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Appl Environ Microbiol. 2010 January; 76(1): 110–118.
Published online 2009 November 6. doi:  10.1128/AEM.01846-09
PMCID: PMC2798658

Tetralin-Induced and ThnR-Regulated Aldehyde Dehydrogenase and β-Oxidation Genes in Sphingomonas macrogolitabida Strain TFA[down-pointing small open triangle]

Abstract

A new cluster of genes has been found downstream of the previously identified thnA2 gene. The gene products are similar to nonacylating aldehyde dehydrogenases (ThnG) and to proteins representing a complete β-oxidation pathway (ThnH to ThnP). ThnG has a nonacylating NAD-dependent pimelic semialdehyde dehydrogenase activity that renders pimelic acid a seven-carbon dicarboxylic acid. For further metabolism via β-oxidation, pimelic acid could be acylated by a constitutive acyl coenzyme A (acyl-CoA) ligase found in Sphingomonas macrogolitabida strain TFA or by ThnH, which would transfer CoA from a previously acylated molecule. The first round of β-oxidation is expected to render glutaryl-CoA and acetyl-CoA. Glutaryl-CoA dehydrogenase (ThnN) would catalyze the oxidation and decarboxylation of glutaryl-CoA and yield crotonyl-CoA, which enters the central metabolism via acetyl-CoA. Mutagenesis studies have shown that these genes are not essential for growth on tetralin or fatty acids, although a thnG disruption mutant showed threefold less pimelic semialdehyde dehydrogenase activity. Transcriptional analysis indicated that these genes are induced by tetralin, subjected to catabolite repression, and regulated by the same regulatory factors previously identified to regulate other thn structural genes. In the present study, transcription initiation upstream of thnH and thnM has been detected by primer extension analysis, and putative promoters were identified by sequence analysis. In addition, binding of the activator ThnR to its putative binding sites at the PH and PM promoter regions has been characterized. These results provide a complete characterization of the biodegradation pathway of tetralin to central metabolites and describe the transcriptional organization of the thn operons in S. macrogolitabida strain TFA.

The Sphingomonas macrogolitabida strain TFA is a gram-negative bacterium that is able to grow on tetralin (1,2,3,4-tetrahydronaphthalene) as the only carbon and energy source (14). This compound is a bicyclic molecule composed of an aromatic and an alicyclic moiety, which shares two carbon atoms. It is produced from naphthalene by catalytic hydrogenation or from anthracene by cracking, and it is widely used as a degreasing agent and organic solvent (6). The toxicity of tetralin is partly due to its lipophilic character, leading to accumulation in cell membranes and resulting in changes in membrane structure and function (32, 33). In addition, tetralin also forms toxic hydroperoxides in the cell (4).

As shown in Fig. Fig.1A,1A, strain TFA metabolizes tetralin through a meta-cleavage pathway that involves an initial dioxygenation step (catalyzed by the enzymatic complex ThnA1A2A3A4), followed by a dehydrogenation step (catalyzed by ThnB) to produce 1,2-dihydroxytetralin (24). A 1,2-dihydroxynaphthalene dioxygenase (ThnC) is responsible for the extradiol cleavage of the catechol derivative (1), followed by hydrolytic cleavage (by ThnD) of the C-C bond that is part of the alicyclic ring of the fission product, resulting in a long dicarboxylic acid with 10 carbon atoms (12). The sequential action of a hydratase (ThnE) and an aldolase (ThnF) generates pimelic semialdehyde and a molecule of pyruvate, which enters the central metabolism (13). Further metabolism of this compound could be through a β-oxidation pathway, which first entails an oxidation and activation with coenzyme A (CoA) to generate pimeloyl-CoA. This reaction could take place by two different methods: via an acylating aldehyde dehydrogenase (ALDH) that could catalyze the reaction to yield pimeloyl-CoA in one step or via the oxidation of pimelic semialdehyde to pimelic acid by a nonacylating ALDH, including its subsequent activation by a CoA-ligase or a CoA-transferase.

FIG. 1.
(A) The tetralin degradation pathway in the S. macrogolitabida strain TFA. Pimelic semialdehyde is shown in a dotted box. (B) Genetic organization of thn genes. A black filled triangle denotes the KIXX cassette insertion in the different mutants. (C) ...

