Abstract
Delftia tsuruhatensis AD9 contains the chromosomally encoded tad gene cluster responsible for the complete metabolism of aniline to TCA cycle intermediates. The tadQTA1A2B genes encode a multi-component aniline dioxygenase, the first enzyme of aniline metabolism, and the tadR gene directly downstream of this gene cluster encodes a putative LysR-type regulatory protein. Inactivation of tadR resulted in the inability to degrade aniline and to grow on aniline. Transcriptional assays using a tadQ promoter (P tadQ )–lacZ fusion revealed that the transcriptional activation of tadQ from P tadQ was dependent on the presence of tadR and aniline, suggesting that tadR encodes a positive regulatory protein for the expression of at least six genes. Induction experiments using the same P tadQ –lacZ fusion showed that, of the 22 chemical compounds, aniline and monochloroanilines activated transcription from P tadQ in wild-type AD9. Sequential deletions of a 1,003-bp region just upstream of tadQ showed that a 148-bp segment upstream of the transcription start site of tadQ, containing one inverted repeat named IR6, was essential for the transcriptional activation of tadQ. Moreover, gel shift assay confirmed the binding of the gene product to the tadQ promoter region. These results clarified the outline of the regulatory mechanism for aniline degradation in AD9.
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Acknowledgments
We thank Dr. Andreas Tauch, University of Bielefeld, Germany, for providing plasmid pK18mobsacB and E. coli strain S17-1 and also Dr. Issay Narumi, Atomic Energy Research Institute, Japan, for providing plasmid pKatCAT5. This work was supported by the Ministry of Science and Technology of China (National Basic Research Program Grant No. 2007CB707805 and National High-Tech Program Grant Nos. 2007AA02Z229 and 2006AA020101) and National Natural Science Foundation of China (Grant Nos. 30470047 and 30770076).
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Communicated by Walter Reineke.
L. Geng and M. Chen contributed equally to this work.
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Geng, L., Chen, M., Liang, Q. et al. Functional analysis of a putative regulatory gene, tadR, involved in aniline degradation in Delftia tsuruhatensis AD9. Arch Microbiol 191, 603–614 (2009). https://doi.org/10.1007/s00203-009-0488-5
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DOI: https://doi.org/10.1007/s00203-009-0488-5