XylS Protein Research Articles

Transcriptional kinetics of the cross-talk between the ortho-cleavage and TOL pathways of toluene biodegradation in Pseudomonas putida mt-2

The TOL plasmid promoters are activated by toluene leading to gene expression responsible for the degradation of the environmental signal. Benzoate is formed as an intermediate, activating the BenR protein of the chromosomal ortho-cleavage pathway that up-regulates the chromosomal PbenA promoter and the TOL Pm promoter resulting in cross-talk between the two networks. Herein, the transcriptional kinetics of the PbenR and PbenA promoters in conjunction with TOL promoters was monitored by real-time PCR during toluene biodegradation of different concentrations in batch cultures. The cross-talk between the two pathways was indicated by the simultaneous maximal expression of the Pm and PbenR promoters, as well as the transcriptional activation from PbenA occurring prior to PbenR, which indicates the potential up-regulation of PbenA by the TOL XylS protein. The repressory effect of toluene on Pr was evident for concentrations higher than 0.3mM suggesting a threshold value for restoring the promoter’s activity, while all the other promoters followed a specific expression pattern, regardless of the initial inducer concentration. Induction of the system with higher toluene concentrations revealed an oscillatory behaviour of Pm, the expression of which remained at high levels until the late exponential phase, demonstrating a novel function of this network.

XylS–Pm Promoter Interactions through Two Helix–Turn–Helix Motifs: Identifying XylS Residues Important for DNA Binding and Activation

The XylS protein is the positive transcription regulator of the TOL plasmid meta-cleavage pathway operon Pm. XylS belongs to the AraC family of transcriptional regulators and exhibits an N-terminal domain involved in effector recognition, and a C-terminal domain, made up of seven α-helices conforming two helix–turn–helix DNA-binding domains. α-Helix 3 and α-helix 6 are the recognition helices. In consonance with XylS structural organization, Pm exhibits a bipartite DNA-binding motif consisting of two boxes, called A and B, whose sequences are TGCA and GGNTA, respectively. This bipartite motif is repeated at the Pm promoter so that one of the XylS monomers binds to each of the repeats. An extensive series of genetic epistasis assays combining mutant Pm promoters and XylS single substitution mutant proteins revealed that α-helix 3 contacts A box nucleotides, whereas α-helix 6 residues contact B box nucleotides. In α-helix 3, Asn246 and Arg242 are involved in specific contacts with the TG dinucleotide at box A, whereas Arg296 and Glu299 contact the second G and T nucleotides at box B. On the basis of our results and of the three-dimensional model of the XylS C-terminal domain, we propose that Ser243, Glu249 and Lys250 in α-helix 3, and Asn299 and Arg302 in α-helix 6 contact the phosphate backbones. Alanine substitutions at the predicted phosphate backbone-contacting residues yielded mutants with low levels of activity, suggesting that XylS–Pm binding specificity not only involves specific amino acid–base interactions, but also relies on secondary DNA structure, which, although at another level, also comes into play. We propose a model in which a XylS dimer binds to the direct repeats in Pm in a head-to-tail conformation that allows the direct interaction of the XylS proximal subunit with the RNA polymerase sigma factor.

Leucines 193 and 194 at the N-terminal domain of the XylS protein, the positive transcriptional regulator of the TOL meta-cleavage pathway, are involved in dimerization.

Members of the AraC/XylS family of transcriptional regulators are usually organized in two domains: a conserved domain made up of 100 amino acids and frequently located at the C-terminal end, involved in DNA binding; and an N-terminal nonconserved domain involved in signal recognition, as is the case for regulators involved in the control of carbon metabolism (R. Tobes and J. L. Ramos, Nucleic Acids Res. 30:318-321, 2002). The XylS protein, which is extremely insoluble, controls expression of the meta-cleavage pathway for alkylbenzoate metabolism. We fused the N-terminal end of XylS to the maltose-binding protein (MBP) in vitro and found in glutaraldehyde cross-linking assays that the protein dimerized. Experiments with a chimeric N-terminal XylS linked to a 'LexA protein showed that the dimer was stabilized in the presence of alkylbenzoates. Sequence alignments with AraC and UreR allowed us to identify three residues, Leu193, Leu194, and Ile205, as potentially being involved in dimerization. Site-directed mutagenesis of XylS in which each of the above residues was replaced with Ala revealed that Leu193 and Leu194 were critical for activity and that a chimera in which LexA was linked to the N terminus of XylSLeu193Ala or XylSLeu194Ala was not functional. Dimerization of the chimeras MBP-N-XylSLeu193Ala and MBP-N-XylSLeu194Ala was not observed in cross-linking assays with glutaraldehyde.

