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Journal of Bacteriology, September 2004, p. 6101-6109, Vol. 186, No. 18
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.18.6101-6109.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Regulation of Tetralin Biodegradation and Identification of Genes Essential for Expression of thn Operons

O. Martínez-Pérez, E. Moreno-Ruiz, B. Floriano, and E. Santero*

Centro Andaluz de Biología del Desarrollo and Departamento de Ciencias Ambientales, Universidad Pablo de Olavide, Seville, Spain

Received 25 May 2004/ Accepted 17 June 2004


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ABSTRACT
 
The tetralin biodegradation genes of Sphingomonas macrogolitabida strain TFA are clustered in two closely linked and divergent operons. To analyze expression of both operons under different growth conditions, transcriptional and translational gene fusions of the first genes of each operon to lacZ have been constructed in plasmids unable to replicate in Sphingomonas and integrated by recombination into the genome of strain TFA. Expression analysis indicated that the transcription of both genes is induced in similar ways by the presence of tetralin. Gene expression in both operons is also subjected to overimposed catabolic repression. Two additional genes named thnR and thnY have been identified downstream of thnCA3A4 genes. ThnR is similar to LysR-type regulators, and mutational analysis indicated that ThnR is strictly required for expression of the thn operons. Unlike other LysR-type regulators, ThnR does not repress its own synthesis. In fact, ThnR activates its own expression, since thnR is cotranscribed with the thnCA3A4 genes. ThnY is similar to the ferredoxin reductase components of dioxygenase systems and shows the fer2 domain, binding a Cys4[2Fe-2S] iron sulfur center, and the FAD-binding domain, common to those reductases. However, it lacks the NAD-binding domain. Intriguingly, ThnY has a regulatory role, since it is also strictly required for expression of the thn operons. Given the similarity of ThnY to reductases and the possibility of its being present in the two redox states, it is tempting to speculate that ThnY is a regulatory component connecting expression of the thn operons to the physiological status of the cell.


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INTRODUCTION
 
The organic solvent tetralin (1,2,3,4-tetrahydronaphthalene) is a bicyclic molecule composed of an aromatic and an alicyclic moiety, which share two carbon atoms. Tetralin is widely used as a degreasing agent and solvent for fats, resins, and waxes, as a substitute for turpentine in paints, lacquers, and shoe polishes, and also in the petrochemical industry in connection with coal liquefaction (19). A concentration of tetralin higher than 100 µM inhibits bacterial growth (44). Its toxicity is partly due to its lipophilic character, which results in its accumulation in the cell membranes, thus leading to changes in their structure and function (46, 47). In addition, tetralin also forms toxic hydroperoxides in the cell (17).

A few bacterial strains which are able to aerobically grow on tetralin as the only carbon and energy source have been isolated (44). By the identification of accumulated intermediates, several reports suggest that some bacteria, such as Pseudomonas stutzeri AS39 (43), initially hydroxylate and further oxidize the alicyclic ring whereas others, such as Corynebacterium sp. strain C125 (45), initially dioxygenate the aromatic ring, thus indicating that aerobic metabolism of tetralin can be performed in different ways. Metabolism of tetralin has been best characterized in Sphingomonas macrogolitabida strain TFA. Biodegradation of tetralin by the strain TFA involves initial oxidation of the aromatic ring to yield 1,2-dihydroxytetralin (1,2-DHT) through reactions catalyzed by a ring-hydroxylating dioxygenase and by a dehydrogenase (37). The catechol intermediate is further metabolized through reactions catalyzed by an extradiol dioxygenase, a hydrolase, a hydratase, and an aldolase, respectively (3, 24, 25). Interestingly, this set of enzymes, typically involved in metabolism of one aromatic ring, is able to cleave both the aromatic and the alicyclic rings of tetralin, which results in the production of pyruvate and pimelic semialdehyde (25). The genes coding for these enzymes have also been identified and shown to cluster together in two closely linked operons, which are divergently transcribed (26, 37) (Fig. 1).



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  		FIG. 1. Schematic representation of the two divergent strain TFA operons, which bear tetralin biodegradation genes. Genes identified in this work are shown in enlargement at the bottom. Chromosomal insertions of plasmids bearing transcriptional or translational lacZ gene fusions to thnB or thnC by a single recombination event are also schematically represented at the top.