In strain TFA, the thn genes that encode the catalytic proteins of this meta-cleavage pathway are organized in two divergent and closely linked operons (14) (Fig. (Fig.1B).1B). Transcription of both operons is coregulated and dependent on two proteins, the LysR-type transcriptional activator (LTTR) ThnR and its coactivator ThnY (21). ThnR binds to ATCA-N7-TGAT palindromic sites present at the promoter region of each operon in order to activate transcription (19). Expression of the thn operons is tetralin inducible and subject to catabolite repression. In addition, a singular and unprecedented method of communication between the tetralin catabolic pathway and its regulatory system has recently been described that prevents gratuitous induction of the thn genes by molecules that cannot be metabolized through the meta-cleavage pathway (20). The key protein in this communication is ThnA3, the ferredoxin that transports electrons to the initial dioxygenase complex, ThnA1A2. In the absence of a suitable substrate for the dioxygenase, ThnA3 would accumulate in its reduced form and act as a negative modulator of the regulatory system.

We describe here the identification and characterization of a new set of genes located downstream of thnA2; this set encodes proteins that could be involved in tetralin catabolism to central metabolic products. Genetic and biochemical characterization was carried out in order to demonstrate the role in tetralin biodegradation. Finally, gene expression studies allowed us to describe the complete the transcriptional organization of thn genes in the S. macrogolitabida strain TFA and to characterize the ThnR-recognized promoters.

MATERIALS AND METHODS

Media and growth conditions.

Escherichia coli strains were routinely grown in Luria-Bertani (LB) medium at 37°C. S. macrogolitabida strains were grown at 30°C in MML-rich medium (1) or MM medium (3) supplied with either tetralin in the vapor phase or 8 or 40 mM β-hydroxybutyrate (βHB) as the carbon sources. Sebacic and pimelic acids were added at 5 and 10 mM, respectively, when used as carbon sources.

Construction of plasmids and strains.

Table Table11 presents the strains, plasmids, and oligonucleotides used in the present study and their main characteristics.

TABLE 1.
Bacterial strains, plasmids, and oligonucleotides used in this study

To overproduce ThnG, the thnG gene was amplified with the primers thnGNdeI and T3, cut with SmaI-NdeI, and cloned into pIZ578 (1), resulting in the pIZ474 plasmid.

To generate the TFA mutant strains, several plasmids were constructed. First, a 2.5-kb EcoRI fragment bearing thn′A1A2GH′ from pIZ604 was cloned into pTZ18R (22). In this EcoRI fragment, a HindIII-cut and Klenow filled-in KIXX cassette was cloned into the NaeI site present in thnG in order to construct pIZ668. pIZ687 contained a 4-kb SacI fragment bearing thn′IJKL′ from pIZ604, cloned into pBluescript II SK(+). pIZ1107 and pIZ1114 were generated by introducing a HindIII-cut and Klenow filled-in KIXX cassette into the NruI site of the thnJ gene on pIZ687 and the Klenow-filled BglII site of the thnK gene on pIZ687, respectively. pIZ1109 contained a 7.3-kb EcoRI fragment bearing thn′A1A2GHIJKL′ from pIZ604 cloned into pTZ18R. pIZ1112 and pIZ1113 were generated by introducing a HindIII-cut and Klenow filled-in KIXX cassette into the EcoRV site of the thnH gene or into the Klenow-filled PstI site of the thnI gene in pIZ1109, respectively. All of these insertions were integrated into the genome of strain TFA by marker exchange, and kanamycin-resistant and ampicillin-sensitive transformants were selected.

To construct a translational thnH-lacZ fusion, a 407-bp fragment containing the intergenic region thnG-thnH and the first codons of thnH was amplified with the thnH15BamHI and thnG3BamHI primers using pIZ1108 as the template. The DNA product was digested with BamHI and cloned into the BamHI site of pMPO521, thus generating pMPO531. An equivalent thnM-lacZ translational fusion (pMPO694) was constructed. Plasmid pIZ111 was used as the template for primers thnLBglII and thnLBglII in order to amplify a 508-bp fragment that was then BglII digested and cloned into the BamHI site of pMPO521.

