Critical nucleotides in the upstream region of the XylS-dependent TOL meta-cleavage pathway operon promoter as deduced from analysis of mutants.

The Pm promoter, dependent on TOL plasmid XylS regulator, which is activated by benzoate effectors, drives transcription of the meta-cleavage pathway for the metabolism of alkylbenzoates. This promoter is unique in that in vivo transcription is mediated by RNA-polymerase with different sigma factors. In vivo footprinting analysis shows that XylS interacted with nucleotides in the -40 to -70 region. In vivo and in vitro methylation of Pm shows extensive methylation of T at position -42 in the bottom strand, suggesting that it represents a key distortion point that may favor XylS/RNA polymerase interactions. Methylation of T-42 was highest in cells bearing XylS and in the presence of an effector. Gs in the -47 to -61 region appeared to be more protected in cells harboring XylS in the presence than in the absence of the effector. Almost 100 mutants in the Pm region between -41 and -78 were generated; transcriptional analysis of these mutants defined the XylS target as two direct repeats with the sequence TGCAN6GGNCA. These motifs cover the -70 to -56 and the -49 to -35 regions. Single point mutations revealed that nucleotides located at -49 to -46 and at -59, -60, -62, and -70 are the most critical for appropriate XylS-Pm interactions.

The Pm promoter, dependent on TOL plasmid XylS regulator, which is activated by benzoate effectors, drives transcription of the meta-cleavage pathway for the metabolism of alkylbenzoates. This promoter is unique in that in vivo transcription is mediated by RNApolymerase with different sigma factors. In vivo footprinting analysis shows that XylS interacted with nucleotides in the ؊40 to ؊70 region. In vivo and in vitro methylation of Pm shows extensive methylation of T at position ؊42 in the bottom strand, suggesting that it represents a key distortion point that may favor XylS/ RNA polymerase interactions. Methylation of T ؊42 was highest in cells bearing XylS and in the presence of an effector. Gs in the ؊47 to ؊61 region appeared to be more protected in cells harboring XylS in the presence than in the absence of the effector. Almost 100 mutants in the Pm region between ؊41 and ؊78 were generated; transcriptional analysis of these mutants defined the XylS target as two direct repeats with the sequence TGCAN 6 GGNCA. These motifs cover the ؊70 to ؊56 and the ؊49 to ؊35 regions. Single point mutations revealed that nucleotides located at ؊49 to ؊46 and at ؊59, ؊60, ؊62, and ؊70 are the most critical for appropriate XylS-Pm interactions.
The TOL plasmid pWW0 of Pseudomonas putida specifies a meta-cleavage pathway for the oxidative catabolism of benzoate and toluates. Genes encoding the TOL meta-cleavage pathway are grouped into a single operon, the expression of which is positively regulated at the level of transcription by the xylS gene product, which is activated by benzoate effectors (1)(2)(3)(4). Stimulation of transcription from the Pm promoter requires a DNA sequence extending to about 80 bp 1 upstream of the transcription initiation point (5)(6)(7). On the basis of genetic data, two regions can be distinguished in the architecture of the Pm promoter: the XylS interaction region, which extends from about Ϫ40 to Ϫ80 bp (Fig. 1); and the downstream RNA polymerase recognition region, which exhibits atypical Ϫ35 and Ϫ10 DNA sequences (Fig. 1).
Transcription from the Pm promoter in the early exponential growth phase is mediated by RNA polymerase with 32 , and later with S , although regardless of the growth phase, expres-sion from Pm remains dependent on XylS, and the transcription initiation point is the same (8,9).
