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Critical Nucleotides in the Upstream Region of the XylS-dependent TOL meta-Cleavage Pathway Operon Promoter as Deduced from Analysis of Mutants

1999; Elsevier BV; Volume: 274; Issue: 4 Linguagem: Inglês

10.1074/jbc.274.4.2286

1083-351X

M.-Mar González-Pérez, Juan L. Ramos, Marı́a-Trinidad Gallegos, Silvia Marqués,

Microbial Natural Products and Biosynthesis

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 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. base pair(s) helix-turn-helix 3-methylbenzoate promoter for the TOLmeta-pathway. The TOL plasmid pWW0 of Pseudomonas putida specifies ameta-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 thexylS gene product, which is activated by benzoate effectors (1Franklin F.C.H. Bagdasarian M. Bagdasarian M.M. Timmis K.N. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 7458-7462Crossref PubMed Scopus (395) Google Scholar, 2Inouye S. Nakazawa A. Nakazawa T. J. Bacteriol. 1981; 148: 413-418Crossref PubMed Google Scholar, 3Ramos J.L. Stolz A. Reineke W. Timmis K.N. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 8467-8471Crossref PubMed Scopus (115) Google Scholar, 4Ramos J.L. Marqués S. Timmis K.N. Annu. Rev. Microbiol. 1997; 31: 341-374Crossref Scopus (271) Google Scholar). Stimulation of transcription from the Pm promoter requires a DNA sequence extending to about 80 bp1 upstream of the transcription initiation point (5Gallegos M.T. Marqués S. Ramos J.L. J. Bacteriol. 1996; 178: 6427-6434Crossref PubMed Google Scholar, 6Kessler B. de Lorenzo V. Timmis K.N. J. Mol. Biol. 1993; 230: 699-703Crossref PubMed Scopus (39) Google Scholar, 7Ramos J.L. Mermod N. Timmis K.N. Mol. Microbiol. 1987; 1: 297-300Crossref Scopus (75) Google Scholar). 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 −40 to −80 bp (Fig. 1); and the downstream RNA polymerase recognition region, which exhibits atypical −35 and −10 DNA sequences (Fig. 1). Transcription from the Pm promoter in the early exponential growth phase is mediated by RNA polymerase with ς32, and later with ςS, although regardless of the growth phase, expression from Pm remains dependent on XylS, and the transcription initiation point is the same (8Marqués S. Gallegos M.T. Ramos J.L. Mol. Microbiol. 1995; 18: 851-857Crossref PubMed Scopus (34) Google Scholar, 9Marqués S. Manzanera M. González-Pérez M.M. Gallegos M.T. Ramos J.L. Mol. Microbiol. 1999; (in press)PubMed Google Scholar). Kessler et al. (6Kessler B. de Lorenzo V. Timmis K.N. J. Mol. Biol. 1993; 230: 699-703Crossref PubMed Scopus (39) Google Scholar) proposed that the XylS binding region in Pm was organized as two homologous 15-bp tandemly imperfect directly repeated motifs (5′-TGCAAPuAAPyGGNTA-3′). The distal one with respect to the RNA polymerase region extends from −70 to −56, and the proximal one extends from −49 to −35 (Fig. 1). Gallegos et al. (5Gallegos M.T. Marqués S. Ramos J.L. J. Bacteriol. 1996; 178: 6427-6434Crossref PubMed Google Scholar) 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. (6Kessler B. de Lorenzo V. Timmis K.N. J. Mol. Biol. 1993; 230: 699-703Crossref PubMed Scopus (39) Google Scholar) might suffice for XylS activation of transcription. These workers found that promoters that had been deleted up to −60 could be activated by constitutive XylS mutants (but not by the wild-type regulator) and that extension of the deletion to −51 prevented transcription. Gallegos et al.(5Gallegos M.T. Marqués S. Ramos J.L. J. Bacteriol. 1996; 178: 6427-6434Crossref PubMed Google Scholar) proposed that the XylS binding site is represented by the motif T(C/A)CAN4TGCA, which appears twice in the promoter sequence, between −46 and −57 and between −67 and −78 (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. Escherichia coli MC4100 was grown at 30 °C in Luria-Bertani medium supplemented, when required, with 100 μg/ml ampicillin, 25 μg/ml kanamycin, or 50 μg/ml streptomycin. The plasmids used in this study, and previously constructed were: pERD103, which is an IncQ plasmid encoding kanamycin resistance (7Ramos J.L. Mermod N. Timmis K.N. Mol. Microbiol. 