Expression And Regulation Of Aniline-Degrading Gene Cluster In Delftia Tsuruhatensis AD9

Delftia. tsuruhatensis AD9 can utilize aniline as the sole carbon source for growth. Compared with the aniline-degrading bacteria reported, AD9 has obvious advantage in the degrading rate of aniline and the tolerant capacity of aniline. We study the function of a putative regulatory gene tadR and the mechanism of regulation of aniline-degrading gene cluster in D. tsuruhatensis AD9 by gene-knockout analysis,β-galactosidase activity, electrophoresis mobility shift assays and real-time PCR system.The sequence analysis of AD9 aniline-degrading gene cluster reveals that there are three operons which regulated by two LysR-type regulators (TadR and OrfS). The amino acid sequence analysis shows that TadR protein shares 97.6% identity with TdnR protein of an aniline degrading strain, Pseudomonas putida UCC22; while the OrfS protein shares 69% amino acid sequence identity with AphT protein of a phenol degrading strain Comamonas testosteroni TA441. Both TdnR and AphT are belong to typical LysR-Type regulators.In this study, we found that knockout tadR from genome led to D. tsuruhatensis AD9 inability to utilize aniline for growth, but ability to utilize catechol as a sole carbon source. The plasmid (pLAF3RtadR) carrying tadR gene could complement the tadR mutant strain to use aniline as a sole carbon source. Our results suggested that the tadR is an important gene for the metabolism pathway of aniline degradation. Electrophoresis mobility shift assay (EMSA) demonstrated that TadR could bind to the promoter of tad genes, suggesting the interaction between TadR and promoter region of tad gene cluster. Comparing the aniline-degrading genes expression between the wild-type AD9 and tadR mutant, the RT-PCR and real-time analysis showed that TadR can activate transcription of tadQTA1A2BRD1C1 and orfS in the presence of aniline.To study the expression of gene, the tadQ-lacZ and tadD2-lacZ fusion vector were constructed and transfered into the wild type strain AD9 and mutant AD91, respectively. The analysis ofβ-galactosidase activity indicated that there are two promoters (PtadQ and PtadD2) in aniline-degrading gene cluster. The tadQTA1A2BRD1C1 encode the aniline dioxygenase, LysR-type regulatory protein and catechol 2,3-dioxygenase, and the tadD2C2EFGIJKL encode the meta-cleavage pathway enzymes for catechol degradation, respectively. Transcriptional activation of the PtadQ is dependent on the presence of TadR as an activator and aniline as an inducer. Other aromatic compounds such as monochlorinated anilines, especially 4-Chloroaniline also could activate transcription from the PtadQ. The PtadD2 could be activated by catechol, muconate and 2-hydroxyaniline in AD9.D. tsuruhatensis AD9 could not degrade any 2-chloroaniline and 3-chloroaniline efficiently. However, the cells could use a very small amount of 4-chloroaniline in late-stationary growth phase. The analysis of catechol 2,3-dioxygenase activity showed that the rate of catechol degradation catalyzed by TadC1 was significant higher than that of TadC2, suggesting that tadC1 encoded catechol 2,3-dioxygenase played a major role in the process of aniline degradation.Deletion analysis of the PtadQ region indicated that the region of the tadQ promoter from 1 to -148 was essential for expression of the aniline dioxygenase gene in AD9. The further deletions resulted in complete loss ofβ-galactosidase activity or aniline dioxygenase activity. The inverted repeat IR6 located within the–89 to–148 promoter region, played an important role for transcriptional activation of the PtadQ. Deletion analysis of the PtadD2 region also suggested that the region upstream containing one inverted repeat IR1, is essential for activation of the PtadD2.