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 differentgrowth conditions, transcriptional and translational gene fusionsof the first genes of each operon to lacZ have been constructedin plasmids unable to replicate in Sphingomonas and integratedby recombination into the genome of strain TFA . Expression analysis indicated that the transcription of both genes is induced insimilar ways by the presence of tetralin . Gene expression inboth 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 activatesits own expression, since thnR is cotranscribed with the thnCA3A4genes . ThnY is similar to the ferredoxin reductase componentsof dioxygenase systems and shows the fer2 domain, binding aCys₄[2Fe-2S] iron sulfur center, and the FAD-binding domain,common to those reductases . However, it lacks the NAD-bindingdomain . Intriguingly, ThnY has a regulatory role, since it isalso strictly required for expression of the thn operons . Giventhe similarity of ThnY to reductases and the possibility ofits being present in the two redox states, it is tempting tospeculate that ThnY is a regulatory component connecting expressionof the thn operons to the physiological status of the cell.

 
The organic solvent tetralin [1,2,3,4-tetrahydronaphthalene]is a bicyclic molecule composed of an aromatic and an alicyclicmoiety, which share two carbon atoms . Tetralin is widely usedas a degreasing agent and solvent for fats, resins, and waxes,as a substitute for turpentine in paints, lacquers, and shoepolishes, and also in the petrochemical industry in connectionwith coal liquefaction [19] . A concentration of tetralin higherthan 100 µM inhibits bacterial growth [44] . Its toxicityis partly due to its lipophilic character, which results inits accumulation in the cell membranes, thus leading to changesin their structure and function [46, 47] . In addition, tetralinalso 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, severalreports suggest that some bacteria, such as Pseudomonas stutzeriAS39 [43], initially hydroxylate and further oxidize the alicyclic ring whereas others, such as Corynebacterium sp . strain C125 [45], initially dioxygenate the aromatic ring, thus indicatingthat aerobic metabolism of tetralin can be performed in differentways . 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 throughreactions 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 onearomatic ring, is able to cleave both the aromatic and the alicyclicrings of tetralin, which results in the production of pyruvateand 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].

 
The success of a catabolic pathway obviously depends on the capability of the enzymes to metabolize a particular compoundor the subsequent intermediates but also depends on an efficientregulatory system . Regulatory proteins and regulated promotersare key elements that control expression of catabolic operonsto assure that the enzymes are only produced under appropriateenvironmental conditions [13] . Thus, the expression of mostcatabolic operons is regulated by specific inducible systemsof control, which allow or activate synthesis of the correspondingenzymes only when the substrate or some intermediate of thepathway 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 tothe metabolic and/or energetic status of the cell [8, 13] . Someglobal controls apparently respond to different stress signalsand may involve the participation of alternate sigma factors[7, 9, 32, 48, 50], although most of them fit within the categoryof carbon catabolite repression, which prevents expression ofcatabolic operons in the presence of preferential carbon andenergy sources . Although carbon catabolite repression appearsto be a conserved phenomenon in bacteria, the molecular mechanismsthat exert the control may be completely different in distantlyrelated bacteria [39, 40] . Several reports of bacteria metabolizing different organic contaminants indicate that the mechanism[s]of carbon catabolite repression of biodegradative operons isdifferent from the cyclic AMP-dependent mechanism, which iswell 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 pathwaysin sphingomonads, although some LysR-type activators have beenrecently described [5, 22, 36] . Carbon catabolite repressionhas not yet been documented for this group of bacteria . Thispaper reports on the regulated expression of the tetralin biodegradationoperons of S . macrogolitabida strain TFA, showing that it isinduced in the presence of the pathway substrate and subjectedto carbon catabolite repression . Characterization of two regulatorygenes whose products are essential for thn gene expression isalso described.

 
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 bycloning a KIXX cassette [from pUC4-KIXX; Pharmacia] excisedwith HindIII into a BglII restriction site, interrupting the241st codon of thnY in pIZ619.

 
Plasmid pIZ1165 was constructed by cloning a 4.5-kb EcoRI fragment from pIZ604 [26] which bore thn'A4RY and 1.35 kb downstreamof thnY in a SalI-PstI-SphI-HindIII-lacking pTZ18R [34] openedwith EcoRI . Then, to construct pIZ601, an EcoRI fragment, harboringthe Km resistance flanked by transcriptional terminators frompUT-miniTn5Km [12], was inserted in pIZ1165 in a PstI site inthnY, 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 orientationsin the SmaI site of pIC552 [31], yielding plasmids pIZ1009 [thnC-lacZtranscriptional fusion] and pIZ1010 [thnB-lacZ transcriptionalfusion].