The success of a catabolic pathway obviously depends on the capability of the enzymes to metabolize a particular compound or the subsequent intermediates but also depends on an efficient regulatory system. Regulatory proteins and regulated promoters are key elements that control expression of catabolic operons to assure that the enzymes are only produced under appropriate environmental conditions (13). Thus, the expression of most catabolic operons is regulated by specific inducible systems of control, which allow or activate synthesis of the corresponding enzymes only when the substrate or some intermediate of the pathway is available.

Additionally, expression of catabolic operons is very frequently subjected to overimposed global regulatory controls, which prevent transcription of catabolic genes under conditions of nutritional excess, thus optimizing gene expression by connecting it to the metabolic and/or energetic status of the cell (8, 13). Some global controls apparently respond to different stress signals and may involve the participation of alternate sigma factors (7, 9, 32, 48, 50), although most of them fit within the category of carbon catabolite repression, which prevents expression of catabolic operons in the presence of preferential carbon and energy sources. Although carbon catabolite repression appears to be a conserved phenomenon in bacteria, the molecular mechanisms that exert the control may be completely different in distantly related bacteria (39, 40). Several reports of bacteria metabolizing different organic contaminants indicate that the mechanism(s) of carbon catabolite repression of biodegradative operons is different from the cyclic AMP-dependent mechanism, which is well characterized for enteric bacteria (2, 9, 11, 15, 33, 38). In addition, more than one global regulatory system may regulate expression of biodegradative genes within the same bacteria (9, 14).

Very little is known about regulation of catabolic pathways in sphingomonads, although some LysR-type activators have been recently described (5, 22, 36). Carbon catabolite repression has not yet been documented for this group of bacteria. This paper reports on the regulated expression of the tetralin biodegradation operons of S. macrogolitabida strain TFA, showing that it is induced in the presence of the pathway substrate and subjected to carbon catabolite repression. Characterization of two regulatory genes whose products are essential for thn gene expression is also described.


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MATERIALS AND METHODS
 
Plasmids and bacterial strains. Plasmids and strains used in this work are listed in Table 1. A 5.3-kb SmaI fragment from pIZ600 (26) was cloned in pTZ18U (34), yielding plasmid pIZ619. pIZ669 was then constructed by cloning a KIXX cassette (from pUC4-KIXX; Pharmacia) excised with HindIII into a BglII restriction site, interrupting the 241st codon of thnY in pIZ619.


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  		TABLE 1. Bacterial strains and plasmids

Plasmid pIZ1165 was constructed by cloning a 4.5-kb EcoRI fragment from pIZ604 (26) which bore thn'A4RY and 1.35 kb downstream of thnY in a SalI-PstI-SphI-HindIII-lacking pTZ18R (34) opened with EcoRI. Then, to construct pIZ601, an EcoRI fragment, harboring the Km resistance flanked by transcriptional terminators from pUT-miniTn5Km (12), was inserted in pIZ1165 in a PstI site in thnY, interrupting it after the 140th codon.

An 1.41-kb EcoRV-ApaI fragment, containing the promoter region, from pIZ608 (26) was cloned between the EcoRV and ApaI sites of the multiple cloning site of pBluescript II SK(+) (Stratagene) to yield pIZ1001.

To obtain translational fusions of the promoter for the thnC or thnB gene to the lacZ reporter gene, plasmids pIZ1002 and pIZ1003 were constructed. An Asp718-EcoRV fragment from pIZ1001 was cloned between the XmaI and EcoRI sites of pJES379 (42), yielding plasmid pIZ1002, which carried the translational thnC-lacZ fusion. Plasmid pIZ1003 carried the translational thnB-lacZ fusion and was constructed by cloning an Asp718-BamHI fragment from pIZ1001 between the EcoRI and BamHI sites of pJES379. The lacZ fusions maintain the first 124 codons of thnC and the first 217 codons of thnB.

To construct the transcriptional thnC-lacZ and thnB-lacZ fusions, an Asp718-EcoRV fragment from pIZ1001 was cloned in both orientations in the SmaI site of pIC552 (31), yielding plasmids pIZ1009 (thnC-lacZ transcriptional fusion) and pIZ1010 (thnB-lacZ transcriptional fusion).

To construct the broad-host-range plasmid pIZ1016, an EagI-SalI fragment, bearing the tac promoter and lacIq from pMM40 (28), was excised from pIZ1015 and cloned between NcoI-SalI sites of pBBR1MCS-5 (29), removing the plasmid's lac promoter. Plasmid pIZ1015 was obtained by cloning an EagI-EcoRI fragment from pMM40, bearing the tac promoter and lacIq, between EagI-ClaI in pBluescript II KS(+) (Stratagene).

