Expression And Functiona Of Genes Encoding Catechol 2,3-dioxygenase In Delftia Tsuruhatensis AD9

Aniline is a refractory organic pollutant, having a carcinogenic effect in humans. Use of microorganism to degrade this kind of compounds has paid attention in the whole world. Our laboratory has previously cloned Chromosome-encoded gene cluster for aniline metabolic pathway from an aniline degrading bacterium, Delftia tsuruhatensis AD9, isolated from the soil surrounding a textile dying plant. The gene cluster consists of 25 open reading frames (ORFs), at least 17 genes involved in aniline degradation for the complete metabolism of aniline to TCA-cycle intermediates, named tadQTAlA2BRD1C1D2C2EFGIJKL, which includes two putative Lys-type regulators {tadR and tadS) and two catechol 2,3-dioxygenase (C23Os) genes (tadC1, tadC2). C23Os are the key enzymes in aniline degradation metabolic pathway. Analysis of the tad gene cluster sequence indicated that the gene cluster has operators, tadC1 and tadC2 are in the position of first and second operator are probably controlled by the regulators TadR and TadS, respectively. tadC1 gene sequence only shared 55% of homology with tadC2, the homology between TadC1 and TadC2 at the amino acid level is only 50.47%. However, the projecting two-three structure of the proteins is very similar.In this study, we examined the ability of resistant and survival to aniline or catechol in D. tsuruhatensis AD9 strain, and found that AD9 strain could grow in present of aniline concentration at 4,500 mg /l, but the growth of AD9 strain was obviously limited at 600 mg /l of catechol. In addition, we found that 2-hydroxymuconic semialdehyde (HMS) accumulated during the early stage of aniline metabolic pathway. Those phenomena showed that catechol 2,3-dioxygenase (C23O) played a important role for further degradation.To analyze the effect of C23Os (TadCl and TadC2) on aniline degradation in AD9 strain, we determined the expression of tadC1 and tadC2 at the transcription level by RT-PCR assay. The results revealed that tadC1 and tadC2 were induced to express by aniline and catechol, respectively. Under the present of aniline inducer, the expression of tadC1 was obviously higher than that of tadC1. To further understand the function of C23Os encoded by tadC1, we amplified the intact tadC1 and tadC2 genes, and constructed into the prokaryotic expression vector (pet28a), resulting the transformants, named pETC1 and pETC2, respectively. The analysis of enzyme activities from the extraction of pETC1 and pETC2 showed that the rate of catechol degradation catalyzed by TadC1 was significant higher than that of TadC2. These results indicated that tadC1 encoded catechol 2,3-dioxygenase played a major role in the process of aniline degradation.