Kessler et al. (6) proposed that the XylS binding region in Pm was organized as two homologous 15-bp tandemly imperfect directly repeated motifs (5Ј-TGCAAPuAAPyGGNTA-3Ј). The distal one with respect to the RNA polymerase region extends from Ϫ70 to Ϫ56, and the proximal one extends from Ϫ49 to Ϫ35 (Fig. 1). Gallegos et al. (5) also studied the organization of the XylS binding sites in the Pm promoter and suggested that sequences shorter than those proposed by Kessler et al. (6) might suffice for XylS activation of transcription. These workers found that promoters that had been deleted up to Ϫ60 could be activated by constitutive XylS mutants (but not by the wild-type regulator) and that extension of the deletion to Ϫ51 prevented transcription. Gallegos et al. (5) proposed that the XylS binding site is represented by the motif T(C/A)CAN 4 -TGCA, which appears twice in the promoter sequence, between Ϫ46 and Ϫ57 and between Ϫ67 and Ϫ78 (Fig. 1).
This study was designed to shed light on the nucleotides in the Pm promoter that are critical for XylS-dependent stimulation of transcription.

MATERIALS AND METHODS
Bacterial Strains, Culture Medium, and Plasmids-Escherichia coli MC4100 was grown at 30°C in Luria-Bertani medium supplemented, when required, with 100 g/ml ampicillin, 25 g/ml kanamycin, or 50 g/ml streptomycin.
The plasmids used in this study, and previously constructed were: pERD103, which is an IncQ plasmid encoding kanamycin resistance (7); pJLR100, which is a pEMBL9 derivative bearing the Pm promoter cloned between the EcoRI and HindIII sites (3); pMD1405, which carries a promoterless ЈlacZ gene and encodes resistance to ampicillin; and pJLR107, which is a pMD1405 derivative bearing the Pm promoter in front of ЈlacZ (3).
DNA Techniques-DNA preparation, digestion with restriction enzymes, analysis by agarose gel electrophoresis, isolation of DNA fragments, ligations, transformations, and sequencing reactions were done according to standard procedures (10).
Methylation Experiments-DNA was methylated in vitro with 2 mM dimethyl sulfate as described (10). For in vivo DNA metylation, E. coli cells bearing the Pm promoter in pMD1405 were exposed to 2 mM dimethyl sulfate for 1 min at 30°C. Cells were processed as described (10). Plasmid DNA was extracted by using the Qiagen Kit (Quiagen, GmbH, Hilden, Germany).
The oligonucleotide 5Ј-CGTCTAAGAAACCATTATTATCAG-3Ј was complementary to the noncoding strand upstream from the Pm promoter region. The first C at the 5Ј-end was located 218 bp from the ϩ1 of the transcription initiation point. The oligonucleotide 5Ј-GGGTCG-GTGAACATCTCGCGCTTGC-3Ј was complementary to the coding strand downstream from the Pm promoter. The first G at the 5Ј-end was located 124 bp from the ϩ1 of the start of the transcript.
For primer extension with Taq-DNA-polymerase, about 2 ϫ 10 5 cpm of the corresponding oligonucleotide end-labeled with 32 P was used. The extension products were separated by electrophoresis on urea-polyacrylamide sequencing gels.
Construction of Pm Mutant Promoters by Polymerase Chain Reac-tion-The Pm mutant promoters were generated by overlap extension polymerase chain reaction mutagenesis as described (5). The internal oligonucleotide primers used for mutagenesis exhibited one or more mismatches with respect to the wild-type sequence. The external oligonucleotide was the so-called M13 reverse primer (5Ј-CAGGAAACAGC-TATGACCATG-3Ј) or a primer complementary to the ␣ fragment of the lacZ gene (5Ј-GATGTGCTGCAAGGCGATTAAGTTA-3Ј). After DNA amplification, the resulting DNA was digested with EcoRI and HindIII, and the 401-bp EcoRI-HindIII Pm mutants were inserted between the EcoRI-HindIII sites of pMD1405 to yield plasmids pMARx (the x indicates the plasmid number from 5D to 296). All the mutant Pm promoters generated in this study were confirmed by DNA sequencing. ␤-Galactosidase Assays-E. coli bearing the wild-type Pm::ЈlacZ or mutant Pm*::ЈlacZ fusions in pMD1405, plus pERD103, were grown overnight on Luria-Bertani medium containing the appropriate antibiotics. Cultures were diluted 100-fold in triplicate in the same medium supplemented or not with 1 mM 3MB. After 4 h of incubation, ␤-galactosidase activity was determined in duplicate in permeabilized cells (5).