A plasmid named pIZ1008 harboring the thnR gene was constructed by cloning a 1.5-kb SacII-PstI fragment from pIZ641 (26) into pBluescript II KS(+). A SacI-PstI fragment from pIZ1008 was inserted into the SalI and PstI sites of pIZ1016, yielding plasmid pIZ1017.

To construct plasmid pIZ698, a 1.35-kb BamHI-NruI fragment harboring thnY was excised from pIZ619 and cloned in the SmaI site of pIZ1016.

Whenever necessary, incompatible cohesive ends were blunted with Klenow polymerase and deoxynucleoside triphosphates or with T4 polymerase and deoxynucleoside triphosphates.

Escherichia coli DH5{alpha} (21) was used for cloning, isolation of DNA for sequencing, and other DNA manipulations.

S. macrogolitabida strain TFA (26) harboring transcriptional or translational fusions of the promoter for the thnB or thnC gene to lacZ (TFA-1002, TFA-1003, TFA-1009, and TFA-1010) were used for ß-galactosidase assays. TFA mutants derivatives T601, T653 (26), T655 (26), T656 (26), T661 (26), T664 (26), and T669 carrying the translational thnC-lacZ fusion (T601-1002, T653-1002, T655-1002, T656-1002, T661-1002, T-664-1002, and T669-1002) were used for complementation experiments or ß-galactosidase assays.

To construct the ThnY– mutant strains T669 and T601, plasmids pIZ669 and pIZ601 were respectively electrotransformed into the wild-type TFA strain, and candidates showing homologous recombination were isolated as previously described (26).

Plasmids pIZ1002, pIZ1003, pIZ1009, and pIZ1010 were transferred to strain TFA and TFA mutants by triparental matings. Since none of these plasmids can replicate in TFA, ampicillin-resistant transconjugants resulted from a single recombination event, leading to integration of the plasmid into the TFA genome.

Preparation of total DNA from strain TFA and Southern blotting. Total DNA from strain TFA was prepared as previously described (20). Southern blot analyses were performed using digoxigenin-dUTP-labeled probes and following the instructions of the manufacturer (Boehringer Mannheim). Total DNA from T669 and T601 was hybridized with a marked 1.35-kb BamHI-NruI fragment, containing thnY, from pIZ619. T669 was also hybridized with a KIXX HindIII probe. An EcoRI fragment, containing the Km resistance gene, was excised from pUT-miniTn5Km, labeled, and hybridized to T601. A 1.41-kb EcoRV-ApaI fragment, containing the promoter region, was marked and used as a probe to check the integration of the transcriptional and translational fusions.

Media and growth conditions. E. coli strains were routinely grown in Luria-Bertani (LB) medium at 37°C. TFA strains were grown at 30°C in MML rich medium (mineral medium [MM] supplemented with 0.2% tryptone and 0.1% yeast extract), LB medium, or MM medium (16) supplied with tetralin in the vapor phase or/and ß-hydroxybutyrate (ßHB) as the carbon and energy source. MM medium containing 8 mM nitrate or 17 mM urea instead of ammonium as a nitrogen source was used in some induction kinetics.

Tetralin induction and carbon catabolite repression assays. Cultures of strains harboring a thnC-lacZ or thnB-lacZ gene fusion integrated into their chromosomes were grown at 30°C in mineral medium containing ßHB as the only carbon and energy source to exponential phase (optical density at 600 nm = 0.8 to 1.0). Then, cells were washed to remove the carbon source and diluted to a final optical density of about 0.1 in MML, LB medium, or MM medium, which could be supplemented with a carbon source, in the absence or the presence of the inducer tetralin in the gas phase. Cultures were grown at 30°C, aliquots were withdrawn at different cell densities, and ß-galactosidase activity was assayed as described by Miller (35).

RNA extraction. RNA extraction was performed as described by Chomczynski and Sacchi (10). Harvested cells were subsequently treated with acid phenol, N-lauryl sarcosine, and guanidinium thiocyanate at 60°C, chloroform, DNase, and proteinase K. RNA was finally recovered after phenol:chloroform:isoamyl alcohol (25:24:1), and chloroform:isoamyl alcohol (24:1) treatment and precipitation with ethanol 96°C and 3 M sodium acetate (pH 5.2).

