INTRODUCTION

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Ralstonia pickettii PKO1 has been investigated by our laboratory for several years as a model microorganism representative of those bacteria capable of metabolizing alkylaromatic hydrocarbons in oxygen-limited (hypoxic) aquifer environments (23, 34, 42, 43). The tbu pathway of R. pickettii PKO1, which encodes enzymes for utilization of benzene, toluene, and related alkylaromatic hydrocarbons as well as enabling this strain to transform trichloroethylene (TCE), has been cloned as a 26.5-kbp DNA fragment designated pRO1957 (41). The genes encoding enzymes for this catabolic pathway have been shown previously to be organized into three operons: the tbuA1UBVA2C and tbuT operon encoding the initial toluene-3-monooxygenase and the transcriptional activator TbuT (6), the tbuD operon encoding phenol/cresol hydroxylase (24, 26), and the tbuWEFGKIHJ operon encoding enzymes of the meta-cleavage pathway for conversion of catechol and methylcatechols to tricarboxylic acid cycle intermediates (25). We have previously shown through physiological analysis as well as through transcriptional fusion analysis of promoter regions that TbuT controls transcription of each of these operons in response to aromatic effector compounds. Moreover, it has been shown that the unique organization of tbuT, which is located immediately downstream of tbuA1UBVA2C, and the associated promoter (PtbuA1), results in a cascade type of up-regulation of tbuT in response to a variety of effector compounds, including toluene and TCE (6). In our efforts to further characterize this unusual mode of gene regulation, we sequenced the DNA region immediately downstream of tbuT and identified an open reading frame whose deduced amino acid sequence shared significant homology with a number of putative outer membrane proteins from aromatic hydrocarbon-degrading bacteria as well as homology to FadL (4), an outer membrane protein needed for uptake of long-chain fatty acids in Escherichia coli. We report here an analysis of the function of this gene, designated tbuX, in toluene utilization.

	MATERIALS AND METHODS

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Bacterial strains, plasmids and culture conditions. The bacterial strains and plasmids used in this study are listed in Table 1. E. coli JM109 and DH5 were used for routine maintenance and construction of plasmids and for construction of fragments used for DNA sequence analysis. The mobilization plasmid pRK2013 (12) carried in E. coli MM294 was used to mobilize plasmid pKRZ1 and its derivatives from E. coli DH5 into Pseudomonas aeruginosa PAO1c via triparental matings. P. aeruginosa transconjugants resulting from triparental matings were selected on the minimal medium of Vogel and Bonner (59) supplemented with 0.5% glucose. E. coli cells containing recombinant plasmids were always maintained on Luria-Bertani (LB) medium (49) supplemented with the antibiotics ampicillin (50 µg/ml), tetracycline (25 µg/ml), or kanamycin (75 µg/ml) as needed. P. aeruginosa PAO1c cells containing recombinant plasmid constructs were maintained on plate count medium (TNA [39]) containing carbenicillin (500 µg/ml), kanamycin (600 µg/ml), or tetracycline (50 µg/ml) as needed. When cells were grown for enzyme assays or for high-pressure liquid chromatography (HPLC) analyses of metabolites, all strains were routinely grown in a basal salts medium (BM [34]) containing (per liter) 2.49 g of Na₂HPO₄, 3.05 g of KH₂PO₄, 0.1995 g of MgSO₄, 0.995 g of CaCl₂, 0.00005 g of FeSO₄, 0.00025 g of NaMoO₄, 1.0 ml of Hunter's trace metal solution (7), 1.0 g of (NH₄)₂SO₄, 1.0 g of KNO₃, 0.1% Casamino Acids (Difco Laboratories, Detroit, Mich.), and 0.25% glucose. When used for enzyme induction experiments, liquid toluene was added directly to BM to a final concentration of 2.8 mM. Growth of liquid cultures and incubation of agar plates was carried out at 37°C except when cultures contained aromatic hydrocarbons, in which case incubations were done at 30°C.

Toluene utilization assays. Cultures were grown in 100 ml of BM with 0.1% Casamino Acids, 0.25% glucose, and 2.8 mM toluene in tightly stoppered 500-ml bottles at 30°C with shaking until they reached the late log phase. Cells were harvested by centrifugation at 10,000 × g at 4°C and were washed twice with 40 mM potassium-sodium phosphate buffer (pH 6.8). Washed cells were transferred to 10 ml of the same buffer containing toluene (2.8 mM) to produce an A₄₂₅ of 1.0. These washed cell suspensions were incubated with shaking at 30°C for 24 h, and 500-µl samples were taken every 4 h, mixed with 500 µl of methanol in 1.5-ml microcentrifuge tubes, and then centrifuged at 4°C for 10 min to remove cells. The resulting supernatants were carefully transferred to autosampler vials and were analyzed by reverse-phase HPLC. Uninoculated bottles, which served as controls, were incubated under the same conditions, and results were corrected for toluene losses from the controls (which were always <5% of the initial toluene concentration). Reverse-phase chromatography was performed with a PhaseSep H4726 column (4.6 by 250 mm) filled with Spherisorb ODS2 (particle diameter, 5 µm) preceded by a Whatman CSKI guard column (6.5 by 65 mm) coupled to a Shimadzu SCL-6B solvent delivery system and a CR501 Chromatopac computing integrator. A methanol-water (90:10) solvent was used at a flow rate of 1 ml/min. Toluene was detected by monitoring at A₂₅₄, and concentration was calculated by comparison with a standard curve as described previously (41).