To monitor the expression level of thn genes in the Δthn mutant strain T690, plasmids bearing the thnH-lacZ or thnM-lacZ gene fusions were introduced by triparental mating and integrated by homologous recombination of the kanamycin resistance genes; the resulting strains were named T690-531 and T690-694, respectively.

The genetic arrangement of all of the constructed strains was confirmed by Southern blot.

Fragments containing the thnG-thnH or the thnL-thnM intergenic region were cloned in pBluescript II SK(+). For thnG-thnH, a 389-bp fragment was amplified with the thnG2 and thnH14 primers and cloned into the EcoRV site, thus yielding pMPO529. For thnL-thnM, the same fragment used for pMPO694 construction was cloned into the BamHI site of pBluescript II SK(+) to generate pMPO532.

Protein overexpression, crude extracts, and native electrophoretic conditions.

For overexpression of thnG, E. coli C41(DE3) was transformed with pIZ474 and pIZ578 was used as a control. The resulting transformants were grown in LB liquid medium at 26°C up to an optical density at 600 nm of 0.7. The transformants were then induced with 1 mM IPTG (isopropyl-β-d-thiogalactopyranoside) overnight (10 to 12 h). The cells were harvested by centrifugation, frozen in liquid nitrogen, broken with aluminum oxide 90 (Merck), and suspended in 0.01 to 0.02 volumes (of the culture) of a buffer containing 20 mM Tris-HCl (pH 8.0) and 100 mM NaCl. The lysate was centrifuged at 12,500 × g for 30 min at 4°C. The supernatant was frozen and stored at −80°C. Gels were stained with GELCODEBlue stain reagent (Pierce).

ALDH activity assays.

ALDH activity in cell extracts was measured as described by Yan and Chen (40). The assay mixture contained 50 mM Tris-HCl buffer (pH 8), 5 mM dithiothreitol, 2 mM NAD+ or NADP+, 10 mM pimelic semialdehyde in dimethyl sulfoxide, and 100 μg of cell extract. To check CoA dependence, this compound was added to the reaction at 0.5 mM.

Activity staining of NAD+-pimelic semialdehyde-dependent aldehyde dehydrogenases in native polyacrylamide gels was performed as described by Schräder et al. (30), by incubating the gels in 100 mM potassium phosphate buffer (pH 7.6) containing 0.08% NAD, 0.04% nitroblue tetrazolium, 0.003% phenazine ethosulfate, and 8 mM pimelic semialdehyde at room temperature in the dark until activity bands were visible.

Tetralin induction assays.

Induction assays with tetralin were performed as described previously (21). The β-galactosidase activity of induced cultures of strains harboring lacZ fusions integrated into the chromosome was assayed as described by Miller (23).

RNA purification.

Total RNA purification was performed essentially as described by Yuste et al. (42). A second DNase I treatment was performed with a DNA-free kit (Ambion).

Once the absence of contaminating DNA was confirmed by PCR amplification, the samples were purified by using RNeasy columns (Qiagen) and RNA integrity was checked by agarose gel electrophoresis.

RT-PCR.

Reverse transcription (RT) of total RNA (5 μg) was carried out by using a high-capacity cDNA archive kit (Applied Biosystems) and random hexamers as primers. To detect specific transcripts, amplification reactions were performed with 10 to 100 ng of cDNA and a 20-cycle PCR program. The primers that were used are listed in Table Table1.1. Amplification of a 455-bp band of the 16S ribosomal gene with primers f27 and r519 (15) was performed to assure integrity of the cDNA.

The RT-PCR products obtained were resolved by 1% agarose gel electrophoresis and visualized by ethidium bromide staining.

Primer extension.

Primer extension reactions were performed as previously described (8) using 20 to 50 μg of RNA as a template, 32P-end-labeled oligonucleotides, and Superscript II reverse transcriptase (Invitrogen). The samples were run on a polyacrylamide sequencing gel. The dried gels were exposed to radiosensitive screens, which were subsequently scanned in a Typhoon 9410 scanner (GE Healthcare).

Electrophoretic mobility shift assays (EMSAs).

