Cloning And Functional Analysis Of Chromosome-encoded Gene Cluster For Aniline Metabolic Pathway In Delftia Tsuruhatensis AD9

AD9 was isolated as an aniline-degrading bacterium from the soil surrounding a textile dying plant. The highest concentration, at which AD9 can grow, is 4,500 mg /l. AD9 can degrade 1,000 mg /l aniline completely within 18h. The 16S rDNA of AD9 (GenBank accession number AY89912) was sequenced and shares high homology with those of several strains belong to Delftia. sp. The G±C content of AD9 is 66.8 mol%, which is very similar to that of D.tsuruhatensis T7 (66.2 mol%) . In addition, the hybridization rate between AD9 and T7 was 83.8%. Together with the result of phenotypic characteristics, AD9 was identified as a strain of D. tsunthatensis.The gene cluster involved in aniline degradation was cloned from the total DNA of strain AD9 into Escherichia coli JM109. By function-based screening, two positive transformants strains contained 9.3-kb and 15.4-kb DNA fragments, respectively. The 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 (tadR1) 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.Aniline oxygenase activity was measured using the cell suspension of E. coli harboring pDAl to confirm whether the cloned AD genes (tadQTA1A2B) are functional. The E. coli cells harboring pDAl showed apparent aniline oxygenase activity (32 ± 3 mgO2 g-dry-wt-1 h-1). The endogenous respiration subtracted was 16 ± 2 mgO2 g-dry-wt-1 h-1. Catechol was detected in the cell suspension as the oxidation product by gas chromatography/mass spectrometry analysis. These results indicate that the recombinant E. coli oxidized aniline to catechol. The recombinant E. coli cell harboring pDB2 extract showed 3.2 units (mg crude protein)-1 of C23O activity.Southern hybridization was carried out using a 384-bp gene probe including a part of the AD genes and the result suggesting that AD9 has the tad gene cluster in a single copy. Furthermore, PFGE result clearly demonstrates that there are no detectable large plasmids in AD9, Southern hybridization with the same gene probe revealed that the tad gene cluster is located on the AD9 chromosome. In this study, we first isolated a chromosome-encoded aniline degradation gene cluster from D. tsuruhatensis AD9, which is responsible for the complete metabolism of aniline to TCA-cycle intermediates via a meta-cleavage pathway. 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.Therefore, it is quite rare that the same catabolic transposon-like structure has been found on both a plasmid of P. putida and a chromosome of D. tsuruhatensis. Consequently, it is very important toanalyze their sequences and gene arrangements to understand their history and transfer mechanisms.A comparison of the chromosome-encoded tad gene cluster with the plasmid-encoded one suggested that the former is the more ancestral type.The phylogenetic analysis of the gene products TadCl and TadC2 illustrates that these two C23Os belong to different phylogenetic branches. The tadC1 and tadC2 genes were expressed by pET28 system. TadC1 and TadC2 showed C23O activity on catechol (100%, 100%, respectively), 3-methylcatechol (56%, 18%), and 4-methylcatechol (28%, <1%). This suggested that TdnC2 has been recruited from another pathway for unsubstituted aniline degradation.Then, the 9.3-kb HindⅢ fragment of pDA1 was subcloned into a broad host range plasmid pVK100 to make a recombinant plasmid pVD. When pVD1 was introduced into the parent strain AD9, the resultant strain could degrade 600mg/L aniline in A15 medium within 16h. This plasmid was introduced by triparental mating into a phenol-degrading bacterium Acinetobacter calcoaceticus PHEA-2, which can assimilate catechol as well as phenol, but not aniline. The resultant strain was able to grow on aniline as a sole carbon source, indicating that the AD genes in pVD1 allowed strain PHEA-2 to convert aniline into catechol.A 1.1kb fragment containing the tad promoter was amplified by PCR from total DNA of strain AD9. The PCR product was cloned into the HindⅢ-BamⅢ sites of pGD926, creating plasmid pGDP . The result of β-Galactosidase assays suggested the tad promoter was obviously induced by aniline.