To construct the broad-host-range plasmid pIZ1016, an EagI-SalI fragment, bearing the tac promoter and lacI^q from pMM40 [28],was excised from pIZ1015 and cloned between NcoI-SalI sitesof pBBR1MCS-5 [29], removing the plasmid's lac promoter . PlasmidpIZ1015 was obtained by cloning an EagI-EcoRI fragment frompMM40, bearing the tac promoter and lacI^q, between EagI-ClaIin 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] intopBluescript 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 bluntedwith Klenow polymerase and deoxynucleoside triphosphates orwith T4 polymerase and deoxynucleoside triphosphates.

Escherichia coli DH5 [21] was used for cloning, isolation ofDNA for sequencing, and other DNA manipulations.

S . macrogolitabida strain TFA [26] harboring transcriptionalor translational fusions of the promoter for the thnB or thnCgene to lacZ [TFA-1002, TFA-1003, TFA-1009, and TFA-1010] wereused for ß-galactosidase assays . TFA mutants derivativesT601, 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, andT669-1002] were used for complementation experiments or ß-galactosidaseassays.

To construct the ThnY^– mutant strains T669 and T601, plasmids pIZ669 and pIZ601 were respectively electrotransformed intothe wild-type TFA strain, and candidates showing homologousrecombination were isolated as previously described [26].

Plasmids pIZ1002, pIZ1003, pIZ1009, and pIZ1010 were transferred to strain TFA and TFA mutants by triparental matings . Sincenone 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 witha marked 1.35-kb BamHI-NruI fragment, containing thnY, frompIZ619 . T669 was also hybridized with a KIXX HindIII probe.An EcoRI fragment, containing the Km resistance gene, was excisedfrom pUT-miniTn5Km, labeled, and hybridized to T601 . A 1.41-kb EcoRV-ApaI fragment, containing the promoter region, was markedand used as a probe to check the integration of the transcriptionaland translational fusions.

Media and growth conditions. E . coli strains were routinely grown in Luria-Bertani [LB] mediumat 37°C . TFA strains were grown at 30°C in MML richmedium [mineral medium [MM] supplemented with 0.2% tryptoneand 0.1% yeast extract], LB medium, or MM medium [16] suppliedwith tetralin in the vapor phase or/and ß-hydroxybutyrate[ßHB] as the carbon and energy source . MM medium containing8 mM nitrate or 17 mM urea instead of ammonium as a nitrogensource 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°Cin mineral medium containing ßHB as the only carbonand energy source to exponential phase [optical density at 600nm = 0.8 to 1.0] . Then, cells were washed to remove the carbonsource and diluted to a final optical density of about 0.1 inMML, LB medium, or MM medium, which could be supplemented witha carbon source, in the absence or the presence of the inducertetralin in the gas phase . Cultures were grown at 30°C,aliquots were withdrawn at different cell densities, and ß-galactosidaseactivity was assayed as described by Miller [35].

RNA extraction. RNA extraction was performed as described by Chomczynski andSacchi [10] . Harvested cells were subsequently treated withacid phenol, N-lauryl sarcosine, and guanidinium thiocyanateat 60°C, chloroform, DNase, and proteinase K . RNA was finallyrecovered after phenol:chloroform:isoamyl alcohol [25:24:1],and chloroform:isoamyl alcohol [24:1] treatment and precipitationwith 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 [AppliedBiosystems] and following the manufacturer's instructions . Differentamounts [0.8 and 4.8 µg] of the obtained cDNA were usedto 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 fragmentfrom thnR with primers thnR-RT1 [5'-CGGTCAAACCGAGTCTGAAGA-3']and thnR-RT2 [5'-ATGGAGCCAACAGCATTTGC-3'] . As an amplificationcontrol, primers f27 and r519 [26] were used to amplify a 500-bp fragment corresponding to 16S rRNA . The PCR program consistedof 5 min at 94°C, 20 cycles of 30 s at 94°C, 30 s at57°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 samplesdid not contain contaminating DNA, PCR amplification was performedusing RNA preparations as templates.

Sequence analysis comparison. The obtained sequence was initially compared using the BLASTpand tBLASTn programs to those in databases [1] . Sequences thatshowed high similarity to that of strain TFA were aligned usingthe 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 tothe DDBJ, EMBL, and GenBank nucleotide sequence databases andannotated as an update of the sequence at accession no. AF157565.

 
Tetralin induction of the thn operons. To easily test expression of the tetralin catabolic operons,transcriptional and translational lacZ gene fusions to thnBand thnC, the first genes of each operon, were constructed asdescribed in Materials and Methods, thus yielding the plasmidspIZ1010 [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 expressionof the gene fusions in the same copy number and the same genomiccontext 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 expressionanalysis.