1987; 1: 297-300Crossref Scopus (75) Google Scholar); pJLR100, which is a pEMBL9 derivative bearing the Pm promoter cloned between the EcoRI and HindIII sites (3Ramos J.L. Stolz A. Reineke W. Timmis K.N. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 8467-8471Crossref PubMed Scopus (115) Google Scholar); pMD1405, which carries a promoterless ′lacZ gene and encodes resistance to ampicillin; and pJLR107, which is a pMD1405 derivative bearing the Pm promoter in front of ′lacZ (3Ramos J.L. Stolz A. Reineke W. Timmis K.N. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 8467-8471Crossref PubMed Scopus (115) Google Scholar). 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$$Google Scholar). DNA was methylated in vitro with 2 mm dimethyl sulfate as described (10$$Google Scholar). 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$$Google Scholar). Plasmid DNA was extracted by using the Qiagen Kit (Quiagen, GmbH, Hilden, Germany). The oligonucleotide 5′-CGTCTAAGAAACCATTATTATCAG-3′ was complementary to the noncoding strand upstream from the Pm promoter region. The first C at the 5′-end was located 218 bp from the +1 of the transcription initiation point. The oligonucleotide 5′-GGGTCGGTGAACATCTCGCGCTTGC-3′ was complementary to the coding strand downstream from the Pm promoter. The first G at the 5′-end was located 124 bp from the +1 of the start of the transcript. For primer extension with Taq-DNA-polymerase, about 2 × 105 cpm of the corresponding oligonucleotide end-labeled with 32P was used. The extension products were separated by electrophoresis on urea-polyacrylamide sequencing gels. The Pm mutant promoters were generated by overlap extension polymerase chain reaction mutagenesis as described (5Gallegos M.T. Marqués S. Ramos J.L. J. Bacteriol. 1996; 178: 6427-6434Crossref PubMed Google Scholar). 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 (5′-CAGGAAACAGCTATGACCATG-3′) or a primer complementary to the α fragment of the lacZ gene (5′-GATGTGCTGCAAGGCGATTAAGTTA-3′). After DNA amplification, the resulting DNA was digested withEcoRI and HindIII, and the 401-bpEcoRI-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. E. coli bearing the wild-type Pm::′lacZ or mutant Pm*::′lacZ 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, β-galactosidase activity was determined in duplicate in permeabilized cells (5Gallegos M.T. Marqués S. Ramos J.L. J. Bacteriol. 1996; 178: 6427-6434Crossref PubMed Google Scholar). The reactivity of guanine residues in the Pm promoter toward dimethyl sulfate was assayedin 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 thein vitro and in vivo methylation pattern is hypermethylation of the T located at −42. This indicates that the DNA was distorted at this point, probably due to the tracks of As in the −41 to −46 region in the top strand (Fig.1). The methylation of T−42 in vivo in cells without XylS was more pronounced thanin 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−42 was highest in the presence of the effector, whereas in the absence of the effector, T−42 appeared to be more protected than in cells without XylS (Fig. 2). In contrast with this behavior was the observation that Gs in the −47 to −61 region appeared to be protected in the presence of the effector. However, G−68 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 −3 to −40 region showed a definite pattern of methylation. The Gs in the −3 to −28 region were more methylated in vitro than in vivo. In particular, G−23 and G−38 were clearly protected in vivo regardless of the presence of XylS and the presence of an effector. 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 (11Gallegos M.T. Schleif R. Bairoch A. Hofmann K. Ramos J.L. Microbiol. Mol. Biol. Rev. 1997; 61: 393-410Crossref PubMed Scopus (657) Google Scholar). For this reason, we decided to carry out initial block scanning mutagenesis assays of the Pm region between −41 and −78. This interval includes all nucleotides previously proposed as important in XylS for Pm recognition (5Gallegos M.T. Marqués S. Ramos J.L. J. Bacteriol. 