In Vivo Methylation Assays Located the XylS Binding Region
Adjacent to the RNA Polymerase Binding Site-The reactivity of guanine residues in the Pm promoter toward dimethyl sulfate was assayed in vivo. DNA protection analysis was done in E. coli bearing only pJLR107 (Pm) or pJLR107 and pERD103 (XylS) and in the absence and in the presence of 3MB. Methylation was done when cells had reached the mid-logarithmic growth phase. As a control, Pm was also methylated in vitro. Representative results are shown in Fig. 2. In the bottom strand, a significant feature of the in vitro and in vivo methylation pattern is hypermethylation of the T located at Ϫ42. This indicates that the DNA was distorted at this point, probably due to the tracks of As in the Ϫ41 to Ϫ46 region in the top strand ( Fig. 1). The methylation of T Ϫ42 in vivo in cells without XylS was more pronounced than in vitro, indicating that this distortion may have been more pronounced in vivo. The methylation pattern of Pm in vivo in cells without XylS was very similar in the absence and in the presence of 3MB. However, the presence of the effector influenced the methylation pattern of Pm in cells bearing the XylS protein. When cells expressed the XylS protein, methylation of T Ϫ42 was highest in the presence of the effector, whereas in the absence of the effector, T Ϫ42 appeared to be more protected than in cells without XylS (Fig.  2). In contrast with this behavior was the observation that Gs in the Ϫ47 to Ϫ61 region appeared to be protected in the presence of the effector. However, G Ϫ68 appeared to be more methylated in the presence of effector (Fig. 2). This set of results suggested that the XylS protein is able to bind to Pm; however, the critical interactions could not be deduced from this assay.
In the top strand in Fig. 2, the Ϫ3 to Ϫ40 region showed a definite pattern of methylation. The Gs in the Ϫ3 to Ϫ28 region were more methylated in vitro than in vivo. In particular, G Ϫ23 and G Ϫ38 were clearly protected in vivo regardless of the presence of XylS and the presence of an effector.
Block Scanning Mutagenesis of the Putative XylS Binding Site at the Pm Promoter-A characteristic of the members of the AraC/XylS family of transcriptional regulators is that they recognize short nucleotide motifs (4 -6 bp) at their cognate promoters (11). For this reason, we decided to carry out initial block scanning mutagenesis assays of the Pm region between Ϫ41 and Ϫ78. This interval includes all nucleotides previously proposed as important in XylS for Pm recognition (5-7) and the region in which in vivo footprinting analyses revealed alterations in the methylation pattern. The interval excluded mutations in the putative Ϫ35 region of the Pm (Fig. 1), which, as shown in the in vivo footprinting analysis, may interfere with recognition of the promoter by the RNA polymerase. A series of 4-bp blocks to induce mutations were introduced randomly by polymerase chain reaction. These included the four TNCA motifs found in this region (Ϫ46/Ϫ49, Ϫ54/Ϫ57, Ϫ67/Ϫ70, and Ϫ75/Ϫ78), the intervening sequences between two adjacent TNCA sequences (Ϫ50/Ϫ53, Ϫ58/Ϫ61, Ϫ62/Ϫ65, Ϫ63/Ϫ66, and Ϫ71/Ϫ74), a sequence between the TNCA sequence closer to the RNA polymerase binding site (Ϫ46/Ϫ49), and the Ϫ41 nucleotide (Ϫ41/Ϫ44). In all cases the mutant promoters (Pm * ) were fused to ЈlacZ in pMD1405. ␤-Galactosidase activity was determined in E. coli MC4100 (Pm * ::ЈlacZ, pERD103) grown in the absence and in the presence of 3MB. The basal level of expression from the Pm* promoters (50 -100 Miller units) was similar to the basal level of expression determined from the Pm wild-type promoter. However, with regard to the induced levels of expression