Chromosome-encoded gene cluster for the metabolic pathway that converts aniline to TCA-cycle intermediates in Delftia tsuruhatensis AD9

Abstract

Delftia tsuruhatensis AD9 was isolated as an aniline-degrading bacterium from the soil surrounding a textile dyeing plant. The gene cluster involved in aniline degradation was cloned from the total DNA of strain AD9 into Escherichia coli JM109. After shotgun cloning, two recombinant E. coli strains showing aniline oxidation activity or catechol meta-cleavage activity were obtained by simple plate assays. These strains contained 9?3 kb and 15?4 kb DNA fragments, respectively. Sequence analysis of the total 24?7 kb region revealed that this region contains a gene cluster (consisting of at least 17 genes, named tadQTA1A2BRD1C1D2C2EFGIJKL) responsible for the complete metabolism of aniline to TCA-cycle intermediates. In the gene cluster, the first five genes (tadQTA1A2B) and the subsequent gene (tadR) were predicted to encode a multi-component aniline dioxygenase and a LysR-type regulator, respectively, while the others (tadD1C1D2C2EFGIJKL) were expected to encode meta-cleavage pathway enzymes for catechol degradation. In addition, it was found that the gene cluster is surrounded by two IS1071 sequences, indicating that it has a class I transposon-like structure. PFGE and Southern hybridization analyses confirmed that the tad gene cluster is encoded on the chromosome of strain AD9 in a single copy. These results suggest that, in strain AD9, aniline is degraded via catechol through a meta-cleavage pathway by the chromosome-encoded tad gene cluster. The tad gene cluster showed significant similarity in nucleotide sequence and genetic organization to the plasmid-encoded aniline degradation gene cluster of Pseudomonas putida UCC22.

Function of a Glutamine Synthetase-Like Protein in Bacterial Aniline Oxidation via γ-Glutamylanilide

“…2). Catechols are known to be the oxidation products from aniline, methylaniline, and chloroaniline (14,18,32,33), but this is the first report to have detected a major amount of catechol from aniline. Next, to prove the presence of ␥-glutamyl intermediates in aniline oxidation, we purified the GS-like protein AtdA1 and showed that it can produce ␥-GA from aniline and L-glutamate in the presence of ATP and MgCl 2 (Fig.…”

“…To date, several AD genes have been cloned and characterized (14,18,23,(30)(31)(32)(33), but the aniline oxidation mechanisms have not been elucidated, that is, the concrete functions of the products of the AD genes in aniline oxidation are unknown. All the AD gene clusters found so far encode GS-like and GAT-like proteins, which have not been found in other dioxygenases, with one exception (34).…”

“…This dioxygenase homolog belongs to group II in the classification of Nam et al (28), based on large-subunit aa sequence analysis, and to type I␣␤ in the later classification of Kweon et al (29), adding information on the electron transfer chain components to large-subunit sequence analysis. Similar AD gene clusters have been cloned from Pseudomonas putida UCC22 (30), Delftia acidovorans 7N (23), Delftia tsuruhatensis AD9 (18), Frateuria sp. strain ANA-18 (31), Delftia sp.…”

“…Simple anilines such as aniline and monosubstituted anilines are known to disappear from the environment mainly via biodegradation (8,9). To understand the mechanisms of aniline biodegradation, many aniline-degrading bacteria have been isolated and characterized (10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25).…”

Role of IncP-1β Plasmids pWDL7:: rfp and pNB8c in Chloroaniline Catabolism as Determined by Genomic and Functional Analyses

“…SRS16, shown to be induced by 3,4-dichloroaniline (3,4-DCA) (5), we also named the operon on pWDL7:rfp and NB8c dca. It was previously shown that aniline dioxygenases consist of multiple components: proteins homologous to glutamine synthetase, glutamine amidotransferase, large and small subunits of the terminal dioxygenase, and a reductase (18,30). In P. putida UCC22 and Delftia tsuruhatensis AD9, where these systems were first described, these components are encoded by, respectively, tdnQ-tadQ, tdnT-tadT, tdnA1-tadA1, tdnA2-tadA2, and tdnB-tadB (18,30).…”