Transcriptional Regulation of the Terephthalate Catabolism Operon in Comamonas sp. Strain E6

摘要

Two almost identical gene clusters, tphR _(I) C _(I) A2 _(I) A3 _(I) B _(I) A1 _(I) and tphR _(II) C _(II) A2 _(II) A3 _(II) B _(II) A1 _(II), are responsible for the conversion of terephthalate (TPA) to protocatechuate in Comamonas sp. strain E6. In the present study, we investigated the transcriptional regulation of the tphR _(II) C _(II) A2 _(II) A3 _(II) B _(II) A1 _(II) gene cluster. Reverse transcription-PCR analysis suggested that the tphR _(II) C _(II) A2 _(II) A3 _(II) B _(II) A1 _(II) genes form two transcriptional units, the tphC _(II) A2 _(II) A3 _(II) B _(II) A1 _(II) catabolism operon and tphR _(II), with the latter encoding an IclR-type transcriptional regulator (ITTR). The transcription start site of the tph _(II) catabolism operon was mapped at 21 nucleotides upstream of the initiation codon of tphC _(II). The lacZ transcriptional fusion experiments showed that tphR _(II) encodes a transcriptional activator of the tph _(II) catabolism operon and that TPA acts as an inducer. On the other hand, TphR_(II) appeared to repress its own transcription regardless of the presence of TPA. The analysis of mutant derivatives of E6 indicated that tphR _(II) is essential for the transcriptional activation of the tph _(II) catabolism operon and the growth on TPA of a tph _(I)-deficient derivative of E6. Purified His-tagged TphR_(II) bound specifically to the tphR _(II)- tphC _(II) intergenic region containing a 21-bp inverted repeat sequence. Alignment of the inverted repeat sequences in the binding sites for TphR_(II) and other members of ITTRs revealed highly conserved nucleotides. The substitution of conserved nucleotides resulted in significantly reduced TPA-dependent transcriptional activation from the tphC _(II) promoter and reduced binding to His-tagged TphR_(II). These results clearly indicate that the conserved nucleotides are required for the inducible expression of the tph _(II) catabolism operon regulated by TphR_(II).