DNA probes for the thnG-thnH and thnL-thnM intergenic regions bearing the putative ThnR binding sites were obtained by PCR. For the thnG-thnH probe, a 157-bp fragment was amplified with the thnH16BamHI and thnG2 primers using pMPO529 as the template and then digested with BamHI. A 199-bp product corresponding to the thnL-thnM intergenic region was obtained with pMPO532 as the template and using thnM1HindIII and thnLMBamHI as the primers. The DNA fragment was then digested with HindIII. The probes were [α-32P]dCTP labeled by Klenow, which filled in the 3′ recessed ends.

EMSAs were performed as previously described (19). The gels were dried and then exposed on a radiosensitive screen. The radioactive bands were visualized with a Typhoon 9410 scanner and analyzed by using ImageQuant software (GE Healthcare). The apparent dissociation constant (Kd) was calculated as the concentration of protein at which 50% of the probe was associated in a complex (18).

Sequencing.

Sequencing was performed using the cosmid pIZ604 as the template (14).

Nucleotide sequence accession number.

The nucleotide sequence has been deposited in GenBank as an update of AF157565.

RESULTS

Cloning and sequence analysis of new thn genes.

A total of 10 putative genes, which we have designated thnG to thnP, have been found in a 14,309-bp DNA sequenced fragment located downstream of thnA2 (Fig. (Fig.1B;1B; Table Table2).2). thnG encodes a protein similar to the nonacylating NAD-dependent aldehyde dehydrogenases that are involved in the biodegradation pathways of aromatic compounds. The four invariant residues (Gly228, Gly281, Glu381, and Phe383; ThnG positions) found in this type of dehydrogenase are also conserved in ThnG (28). Gly228, Glu381, and Phe383 are required for binding of the nicotinamide ring of NAD, whereas Gly281 places the catalytic residue Cys284 correctly for the activity (28). In addition, 9 of the 11 residues found in more than 95% of the ALDHs are also present in ThnG; these include Gly145, Asn154, Pro156, Lys245, Gly252, Pro385, Gly431, Asn436, and Gly449. No significant homology was found with the CoA-acylating aldehyde dehydrogenases, which have four invariant residues (37) that are absent in ThnG. The ThnG function could be to catalyze the oxidation of pimelic semialdehyde to pimelic acid (Fig. (Fig.1C).1C). thnH encodes a product similar to proteins that belong to a new family of CoA transferases known as the “CaiB/BaiF” protein family (11). These enzymes catalyze the reversible transfer of CoA groups from CoA thioesters to free acids in order to activate them for further metabolism. This protein might catalyze the activation of pimelic acid to pimeloyl-CoA (Fig. (Fig.1C).1C). thnI encodes a protein homologous to the acetyl-CoA acetyl transferases, which catalyze the cleavage of a 3-oxoacyl-CoA to generate acetyl-CoA and a CoA-activated fatty acid that is shorter than the original substrate by two carbon atoms. In the case of pimeloyl-CoA, the resulting product would be glutaryl-CoA (10) (Fig. (Fig.1C).1C). thnJ and thnK encode products similar to the large and small subunits of pimeloyl-CoA dehydrogenases, respectively. These products might catalyze the oxidation of pimeloyl-CoA to the corresponding 2,3-dehydroacyl-CoA acid (10) (Fig. (Fig.1C).1C). thnL is similar to proteins with enoyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase domains located at their N and C termini, respectively, which could catalyze the two subsequent steps of the β-oxidation pathway (41) (Fig. (Fig.1C),1C), thus generating the corresponding 3-oxoacyl-CoA acid. thnM encodes a protein homologous to the TonB-dependent receptors, which seem to be involved in iron transport. thnN codes for a protein similar to the putative glutaryl-CoA dehydrogenases. This enzyme could catalyze the oxidative decarboxylation of glutaryl-CoA to crotonyl-CoA (16, 39), which can be further converted to acetyl-CoA (Fig. (Fig.1C).1C). The last two genes, thnO and thnP, encode proteins similar to the α- and β-subunits of flavoproteins, respectively, which serve as specific electron acceptors for primary dehydrogenases of the β-oxidation pathway and transfer electrons to the respiratory chain (38).