XylS activator and RNA polymerase binding sites at the Pm promoter overlap

Transcription from the TOL plasmid meta-cleavage pathway operon, Pm, depends on the XylS protein being activated by a benzoate effector. The XylS binding sites are two imperfect 5′-TGCAN 6GGNTA-3′ direct repeats located between positions −70/−56 and −49/−35 [González-Pérez et al. (1999) J. Biol. Chem. 274, 2286–2290]. An intrinsic bending of 40°, which is not essential for transcription, is centered at position −43. We have determined the potential overlap between the XylS and RNA polymerase binding sites. The insertion of 2 or more bp between C and T at positions −37 and −36 abolished transcription activation by the wild-type XylS and by XylSS229I, a mutant with increased affinity for the XylS binding sites. In contrast, a 1-bp insertion at −37 was permissible, although when in addition to the 1-bp insertion at −37 the mutant promoter had a point mutation at the XylS binding site (C−47→T), transcription was abolished with the wild-type XylS protein, but not with XylSS229I. The overlap between the proximal XylS binding site and the −35 region recognized by RNA polymerase at positions −35 and −36 appears to be critical for transcription.

Residues 137 and 153 at the N Terminus of the XylS Protein Influence the Effector Profile of This Transcriptional Regulator and the ς Factor Used by RNA Polymerase to Stimulate Transcription from Its Cognate Promoter

The 321-residue XylS and XylS1 proteins, encoded by the pWW0 and pWW53 plasmids respectively, differ in only 5 residues at positions 4, 53, 90, 137, and 153. As a result, the effector profile of XylS is wider than that of XylS1, and XylS mediates higher levels of transcription from its cognate-regulatable promoter than does XylS1. We generated a series of XylS-pWW0 mutants and found that the single mutants Asp-137-->Glu and His-153-->Asn exhibited an activation pattern different from that of the wild-type regulator. In the double-mutant XylSD137E,H153N the effector profile for benzoates was similar to that of XylS1. This suggests that these two residues are crucial for effector recognition and regulator activation to stimulate transcription. XylS-dependent transcription from its cognate promoter is mediated by RNA polymerase with sigma(32) or sigma(38), whereas XylS1 uses RNA polymerase with sigma(32) or sigma(70). We also found that point mutations at positions 137 and 153 of XylS led RNA polymerase to mediate transcription with sigma(70) rather than with sigma(38), as demonstrated by primer extension analysis in a sigma(70)-thermosensitive background proficient and deficient in sigma(38). This suggests that a positive transcriptional regulator can choose the RNA polymerase complex that mediates transcription from a given promoter.

Interactions of the XylS regulators with the C-terminal domain of the RNA polymerase α subunit influence the expression level from the cognate Pm promoter

The Pseudomonas putida meta-cleavage operon encodes the enzymes for the catabolism of alkylbenzoates. Activation of meta-operon transcription is mediated by the XylS protein which, upon activation by effectors, binds two sites between −70 and −35 with respect to the main transcription initiation point at the Pm promoter. Two naturally occurring regulators, XylS and XylS1, that differ by only five amino acids, have been analyzed with regard to potential interactions of these positive regulators with the C-terminal domain of the α subunit of RNA polymerase (α-CTD). For these studies we expressed a derivative of α deprived of the entire C-terminal domain (α-Δ235) and found that expression from Pm with XylS or XylS1 was significantly decreased. To discern whether α-CTD activation depended on interactions with DNA and/or XylS proteins we tested a large collection of alanine substitutions within α-CTD. Most substitutions that had an effect on XylS and XylS1-dependent transcription were located in or adjacent to helix 1 and 4, which are known to be involved in α-CTD interactions with DNA. Two alanine substitutions in helix 3 (residues 287 and 291) identified a putative region of α-CTD/XylS regulator interactions.