1996; 178: 6427-6434Crossref PubMed Google Scholar, 6Kessler B. de Lorenzo V. Timmis K.N. J. Mol. Biol. 1993; 230: 699-703Crossref PubMed Scopus (39) Google Scholar, 7Ramos J.L. Mermod N. Timmis K.N. Mol. Microbiol. 1987; 1: 297-300Crossref Scopus (75) Google Scholar) and the region in which in vivofootprinting analyses revealed alterations in the methylation pattern. The interval excluded mutations in the putative −35 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 (−46/−49, −54/−57, −67/−70, and −75/−78), the intervening sequences between two adjacent TNCA sequences (−50/−53, −58/−61, −62/−65, −63/−66, and −71/−74), a sequence between the TNCA sequence closer to the RNA polymerase binding site (−46/−49), and the −41 nucleotide (−41/−44). In all cases the mutant promoters (Pm*) were fused to ′lacZ in pMD1405. β-Galactosidase activity was determined in E. coli MC4100 (Pm*::′lacZ, 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 (TableI): 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 wild-type or near wild-type XylS-dependent inducible β-galactosidase activity,i.e. ≥80% of wild-type levels.Table IBlock scanning mutagenesis of the upstream region of the Pm promoter and its effect on its transcriptional activityPromoterβ-Galactosidase activityActivity of wild-typeunits%Pm wild-type8900100Pm 250 (−44/−41 AAAA → CCCT)385043Pm 251 (−45/−42 AAAA → TCAA)560062Pm 252 (−45/−42 AAAA → CCAA)555062Pm 206 (−49/−46 TGCA → GTCA)170019Pm 207 (−49/−46 TGCA → GAGG)5506Pm 208 (−49/−46 TGCA → GTGT)3504Pm 209 (−49/−46 TGCA → ACAG)5006Pm 253 (−49/−46 TGCA → GGCG)120013Pm 254 (−49/−46 TGCA → GGCC)6007Pm 255 (−49/−46 TGCA → TCGA)5005Pm 230 (−53/−50 GGAG → CACC)813091Pm 257 (−53/−50 GGAG → AAAG)860097Pm 212 (−57/−54 TACA → GCAG)210024Pm 213 (−57/−54 TACA → GTTT)800089Pm 259 (−61/−58 CGGA → CCAA)350039Pm 260 (−61/−58 CGGA → CTCA)270030Pm 261 (−61/−58 CGGA → CGCA)450051Pm 264 (−66/−63 GAAA → GAGT)440050Pm 267 (−66/−63 GAAA → CTAA)650073Pm 268 (−66/−63 GAAA → GGAA)9900111Pm 222 (−70/−67 TGCA → ACGT)560063Pm 219 (−70/−67 TGCA → AACA)220025Pm 220 (−70/−67 TGCA → AGCA)290033Pm 233 (−74/−71 GCCT → TCCG)8900100Pm 226 (−78/−75 TCCA → GATT)740083Pm 245 (−49/−46, TGCA → AGGA; −57/−54 TACA → TTGG)601Pm 244 (−57/−54 TACA → GTTT; −67/−70, TGCA → ACGT)150016E. coli MC4100 bearing the indicated Pm*:′lacZfusion and xylS in pERD103 were grown on LB medium with 3MB as described under "Materials and Methods." In the absence of 3MB, basal activity was 50–150 Miller units. The data correspond to induced levels and are averages of at least six independent determinations, with standard deviations below 20% of the given values. Open table in a new tab E. coli MC4100 bearing the indicated Pm*:′lacZfusion and xylS in pERD103 were grown on LB medium with 3MB as described under "Materials and Methods." In the absence of 3MB, basal activity was 50–150 Miller units. The data correspond to induced levels and are averages of at least six independent determinations, with standard deviations below 20% of the given values. 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 −46/−49 (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 −41/−45, the TACA sequence between −54/−57, the CGGA sequence between −58/−61, and the TGCA sequence between −67/−70 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 −46/−49 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 −50/−53, −62/−65, −63/−66, −71/−74, and −75/−78. 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 −46/−49 (TGCA→AGGA) with a block of mutations at −54/−57 (TACA→TTGG) resulted in a mutant Pm promoter (Pm 245) that had no activity at all (Table I). At the −54/−57 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.−67/−70 (TGCA → 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. 