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

ABSTRACT: 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).

SUBMITTER: Kasai D

PROVIDER: S-EPMC2937507 | biostudies-literature | 2010 Sep

REPOSITORIES: biostudies-literature

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

Kasai Daisuke D Kitajima Masahiro M Fukuda Masao M Masai Eiji E

Applied and environmental microbiology 20100723 18

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)A ...[more]

PMID: 20656871

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Project description:UnlabelledThe Bacillus subtilis rhaEWRBMA (formerly yuxG-yulBCDE) operon consists of four genes encoding enzymes for l-rhamnose catabolism and the rhaR gene encoding a DeoR-type transcriptional regulator. DNase I footprinting analysis showed that the RhaR protein specifically binds to the regulatory region upstream of the rhaEW gene, in which two imperfect direct repeats are included. Gel retardation analysis revealed that the direct repeat farther upstream is essential for the high-affinity binding of RhaR and that the DNA binding of RhaR was effectively inhibited by L-rhamnulose-1-phosphate, an intermediate of L-rhamnose catabolism. Moreover, it was demonstrated that the CcpA/P-Ser-HPr complex, primarily governing the carbon catabolite control in B. subtilis, binds to the catabolite-responsive element, which overlaps the RhaR binding site. In vivo analysis of the rhaEW promoter-lacZ fusion in the background of ccpA deletion showed that the L-rhamnose-responsive induction of the rhaEW promoter was negated by the disruption of rhaA or rhaB but not rhaEW or rhaM, whereas rhaR disruption resulted in constitutive rhaEW promoter activity. These in vitro and in vivo results clearly indicate that RhaR represses the operon by binding to the operator site, which is detached by L-rhamnulose-1-phosphate formed from L-rhamnose through a sequence of isomerization by RhaA and phosphorylation by RhaB, leading to the derepression of the operon. In addition, the lacZ reporter analysis using the strains with or without the ccpA deletion under the background of rhaR disruption supported the involvement of CcpA in the carbon catabolite repression of the operon.ImportanceSince L-rhamnose is a component of various plant-derived compounds, it is a potential carbon source for plant-associating bacteria. Moreover, it is suggested that L-rhamnose catabolism plays a significant role in some bacteria-plant interactions, e.g., invasion of plant pathogens and nodulation of rhizobia. Despite the physiological importance of L-rhamnose catabolism for various bacterial species, the transcriptional regulation of the relevant genes has been poorly understood, except for the regulatory system of Escherichia coli. In this study, we show that, in Bacillus subtilis, one of the plant growth-promoting rhizobacteria, the rhaEWRBMA operon for L-rhamnose catabolism is controlled by RhaR and CcpA. This regulatory system can be another standard model for better understanding the regulatory mechanisms of L-rhamnose catabolism in other bacterial species.

Dataset Information

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

Publications

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

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