Mutational analysis of the highly conserved C-terminal residues of the XylS protein, a member of the AraC family of transcriptional regulators

The XylS protein of the TOL plasmid of Pseudomonas putida belongs to the so-called AraC/XylS family of regulators, that includes more than 100 different bacterial proteins. A conserved stretch of about 100 amino acids is present at the C-terminal end. This conserved region is believed to contain seven α-helices, including two helix-turn-helix (HTH) DNA binding motifs (α 2-T-α 3 and α 5-Tα- 6), connected by a linker α-helix (α 4), and two flanking α-helices (α 1 and α 7). The second HTH motif is the region with the highest homology in the proteins of the family, with certain residues showing almost 90% identity. We have constructed XylS single mutants in the most conserved residues and have analysed their ability to stimulate transcription from its cognate promoter, Pm, fused to ′ lacZ. The analysis revealed that mutations in the α 5-helix conserved residues had little effect on the XylS transcriptional activity, whereas the distribution of polarity in the α 6-helix was important for the activity. The strongest effect of the mutations was observed in conserved residues located outside the DNA binding domain, namely, Gly-290 in the turn between the two helices, Pro-309 located downstream of α 6, and Leu-313, in the small last helix α 7, that seems to play an important role in the activation of RNA-polymerase. Our analysis shows that conservation of amino acids in the family reflects structural requirements rather than functionality in specific DNA interactions.

Functional domains of the TOL plasmid transcription factor XylS.

The alkylbenzoate degradation genes of Pseudomonas putida TOL plasmid are positively regulated by XylS, an AraC family protein, in a benzoate-dependent manner. In this study, we used deletion mutants and hybrid proteins to identify which parts of XylS are responsible for the DNA binding, transcriptional activation, and benzoate inducibility. We found that a 112-residue C-terminal fragment of XylS binds specifically to the Pm operator in vitro, protects this sequence from DNase I digestion identically to the wild-type (wt) protein, and activates the Pm promoter in vivo. When overexpressed, that C-terminal fragment could activate transcription as efficiently as wt XylS. All the truncations, which incorporated these 112 C-terminal residues, were able to activate transcription at least to some extent when overproduced. Intactness of the 210-residue N-terminal portion was found to be necessary for benzoate responsiveness of XylS. Deletions in the N-terminal and central regions seriously reduced the activity of XylS and caused the loss of effector control, whereas insertions into the putative interdomain region did not change the basic features of the XylS protein. Our results confirm that XylS consists of two parts which probably interact with each other. The C-terminal domain carries DNA-binding and transcriptional activation abilities, while the N-terminal region carries effector-binding and regulatory functions.

Protein binding in vivo to OP2 promoter of the Pseudomonas putida TOL plasmid.

The transcription of OP2 encoding enzymes for m-toluate catabolism on the Pseudomonas putida TOL plasmid is activated by basal-level XylS protein in the presence of m-toluate or by overproduced XylS protein in the absence of m-toluate. In this study, in vivo dimethyl sulfate (DMS) footprinting was performed to understand the mechanism of transcriptional regulation of OP2 promoter by XylS. In the presence of overproduced XylS without m-toluate, several protected nucleotides were observed, indicating the binding of RNA polymerase to DNA. However, the protection was canceled upon addition of m-toluate. These results suggest that RNA polymerase is retained by XylS on the OP2 promoter in the absence of inducer, and is released by m-toluate binding to XylS, concomitant with transcription.

Transcriptional control of the Pseudomonas TOL plasmid catabolic operons is achieved through an interplay of host factors and plasmid-encoded regulators.

The xyl genes of Pseudomonas putida TOL plasmid that specify catabolism of toluene and xylenes are organized in four transcriptional units: the upper-operon xylUWCAMBN for conversion of toluene/xylenes into benzoate/alkylbenzoates; the meta-operon xylXYZLTEGFJQKIH, which encodes the enzymes for further conversion of these compounds into Krebs cycle intermediates; and xylS and xylR, which are involved in transcriptional control. The XylS and XylR proteins are members of the XylS/AraC and NtrC families, respectively, of transcriptional regulators. The xylS gene is constitutively expressed at a low level from the Ps2 promoter. The XylS protein is activated by interaction with alkylbenzoates, and this active form stimulates transcription from Pm by sigma70- or sigmaS-containing RNA polymerase (the meta loop). The xylR gene is also expressed constitutively. The XylR protein, which in the absence of effectors binds in a nonactive form to target DNA sequences, is activated by aromatic hydrocarbons and ATP; it subsequently undergoes multimerization and structural changes that result in stimulation of transcription from Pu of the upper operon. This latter process is assisted by the IHF protein and mediated by sigma54-containing RNA polymerase. Once activated, the XylR protein also stimulates transcription from the Ps1 promoter of xylS without interfering with expression from Ps2. This process is assisted by the HU protein and is mediated by sigma54-containing RNA polymerase. As a consequence of hyperexpression of the xylS gene, the XylS protein is hyperproduced and stimulates transcription from Pm even in the absence of effectors (the cascade loop). The two sigma54-dependent promoters are additionally subject to global (catabolite repression) control.