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−51 → G, A−64 → C, G−65 → A, A−75 → C, C−77 → G, and T−78 → G. These mutations, as expected, had little or no effect (<20%) on XylS-dependent transcription from Pm (not shown). Of the less critical boxes (−41/−44, −54/−57, −58/−61, and −67/−70), some of the substitutions (for example, A−42→ T, A−44 → G, A−54 → G, A−54 → T, G−69 → C, and G−69→ T) did not significantly affect activity from Pm (TableII); others had an intermediate effect, reducing the induced XylS-dependent activation of Pm by 20–50%. This effect was observed for the changes C−55→ A, C−55 → G, A−67 → C, and A−67 → T (Table II). Within these sequences, the change T−70 → G resulted in loss of almost 90% of the activity (Table II).Table IISingle point mutations at the Pm promoterPromoterLocation and base changedβ-Galactosidase activityActivity of wild-typeunits%PmWild-type8900100Pm270−42A → T810091Pm 5D−44A → G620070Pm201−46A → T360040Pm203−47C → G185021Pm204−47C → T370042Pm202−48G → C140016Pm205−48G → A9300104Pm200−49T → G205023Pm272−54A → G725081Pm273−54A → T770087Pm274−54A → C550062Pm275−55C → A410046Pm276−55A → G615069Pm277−56C → C9700109Pm108−57T → G955040Pm278−59G → C180020Pm279−60G → T160018Pm280−60G → A7008Pm282−62G → C1001Pm283−63A → C636071Pm287−67A → C450051Pm4−68C → G215024Pm216−69G → C9200103Pm217−69G → T9000101Pm215−70T → G100011Conditions were as in the legend for Table I except that the mutant Pm promoters were those indicated below. Open table in a new tab Conditions were as in the legend for Table I except that the mutant Pm promoters were those indicated below. In the −58/−61 box, the G−59 → C, G−60→ T, and G−60 → A changes had a significant effect on transcription, as shown by the finding that β-galactosidase activity was less than 20% of that seen with the wild-type Pm promoter. A surprising finding was that the G−62 → C change resulted in a mutant Pm promoter that lacked activity. In the critical −46/−49 set of bases, single bp substitutions had a significant effect on activity. The changes A−46 → T, C−47 → G or C−47 → T, G−48→ C, and T−49 → G resulted in a 60–85% decrease in activity. However, the change G−48 → A had little or no effect on transcriptional activity (Table II). When we compared the critical sequences proposed by Kessler et al. (6Kessler B. de Lorenzo V. Timmis K.N. J. Mol. Biol. 1993; 230: 699-703Crossref PubMed Scopus (39) Google Scholar) and Gallegoset al. (5Gallegos M.T. Marqués S. Ramos J.L. J. Bacteriol. 1996; 178: 6427-6434Crossref PubMed Google Scholar), we found that they had the two TGCA submotifs at −46/−49 and −67/−70 in common (Fig. 1). The hypothesis of Gallegoset al. (5Gallegos M.T. Marqués S. Ramos J.L. J. Bacteriol. 1996; 178: 6427-6434Crossref PubMed Google Scholar) suggested that the TACA submotif between −54/−57 was critical for transcription activation from Pm, whereas Kessler et al. (6Kessler B. de Lorenzo V. Timmis K.N. J. Mol. Biol. 1993; 230: 699-703Crossref PubMed Scopus (39) Google Scholar) suggested that only the −57/−56 nucleotides were of importance (Fig. 1). Our results with single point mutations in the −54 to −57 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 −54/−55, −55/−56, −56/−57, and −54/−57 positions. We found that the −54/−55 changes (CA → AC or CA → GT) did not influenced the level of β-galactosidase activity from the mutant Pm promoters. In contrast, the −57/−56 TA → AT change almost completely prevented activity (99% decrease). The combination of mutations at −55/−56 and −54/−57 had an intermediate effect, with activities in the range of 15–38% when the mutation involved the −57 position, and in the range of 40–80% when the changed involved the −56 position (not shown). From these results, we deduced that the −57/−56 bases are critical for the activity of Pm. The XylS protein (12Inouye S. Nakazawa A. Nakazawa T. Gene. 1986; 44: 235-242Crossref PubMed Scopus (40) Google Scholar, 13Mermod N. Ramos J.L. Bairoch A. Timmis K.N. Mol. Gen. Genet. 1987; 207: 349-354Crossref PubMed Scopus (43) Google Scholar, 14Spooner R.A. Bagdasarian M. Franklin F.C.H. J. Bacteriol. 1987; 169: 3581-3586Crossref PubMed Google Scholar) 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 (11Gallegos M.T. Schleif R. Bairoch A. Hofmann K. Ramos J.L. Microbiol. Mol. Biol. Rev. 1997; 61: 393-410Crossref PubMed Scopus (657) Google Scholar). 