TABLE 2.
Database comparison of the deduced Thn proteins

Role of ThnG: mutant phenotype and characterization of its enzymatic activity.

The TFA T-668 mutant was obtained by insertion of a nonpolar KIXX cassette into the NaeI site of thnG, resulting in truncation of the gene beyond codon 169. In contrast to the inability of the thn insertion mutants to grow on tetralin (14), T-668 was still able to use this compound as a carbon and energy source. Cell extracts of TFA and T-668 mutant strains were assayed for ALDH activity dependent on pimelic semialdehyde and NAD. Consistent with its phenotype, strain T-668 showed reduced but clearly evident ALDH activity (43.5 ± 11.5 U mg−1 for TFA versus 14.7 ± 5.2 U mg−1 for T-668). A form of ThnG bearing a His6 tag at its N terminus was overproduced in E. coli C41 to characterize its activity in detail. ALDH activity ranging from 1,500 to 2,000 U mg of total protein−1 was detected in crude extracts of the C41 (pIZ474) strain that overproduces ThnG-His6. No activity was detected when NADP was used as the oxidant instead of NAD. Crude extracts of C41 bearing the control plasmid pIZ578 showed no pimelic semialdehyde-dependent ALDH activity. ThnG activity was not affected by the addition of 0.5 mM coenzyme A or dithiothreitol to the assay mixture, which indicated that the ALDH activity of ThnG is not acylating.

To test the presence of additional pimelic semialdehyde dependent ALDH activities in TFA that might account for the growth of strain T-668 on tetralin, enzymatic assays were carried out using nondenaturing gels (30). Crude extracts of TFA wild-type and T-668 mutant cells grown on tetralin were subjected to native electrophoresis and pimelic semialdehyde dependent ALDH activity was detected in gels. In addition, a crude extract from E. coli C41 overexpressing His-tagged ThnG was included as a control. As shown in Fig. Fig.2,2, a predominant activity band was obtained in extracts of the E. coli strain overproducing ThnG-His6. A weaker activity band that migrated faster was present in strain TFA but absent in the T-668 mutant, indicating that this band represents ThnG activity. Additional weaker ALDH activity bands that migrated more slowly were detected in both TFA and the T-668 mutant. This pimelic semialdehyde dehydrogenase activity may account for the residual activity found in the cell extracts of the T-668 mutant and for its growth on tetralin.

FIG. 2.
Pimelic semialdehyde dependent ALDH activity in nondenaturing gels. Cell extracts (20 μg) of E. coli C41(pIZ578) (lane 1), E. coli C41(pIZ474) (lane 2), S. macrogolitabida wild type (lane 3), and S. macrogolitabida T-668 mutant (lane 4) were assayed. ...

The β-oxidation genes are not essential for growth on tetralin.

In order to elucidate the role of the genes found downstream of thnG, mutations were constructed by insertion of a nonpolar KIXX cassette into thnH, thnI, thnJ, and thnK (T-1112, T-1113, T-1107 and T-1114, respectively) (see Fig. Fig.1B)1B) and introduced into the TFA genome. Mutant strains were characterized by their capacity for growth on sebacic acid, tetralin, pimelic acid or tetralin plus pimelic acid. In all of the tests, the mutant strains grew similarly to the wild type (data not shown), and their growth rate and final cell density were unaffected. This indicates that the expression of these newly discovered thn genes is not essential in TFA for growth on tetralin or on the dicarboxylic pimelic or sebacic acids.

Tetralin induction and coordinated expression of the new thn genes.

