpR and XylR in these derivatives are illustrated in Fig. 6A. In the XDX-1 and -2 derivatives, DmpR A-domain residues are flanked by XylR residues, while in the DXD-1 and -2 derivatives, XylR A-domain residues are flanked by DmpR residues. Of these four derivatives, only one, DXD-1, gave detectable responses to any of the aromatic effectors, again supporting the idea that a high proportion of mosaic A-domains are non-aromatic responsive, even when junction points that are productive in formation of hybrid A-domains are used (Fig. 6C and data not shown). Furthermore, the DXD-1 derivative had a XylR-like aromatic effector response profile, as would be predicted from the results of rounds 1 and 2. 

View larger version (25K): [in this window] [in a new window]   FIG. 6.   Analysis of mosaic DmpR/XylR regulators. (A) Schematic illustration of the relative contributions of DmpR (open boxes) and XylR (shaded boxes) amino acid sequences to the A-domains of the indicated derivatives. Western analysis (B) and aromatic response profile (C) of DXD-1, as described in the legend to Fig. 3. DXD-1 is expressed at about 50% of the level of DmpR-wt (wild type). LU, luciferase units.

Concluding remarks. In this report we have demonstrated, using hybrid A-domains of DmpR and XylR, that the amino acid residues 110 to 186 are intimately involved in mediating the distinct effector profiles of these two regulators. The aromatic-sensing properties of natural regulators of catabolic pathways have been used in biosensor applications (2, 9, 11, 13, 19, 33). The identification of a subregion of the A-domain that is primarily responsible for determining effector specificity provides a target for directed mutagenesis to modify sensitivity and expand the effector specificity of this class of regulators for greater utility in such biosensor systems. The DNA-shuffling approach used here was designed to be unbiased, and identification of functional derivatives by screening did not involve any positive selection for a desired property. Nevertheless, derivatives with novel response abilities and broadened or narrowed response profiles were identified, demonstrating the power of the approach to generate variants with different response properties. Previous work has shown that the affinity of DmpR for four of its natural effectors tested determines the concentration at which a response can be detected but not the magnitude of the response (18). The finding that a hybrid derivative can have enhanced binding affinity for phenol but be completely deficient in transcriptional activation has important mechanistic implications and suggests that the conformation of the A-domain upon binding of the aromatic effector is crucial for productive interaction with the transcriptional apparatus. Hence, novel or enhanced binding affinities will only lead to productive in vivo transcriptional responses if the alterations made are also compatible with the release of interdomain repression and subsequent interaction of the C-domain with RNA polymerase.