Reverse transcription and PCR amplification. RNA (2 µg) was retrotranscribed using a TaqMan kit (Applied Biosystems) and following the manufacturer's instructions. Different amounts (0.8 and 4.8 µg) of the obtained cDNA were used to amplify a 101-bp fragment from thnB with the primers thnB-RT1 (5'-AGGTCGGCGTACTTGAAGTC-3') and thnB-RT2 (5'-AGCAAAGCTCGCAACGCT-3'), a 142-bp fragment from thnC with primers thnC-RT1 (5'-CAGCCGTCCATCCTGAGATAG-3') and thnC-RT2 (5'-AAGGCAAGTGTCACGGAACTC-3'), and a 136-bp fragment from thnR with primers thnR-RT1 (5'-CGGTCAAACCGAGTCTGAAGA-3') and thnR-RT2 (5'-ATGGAGCCAACAGCATTTGC-3'). As an amplification control, primers f27 and r519 (26) were used to amplify a 500-bp fragment corresponding to 16S rRNA. The PCR program consisted of 5 min at 94°C, 20 cycles of 30 s at 94°C, 30 s at 57°C, and 30 s at 72°C, and 5 min of elongation at 72°C. Samples were then run in an 8% acrylamide:bisacrylamide (29:1) gel and stained with ethidium bromide. To ensure that RNA samples did not contain contaminating DNA, PCR amplification was performed using RNA preparations as templates.

Sequence analysis comparison. The obtained sequence was initially compared using the BLASTp and tBLASTn programs to those in databases (1). Sequences that showed high similarity to that of strain TFA were aligned using the CLUSTALW program (49) and default parameters. A distance matrix and a phylogenetic tree was constructed by the neighbor-joining method (41) and visualized using the TreeView program.

Nucleotide sequence accession number. The nucleotide sequence reported here has been submitted to the DDBJ, EMBL, and GenBank nucleotide sequence databases and annotated as an update of the sequence at accession no. AF157565.


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RESULTS
 
Tetralin induction of the thn operons. To easily test expression of the tetralin catabolic operons, transcriptional and translational lacZ gene fusions to thnB and thnC, the first genes of each operon, were constructed as described in Materials and Methods, thus yielding the plasmids pIZ1010 (transcriptional thnB-lacZ fusion), pIZ1003 (translational thnB-lacZ fusion), pIZ1009 (transcriptional thnC-lacZ fusion), and pIZ1002 (translational thnC-lacZ fusion). Since none of these plasmids can replicate in strain TFA, strain derivatives bearing each plasmid integrated into the genome by a single recombination event were directly selected as ampicillin-resistant transconjugants (Fig. 1). This approach allows testing expression of the gene fusions in the same copy number and the same genomic context as the original genes. After confirmation by Southern blot analysis that transconjugants harbored the appropriate plasmid integrated in the right genomic region, four of them, each bearing a different plasmid, were selected for expression analysis.

Each strain was grown to exponential phase in mineral medium containing ßHB as the only carbon and energy source. Growing cells were then washed and resuspended in mineral medium with tetralin in the gas phase, and expression of the thn operons was measured by testing ß-galactosidase activity in samples taken at time intervals. As shown in Fig. 2, cells growing on ßHB did not express any of the gene fusions (t = 0 h). Similar results were obtained with cultures grown up to stationary phase (data not shown). However, expression of all gene fusions was evident shortly after the cells were transferred to growing conditions on tetralin as the only carbon and energy source, thus showing that expression of both tetralin biodegradation operons is not constitutive but induced by the presence of the pathway substrate. Although activity obtained from the transcriptional fusions stabilized a little earlier, both transcriptional and translational gene fusions were induced in similar ways and achieved similar induction ratios (120-fold and 180-fold induction for thnB and thnC, respectively), thus indicating that regulation was exerted at a transcriptional level. According to the maximal levels of expression, it appears that activity of the thnC promoter is slightly stronger than that of the thnB promoter.



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  		FIG. 2. Tetralin induction of lacZ fusions to thn genes. The ß-galactosidase activity of strains bearing a transcriptional thnB-lacZ fusion ({blacksquare}), a translational thnB-lacZ fusion ({square}), a transcriptional thnC-lacZ fusion (•), or a translational thnC-lacZ fusion ({circ}) after the strains were transferred to mineral medium with tetralin as the only carbon and energy source is shown.