Total RNA of TFA was isolated from cells grown on tetralin plus 8 mM βHB (inducing conditions for thn genes) and on 40 mM βHB (noninducing conditions). Semiquantitative RT-PCR was performed to analyze the expression of the new set of genes in both conditions and to define the putative transcriptional units (Fig. (Fig.3).3). The oligonucleotides that were used to amplify intergenic and intragenic sequences are shown in Table Table1.1. The results show that all of the genes were tetralin-induced because amplification was obtained only with cDNA samples from tetralin-grown cells. thnG and the first five thn genes involved in β-oxidation (thnH-thnL) seemed to be part of the thnBDEFA1A2 operon (see Fig. Fig.1B)1B) (14, 25) because amplification of the thnA2-thnG intergenic region and the five subsequent intergenic regions was positive in tetralin-grown cells. The only intergenic region that failed to yield an amplified product was thnL-thnM, which suggests that genes from thnM to thnP are organized in a completely different operon. Expression of this new operon seems to also be dependent on the transcriptional activator ThnR (20, 21) because no amplification of the thnM-thnN intergenic region was obtained using cDNA from the mutant T1032 that bears a deletion of thnR (Fig. (Fig.33).

FIG. 3.
Transcriptional analysis of thn genes by RT-PCR. RNA samples from TFA (wild type) and the T1032 (ΔthnR) mutant grown on tetralin plus 8 mM βHB (βH8T) or on 40 mM βHB (βH40) were used. A control PCR using total DNA ...

Transcriptional organization of the new thn genes.

Despite the presence of an RNA transcript covering the 3′ end of thnG and the 5′ end of thnH, in silico analysis of the thnG-thnH intergenic region showed the presence of a putative ThnR binding site (19), which suggested the existence of an additional tetralin-inducible promoter. To detect whether there is promoter activity that responds to tetralin in this region, a translational thnH::lacZ fusion (pIZ531) to the codon 58 of thnH that contained up to 221 bp upstream of the initiation codon was constructed and integrated into the genome of the T690 strain (Δthn) by homologous recombination between the kanamycin resistance gene present on both DNA molecules. To analyze the expression of the thnMNOP operon, a translational thnM::lacZ fusion (pIZ694) from codon 21 to 431 bp upstream of the initiation codon of thnM was also constructed and similarly integrated into the T690 genome. The expression level of the fusions in this strain, lacking the thn genes, was tested under several growth conditions, and the results are summarized in Fig. Fig.4.4. Very low expression levels of the thnH::lacZ gene fusion were observed when the cultures were grown in the absence of tetralin, but substantial expression was evident when the cultures were grown under inducing conditions. This indicated that a promoter with significant transcriptional activity does exist at the thnH 5′ region and that expression of the promoter is strictly dependent on ThnR and ThnY (only detected in the strain T690 bearing pIZ1158). When a sufficient amount of the additional carbon source was present (βHB 40 mM), tetralin induction was completely abolished, which indicated that this promoter is also subject to strong catabolite repression. Unlike thnH::lacZ, the thnM::lacZ gene fusion showed a high expression level in 8 mM βHB compared to 40 mM βHB in the absence of tetralin. However, the presence of tetralin and 8 mM βHB resulted in a fivefold induction of expression. In addition, under catabolite repression conditions (40 mM βHB and tetralin), expression was reduced to the basal level in the absence of the inducer and 8 mM βHB. Expression of the thnM::lacZ gene fusion was also strictly dependent on ThnR and ThnY. In spite of the different expression levels of the two lacZ gene fusions under noninducing conditions, these results confirmed the presence of promoters for the thnH and thnM genes and showed that they are regulated by ThnR and ThnY in the same way that the thnC and thnB promoters are, i.e., they are induced by tetralin and subjected to catabolite repression by βHB (21).

FIG. 4.
Expression of thnH-lacZ and thnM-lacZ translational gene fusions. The fusions were integrated into the Δthn strain T690, which also harbored pIZ1016 (empty plasmid) or pIZ1158 (expressing the regulatory thnRY genes from Ptac). Cells were grown ...

Determination of the 5′ end of the thnH and thnM transcripts and analysis of the promoter sequences.

Primer extension assays were carried out in order to determine the 5′ end of the thnH and thnM transcripts. To avoid any interference from the transcript that carries the 5′ region of thnH from the thnB promoter, total RNA was isolated from T690 cells with the thnH::lacZ or thnM::lacZ fusions integrated into the chromosome plus pIZ1158, which were grown on tetralin. Primer extension reactions were carried out using the oligonucleotides orf1PEX for thnH and thnMPEX for thnM (see Table Table1).1). Although there was a substantial amount of background bands, prominent bands were obtained only under inducing conditions (Fig. (Fig.5A),5A), which led to mapping of the 5′ ends at 72 and 191 nucleotides upstream of the translation initiation codons of thnH and thnM, respectively. Consensus −10 and −35 promoter sequences were found at the expected positions but were different from the E. coli established promoter consensus (Fig. (Fig.5B).5B). Putative ThnR binding sites were detected and centered at −64 in both promoter regions, whose sequences were quite similar to those previously identified as ThnR binding sites at the thnB and thnC promoter regions (19) (Fig. (Fig.5B5B).

