A gene designated thnD, which is required for biodegradation of the organic solvent tetralin by Sphingomonas macrogoltabidus strain TFA, has been identified. Sequence comparison analysis indicated that thnD codes for a carbon-carbon bond serine hydrolase showing highest similarity to hydrolases involved in biodegradation of biphenyl. An insertion mutant defective in ThnD accumulates the ring fission product which results from the extradiol cleavage of the aromatic ring of dihydroxytetralin. The gene product has been purified and characterized. ThnD is an octameric thermostable enzyme with an optimum reaction temperature at 65°C. ThnD efficiently hydrolyzes the ring fission intermediate of the tetralin pathway and also 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid, the ring fission product of the biphenyl meta-cleavage pathway. However, it is not active towards the equivalent intermediates of meta-cleavage pathways of monoaromatic compounds which have small substituents in C-6. When ThnD hydrolyzes the intermediate in the tetralin pathway, it cleaves a C-C bond comprised within the alicyclic ring of tetralin instead of cleaving a linear C-C bond, as all other known hydrolases of meta-cleavage pathways do. The significance of this activity of ThnD for the requirement of other activities to mineralize tetralin is discussed.

A genomic region involved in tetralin biodegradation was recently identified in Sphingomonas strain TFA. We have cloned and sequenced from this region a gene designated thnC, which codes for an extradiol dioxygenase required for tetralin utilization. Comparison to similar sequences allowed us to define a subfamily of 1,2-dihydroxynaphthalene extradiol dioxygenases, which comprises two clearly different groups, and to show that ThnC clusters within group 2 of this subfamily. 1,2-Dihydroxy-5,6,7,8-tetrahydronaphthalene was found to be the metabolite accumulated by a thnC insertion mutant. The ring cleavage product of this metabolite exhibited behavior typical of a hydroxymuconic semialdehyde toward pH-dependent changes and derivatization with ammonium to give a quinoline derivative. The gene product has been purified, and its biochemical properties have been studied. The enzyme is a decamer which requires Fe(II) for activity and shows high activity toward its substrate (V max , 40.5 U mg ؊1 ; K m , 18.6 M). The enzyme shows even higher activity with 1,2-dihydroxynaphthalene and also significant activity toward 1,2-dihydroxybiphenyl or methylated catechols. The broad substrate specificity of ThnC is consistent with that exhibited by other extradiol dioxygenases of the same group within the subfamily of 1,2-dihydroxynaphthalene dioxygenases.Tetralin (1,2,3,4-tetrahydronaphthalene) is an organic solvent 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 (12). Tetralin is very toxic to bacteria because of its accumulation in the cell membranes, which leads to changes in structure and function (39,40), and also because of the formation of highly toxic hydroperoxides (11).Tetralin is a bicyclic molecule composed of an aromatic moiety and an alicyclic moiety sharing two carbon atoms. Just a few bacterial strains able to grow on tetralin as the only carbon and energy source have been reported (37), and very little is known about the utilization of this molecule by bacteria. Identification of accumulated intermediates during growth on tetralin suggests that some bacteria, such as Pseudomonas stutzeri AS39, initially hydroxylate and further oxidize the alicyclic ring (35), while others, such as Corynebacterium sp. strain C125, initially dioxygenate the aromatic ring, which is subsequently cleaved in the extradiol position (38). In spite of previous reports showing modification and utilization of tetralin (35,38,41,42), a complete biodegradation pathway has not yet been elucidated.Key enzymes in the pathways of aromatic compounds are the metal-dependent ring cleavage dioxygenases, which act on the corresponding catechol-type derivatives, cleaving them at the intradiol position (ortho cleavage) or the extradiol position (meta cleavage) (18). While intradiol dioxygenases typically depend on Fe(III), most extradiol dioxygenases depend on Fe(II), although one m...

Five genes involved in the two initial steps of the tetralin biodegradation pathway of Sphingomonas macrogolitabida strain TFA have been characterized. ThnA1A2 and ThnA3A4, components of the ring-hydroxylating dioxygenase, were encoded in divergently transcribed operons. ThnA1, ThnA2, and ThnA3 were essential for tetralin ring-hydroxylating dioxygenase activity. ThnB was identified as a dehydrogenase required for tetralin biodegradation.The organic solvent tetralin (1,2,3,4-tetrahydronaphthalene) is widely used as a degreasing agent and solvent for fats, resins, and waxes and as a substitute for turpentine in paints, lacquers, and shoe polishes; tetralin is also used in the petrochemical industry in connection with coal liquefaction (7). Tetralin is a bicyclic molecule composed of an aromatic moiety and an alicyclic moiety, which share two carbon atoms.A few bacterial strains able to aerobically grow on tetralin as the only carbon and energy source have been described previously (22). Several reports suggest that some bacteria initially hydroxylate and further oxidize the alicyclic ring, while others initially dioxygenate the aromatic ring, thus indicating that tetralin may be aerobically metabolized in different ways (21,23). Sphingomonas macrogolitabida strain TFA (formerly Sphingopyxis macrogoltabida) (9,26,27) is able to grow on tetralin as the only source of carbon and energy (10). Previous work with this strain characterized a meta-cleavage and subsequent reactions, which cleave both the aromatic and the alicyclic rings of tetralin, thus yielding pyruvate and pimelic acid semialdehyde (2,8,9). Four genes (thnC, thnD, thnE, and thnF) coding for these enzymes have been identified and have been shown to be located in two closely linked operons, which are divergently transcribed (Fig. 1). These data suggested that biodegradation of tetralin by strain TFA involves initial oxidation of the aromatic ring to yield 1,2-dihydroxytetralin (1,2-DHT), through reactions presumably catalyzed by a ringhydroxylating dioxygenase followed by a dehydrogenase. However, these enzymes or the genes coding for them had not yet been identified. In this work, we describe the structural and functional characterization of the genes encoding the enzyme components involved in the first two steps of tetralin biodegradation.In previous work, a collection of mutants unable to grow on tetralin as the only carbon and energy source had been constructed (10). Some of them were located in genomic regions that were very close but outside of the previously sequenced DNA fragments. In an attempt to fully characterize the tetralin biodegradation pathway of strain TFA, three DNA regions surrounding the previously identified genes were sequenced (Fig. 1). Putative gene products encoded by the new sequences were initially compared to those in the databases with the Gapped BLAST program (1). For each open reading frame, sequences showing high similarity to that of strain TFA were aligned by using the CLUSTAL X program (25) with default parameters. A ph...

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 ␤-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 ␤-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 ␤-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 crotonylCoA, 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 P H and P M 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 gramnegative 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. 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 ...