Functional analysis of a putative regulatory gene, tadR, involved in aniline degradation in Delftia tsuruhatensis AD9

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.