FIG. 5.
(A) Primer extension analysis of the thnH and thnM promoters. RNA from cells grown on 8 mM βHB plus tetralin (βH8T) and on 40 mM βHB (βH40) was isolated and retrotranscribed using orf1PEX and thnMPEX oligonucleotides. A ...

ThnR binding to thnH and thnM promoter sequences.

EMSAs were carried out in order to demonstrate the binding of ThnR to the thnH and thnM promoters. Purified ThnR-His tagged protein (19) and DNA fragments containing the thnG-thnH and thnL-thnM intergenic regions were used in gel shift assays. In both cases, a high-molecular-weight complex was detected when increasing amounts of ThnR were added to the binding reactions (Fig. (Fig.6).6). The binding affinity of ThnR to each promoter was calculated from these assays, resulting in a Kd of 60.1 ± 5.6 nM for the thnH binding site and 69.6 ± 9.9 nM for the thnM binding site.

FIG. 6.
ThnR binding to the thnH and thnM promoter regions. EMSA analysis was carried out with a ThnR-His tag protein and radioactively labeled promoter probes. Increasing amounts of ThnR (0, 25, 50, 75, 100, and 200 nM) resulted in the disappearance of the free ...

DISCUSSION

The tetralin catabolic pathway in the S. macrogolitabida strain TFA renders pyruvate and pimelic semialdehyde as products (13). Pyruvate directly enters the central metabolism, but pimelic semialdehyde must be further oxidized. A pathway for pimelic acid degradation in which pimeloyl-CoA is formed and then degraded by β-oxidation to glutaryl-CoA and acetyl-CoA has been proposed (5) and characterized in several bacteria (10, 27). In strain TFA, the conversion of pimelic semialdehyde to pimeloyl-CoA seems to take place in two different steps: first, the oxidation of pimelic semialdehyde to pimelic acid by a nonacylating pimelic semialdehyde dehydrogenase (ThnG) and, second, the activation of pimelic acid by a CoA transferase (ThnH). In addition to the in silico analysis of ThnG, which revealed an absence of key amino acids that are typically present in acylating aldehyde dehydrogenases (37), the in-gel enzymatic activity demonstrated that the aldehyde dehydrogenase activity of this protein was independent of CoA-SH. However, ThnG is not essential for growth on tetralin in TFA because a thnG mutant grew on this compound. This can be explained, in part, by the formation of pyruvic acid in the previous step, which could sustain cell growth to some extent, and by the presence of additional aldehyde dehydrogenases with activity toward pimelic semialdehyde, as shown by the in-gel assays and the activity measurements. The presence of different ALDHs in the same bacterial genome is common, as shown by the recent update of the ALDH family (34). Also, overlapping substrate specificities have been described previously in other ALDHs that are involved in the biodegradation of aromatic compounds, such as XylG, which oxidizes 2-hydroxymuconic semialdehyde and benzaldehyde (17), allowing functional substitution of a particular missing ALDH by an alternative one that is present in the cell.

Further metabolism of pimelic acid might take place by a β-oxidation pathway. Downstream of thnG, a cluster of genes was found whose products have homology to β-oxidation enzymes. This cluster lacks a pimeloyl-CoA synthetase gene, unlike what was recently found in the tetralin degrader Rhodococcus sp. strain TFB (thnQ) (36). Instead, the thnH gene, whose product has a domain similar to that found in members of the family III of CoA-transferases (11), is present. Thus, it appears that activation of pimelic acid for β-oxidation occurs by transfer of the coenzyme A group from a CoA-thioester rather than by de novo acylation. Conversion of pimeloyl-CoA to crotonyl-CoA could take place by the sequential action of ThnJK, ThnL, ThnI, and ThnN. The electrons resulting form the oxidation reactions would reach the main respiratory chain via ThnO and ThnP, which might be the two subunits of an electron transfer flavoprotein that accepts electrons from the FAD cofactor of acyl-CoA dehydrogenases and transfers them to the ubiquinone pool (38).