Expression of the TOL plasmid xylS gene in Pseudomonas putida occurs from a alpha 70-dependent promoter or from alpha 70- and alpha 54-dependent tandem promoters according to the compound used for growth.

Growth of Pseudomonas putida (pWWO) on alkylbenzoates requires the expression of the meta pathway operon, which is mediated by the XylS protein after binding of a benzoate effector. Alternatively, in cells growing on toluene or its aromatic alcohols, overexpression of xylS mediated by XylR activated by these compounds leads to overproduction of the XylS regulator, which even in the absence of benzoate effectors stimulates transcription from the meta cleavage pathway operon promoter. We show here that in bacteria growing on glycerol or alkylbenzoates, the xylS gene is expressed at a low but constitutive level from a newly found sigma 70-dependent promoter called Ps2. The amount of XylS protein made from the transcript originated from Ps2 was sufficient to allow high levels of expression from the meta cleavage pathway operon promoter when the cells were grown in the presence of 3-methylbenzoate. The transcription initiation point of the transcript generated from Ps2 mapped 9 bp upstream from the proposed ATG of the xylS gene; this transcript contains the ribosome-binding site. The Ps2 promoter was located 110 bp downstream from a previously described sigma54-dependent promoter located upstream from the xylS open reading frame, now called Ps1. In cells growing on toluene or benzyl alcohols, the XylS regulator is overproduced as a consequence of increased expression of the gene through the effect of the two promoters working in tandem: the newly found sigma 70-dependent promoter, whose expression is XylR and toluene independent, and the sigma 54-dependent promoter, whose expression is dependent on XylR activated by its effectors. This expression pathway of the xylS gene explains why sigma 54-deficient P. putida bearing the wild-type TOL plasmid, or the wild-type P. putida strain bearing a TOL plasmid with a knocked-out xylR gene, can grow on alkylbenzoates. Until now this has been one of the unresolved paradoxes in the transcriptional control of the TOL meta cleavage pathway.

Biosensing of benzene derivatives in the environment by luminescent Escherichia coli

Sensitive and convenient biosensing of environmental pollutants has been developed by fusing a gene of firefly luciferase to the TOL plasmid. TOL plasmid of Pseudomonas putida encodes a series of enzymes for degradation of benzene and its derivatives. The expression of these enzymes is controlled with the regulating proteins xylR and xylS, whose promoters are activated in the presence of aromatic compounds. The structural gene of firefly luciferase, as a reporter enzyme, was inserted under the control of the promoter of xylS protein, and gene fusion plasmid pTSN316 was constructed. The recombinant Escherichia coli transformed with this plasmid was applied to the environmental biosensing of benzene derivatives. The expression of luciferase was induced in the presence of aromatic compounds and the lower detection limit for m-xylene was 5 μM.

The organization of the Pm promoter of the TOL plasmid reflects the structure of its cognate activator protein XylS.

The toluate catabolic operon carried by the TOL plasmid pWW0 of Pseudomonas putida is positively regulated by the benzoate-responsive XylS protein which, when activated, stimulates transcription from the operon promoter Pm. In this study, the mode in which XylS effects the activity of the Pm promoter was examined in vivo by genetic analysis of both protein and promoter variants. Substitution of His31Asp/Ser32Pro,Leu113Pro,Phe214Leu/Glu215A sp/Arg216Pro or Thr312Pro, all predicted to disrupt the secondary structure, renders XylS inactive and unable to compete with the native protein for activation of Pm. In contrast, Pro substitutions at Ser64 or Gly160 had no or only minor effects on XylS activity, and a protein with residual capacity to activate Pm resulted when Glu11 was altered to Pro. None of a collection of truncated variants, of XylS protein deleted for more than 10 terminal amino acids retained activity or were able to negatively interact the wild-type XylS protein. These data indicate that amino- and carboxy-terminal portions of the protein sequence depend on each other for the maintenance of their functional structure, rather than being arranged as independent domains. Pm promoter derivatives, in which the two half-sites of the direct repeat sequence that confers responsiveness to XylS were separated and/or inverted by one-half or one complete DNA helix turn, lost responsiveness in vivo. These results favour a model for XylS-mediated activation of Pm in which a tandem dimer is the protein form that binds the Pm promoter.