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 mediumwith tetralin in the gas phase, and expression of the thn operonswas measured by testing ß-galactosidase activity insamples taken at time intervals . As shown in Fig . 2, cells growingon ßHB did not express any of the gene fusions [t= 0 h] . Similar results were obtained with cultures grown upto stationary phase [data not shown] . However, expression ofall gene fusions was evident shortly after the cells were transferredto growing conditions on tetralin as the only carbon and energysource, thus showing that expression of both tetralin biodegradationoperons is not constitutive but induced by the presence of thepathway substrate . Although activity obtained from the transcriptionalfusions stabilized a little earlier, both transcriptional andtranslational gene fusions were induced in similar ways andachieved similar induction ratios [120-fold and 180-fold inductionfor thnB and thnC, respectively], thus indicating that regulationwas exerted at a transcriptional level . According to the maximallevels of expression, it appears that activity of the thnC promoter is slightly stronger than that of the thnB promoter.

 
Carbon catabolite repression of the thn operons. To test the effect of availability of a readily metabolizedcarbon source on the induction of thn operons, similar inductionkinetics by tetralin were carried out using mineral medium containingdifferent concentrations of ßHB, which allows a highergrowth 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 ina proportional delay and a reduced level of tetralin induction,thus showing that availability of ßHB prevented expressionof the thn operons . A result similar to that shown in the presenceof 40 mM ßHB was obtained using 10 mM of differentfatty acids [hexanoic acid, octanoic acid, sebacic acid, orsuberic acid] [data not shown], thus indicating that other carbonsources have the same repressing effect . The rich medium MMLalso prevented tetralin induction, although significant expressionwas observed at the end of the induction . Full repression wasobtained only in LB medium, which contains fivefold higher concentrationsof tryptone and yeast extract than MML . Cultures similar tothose used for the induction kinetics but lacking tetralin inthe gas phase did not induce thnC expression at all [data notshown], which confirms that thn genes are not simply inducedby carbon-limited growth conditions but their expression isalso strictly dependent on the presence of the specific inducerof the pathway.

 
Kinetics of induction by tetralin in a mineral medium containing8 mM ßHB and nitrate or urea as the nitrogen sourceinstead of ammonium were also carried out . The use of urea ornitrate instead of ammonium as a nitrogen source significantlyreduced the growth rate [data not shown], which indicated thatnitrogen availability was the growth-limiting factor under theseconditions . Although the concentration of ßHB wasnot high enough to prevent induction of thnC-lacZ in a mediumcontaining 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 lowgrowth rate per se allows induction of thnC by tetralin.

 
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 putativeORFs located downstream of thnA4 and in the same orientationand the partial sequence of a third ORF located 650 bp awayin 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 comparisonof amino acid sequences of similar LysR-type activators is shownin Fig . 5 . Although a number of NahR activators from differentstrains have been removed from the figure for simplicity, thedendrogram indicates that ThnR diverged early from a branchwhere the activators of naphthalene biodegradation genes [NagR/NahR]cluster together, which suggests a possible evolutionary relationshipbetween ThnR and activators of naphthalene-biodegradative operons.

 
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-Dalgarnosequence, which suggests that it is not translated to high levels.BLAST comparison of the putative product to those in the databasesshowed significant similarity to ferredoxin reductases, whichare 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 ferredoxinreductase contains three domains . An NAD-1 binding domain, anFAD-6-binding domain, and a fer2 domain, which binds a chloroplast-typeCys₄[2Fe-2S] iron sulfur center, are recognizable by sequenceanalysis of their C termini . ThnY showed the existence of thefer2 and the FAD-6-binding domains in an arrangement similarto that shown by other ferredoxin reductases . However, the NAD-1-bindingdomain was not detected . BLAST analysis of the C-terminal regioncovering 40% of ThnY, where the NAD-binding domain should be,showed similarity to the corresponding regions of the ferredoxinreductases [29 to 31% identity to the most similar sequences].However, this similarity was clearly lower than that shown bythe 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 originallya 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 electronsto the ferredoxin ThnA3, as tested by tetralin dioxygenase activityassays [data not shown].

Expression of the thn operons requires ThnR and ThnY. A collection of KIXX insertion mutants of strain TFA, unableto grow on tetralin as the only carbon and energy source, waspreviously constructed [26] . Sequencing has revealed that mutant strain T656 contains the K1 KIXX insertion at the 69th codonof 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 codonof thnY, while mutant strain T601 bears a polar kanamycin resistance cassette insertion, flanked by transcription terminators, inits 140th codon . None of these mutants were able to grow usingtetralin as the only carbon and energy source, thus suggestingthat ThnY is also required for tetralin utilization.