Deletion mutagenesis and general molecular techniques. For construction of a DNA fragment lacking tbuX, plasmid pRO1959 (Table 1) was digested with restriction endonucleases ClaI and BamHI. The resultant 10-kb ClaI-BamHI DNA fragment (which when cloned into vector pRO1727 was designated pRO1966 [Table 1]) was extracted following electrophoretic separation in a 1.2% agarose gel, partially digested with PvuII (Fig. 1), and then ligated with ClaI- and EcoRV-digested vector plasmid pRO1727. The ligation mixture was introduced into P. aeruginosa PAO1c by electroporation using the method of Smith and Iglewski (56), and electrotransformants were selected on TNA medium containing carbenicillin (500 µg/ml). Plasmids with the expected deletion were confirmed by ClaI and BamHI digestion patterns of purified DNAs obtained from selected clones. The desired tbuX deletion construct was designated pHYK1000 (Table 1). Other DNA restriction digests, ligations, and transformation procedures were performed according to previously published methods (38, 40, 49).

View larger version (40K): [in this window] [in a new window]   FIG. 1.   Physical and genetic maps of the 9.4-kb ClaI-BamHI fragment of pRO1966. The scale bar represents 0.5 kbp. The large arrows represent the locations and orientations of the genes (identified at the top) which comprise the toluene-3-monooxygenase operon (tbuA1UBVA2C and the positive regulator tbuT) and the tbuX gene. The lollipop symbol between the tbuT and tbuX genes indicates the location of a putative transcriptional terminator. The location of the toluene-3-monooxygenase promoter, PtbuA1, is also shown. Below the restriction map, the putative tbuX promoter region with relevant flanking DNA is expanded. The putative TbuT-binding site is demarcated by the horizontal hatched bar. The 12 and 24 sequences of the tbuX promoter, PtbuX, are boxed and labeled accordingly. The putative ribosome-binding site is boxed and labeled rbs. The first nine amino acids from the deduced amino acid sequence of the tbuX gene (labeled TbuX) are shown in one-letter code beneath the corresponding DNA coding region. The underlined C residue with a +1 designation identifies the tbuX transcriptional start site as determined by primer extension analyses. The BclI restriction endonuclease recognition sequence is shown in italics. The location and lengths of various sets of inverted repeats are shown by pairs of arrows.

DNA sequence analysis. Nucleotide sequencing was initially carried out manually by the dideoxy-chain termination method of Sanger et al. (50), with a Sequenase version 1.0 kit (U.S. Biochemical, Cleveland, Ohio), [-³²P]dATP, and T7, T3, or specific synthetic oligonucleotide primers, as previously described (6). Later sequencing efforts were carried out using an ABI 373A automated sequencer. For PCR amplification of fragments to be sequenced, a total 10-µl reaction mixture containing 0.2 µg of template DNA, 1.6 pmol of primer, and 1 U of Amplitaq FS (Gibco BRL, Gaithersburg, Md.) was used for 25 cycles of 10 s at 96°C, 5 s at 50°C, and 4 min at 60°C. Sequence analysis was done with Lasergene software (DNA STAR, Inc., Madison, Wis.), MacVector version 4.5.3 (Oxford Molecular, Campbell, Calif.), and the Genetics Computer Group (GCG; University of Wisconsin, Madison) software package, version 8.1. Searches of the GenBank database and pairwise sequence comparisons were carried out with GCG programs TFASTA and BESTFIT, respectively. Similarity searches were also performed with the BLAST program and the National Center for Biotechnology Information databases. Nucleotide sequence alignments were done by using the GCG multiple sequence alignment program PILEUP or the Lasergene program MEGALIGN.