Carbon catabolite repression of the thn operons. To test the effect of availability of a readily metabolized carbon source on the induction of thn operons, similar induction kinetics by tetralin were carried out using mineral medium containing different concentrations of ßHB, which allows a higher growth rate than tetralin, and two different complex-rich media. As shown for the thnC-lacZ translational fusion in Fig. 3, increasing the concentration of carbon in the mineral medium resulted in a proportional delay and a reduced level of tetralin induction, thus showing that availability of ßHB prevented expression of the thn operons. A result similar to that shown in the presence of 40 mM ßHB was obtained using 10 mM of different fatty acids (hexanoic acid, octanoic acid, sebacic acid, or suberic acid) (data not shown), thus indicating that other carbon sources have the same repressing effect. The rich medium MML also prevented tetralin induction, although significant expression was observed at the end of the induction. Full repression was obtained only in LB medium, which contains fivefold higher concentrations of tryptone and yeast extract than MML. Cultures similar to those used for the induction kinetics but lacking tetralin in the gas phase did not induce thnC expression at all (data not shown), which confirms that thn genes are not simply induced by carbon-limited growth conditions but their expression is also strictly dependent on the presence of the specific inducer of the pathway.



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  		FIG. 3. Carbon catabolite repression of tetralin biodegradation genes. The results of tetralin gene induction in the strain bearing the translational thnC-lacZ fusion while growing in mineral medium supplemented with 8 mM (•), 20 mM ({diamondsuit}), or 40 mM ({blacksquare}) ß-hydroxybutyrate, in rich MML medium ({square}), or in LB medium ({circ}) are shown.

Kinetics of induction by tetralin in a mineral medium containing 8 mM ßHB and nitrate or urea as the nitrogen source instead of ammonium were also carried out. The use of urea or nitrate instead of ammonium as a nitrogen source significantly reduced the growth rate (data not shown), which indicated that nitrogen availability was the growth-limiting factor under these conditions. Although the concentration of ßHB was not high enough to prevent induction of thnC-lacZ in a medium containing ammonium as the nitrogen source (Fig. 3 and 4), further limitation of growth by urea or nitrate prevented thnC-lacZ expression (Fig. 4). The use of these nitrogen sources did not prevent induction of thnC-lacZ expression in a medium containing tetralin as the only carbon and energy source (data not shown). These data clearly indicate that carbon limitation but not low growth rate per se allows induction of thnC by tetralin.



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  		FIG. 4. Effect of nitrogen limitation on catabolic repression of tetralin biodegradation genes. The results of tetralin gene induction in the strain bearing the translational thnC-lacZ fusion during growth in mineral medium with 8 mM ß-hydroxybutyrate and ammonium (•), urea ({diamondsuit}), or nitrate ({blacksquare}) as the nitrogen source are shown.

Identification of genes required for thn gene expression. Previous DNA sequencing identified structural thn genes encoding enzymes of the tetralin catabolic pathway. Further sequencing of 3.9 kb has allowed identification of two additional putative ORFs located downstream of thnA4 and in the same orientation and the partial sequence of a third ORF located 650 bp away in the opposite orientation (Fig. 1). Comparison of this partial ORF to those in the databases showed a high level of similarity to CopB, which is involved in copper resistance; therefore, this partial ORF is apparently not involved in tetralin biodegradation.

However, the product putatively encoded by the ORF just downstream of thnA4 showed high similarity to known LysR-type activators of operons involved in biodegradation of different aromatic pollutants; therefore, this ORF was named thnR. ThnR showed highest similarity to DntR from Burkholderia sp. strain DNT and to NagR from Ralstonia sp. strain U2 (45% identity along the molecules) (52). A dendrogram resulting from the comparison of amino acid sequences of similar LysR-type activators is shown in Fig. 5. Although a number of NahR activators from different strains have been removed from the figure for simplicity, the dendrogram indicates that ThnR diverged early from a branch where the activators of naphthalene biodegradation genes (NagR/NahR) cluster together, which suggests a possible evolutionary relationship between ThnR and activators of naphthalene-biodegradative operons.