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 functionin 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 (15Brunelle A. Schleif R. J. Mol. Biol. 1989; 209: 607-622Crossref PubMed Scopus (73) Google Scholar, 16Caswell R. Williams J. Lyddiatt A. Busby S. Biochem. J. 1992; 287: 493-499Crossref PubMed Scopus (21) Google Scholar, 17Egan S.M. Schleif R.F. J. Mol. Biol. 1993; 234: 87-98Crossref PubMed Scopus (90) Google Scholar, 18Eustance R.J. Bustos S.A. Schleif R. J. Mol. Biol. 1994; 242: 330-338PubMed Google Scholar, 19Fawcett W.P. Wolf Jr., R.E. J. Bacteriol. 1995; 177: 1742-1750Crossref PubMed Google Scholar, 20Li Z. Demple B. Mol. Microbiol. 1996; 20: 937-945Crossref PubMed Scopus (80) Google Scholar, 21Lobell R.B. Schleif R.F. Science. 1990; 250: 528-532Crossref PubMed Scopus (188) Google Scholar). 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 −70 position in the 5′-end to at least the −41 position in the 3′ end. Our mutational analysis revealed that within this stretch the XylS recognition sequence seems to be TGCAN6GGNTA, which appears twice in the Pm promoter, between −70 and −56 and between −49 and −35 (Fig. 1). In favor of this proposal is the observation that a tagged XylS-protein immunoadsorbed onto glass beads produced footprintsin vitro, which showed protection of the Gs within the above direct repeat at −48, −59, −60, and −69 (22Kaldalu N. Mandel T. Ustav M. Mol. Microbiol. 1996; 20: 569-579Crossref PubMed Scopus (25) Google Scholar). This is in agreement with our in vivo results. The AraC protein—the best characterized regulator of the family—stimulates transcription as a dimer (23Bustos S.A. Schleif R.F. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5638-5642Crossref PubMed Scopus (117) Google Scholar, 24Menon K.P. Lee N.L. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 3708-3712Crossref PubMed Scopus (31) Google Scholar, 25Schleif R. McKnight S.L. Yamamoto K.R. Transcriptional Regulation. 2. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1992: 643-665Google Scholar). The consensus sequence for AraC-activable promoters is a direct repetition of the TAGCN7TCCATA motif: each AraC monomer recognizes one of the direct repeats (26Brunelle A. Schleif R. J. Mol. Biol. 1989; 209: 607-622Crossref PubMed Scopus (74) Google Scholar, 27Carra J.H. Schleif R.F. EMBO J. 1993; 12: 35-44Crossref PubMed Scopus (104) Google Scholar, 28Lobell R.B. Schleif R.F. J. Mol. Biol. 1991; 218: 45-54Crossref PubMed Scopus (62) Google Scholar, 29Reeder T. Schleif R. J. Mol. Biol. 1993; 231: 205-218Crossref PubMed Scopus (46) Google Scholar). 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 (11Gallegos M.T. Schleif R. Bairoch A. Hofmann K. Ramos J.L. Microbiol. Mol. Biol. Rev. 1997; 61: 393-410Crossref PubMed Scopus (657) Google Scholar). 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 (15Brunelle A. Schleif R. J. Mol. Biol. 1989; 209: 607-622Crossref PubMed Scopus (73) Google Scholar) 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. (30Niland P. Hühne R. Müller-Hill B. J. Mol. Biol. 1996; 264: 667-674Crossref PubMed Scopus (56) Google Scholar), 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 5′-TAGC submotif with one of the HTH motifs, and the 3′-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 −70 to −56) 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 RNA-polymerase (Fig. 3). Mutations at the −46/−49 TGCA submotif result in mutants in which the capacity to activate transcription 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 (−67/−70) 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 (5Gallegos M.T. Marqués S. Ramos J.L. J. Bacteriol. 1996; 178: 6427-6434Crossref PubMed Google Scholar). This is because one of the XylS mutant monomers binds to the downstream motif (−49/−35); 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 −60/−56 sequences is seriously impaired, because failure of one of the monomers to bind correctly prevents dimer stabilization at Pm.