PCR. PCRs for amplification of a DNA fragment containing the putative tbuX promoter region were carried out in 100-µl volumes using a GeneAmp kit (Perkin-Elmer, Branchburg, N.J.) and thermal cycler 480 (Perkin-Elmer Cetus, Norwalk, Conn.) essentially as previously described (6. A. M. Byrne and R. H. Olsen, Abstr. 96th Gen. Meet. Am. Soc. Microbiol. 1996, abstr. Q-356, p. 447, 1996). The following oligonucleotide primers, synthesized by the DNA Core facility at the University of Michigan, were used: 5'-GGCGCTCGAGCGAGGGCGGGATGGGCTGG (nucleotides 455 to 437 [Fig. 1]) and 5'-AGTTCCCGGGGGCCGCGCGAACGTAGTTGC (nucleotides 1 to 20 [Fig. 1]). The incorporation of an XhoI or SmaI restriction endonuclease site at the 5' ends (underlined) of the primers allowed for directional cloning of the PCR product into SalI-SmaI cleaved pKRZ1 vector, resulting in plasmid p454X/S (Table 1).

Protein analysis. Cells of P. aeruginosa PAO1c carrying pRO1966 or pHYK1000 (Table 1) were grown overnight at 37°C with shaking in 50 ml of BM with 0.1% Casamino Acids, 0.25% glucose, 500 µg of carbenicillin per ml, and 1 mM toluene. Cells harvested by centrifugation at 4°C were washed twice in 10 ml of 40 mM sodium-potassium phosphate buffer (pH 6.8) and then transferred in 10 ml of this buffer containing 2.8 mM toluene. The cells were incubated with shaking at 30°C for 12 h to ensure tbu pathway gene expression. Following this induction period, cells were harvested by centrifugation at 4°C. The harvested cells were lysed directly in a lysis buffer consisting of 25% glycerol, 14.4 mM -mercaptoethanol, 2% sodium dodecyl sulfate (SDS), 60 mM Tris-HCl (pH 6.8), and 0.1% bromophenol blue. Lysis was achieved by incubation at 95°C for 5 min. The cell debris was removed by centrifugation at 14,000 × g for 20 min, and the supernatant was used for SDS-polyacrylamide gel electrophoresis (SDS-PAGE) using a Mini-Protean II system (Bio-Rad, Hercules, Calif.) according to the method of Laemmli (30). Gels were run for 3 h at 120 V through a 5% acrylamide stacking gel and 15% acrylamide separating gel. Prestained protein molecular weight standards (Gibco BRL) were used for molecular mass estimation. Gels were stained with Coomassie brilliant blue, destained in methanol-glacial acetic acid-water (1:1:8, vol:vol:vol), blotted onto Whatman 3-MM paper, and dried under vacuum at 70°C.

In vivo analysis of promoter activity. Promoter activity, assessed by in vivo transcomplementation, was determined by assaying -galactosidase activity in cells of P. aeruginosa PAO1c essentially as described previously (6, 40). P. aeruginosa PAO1c cells harboring pKRZ1 and its derivatives together with pRO1614 and its derivatives were grown overnight in LB broth containing kanamycin (600 µg/ml) and tetracycline (50µg/ml) to maintain plasmids. Cells were subsequently diluted in the same medium and grown for 8 h in the absence or presence of toluene. -Galactosidase activity was assayed as described by Miller (35) except that cells were permeabilized by addition of chloroform and SDS. Reported -galactosidase activity values, which represent the average of at least three independent experiments, are expressed in units as specified by Miller (35).

RNA preparation and primer extension analysis. Total RNA was isolated from toluene-induced and uninduced cells of P. aeruginosa PAO1c carrying p454X/S in trans with pHYK1001, which has a 3.1-kb EcoRI-StuI DNA fragment expressing tbuT carried on plasmid pRO1614 (Table 1). Cells were grown under the conditions used for -galactosidase assays, described above. Total RNA was extracted from 2 ml of the cultures using Trizol reagent (Gibco BRL) according to the manufacturer's recommended protocol, except that after the cells were mixed with the Trizol reagent, the suspension was incubated at 68°C for 10 min and then allowed to cool to room temperature. The remainder of the RNA extraction protocol was carried out as previously described (6, 27, 40). The concentration and purity of the RNA were estimated from the A₂₆₀/A₂₈₀ ratio and from visual inspection of RNA bands following electrophoretic analysis.

Chemicals and reagents. All chemicals used in this study were of the highest purity commercially available. Aromatic hydrocarbons were purchased from Sigma Chemical Co. (St. Louis, Mo.). Components for cell growth were purchased from Difco, Aldrich Chemical Co. (St. Louis, Mo.), and Gibco BRL. Enzymes and reagents used for nucleic acid manipulations were purchased from Promega, Gibco BRL, and Stratagene (La Jolla, Calif.).

Nucleotide sequence accession number. The sequence data in this report have been submitted to the GenBank data library under accession no. AF100782.

