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Effects exerted by transcriptional regulator PcaU from Acinetobacter sp. strain ADP1.

ABSTRACT: Protocatechuate degradation is accomplished in a multistep inducible catabolic pathway in Acinetobacter sp. strain ADP1. The induction is brought about by the transcriptional regulator PcaU in concert with the inducer protocatechuate. PcaU, a member of the new IclR family of transcriptional regulators, was shown to play a role in the activation of transcription at the promoter for the structural pca genes, leaving open the participation of additional activators. In this work we show that there is no PcaU-independent transcriptional activation at the pca gene promoter. The minimal inducer concentration leading to an induction response is 10(-5) M protocatechuate. The extent of expression of the pca genes was observed to depend on the nature of the inducing carbon source, and this is assumed to be caused by different internal levels of protocatechuate in the cells. The basal level of expression was shown to be comparatively high and to vary depending on the noninducing carbon source independent of PcaU. In addition to the activating function, in vivo results suggest a repressing function for PcaU at the pca gene promoter in the absence of an elevated inducer concentration. Expression at the pcaU gene promoter is independent of the growth condition but is subject to strong negative autoregulation. We propose a model in which PcaU exerts a repressor function both at its own promoter and at the structural gene promoter and in addition functions as an activator of transcription at the structural gene promoter at elevated inducer concentration.

SUBMITTER: Trautwein G

PROVIDER: S-EPMC94953 | biostudies-literature | 2001 Feb

REPOSITORIES: biostudies-literature

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Effects exerted by transcriptional regulator PcaU from Acinetobacter sp. strain ADP1.

Trautwein G G Gerischer U U

Journal of bacteriology 20010201 3

Protocatechuate degradation is accomplished in a multistep inducible catabolic pathway in Acinetobacter sp. strain ADP1. The induction is brought about by the transcriptional regulator PcaU in concert with the inducer protocatechuate. PcaU, a member of the new IclR family of transcriptional regulators, was shown to play a role in the activation of transcription at the promoter for the structural pca genes, leaving open the participation of additional activators. In this work we show that there i ...[more]

PMID: 11208784

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Project description:The two-component anthranilate 1,2-dioxygenase of the bacterium Acinetobacter sp. strain ADP1 was expressed in Escherichia coli and purified to homogeneity. This enzyme converts anthranilate (2-aminobenzoate) to catechol with insertion of both atoms of O(2) and consumption of one NADH. The terminal oxygenase component formed an alpha(3)beta(3) hexamer of 54- and 19-kDa subunits. Biochemical analyses demonstrated one Rieske-type [2Fe-2S] center and one mononuclear nonheme iron center in each large oxygenase subunit. The reductase component, which transfers electrons from NADH to the oxygenase component, was found to contain approximately one flavin adenine dinucleotide and one ferredoxin-type [2Fe-2S] center per 39-kDa monomer. Activities of the combined components were measured as rates and quantities of NADH oxidation, substrate disappearance, product appearance, and O(2) consumption. Anthranilate conversion to catechol was stoichiometrically coupled to NADH oxidation and O(2) consumption. The substrate analog benzoate was converted to a nonaromatic benzoate 1,2-diol with similarly tight coupling. This latter activity is identical to that of the related benzoate 1, 2-dioxygenase. A variant anthranilate 1,2-dioxygenase, previously found to convey temperature sensitivity in vivo because of a methionine-to-lysine change in the large oxygenase subunit, was purified and characterized. The purified M43K variant, however, did not hydroxylate anthranilate or benzoate at either the permissive (23 degrees C) or nonpermissive (39 degrees C) growth temperatures. The wild-type anthranilate 1,2-dioxygenase did not efficiently hydroxylate methylated or halogenated benzoates, despite its sequence similarity to broad-substrate specific dioxygenases that do. Phylogenetic trees of the alpha and beta subunits of these terminal dioxygenases that act on natural and xenobiotic substrates indicated that the subunits of each terminal oxygenase evolved from a common ancestral two-subunit component.

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Effects exerted by transcriptional regulator PcaU from Acinetobacter sp. strain ADP1.

Publications

Effects exerted by transcriptional regulator PcaU from Acinetobacter sp. strain ADP1.

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