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  		FIG. 5. Dendrogram showing the best tree obtained by the neighbor-joining method from the alignment of 17 sequences showing significant similarity to that of ThnR. The ThnR sequence is boxed. GenBank accession numbers for other sequences are as follows: for NagR (Ralstonia sp. strain U2), AF036940.2; for NbzR (Comamonas sp. strain JS765), AY223675.1; for NahR (Pseudomonas putida AN10), AF039534.1; for NahR (P. putida pNAH7), A32837; for HybR (Pseudomonas aeruginosa), AF087482.1; for MidR (Ralstonia sp. strain TAL1145), AF312768.2; for PcpR (Sphingomonas chlorophenolica ATCC39723), U12290.2; for PnbR (P. putida TW3), AF292094.1; for SalR (Acinetobacter sp. strain ADP1), AF150928.2; for CatR (P. putida), A35118; for AphT (Comamonas testosteroni TA441), BAA88500; for BenM (Acinetobacter sp. strain ADP1), AAC46441; for ClcR (P. putida pAC27), A40641; for PhnS (Burkholderia sp. strain RP007), AAD09867; for TcbR (Pseudomonas sp. strain P51), A38861; for HcaR (E. coli K-12), Q47141.

The start codon of another ORF (which was named thnY) which putatively encodes a product 324 amino acids long is seven nucleotides downstream of the stop codon of thnR. Unlike other thn genes, the start codon of thnY is not preceded by an evident Shine-Dalgarno sequence, which suggests that it is not translated to high levels. BLAST comparison of the putative product to those in the databases showed significant similarity to ferredoxin reductases, which are components of systems of electron transfer to dioxygenases or monooxygenases of different aromatic pollutants. The putative product showed highest (36%) identity to the ferredoxin reductase component of naphthalene dioxygenases from different strains, including Ralstonia sp. strain U2 (52). This type of ferredoxin reductase contains three domains. An NAD-1 binding domain, an FAD-6-binding domain, and a fer2 domain, which binds a chloroplast-type Cys4[2Fe-2S] iron sulfur center, are recognizable by sequence analysis of their C termini. ThnY showed the existence of the fer2 and the FAD-6-binding domains in an arrangement similar to that shown by other ferredoxin reductases. However, the NAD-1-binding domain was not detected. BLAST analysis of the C-terminal region covering 40% of ThnY, where the NAD-binding domain should be, showed similarity to the corresponding regions of the ferredoxin reductases (29 to 31% identity to the most similar sequences). However, this similarity was clearly lower than that shown by the N-terminal region covering 60% of the protein (38 to 40% identity to the most similar sequences). In addition, pairwise BLAST of the C-terminal region of ThnY and the consensus NAD-1-binding domain showed no significant alignment. Multialignment of C-terminal regions of ferredoxin reductases and the consensus NAD-1-binding domain showed two blocks of highly conserved residues. Interestingly, two conserved residues of each block were absent from the sequence of ThnY. These data clearly suggest that ThnY was originally a ferredoxin reductase whose NAD-binding domain has degenerated; therefore, it is not expected that ThnY could bind NAD/NADH. However, it still keeps some capacity to transfer electrons to the ferredoxin ThnA3, as tested by tetralin dioxygenase activity assays (data not shown).

Expression of the thn operons requires ThnR and ThnY. A collection of KIXX insertion mutants of strain TFA, unable to grow on tetralin as the only carbon and energy source, was previously constructed (26). Sequencing has revealed that mutant strain T656 contains the K1 KIXX insertion at the 69th codon of thnR, which suggests that ThnR is required for growth on tetralin. Two additional insertion mutants have been constructed. Mutant T669 bears a nonpolar KIXX insertion at the 241st codon of thnY, while mutant strain T601 bears a polar kanamycin resistance cassette insertion, flanked by transcription terminators, in its 140th codon. None of these mutants were able to grow using tetralin as the only carbon and energy source, thus suggesting that ThnY is also required for tetralin utilization.

The translational thnC-lacZ fusion was integrated into the genome of mutants T656, T669, and T601. As shown in Table 2, none of these mutants were able to induce thnC expression in response to tetralin. thnR and thnY were cloned separately in pIZ1016 so that transcription of both genes proceeded from the isopropyl-ß-D-thiogalactopyranoside (IPTG)-inducible tac promoter, thus yielding pIZ1017 and pIZ698, respectively. Mutant T656 transformed with pIZ1017 was able to grow on tetralin. In the absence of IPTG, partial induction of thnC by tetralin was observed, thus suggesting that plasmid-driven transcription of thnR was sufficiently high even in the absence of IPTG. However, maximal levels of thnC induction were achieved only by adding IPTG (Table 2). Similar positive complementation was observed in the mutants T601 and T669 transformed with pIZ698 (Table 2). Transformation of T656 with pIZ698 or T669 and T601 with pIZ1017 did not result in a change of the mutant phenotype (data not shown). Taken together, these data clearly show that the mutant phenotype of each insertion is due to lack of the corresponding product and not to potential effects preventing expression of the neighbor gene. Therefore, both ThnR and ThnY are required for expression of tetralin biodegradation genes.