	RESULTS

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Identification and nucleotide sequence analysis of the tbuX gene of R. pickettii PKO1. Our previous studies on regulation of the tbu pathway genes from R. pickettii PKO1 reported an unusual organization of tbuT, the gene encoding the transcriptional activator, TbuT. The unusual feature involves the organization of the toluene-3-monooxygenase genes (tbuA1UBVA2) and the regulator tbuT with respect to PtbuA1. This organization allows for a cascade type of regulation where the presence of an effector molecule (an inducer) and TbuT results in induced expression of tbuA1UBVA2C and simultaneously increases expression of tbuT via readthrough transcription through tbuA1UBVA2C. This further elevates expression of tbuA1UBVA2C if additional effector molecules are present (6). In our efforts to further characterize the overall regulation of the tbu pathway of strain PKO1, we sequenced the region 3' of the translational stop of tbuT and in the process identified an incomplete open reading frame whose deduced amino acid sequence shared significant homology with several putative membrane proteins from other aromatic hydrocarbon-degrading bacteria. To further explore this DNA region, we determined the complete nucleotide sequence of a 2,093-bp SmaI-EcoRI fragment of pRO1966 (Fig. 1), which is a 10-kb ClaI-BamHI subclone of pRO1957 that contains the genes encoding toluene-3-monooxygenase and the transcriptional activator, TbuT (5). Analysis of the nucleotide sequence revealed a 1,374-bp open reading frame that we have designated tbuX. The tbuX gene is encoded on the opposite DNA strand from tbuT and is located 63 bp downstream of the tbuT stop codon (Fig. 1). This open reading frame encodes a putative peptide of 458 amino acids with an estimated molecular mass of 47.8 kDa and estimated pI of 8.7. Determination of the probable 5' end of tbuX was based on the identification of a putative ribosome-binding site (5'-AAGGAGA-3') and the extensive homology with genes from other organisms (as discussed below). As found for the other genes of the tbu pathway, the tbuX coding region is G+C rich (63.8%) and accordingly displays a preferential use of codons with either a G or a C residue in the third position.

View larger version (75K): [in this window] [in a new window]   FIG. 2.   Multiple sequence alignment of TbuX from R. pickettii PKO1, PhlX from R. eutropha JMP134 (GenBank accession no. AF065891 [unpublished]), CymD from P. putida F1 (10), PorA from Pseudomonas sp. strain Y2 (58), IpbH from P. putida RE204 (GenBank accession no. AF006691 [unpublished]), CumH from P. fluorescens IP01 (14), TodX from P. putida F1 (60), XylN from P. putida PaW1 (GenBank accession no. D63341 [unpublished]), and FadL from E. coli (4). The amino acid residues conserved in all nine sequences are indicated in boldface and marked with daggers. Residues conserved in TbuX, PhlX, CymD, PorA, IpbH, CumH, TodX, and XylN are indicated with asterisks. Gaps are represented by dashes and were introduced to maximize the alignment. The multiple sequence alignment analysis was carried out using the Clustal method within the MEGALIGN program of Lasergene.

Analysis of the tbuX promoter and regulatory region. Examination of the nucleotide sequence upstream of the tbuX gene revealed a promoter containing the invariant 24GG/12GC sequence unique to ⁵⁴ (rpoN)-dependent promoters (Fig. 3A). A putative TbuT-binding site was also identified approximately 150 bp upstream of the putative tbuX promoter (Fig. 3B) based on its similarity to the promoter for tbuA1. Comparisons of the nucleotide sequences of both the putative tbuX promoter and the putative TbuT-binding site upstream of tbuX with similar sequences found within the Pu promoter of the upper TOL operon from P. putida mt-2 (47), the Po promoter of the dmp operon from Pseudomonas sp. strain CF600 (57), the Ptbm promoter upstream of the toluene and benzene monooxygenase operon from Burkholderia cepacia JS150 (20), the PtbuD promoter upstream of the phenol/cresol hydroxylase structural gene (40), and the PtbuA1 promoter upstream of the toluene-3-monooxygenase structural genes (6) are shown in Fig. 3B. Interestingly, the spacing between the islands of homology observed in the putative TbuT-binding site upstream of tbuX is similar to that observed in the putative DmpR-binding site upstream of its cognate promoter Po.

View larger version (23K): [in this window] [in a new window]   FIG. 3.   DNA sequence similarities among regions within and upstream of the PtbuX promoter sequence; promoters and UASs with homology to PtbuX. Nucleotide sequence identities among the compared sequences are indicated in boldface. The consensus sequences established from comparison of the region within and upstream of the PtbuX, PtbuD, Po, Ptbm, Pu, and PtbuA1 sequences are displayed below each alignment. (A) DNA sequence alignment of the 54-dependent promoter sequences of PtbuX, PtbuA1, PtbuD, Pu, and Po. The 24 and 12 sequences within the consensus sequence are labeled accordingly. (B) DNA sequence alignment of the region upstream of tbuX with palindromic regions identified as the XylR-binding site upstream of Pu and those proposed for DmpR binding upstream of Po, TbuT binding upstream of tbuD and tbuA1, and TbmR binding upstream of tbmA. Gaps, indicated by dashes, were introduced to maximize homology.

