Functional analysis of a putative regulatory gene, tadR , involved in aniline degradation in Delftia tsuruhatensis AD9
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ORIGINAL PAPER
Functional analysis of a putative regulatory gene, tadR, involved in aniline degradation in Delftia tsuruhatensis AD9 Lizhao Geng · Ming Chen · Quanfeng Liang · Wei Liu · Wei Zhang · Shuzhen Ping · Wei Lu · Yongliang Yan · Weiwei Wang · Masahiro Takeo · Min Lin
Received: 25 July 2008 / Revised: 18 May 2009 / Accepted: 25 May 2009 / Published online: 7 June 2009 © Springer-Verlag 2009
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 Wrst 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 (PtadQ)–lacZ fusion revealed that the transcriptional activation of tadQ from PtadQ 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
PtadQ–lacZ fusion showed that, of the 22 chemical compounds, aniline and monochloroanilines activated transcription from PtadQ in wild-type AD9. Sequential deletions of a 1,003-bp region just upstream of tadQ showed that a 148bp 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 conWrmed the binding of the gene product to the tadQ promoter region. These results clariWed the outline of the regulatory mechanism for aniline degradation in AD9.
Communicated by Walter Reineke.
Introduction
L. Geng and M. Chen contributed equally to this work.
Aniline and its derivatives have been used widely as materials for the synthesis of chemical products such as herbicides and dyes, and they have been released into the environment through industrial and agricultural uses during the past century (Lyons et al. 1985). They have been reported to exhibit signiWcant toxic and mutagenic eVects on mammalian cells, Wsh, aquatic invertebrates, and bacteria even at low concentrations (Timourian et al. 1982; Chung et al. 1997; Bhunia et al. 2003). Therefore, anilines are considered to be important environmental pollutants (Meyer 1981; Chung et al. 1997). Many researchers have paid attention to and extensively studied their fates in the environment. Consequently, it was found that biodegradation was the most signiWcant removal mechanism for aniline in the environment (Lyons et al. 1984; Lyons et al. 1985). To date, many aniline-degrading bacteria have been isolated to clarify the biodegradation mechanism, but only
L. Geng · M. Chen · W. Liu · W. Zhang · S. Ping · W. Lu · Y. Yan · W. Wang · M. Lin (&) Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, 12 Zhongguancun Nandajie, 100081 Beijing, People’s Republic of Chi
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