Analysis of the mRNA structure of the Pseudomonas putida TOL meta fission pathway operon around the transcription initiation point, the xylTE and the xylFJ regions

The 13 genes encoded by the meta-cleavage operon (approx. 11 kb) of Pseudomonas putida TOL plasmid pWW0 are transcribed from a single promoter, Pm. In P. putida, transcription from Pm was strictly dependent on the presence of effector-activated XylS protein. Three regions of the transcript were analyzed in the wild-type strain of P. putida (pWW0) by S1 nuclease protection and primer extension analyses. A major point of transcription initiation was found in the most 5′-end of the operon, which defined a −10 −35 promoter. Another main mRNA 5′-end point located within the coding sequence of the first gene of the operon was detected and identified as a stable endonucleolytic cleavage product, which rendered this gene inactive for further translation. The estimated half-life of this leader region was 2 min. Analyses of the transcripts at xylLTE and xylFJ regions revealed the presence of several relatively stable mRNA products with 5′-ends located along these regions, which arose from specific endonucleolytic mRNA cleavage. The degradation of the stable intermediate products observed after the addition of rifampicin was similar for all the products, presenting a delay of approx. 10 min, followed by an exponential decay, with a half-life that ranged between 6 and 10 min.

Identification of critical amino-terminal regions of XylS. The positive regulator encoded by the TOL plasmid

The XylS protein is the positive regulator of the promoter controlling the meta-cleavage pathway (Pm) for catabolism of certain alkylbenzoates on the TOL plasmid of Pseudomonas. Transcription from Pm is mediated by XylS either in the presence of benzoate effectors or through XylS hyperproduction. Two regions of the NH2 terminus of XylS (residues 37-45) had been predicted to be involved in effector control of XylS transcriptional activation. Different methods were used to induce mutations in this region, including genetic selections (where Pm controlled a tetracycline resistance gene), bisulfite mutagenesis at a unique restriction site, and extensive oligonucleotide mutagenesis at residues 41 and 45. The mutants fell into four classes based on their phenotypes with respect to effector-mediated activation of a Pm-lacZ fusion: (a) effector profiles similar to wild type, (b) no Pm stimulation with benzoates, (c) altered effector specificity, and (d) higher basal Pm activities, in some cases including changes in effector specificity. In some mutants, higher basal Pm activity was apparently due to mutations that increased XylS stability. Substitutions at Arg-41 resulted in all four mutant phenotypes, indicating that this is a critical residue in XylS for effector stimulation of transcription activity.

XylS domain interactions can be deduced from intraallelic dominance in double mutants of Pseudomonas putida.

The XylS protein is the positive regulator of the TOL plasmid-encoded meta-cleavage pathway for the metabolism of alkylbenzoates in Pseudomonas putida. This protein is activated by a variety of benzoate analogues. To elucidate the functional domains of the regulator and their interactions, several fusions of the XylS C-terminus to MS2 polymerase and of the N-terminus to beta-galactosidase were constructed but all are inactive. In addition, 15 double mutant xylS genes were constructed in vitro by fusing parts of various mutant genes to produce mutant regulators exhibiting C-terminal and N-terminal amino acid substitutions. The phenotypic properties of the parental single mutant genes, and those of the double mutant genes, suggest that the C-terminal region is involved in binding to DNA sequences at the promoter of the meta-cleavage pathway operon, and that the benzoate effector binding pocket includes critical residues present at both the N-terminal and C-terminal ends of the protein. The intraallelic dominance of the Ile229 (Ser229-->Ile) and Val274 (Asp274-->Val) substitutions over the N-terminal His41 (Arg41-->His) substitution, and the intraallelic dominance of Thr45 (Arg45-->Thr) over Ile229 and Val274, support the proposal that these two regions of the regulator interact functionally. Combination of the Leu88 (Trp88-->Leu) and Arg256 (Pro256-->Arg) substitutions did not suppress the semiconstitutive phenotype conferred by Leu88, but resulted in a protein with altered ability to recognize benzoates. In contrast, the Leu88 semiconstitutive phenotype was suppressed by Val288 (Asp288-->Val), and the double mutant was susceptible to activation by benzoates.(ABSTRACT TRUNCATED AT 250 WORDS)

Mutations leading to constitutive expression from the TOL plasmid meta-cleavage pathway operon are located at the C-terminal end of the positive regulator protein XylS

The XylS protein is the positive activator of the TOL plasmid meta-cleavage pathway operon for the metabolism of alkylbenzoates in Pseudomonas putida. The regulator stimulates transcription from the TOL meta pathway operon promoter (Pm) when activated by benzoate effectors or in the absence of effectors when overproduced. xylS mutant alleles that encode regulators which constitutively mediate expression from Pm were isolated and characterized. The mutant proteins all exhibit single amino acid substitutions adjacent to putative alpha-helix-turn-alpha-helix domains at their C-terminal ends. The XylS mutant proteins can still be partially activated by the usual and unusual benzoate effectors for the wild-type regulator and when activated stimulate higher levels of transcription from Pm.