Transcriptional analysis of tbuX. A transcriptional start site for tbuX was mapped by primer extension analysis just downstream of the ⁵⁴-dependent promoter sequence (Fig. 1). This single transcript was observed only from the primer extension reaction using total RNA extracted from toluene-induced cells carrying p454X/S and TbuT (Fig. 4, lane 6). These results provide evidence that the 24/12 promoter sequence, designated PtbuX, is the TbuT-dependent toluene-inducible promoter identified in the tbuX-lacZ fusion studies, described above. The absence of a similar transcript from RNA extracted from uninduced cells carrying p454X/S and TbuT (Fig. 4, lane 3) suggests that the constitutive expression observed from the tbuX-lacZ fusion studies may not be from a leaky PtbuX promoter but rather from a second promoter elsewhere upstream of tbuX.

View larger version (111K): [in this window] [in a new window]   FIG. 4.   Determination of the 5' end of the tbuX transcript by primer extension analysis. RNA was isolated from P. aeruginosa PAO1c(pKRZ, pHYK1001), P. aeruginosa PAO1c(p454X/S, pRO1614), and P. aeruginosa PAO1c(p454X/S, pHYK1001) grown in the absence (lanes 1 to 3, respectively) and presence (lanes 4 to 6, respectively) of toluene. A sequence ladder using the same primer and p454X/S is also shown (A, T, C, and G). To the left, an expanded view of the nucleotide sequence surrounding the transcriptional start site (marked with an asterisk) is shown.

Analysis of PtbuX promoter activity. Given the differences observed in -galactosidase levels for the tbuX-lacZ fusion in contrast to the tbuA1- and tbuD-lacZ fusions with toluene as an inducer (Table 2), we set out to investigate the possibility of differential promoter response from PtbuX and PtbuA1 for a range of effector compounds. These experiments were carried out using P. aeruginosa PAO1c carrying either p454X/S (the tbuX-lacZ fusion) or p352X/S (the PtbuA1-lacZ fusion) in trans with TbuT expressed from pHYK1001, as described above. Comparisons of promoter activity obtained from PtbuX and PtbuA1 in response to the presence of various effector compounds revealed that PtbuX is a stronger promoter (Fig. 5). Although the differences in promoter activity (PtbuX versus PtbuA1) in response to different effectors varied between 2- and 12-fold, in general the relative activation profiles from the various effectors were maintained. The rank order for effector activation of PtbuX was toluene > benzene > ethylbenzene > TCE > o-xylene > phenol > o-cresol > m-cresol > m-xylene > p-xylene > p-cresol. The rank order for PtbuA1 activation was toluene > benzene > ethylbenzene > TCE > o-cresol > m-cresol > phenol > o-xylene > m-xylene > p-cresol > p-xylene. Overall, the effector activation profile was similar for the two promoters with the exceptions of phenol and o-xylene. For PtbuX, o-xylene and phenol were better effectors than o- or m-cresol, whereas for PtbuA1 o- and m-cresol were better effectors.

View larger version (30K): [in this window] [in a new window]   FIG. 5.   Activation of the PtbuX and PtbuA1 promoters by TbuT in response to the presence of effectors. P. aeruginosa PAO1c cells containing pHYK1001 and either the PtbuX-lacZ transcriptional fusion plasmid p454X/S or the PtbuA1-lacZ transcriptional fusion plasmid p352X/S were grown and treated as described in Materials and Methods. The histogram represents the accumulation of -galactosidase after 8 h of exposure of the cultures to the different effectors. Values are averages of duplicate determinations from three independent experiments. The variability between triplicate values did not exceed 10%. Abbreviations for effector compounds: Ben, benzene; Tol, toluene; Eth, ethylbenzene; oXyl, o-xylene; mXyl, m-xylene; pXyl, p-xylene; TCE, trichloroethylene; Phl, phenol; oCr, o-cresol; mCr, m-cresol; pCr, p-cresol.

Effect of tbuX deletion on expression of the tbu pathway genes. To elucidate a possible functional role of tbuX in utilization of toluene by the toluene-3-monooxygenase encoded by tbuA1UBVA2C, a tbuX-deletion derivative of pRO1966 (Table 1) was constructed as described in Materials and Methods. The deletion derivative, designated pHYK1000, was introduced into P. aeruginosa PAO1c, and the cells carrying either pHYK1000 or pRO1966 were then analyzed for toluene utilization and protein synthesis.

View larger version (11K): [in this window] [in a new window]   FIG. 6.   Toluene utilization (A) and m-cresol production (B) for cells of P. aeruginosa PAO1c carrying pRO1966 (tbuX+) or pHYK1000 (tbuX).