of the mutant promoters, three classes of Pm mutants were found (Table I): 1) mutant promoters that exhibited less than 20% of the wild-type activity; 2) mutant Pm promoters with a level of XylS-dependent expression between 65 and 25% of the expression measured with the wild-type promoter; and 3) mutant Pm promoters that conserved wildtype or near wild-type XylS-dependent inducible ␤-galactosidase activity, i.e. Ն80% of wild-type levels. The mutations that resulted in the largest reduction in transcription, i.e. a decrease equal to or greater than 80% of the wild-type activity, were any random substitution of the TGCA sequence between Ϫ46/Ϫ49 (Table I). This suggests that these nucleotides are critical for XylS-dependent transcription activation of Pm. The substitution by random sequences of the AAAAA sequence located at Ϫ41/Ϫ45, the TACA sequence between Ϫ54/Ϫ57, the CGGA sequence between Ϫ58/Ϫ61, and the TGCA sequence between Ϫ67/Ϫ70 resulted in a significant decrease in XylS-dependent transcription activation of Pm. The activity of most of the mutant Pm promoters at these locations ranged from approximately 25 to about 65% of the activity of the wild-type promoter (Table I), although certain substitutions had little effect. These results suggest that these sets of bases are less critical than those at the Ϫ46/Ϫ49 region; however, they may play a direct role in the recognition of the Pm DNA sequences by XylS, or they may contribute to the overall affinity for Pm. We cannot rule out other effects.
The third group of mutations, i.e. those that had no effect (or little effect) on transcription from Pm, were found to correspond to the locations of Ϫ50/Ϫ53, Ϫ62/Ϫ65, Ϫ63/Ϫ66, Ϫ71/ Ϫ74, and Ϫ75/Ϫ78.
We also investigated whether the combination of different blocks of mutations had a synergistic effect on XylS-dependent transcription activation from the mutant promoters. The combination of a block of mutations in Ϫ46/Ϫ49 (TGCA3 AGGA) with a block of mutations at Ϫ54/Ϫ57 (TACA3 TTGG) resulted in a mutant Pm promoter (Pm 245) that had no activity at all (Table I).
At the Ϫ54/Ϫ57 block some substitutions had little effect on XylS-dependent transcription from Pm (i.e. Pm 213, 11% reduction), whereas other substitutions at the same block had a clear effect (i.e. Pm 212, 78% reduction). In Pm 244, we combined the block of mutations in Pm 213 with a mutant block that had a moderate effect on the level of expression from Pm (i.e. Ϫ67/Ϫ70 (TGCA 3 ACGT)). This combination had cumulative effects (Table I). These results confirmed the essential role of the nucleotides at these positions in XylS-dependent transcription activation from Pm. The transcription initiation point of the mRNA generated from a number of the above Pm mutants promoters was the same that that determined for the wild-type promoter (not shown). This suggests that the mutations analyzed affected the strength of transcription from the mutant promoters.

Single Point Mutations within the Set of Blocks That Showed or Did Not Show Any Effect on Transcription Activation from
Pm-The above series of assays suggested critical, less critical, and irrelevant blocks for the XylS-dependent transcription activation from Pm. We expected that single mutations in irrelevant sets of sequences would have no effect at all on transcriptional activity from Pm, whereas single mutations in the critical and important blocks of sequences would have an effect. A number of bases at the noncritical region were selected to introduce single point mutations: A Ϫ51 3 G, A Ϫ64 3 C, G Ϫ65 3 A, A Ϫ75 3 C, C Ϫ77 3 G, and T Ϫ78 3 G. These mutations, as expected, had little or no effect (Ͻ20%) on XylS-dependent transcription from Pm (not shown).