Signal-regulator interactions, genetic analysis of the effector binding site of xyls, the benzoate-activated positive regulator of Pseudomonas TOL plasmid meta-cleavage pathway operon

This study reports a genetic analysis of the interactions between a positive regulator of gene expression and its effector molecules. Transcription of the TOL plasmid meta-cleavage pathway operon is specifically stimulated by the XylS protein positive regulator either through activation of this regulator by benzoate effectors or through its hyperproduction. One xylS mutant that exhibits constitutive expression of the operon promoter has been characterized, together with six mutants encoding altered XylS proteins that recognize as effectors benzoate analogues that are non-effectors for the XylS wild-type protein. The changes in two mutant regulators are located at the N-terminal end of the protein, within a putative β-pleated domain. These mutant proteins exhibit a markedly increased affinity for normal benzoate effectors, with K′ s values fivefold to 60-fold lower than those of the wild-type XylS protein. They are additionally activated by new effectors having certain substituents at position 2, 3 and 4 of the aromatic ring. Two other mutant proteins recognize new effectors having substituents at position 4 and 5 of the aromatic ring, and contain mutations at their C-terminal end within a putative α-helix-rich domain. Three other mutations, one of which leads to constitutive expression from Pm, each result in an amino acid change in the central region of the regulator. These findings suggest but do not prove that the effector binding pocket of the XylS protein may be composed of two or more non-contiguous segments of its primary structure. The XylS protein exhibits homology with the AraC protein of Escherichia coli, a protein that stimulates transcription from ara promoters when it is activated by arabinose or benzoate. Mutations influencing effector activation of the XylS protein characterized in this study are all located in regions exhibiting a high degree of homology with the corresponding aligned sequence of AraC protein.

Regulatory circuits controlling transcription of TOL plasmid operon encoding meta‐cleavage pathway for degradation of alkylbenzoates by Pseudomonas

TOL plasmid pWWO of Pseudomonas putida contains two operons that specify a pathway for the degradation of aromatic hydrocarbons. The 'upper' operon encodes enzymes for the oxidation of toluene to benzoate and xylenes to toluates, whereas the meta-cleavage operon specifies the further oxidation of benzoate and toluates. Transcription of the upper pathway operon is positively regulated by the XylR protein, which is activated by toluene/xylenes and their alcohol catabolic products, in combination with the NtrA protein, a sigma factor. Expression of the meta-operon is positively controlled by the XylS protein which is activated by meta-pathway substrates, and is independent of NtrA protein. Expression of the meta pathway is also induced by toluene/xylene-activated XylR protein via a cascade regulatory system in which this protein in combination with NtrA protein stimulates transcription from the xylS gene promoter. Hyper-production of XylS protein in turn provokes high level expression of the meta-operon, which is independent of meta-pathway substrates. The two promoters, which are activated by the XylR and NtrA proteins, the upper pathway promoter and the xylS gene promoter, exhibit three regions of homology centred at -12(5'-TTGCATG-3'), -24(5'-TGGCPuT-3') and -45(5'-TAAAATAAGPuPuCGPuTC-3'), with respect to their principal transcription initiation points. The possible physiological significance of activated XylR-protein-induced expression of the meta-operon through amplification of XylS protein levels is considered.

Nucleotide sequence of the regulatory gene xylS on the Pseudomonas putida TOL plasmid and identification of the protein product

The xylS gene is a regulatory gene which positively controls expression of the genes on the TOL plasmid for degradation enzymes of benzoate or m-toluate in Pseudomonas putida. Cloning of the gene in Escherichia coli and determination of the nucleotide sequence revealed an open reading frame of 963 bp which corresponds to a protein with an M r of 36 502. The xylS gene was recloned onto a tac-promoter vector, and the product was identified by the maxicell procedure as a protein with an approximate M r of 37000. The predicted amino acid sequence of XylS protein showed a basic character and contained a region similar to those in other DNA-binding proteins.