View larger version (111K): [in this window] [in a new window]   FIG. 7.   SDS-PAGE profile of soluble cellular proteins of P. aeruginosa PAO1c carrying pRO1966 (lanes 2 and 4) or pHYK1000 (lanes 3 and 5), grown on 0.1% Casamino Acids and 0.25% glucose (lanes 2 and 3) or 0.1% Casamino Acids, 0.25% glucose, and 1 mM toluene (lanes 4 and 5). Sizes of molecular weight markers are shown to the left; arrows indicate the positions of TbuA1UBVA2C and TbuT components, as described in the text.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

In this study, we have identified and characterized a new gene, tbuX, associated with the tbu pathway for toluene and benzene utilization in R. pickettii PKO1. The tbuX gene was identified downstream of and on the DNA strand opposite that of tbuT, the NtrC-like positive regulator that controls transcription of the tbu regulon (6). Analysis of the deduced amino acid sequence of TbuX demonstrated that this protein shared significant identity with a group of proteins associated with pathways for hydrocarbon degradation in a variety of gram-negative bacteria. These pathways included styrene utilization in Pseudomonas sp. strain Y2; isopropylbenzene utilization in P. putida RE204 and P. fluorescens IP01; toluene utilization in P. putida PaW1 which carries the TOL plasmid pWW0; two separate pathways in P. putida F1, one for toluene and one for p-cymene utilization; and phenol utilization in R. eutropha JMP134. The group of homologous proteins found in these organisms all shared a low but significant level of similarity to FadL, an outer membrane protein required for binding and transport of long-chain fatty acids in E. coli. Except for FadL, none of the members of this group has been analyzed in detail with regard to subcellular location. However, the provisional identification of TbuX as an outer membrane protein in R. pickettii PKO1 appears to be reasonable given the following observations derived from comparative sequence analysis: (i) the presence of a putative signal sequence (37) (residues 1 to 23 [VKGNYVRAATAICVAICGTHARA]) containing a charged amino acid (K, position 2) followed by a mostly hydrophobic segment and a peptidase cleavage site adjacent to three conserved residues (ARA, positions 21 to 23); (ii) the absence of cysteinyl residues in the putative mature peptide; and (iii) an abundance of hydrophobic amino acids (37.4% by frequency) in the putative mature peptide (16). In addition, comparative hydropathy analyses of TbuX, PhlX, CymD, PorA, IpbH, CumH, TodX, and XylN using the algorithms of Hopp and Woods (data not shown) revealed the presence of similar regions of hydrophobicity in each of these proteins, as previously demonstrated for FadL (4, 28). It is noteworthy that the putative outer membrane protein with which TbuX shares the greatest overall similarity is PhlX, which is associated with a catabolic pathway for phenol degradation in R. eutropha JMP134 (22). The tbu pathway of R. pickettii PKO1 can accommodate both hydroxylated aromatic compounds such as phenol and cresols as well as unactivated aromatic hydrocarbons such as benzene and toluene as both effectors and substrates (5, 24, 25, 41). It is possible that the similarities between PhlX and TbuX relate, in part, to their potential roles in phenol utilization in these two strains, in contrast to the other members of this protein group (CymD, PorA, IpbH, CumH, TodX, and XylN) which are all associated with pathways for utilization of unactivated aromatic hydrocarbons. In this regard, it would also be of interest to determine whether PhlX and the associated phl pathway of strain JMP134 can accommodate toluene and benzene as effectors and as substrates.

Deletion of TbuX severely affected toluene utilization in cells of P. aeruginosa PAO1c carrying tbu pathway genes (Fig. 6). This result can be explained by a model in which TbuX plays a role in the entry of toluene into the cell. A possible scenario might involve the facilitated passage of toluene through the outer membrane as a consequence of interaction with the TbuX protein. Such facilitated entry could provide toluene to TbuT, which in turn would result in activation of the various tbu pathway promoters. Wang et al. (60) have suggested that TodX, a homolog of TbuX that occurs in P. putida F1, may function as a facilitator of toluene entry into this strain. A necessary feature of this model is the constant presence of the TbuX protein in the cell's outer membrane as a consequence of a low basal level of expression. This prediction is consistent with our observations on the behavior of the PtbuX promoter, namely that a significant level of PtbuX promoter activity was observed when cells were grown in the absence of the effector, toluene, or when the cells did not carry the transcriptional activator, TbuT (Table 2), suggesting that there may be partially constitutive expression of tbuX. Our transcriptional analysis using primer extension did not reveal any message initiating from the major TbuT-dependent and toluene-responsive tbuX promoter, PtbuX, when analyzed in the absence of TbuT or toluene (Fig. 4, lanes 2 and 3); therefore, it is possible that the low basal level of tbuX expression is dependent on a second weakly constitutive promoter located further upstream and that was not contained in p454X/S. Inspection of the DNA sequence 5' of the putative translational start of TbuX to the BamHI site at the right-hand terminus of the pRO1966 clone (Fig. 1) did not reveal any obvious ⁷⁰-like promoter (17), which might be expected to allow for partially constitutive expression; therefore, additional research will be required to further elucidate this.