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  		TABLE 2. Expression of the thnC-lacZ translational fusion in thnR or thnY mutants and complementation of the regulatory phenotypea

Given the sequence similarity of ThnY to ferredoxin reductases and its residual activity, ThnY requirement for thn operons induction might be explained if the real function of ThnY were to participate in some reaction of the tetralin biodegradation pathway and if some product of its metabolism, rather than tetralin by itself, were the real inducer. To test whether tetralin has to be metabolized to induce the thn operons, thnC-lacZ fusions, two in each operon, were constructed in four KIXX insertion mutants, each lacking one of the activities required for the first four reactions of the tetralin pathway. Expression of thnC in these mutants after growth under inducing conditions (8 mM ßHB plus tetralin) was monitored. As shown in Fig. 6, all mutants, including the one lacking ferredoxin that is essential for tetralin dioxygenase activity (37), expressed thnC to levels even higher than that obtained with the wild-type strain. As for the wild-type strain, expression of thnC in these mutants is dependent on the presence of tetralin (data not shown). These results indicate that tetralin by itself, and not any of its metabolic products, is the real inducer.



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  		FIG. 6. Tetralin induction of the translational thnC-lacZ fusion in different thn mutants. ß-Galactosidase activity of strains bearing a translational thnC-lacZ fusion was measured 20 h after transferring them to mineral medium with 8 mM ß-hydroxybutyrate and tetralin.

Expression of thnR is coregulated with other thn genes. Earlier genetic complementation analysis showed that the mutant T656 strain bearing the K1 insertion (thnR::KIXX) could not be complemented by the cosmid pIZ629, which carries the whole thn region but bears a polar mini-Tn5Km insertion in thnC (26). This data clearly suggested that thnR is transcribed from a promoter located upstream from the polar insertion in thnC, although interpretation was not possible until the K1 insertion had been precisely located within thnR. In turn, this indicates that ThnR, unlike other LysR-type regulators, does not repress its own synthesis. Actually, ThnR should activate its own transcription because ThnR is required to transcribe thnC in the presence of tetralin.

Coregulation of thnR, thnB, and thnC under inducing (8 mM ßHB plus tetralin) and noninducing (40 mM ßHB with no tetralin) conditions was analyzed by reverse transcription and PCR amplification. As shown in Fig. 7, no mRNA corresponding to any of these genes was detected under noninducing conditions. However, amplification of fragments of each of these genes was evident after reverse transcription of mRNA isolated from cultures grown under inducing conditions, which clearly indicates that transcription of thnR is regulated just as transcription of thnB and thnC is.



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  		FIG. 7. Reverse transcription-PCR of thnB, thnC, thnR, and ribosomal 16S genes. Two different amounts (0.8 and 4.8 µg) of cDNA obtained by retrotranscription of RNA isolated from strain TFA growing in MM-8 mM ßHB-tetralin (lanes 2 and 4) or MM-40 mM ßHB (lanes 3 and 5) were used. Amplification of the 16S ribosomal gene was used as a control to ensure equivalent amounts of cDNA between different growth conditions. Lane 1, 1-kb Plus DNA ladder (GibcoBRL).


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DISCUSSION
 
In an effort to understand how the ability to metabolize tetralin is expressed in Sphingomonas, a number of gene fusions were constructed and integrated by recombination into the genomic region containing the original genes. Analysis of expression of these gene fusions revealed that the two operons bearing tetralin biodegradation genes are regulated at the transcriptional level (Fig. 2) and that transcription of the thn operons strictly requires the presence of tetralin in the culture medium. Induction of catabolic gene expression by the substrate or by intermediates of the pathway is the most common and efficient way of adapting the metabolic capabilities of a bacteria to the opportunities offered by the environment.

Induction of the thn operons by tetralin is repressed under carbon-sufficient conditions such as undefined rich medium or mineral medium containing preferential carbon sources (Fig. 3). This clearly indicates that the thn operons are also regulated by a physiological control system which prevents expression of tetralin biodegradation capability when it is dispensable, thus improving adaptation of metabolic capabilities of the bacteria to their nutritional and energetic needs. Induction of the thn operons by tetralin does not take place under other growth-limiting conditions, such as nitrogen limitation (Fig. 4), but only under carbon-limiting conditions. Thus, limitation of growth rate per se is not responsible for the expression levels of thn operons, as previously shown in other systems such as the alkane degradation genes (14, 51); therefore, the global regulation system controlling expression of thn genes is a true carbon catabolite repression system.