The translational thnC-lacZ fusion was integrated into the genomeof mutants T656, T669, and T601 . As shown in Table 2, none ofthese mutants were able to induce thnC expression in responseto tetralin . thnR and thnY were cloned separately in pIZ1016so 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 ableto grow on tetralin . In the absence of IPTG, partial inductionof 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 complementationwas observed in the mutants T601 and T669 transformed with pIZ698[Table 2] . Transformation of T656 with pIZ698 or T669 and T601with pIZ1017 did not result in a change of the mutant phenotype[data not shown] . Taken together, these data clearly show thatthe mutant phenotype of each insertion is due to lack of thecorresponding product and not to potential effects preventingexpression of the neighbor gene . Therefore, both ThnR and ThnYare required for expression of tetralin biodegradation genes.

 
Given the sequence similarity of ThnY to ferredoxin reductasesand its residual activity, ThnY requirement for thn operons induction might be explained if the real function of ThnY wereto participate in some reaction of the tetralin biodegradationpathway and if some product of its metabolism, rather than tetralinby itself, were the real inducer . To test whether tetralin hasto 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 thefirst four reactions of the tetralin pathway . Expression ofthnC in these mutants after growth under inducing conditions[8 mM ßHB plus tetralin] was monitored . As shown inFig . 6, all mutants, including the one lacking ferredoxin thatis 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 anyof its metabolic products, is the real inducer.

 
Expression of thnR is coregulated with other thn genes. Earlier genetic complementation analysis showed that the mutantT656 strain bearing the K1 insertion [thnR::KIXX] could notbe complemented by the cosmid pIZ629, which carries the wholethn 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 ßHBplus tetralin] and noninducing [40 mM ßHB with no tetralin] conditions was analyzed by reverse transcription andPCR 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.

 
In an effort to understand how the ability to metabolize tetralinis expressed in Sphingomonas, a number of gene fusions were constructed and integrated by recombination into the genomicregion containing the original genes . Analysis of expressionof these gene fusions revealed that the two operons bearingtetralin biodegradation genes are regulated at the transcriptionallevel [Fig . 2] and that transcription of the thn operons strictly requires the presence of tetralin in the culture medium . Inductionof catabolic gene expression by the substrate or by intermediatesof the pathway is the most common and efficient way of adaptingthe metabolic capabilities of a bacteria to the opportunitiesoffered by the environment.

Induction of the thn operons by tetralin is repressed under carbon-sufficient conditions such as undefined rich medium ormineral medium containing preferential carbon sources [Fig.3] . This clearly indicates that the thn operons are also regulated by a physiological control system which prevents expressionof tetralin biodegradation capability when it is dispensable,thus improving adaptation of metabolic capabilities of the bacteriato 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 undercarbon-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 controllingexpression of thn genes is a true carbon catabolite repressionsystem.

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 relatedto activators of naphthalene biodegradation genes, particularlyto 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 divergentfrom the activated operon and that the regulator constitutivelyrepresses its own transcription in a feedback circuit, whichmaintains the concentration of the activator at levels justhigh enough to allow expression of the operon whose transcriptionactivates under the appropriate conditions . Two interestingaspects are that thnR is cotranscribed with the thnCA3A4 genesand that ThnR does not appear to repress their own synthesis[compare basal expression levels in Table 2] . In fact, ThnRappears to activate its own expression in a positive circuitresponsive 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 allowa faster switch-on or switch-off of the system in response tothe 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 differentsteps of the tetralin degradation pathway indicates that theactual inducer of thn operons is tetralin itself [Fig . 6]; therefore,the requirement for ThnY cannot be due to lack of an inducerwhose production required ThnY . Additionally, heterologous expressionof ThnR in both TFA and E . coli strains did not relieve a strictrequirement of ThnY for activation [data not shown], which suggeststhat 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 instancesLysR-type regulated systems are very simple and involve a singleregulatory component, the activator, which is able to directlysense the effector and to regulate transcription . In some systems,an additional regulatory protein has been shown to modulatethe activity of the activator by binding to it and thus preventingits function [23] . However, to our knowledge, this is the firstreport of a LysR-type activator that requires an auxiliary proteinto 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 exertits regulatory role, and what is it sensing . Considering itsamino acid sequence, it is really unlikely that ThnY plays adirect 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 ThnYmight be in an oxidized or a reduced form, it is tempting tospeculate that its activity may depend on its redox status [4],thus providing a way of connecting expression of thn operonsto the physiological state of the cell . ThnY might be a componentthrough 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 physiologicalsense, since the degradation pathway is strictly dependent onoxygen.

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

 
* 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@dex.upo.es .

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