Transcriptional regulation of the terephthalate catabolism operon in Comamonas sp. strain E6

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).