The thnR gene, coding for a LysR-type transcriptional activator, has been identified by sequencing downstream of the thnA3A4 genes, and mutational analysis indicated that ThnR is strictly required for expression of tetralin biodegradation genes. Sequence comparison suggested that ThnR may be evolutionarily related to activators of naphthalene biodegradation genes, particularly to NagR (52). Although it is not formally proven, functional and sequence comparison data strongly suggest that ThnR is the activator of thn genes in response to tetralin.

The most common arrangement is that the gene coding for the LysR-type activator is located very closed to and divergent from the activated operon and that the regulator constitutively represses its own transcription in a feedback circuit, which maintains the concentration of the activator at levels just high enough to allow expression of the operon whose transcription activates under the appropriate conditions. Two interesting aspects are that thnR is cotranscribed with the thnCA3A4 genes and that ThnR does not appear to repress their own synthesis (compare basal expression levels in Table 2). In fact, ThnR appears to activate its own expression in a positive circuit responsive to tetralin, just like thnB or thnC expression (Fig. 7). Although this is unusual, there are precedents of similar situations in other LysR-type activators such as lrhA, required for flagella, motility, and chemotaxis in E. coli (30), or alkS, required for alkane biodegradation, and it is thought to allow a faster switch-on or switch-off of the system in response to the inducer (6).

ThnR is necessary but not sufficient for transcription of thn genes. Mutational and complementation analysis clearly indicated that ThnY, encoded downstream of thnR, is also strictly required (Table 2). Expression of thnC-lacZ in mutants blocked in different steps of the tetralin degradation pathway indicates that the actual inducer of thn operons is tetralin itself (Fig. 6); therefore, the requirement for ThnY cannot be due to lack of an inducer whose production required ThnY. Additionally, heterologous expression of ThnR in both TFA and E. coli strains did not relieve a strict requirement of ThnY for activation (data not shown), which suggests that ThnR cannot activate by itself even when overproduced. Thus, ThnY should be considered an auxiliary regulatory protein. Again, this is an unusual situation because in most instances LysR-type regulated systems are very simple and involve a single regulatory component, the activator, which is able to directly sense the effector and to regulate transcription. In some systems, an additional regulatory protein has been shown to modulate the activity of the activator by binding to it and thus preventing its function (23). However, to our knowledge, this is the first report of a LysR-type activator that requires an auxiliary protein to activate transcription.

Involvement of accessory regulatory proteins increases the versatility of the response of regulated systems. Implication of ThnY in activation of the thn operons and the fact that is similar to ferredoxin reductases raises a number of intriguing issues, such as what is the real function of ThnY, how does it exert its regulatory role, and what is it sensing. Considering its amino acid sequence, it is really unlikely that ThnY plays a direct role in the process of transcriptional activation. Rather, ThnY may be required for ThnR (or an additional undefined regulator) to adopt or maintain an appropriate configuration. Since ThnY might be in an oxidized or a reduced form, it is tempting to speculate that its activity may depend on its redox status (4), thus providing a way of connecting expression of thn operons to the physiological state of the cell. ThnY might be a component through which catabolic repression of thn operons is exerted. Alternatively, ThnY might sense oxygen through its FAD-binding domain, like the oxygen sensor NifL (27), which would make physiological sense, since the degradation pathway is strictly dependent on oxygen.


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ACKNOWLEDGMENTS
 
This work was supported by the Spanish Comisión Interministerial de Ciencia y Tecnología, grant BIO2002-03621, by a fellowship of the Spanish Ministerio de Educación to O. M.-P., and by a fellowship of Fundación Cámara to E. M.-R.


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FOOTNOTES
 
* Corresponding author. Mailing address: Centro Andaluz de Biología del Desarrollo, Universidad Pablo de Olavide, ctra. Utrera Km 1, 41013 Sevilla, Spain. Phone: 34-95-4349386. Fax: 34-95-4349376. E-mail: esansan{at}dex.upo.es. Back


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Journal of Bacteriology, September 2004, p. 6101-6109, Vol. 186, No. 18
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.18.6101-6109.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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