In addition to affecting toluene utilization, deletion of tbuX also affected expression of peptides necessary for toluene utilization (Fig. 7). Analysis of the expression pattern for soluble proteins obtained from toluene-grown cells of P. aeruginosa PAO1c carrying pHYK1000, which has been deleted for tbuX, shows (Fig. 7, lane 5) the absence of bands at estimated molecular masses of 62, 57, and 37 kDa. In contrast, protein pattern analysis for toluene-grown P. aeruginosa PAO1c carrying pRO1966, which contains tbuA1UBVA2C, tbuT, and tbuX (Fig. 1), clearly shows (Fig. 7, lane 4) the presence of novel peptide bands with these estimated molecular masses. Peptides of these sizes would correspond to the predicted molecular masses for TbuT (65.2 kDa) and TbuA1 (57.6 kDa) and for TbuA2 (37.5 kDa) and TbuC (36.1 kDa), which are similar to one another in size. The other peptides that would be expressed from pRO1966, TbuB, TbuU, and TbuV, would not be expected to be detectable in this analysis since the molecular masses of these components are 12.3, 9.6, and 11.7 kDa, respectively. These results are consistent with the toluene-3-monooxygenase peptide expression patterns that we have previously determined from related work on regulation of the tbu pathway (5, 24, 40, 41), and they support the model that deletion of tbuX impaired the ability of toluene to enter the cell, which in turn negatively affected the expression of the toluene-3-monooxygenase structural genes as a result of the lack of effector binding to the transcriptional activator, TbuT. Therefore, Pseudomonas cells harboring pHYK1000 could not utilize toluene even though the toluene-3-monooxygenase structural genes and the transcriptional regulatory gene were present.

PtbuX is a stronger TbuT-dependent and toluene-responsive promoter than is PtbuA1. This finding is also consistent with our overall model of TbuX as a participant in facilitated entry of toluene into the cell, inasmuch as rapid and high-level synthesis of TbuX from a strong promoter would allow for more toluene entry and hence would provide more substrate for toluene-3-monooxygenase which is transcribed from the comparatively weaker PtbuA1 promoter. Such a regulatory scheme might be part of a general mechanism by which carbon flow could be controlled during toluene utilization by strain PKO1. In this regard, the tbu regulon appears to be unique among bacterial regulatory systems in that the binding of a single transcriptional activator, TbuT, to multiple promoter-upstream regions of different architecture (Fig. 3B) appears to allow for differential promoter response. The catabolic pathways that have been studied in the greatest detail and with which the tbu pathway shares the greatest homology with regard to transcriptional activators are the xyl pathway of P. putida PaW1 carrying the TOL plasmid, pWW0 (1, 2, 9, 19), and dmp pathway of Pseudomonas sp. strain CF600 (55). These pathways, although they show some similarity in overall mode of regulation to the tbu pathway, are substantially different in the detailed mechanisms by which the transcriptional units are controlled. The xyl regulatory system is controlled by two separate transcriptional activators, XylR (an NtrC-like activator) and XylS (a member of the AraC family of activators), that respond to separate effectors and that bind to separate and distinct promoter-upstream regions for each operon (48). In contrast, the dmp pathway is controlled by a single NtrC-like activator, DmpR, that binds to a single promoter-upstream region that controls the single dmp operon (55).

There seems to be some similarity in regulatory organization between the tbu pathway of strain PKO1 and the tbm pathway of B. cepacia JS150, inasmuch as the tbm system appears to controlled by one transcriptional activator, TbmR, that regulates expression of two different monooxygenases expressed from divergent promoters (20, 21), but this regulatory system has not yet been investigated in sufficient detail to determine the extent of its similarity to the tbu model system. Other catabolic pathways that have significant homology to the tbu pathway with respect to the structural genes, such as the pathways for toluene utilization in Pseudomonas mendocina KR1 (61) or in B. cepacia G4 (51-53), have not yet been investigated with regard to operonic organization or gene regulation. Therefore, it is not clear what regulatory paradigm would be expected to occur in these organisms, although data from an analysis of the kinetics of toluene degradation indicate that there are similarities between strains G4 and PKO1 with respect to the effect of oxygen availability on the rate of toluene degradation for the two strains (32). This suggests that these strains might share similar features for regulation of their toluene biodegradative pathways. The overall picture that is emerging from comparative analysis of these regulatory systems suggests that regulatory units have evolved independently of catabolic genes (11) and that regulatory units can be recruited in a modular fashion. Further evidence for the latter observation comes from our recent work on comparative organization of the regulatory systems controlling the phenol hydroxylase in R. pickettii PKO1 and the toluene/benzene-2-monooxygenase in Burkholderia sp. strain JS150, in which it appears that a regulatory module and associated promoter structure has been recruited, in the case of strain PKO1, to be associated with a unit peptide phenol hydroxylase, whereas in strain JS150 a similar regulatory module is associated with a multicomponent toluene/benzene-2-monooxygenase (40).