Of the less critical boxes (Ϫ41/Ϫ44, Ϫ54/Ϫ57, Ϫ58/Ϫ61, and Ϫ67/Ϫ70), some of the substitutions (for example, A Ϫ42 3 T, A Ϫ44 3 G, A Ϫ54 3 G, A Ϫ54 3 T, G Ϫ69 3 C, and G Ϫ69 3 T) did not significantly affect activity from Pm (Table II); others had an intermediate effect, reducing the induced XylS-dependent activation of Pm by 20 -50%. This effect was observed for the changes C Ϫ55 3 A, C Ϫ55 3 G, A Ϫ67 3 C, and A Ϫ67 3 T (Table  II). Within these sequences, the change T Ϫ70 3 G resulted in loss of almost 90% of the activity (Table II).
In the Ϫ58/Ϫ61 box, the G Ϫ59 3 C, G Ϫ60 3 T, and G Ϫ60 3 A   Ϫ42 Ϫ46 Ϫ47 C 3 T 3700 42 Pm202 Ϫ48 Ϫ48 Ϫ49 Ϫ54 Ϫ54 Ϫ54 Ϫ55 Ϫ55 Ϫ56 Ϫ57 Ϫ59 G 3 C 1800 20 Pm279 Ϫ60 Ϫ62 Ϫ63 Ϫ67 Ϫ69 Ϫ69 changes had a significant effect on transcription, as shown by the finding that ␤-galactosidase activity was less than 20% of that seen with the wild-type Pm promoter. A surprising finding was that the G Ϫ62 3 C change resulted in a mutant Pm promoter that lacked activity.
In the critical Ϫ46/Ϫ49 set of bases, single bp substitutions had a significant effect on activity. The changes A Ϫ46 3 T, C Ϫ47 3 G or C Ϫ47 3 T, G Ϫ48 3 C, and T Ϫ49 3 G resulted in a 60 -85% decrease in activity. However, the change G Ϫ48 3 A had little or no effect on transcriptional activity (Table II).
Role of the Bases at Ϫ54/Ϫ57-When we compared the critical sequences proposed by Kessler et al. (6) and Gallegos et al. (5), we found that they had the two TGCA submotifs at Ϫ46/ Ϫ49 and Ϫ67/Ϫ70 in common (Fig. 1). The hypothesis of Gallegos et al. (5) suggested that the TACA submotif between Ϫ54/Ϫ57 was critical for transcription activation from Pm, whereas Kessler et al. (6) suggested that only the Ϫ57/Ϫ56 nucleotides were of importance (Fig. 1). Our results with single point mutations in the Ϫ54 to Ϫ57 region showed some effect in some positions, but in no case was the effect large enough to fully impede transcription (Table II). To elucidate the possible role of these four nucleotides, we generated the set of changes involving the Ϫ54/Ϫ55, Ϫ55/Ϫ56, Ϫ56/Ϫ57, and Ϫ54/Ϫ57 positions. We found that the Ϫ54/Ϫ55 changes (CA 3 AC or CA 3 GT) did not influenced the level of ␤-galactosidase activity from the mutant Pm promoters. In contrast, the Ϫ57/Ϫ56 TA 3 AT change almost completely prevented activity (99% decrease). The combination of mutations at Ϫ55/Ϫ56 and Ϫ54/ Ϫ57 had an intermediate effect, with activities in the range of 15-38% when the mutation involved the Ϫ57 position, and in the range of 40 -80% when the changed involved the Ϫ56 position (not shown). From these results, we deduced that the Ϫ57/Ϫ56 bases are critical for the activity of Pm. DISCUSSION The XylS protein (12)(13)(14) belongs to the AraC/XylS family of regulators that comprises more than 100 different proteins involved in transcription stimulation of several cell processes, such as carbon metabolism, pathogenesis, and response to alkylating agents in bacteria (11). Members of the family for which in vitro or in vivo footprinting assays are available (AraC, RhaR, RhaS, MelR, SoxS, and Ada) have at least two features in common: they function in vivo as a dimer, and the stretch of nucleotides covered by a monomer of the regulatory protein at the regulated promoter is between 15 and 20 bp long. However, within this set of bases, short motifs seem to confer critical base recognition for DNA-protein interactions (15)(16)(17)(18)(19)(20)(21). This seems also to be the case for Pm/XylS interactions. Our in vivo footprinting analysis and the analysis of transcriptional activity from wild-type and mutant Pm promoters showed that critical nucleotides extended from the Ϫ70 position in the 5Јend to at least the Ϫ41 position in the 3Ј end. Our mutational analysis revealed that within this stretch the XylS recognition sequence seems to be TGCAN 6 GGNTA, which appears twice in the Pm promoter, between Ϫ70 and Ϫ56 and between Ϫ49 and Ϫ35 (Fig. 1). In favor of this proposal is the observation that a tagged XylS-protein immunoadsorbed onto glass beads produced footprints in vitro, which showed protection of the Gs within the above direct repeat at Ϫ48, Ϫ59, Ϫ60, and Ϫ69 (22). This is in agreement with our in vivo results.