A gene apparently unrelated to β-oxidation genes, thnM, was found between thnL and thnN. This gene encodes an outer membrane protein with a C-terminal conserved domain, similar to the TonB-dependent receptors (2). Proteins similar to ThnM are part of the TonB system, which is an energy transduction complex that delivers energy from the cytoplasmic membrane to the outer membrane and is involved in the active transport of different types of ligands across the outer membrane. Although we do not know the role of this protein in tetralin metabolism, it is interesting that the TonB system is required for the functioning of efflux pumps that are involved in tolerance to solvents and drugs in Pseudomonas putida DOT-T1E (7). Tetralin is toxic to bacteria at concentrations above 15 μM (31), and thus it is possible that ThnM plays a detoxifying role in TFA.

To demonstrate the implication of these genes in tetralin metabolism, nonpolar insertion mutants on thnH, thnJ, thnK, and thnL were constructed. None was impaired or affected for growth on tetralin, pimelic, or sebacic acids, indicating that a second set of β-oxidation genes is present to efficiently carry out dicarboxylic acid oxidation. Olivera et al. (26) described metabolic redundancy in P. putida, in which two different pathways are involved in β-oxidation of n-alkanoic and n-phenylalkanoic acids. In Rhodopseudomonas palustris, deletion of the pim genes, which seem to be involved in the conversion of odd-chain dicarboxylic acids to glutaryl-CoA and the conversion of even-chain dicarboxylic acids to succinyl-CoA, did not dramatically affect growth of this bacterium on dicarboxylic acids, indicating the existence of other proteins that can take over the degradation of these compounds (10).

Expression of these genes follows the same pattern as that of the catabolic genes, whose transcription starts from the thnB or thnC promoters. The genes are induced in the presence of tetralin and subjected to catabolite repression by βHB, and their expression is strictly dependent on ThnR and ThnY. The primer extension assays and the β-galactosidase activity of the lacZ translational fusions clearly show the presence of two new thn promoters, PH and PM, which can drive transcription of all of these genes. In spite of the functional redundancy of these genes, their regulated expression indicates that they play a role in tetralin catabolism.

In silico analysis of the thnH and thnM promoters, and comparison to those previously identified, showed the presence of putative ThnR binding sites that match the consensus T-N11-A for LTTRs (19). As in the case of the thnB promoter region, and as usual for LTTRs, these binding sites are located −64 bp from the transcription start point (Fig. (Fig.5B).5B). The palindromic sequences containing the LysR binding consensus in PH and PM are not perfect, and a mismatch is present in the most distal subsites of each promoter region. However, these mismatches do not affect ThnR binding affinity because purified ThnR binds to PH and PM with an affinity similar to that of the thnB promoter (19). Examples of degenerate half sites can also be found in promoters that are recognized by OxyR (35), which do not affect the binding of the regulator. In addition, LTTRs bind to additional sequences located closer to the promoters, and these additional contacts also contribute to the LTTR binding affinity (29). Sequence similarities downstream of the ThnR binding sites are shared by the PH, PM, and PB promoters but not by PC. This fact and the relative location of its ThnR primary binding site make the PC promoter distinct from the others.

The data presented here complete the characterization of the tetralin biodegradation pathway and the transcriptional organization of the genes involved in its biodegradation in S. macrogolitabida strain TFA.

Acknowledgments

We thank Laura Ledesma and Francisca Reyes for setting up the expression assay in the T-690 strain, Guadalupe Martín and Nuria Pérez for their technical help, and all of the members of the laboratory for their insights and suggestions.

This study was supported by the Spanish Ministry of Science and Innovation, grants BIO2008-01805 and CSD2007-00005, and by the Andalusian government, grants P05-CVI-131 and P07-CVI-2518.

Footnotes

[down-pointing small open triangle]Published ahead of print on 6 November 2009.

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