A central part of our studies on tbuX have focused on regulation of this operon as part of the overall tbu regulon in strain PKO1. Consistent with previous observations, we have shown that expression from PtbuX is dependent on the presence of the regulatory protein, TbuT, and appropriate effector compounds. Sequence analyses have suggested that the PtbuX promoter is dependent on the alternative sigma factor, ⁵⁴, and that activation of PtbuX is dependent on a DNA region 178 bp upstream of the transcription start site. Taken together, these findings indicate that the organization and regulation of the tbuX operon are similar to what has been found previously for the other operons in the tbu regulon (5, 6, 25-27, 40). The tbu pathway promoters are regulated by TbuT, which is an NtrC-like transcriptional activator that belongs to the large family of ⁵⁴-dependent activators (29, 33, 36, 54). These transcriptional activators function by binding to DNA upstream activation sequences (UAS) located 100 to 200 bp upstream of the promoters that they regulate. For the tbu pathway UAS shown in Fig. 3B, these sequences are found approximately 150 to 200 bp upstream of the transcriptional start site for each promoter. The tbu UAS regions have significant sequence homology to the palindromic regions upstream of Pu, the upper pathway promoter from the TOL plasmid, to which the transcriptional activator XylR has been shown to bind (9, 19). The sequences recognized by XylR in the Pu UAS region include the motif 5'-TTGANCAAATC-3' (9). A sequence highly similar to this is present within each arm (Fig. 3B) of the inverted repeat upstream of PtbuX; thus, it is likely that this is the target site for TbuT binding for the PtbuX promoter. It is of interest that the spacing between the conserved inverted repeats in the PtbuX UAS region is similar to that found for the UAS region of the PtbuD promoter and not the PtbuA1 promoter (Fig. 3B). This difference in UAS palindrome architecture is correlated with promoter activity; i.e., greater promoter activity is observed when tbuT is in trans with promoter/UAS 'lacZ fusions of PtbuX or PtbuD than when it is in trans with fusions of PtbuA1. This suggests that promoter function can be affected by the structure of the site to which the transcriptional activator binds. Such an effect was seen previously by Leahy and coworkers (31) for cross-regulation of the PtbuA1 promoter of R. pickettii PKO1 and the PtbmA promoter of B. cepacia JS150 by either TbuT or TbmR. These researchers observed that the cognate and noncognate activators were capable of transcriptionally activating each promoter in response to the same set of effectors, but that the magnitude of the transcriptional response was greater for PtbmA than for PtbuA1 for each of the effectors tested. As shown in Fig. 3B, PtbmA is similar in architecture to PtbuX and not to PtbuA1.

As indicated above, members of the ⁵⁴-dependent family of promoters require an interaction between the transcriptional activator bound at the UAS and RNA polymerase bound at the 24/12 region in order for an open transcriptional complex to form. This process necessarily requires DNA bending in order to achieve close physical contact between the UAS-bound activator and the promoter-bound ⁵⁴-RNA polymerase (45). In some ⁵⁴-dependent promoters, the required DNA bending is mediated by specific DNA-bending proteins such as integration host factor (IHF), which recognizes and binds to specific DNA sequences and induces sharp bends in the DNA. IHF-dependent DNA bending has been shown to be essential for promoter function for the XylR-dependent Pu promoter (1, 9, 46) as well as the DmpR-dependent Po promoter (55, 57). Moreover, in the case of the Pu promoter, IHF binding has been shown to specifically stimulate recruitment of RNA polymerase to the 24/12 promoter region as a consequence of interaction of the carboxy-terminal domain of the  subunit of RNA polymerase with specific sequences upstream of the IHF-binding site (3). Inspection of the DNA sequence between the putative TbuT-binding site and the 24/12 promoter region of the PtbuX promoter did not reveal a consensus IHF-binding site; therefore, it seems unlikely that IHF could contribute to the necessary promoter architecture for PtbuX. However, protein-assisted DNA bending necessary for ⁵⁴ promoter function can be achieved by mechanisms that are independent of IHF, as has been shown for the XylR-dependent Ps promoter of the TOL plasmid of P. putida (13, 44). With this promoter, the combined effects of intrinsically curved DNA and HU protein-mediated DNA bending have been shown to be necessary to achieve full transcriptional activity. Since the DNA region between the putative TbuT-binding site and the 24/12 promoter region of the PtbuX promoter contains a greater number of dT stretches (Fig. 1) than found in the rest of the tbuX operon, it is possible that PtbuX is similar to Ps, relying on both the intrinsic curvature of promoter DNA and a nonspecific DNA-bending protein, such as HU, to achieve the promoter architecture required for transcription.