The AraC protein-the best characterized regulator of the family-stimulates transcription as a dimer (23)(24)(25). The consensus sequence for AraC-activable promoters is a direct repetition of the TAGCN 7 TCCATA motif: each AraC monomer recognizes one of the direct repeats (26 -29).
At the C-terminal end, the regulators of the AraC/XylS family show a highly conserved stretch of about 100 amino acids that seems to be involved in DNA binding and probably in interactions with RNA polymerase (11). One characteristic of members of this family is that they exhibit two possible HTH DNA binding motifs (located at 228 -251 and 281-305 in XylS and at 198 -217 and 246 -264 in AraC). Brunelle and Schleif (15) analyzed these possible HTH motifs with substitutions of several amino acids that should contact DNA in an HTH structure and found evidence for the first one. Niland et al. (30), using synthetic oligonucleotides, systematically substituted the bases in the AraC recognition sequence and did gel retardation assays with mutant AraC in each of the possible HTH elements. They showed that the mutant AraC in each of the HTH motifs exhibited altered DNA binding properties. On the basis of their results, these authors proposed that each AraC monomer binds the 5Ј-TAGC submotif with one of the HTH motifs, and the 3Ј-TCCATA submotif with the second HTH.
If XylS contacts DNA via the two possible HTH elements, all mutant Pm promoters generated in different laboratories can be explained by the following model (Fig. 3): the XylS protein recognizes two submotifs in Pm, TGCA and GGNTA, which are separated from each other by six nucleotides. Each submotif is recognized by the recognition helix of one of the HTH elements of XylS. For the wild-type protein, recognition of direct sequences leads to the formation of a dimer. One monomer recognizes the upstream motif (from Ϫ70 to Ϫ56) with the two HTH DNA binding elements; the second monomer recognizes mainly the TGCA submotif and interacts with the downstream sequence, where it may compete for binding with the RNApolymerase (Fig. 3). Mutations at the Ϫ46/Ϫ49 TGCA submotif result in mutants in which the capacity to activate transcrip-

Tandem Repeats as XylS Targets
tion is impaired because they cannot be contacted properly by XylS. Failure of one of the XylS monomers to interact with this submotif prevents dimer formation and leads to a nonactivable mutant Pm promoter. Pm mutants at the distal TGCA (Ϫ67/ Ϫ70) submotif can be activated weakly by the wild-type XylS protein as a result of the formation of an unstable dimer; however, they can still be induced to a high level of activation by mutant XylS proteins with higher affinity for target sequences than the wild-type regulator (5). This is because one of the XylS mutant monomers binds to the downstream motif (Ϫ49/Ϫ35); and because of the mutation in the XylS, the second monomer is still able to interact well with the GGNTA submotif, and this suffices for dimer formation. The transcriptional activity of mutant Pm promoters with altered GGNTA at Ϫ60/ Ϫ56 sequences is seriously impaired, because failure of one of the monomers to bind correctly prevents dimer stabilization at Pm.