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Characterization of TsaR, an oxygen-sensitive LysR-type regulator for the degradation of p-toluenesulfonate in Comamonas testosteroni T-2.

ABSTRACT: TsaR is the putative LysR-type regulator of the tsa operon (tsaMBCD) which encodes the first steps in the degradation of p-toluenesulfonate (TSA) in Comamonas testosteroni T-2. Transposon mutagenesis was used to knock out tsaR. The resulting mutant lacked the ability to grow with TSA and p-toluenecarboxylate (TCA). Reintroduction of tsaR in trans on an expression vector reconstituted growth with TSA and TCA. The tsaR gene was cloned into Escherichia coli with a C-terminal His tag and overexpressed as TsaR(His). TsaR(His) was subject to reversible inactivation by oxygen, which markedly influenced the experimental approaches used. Gel filtration showed TsaR(His) to be a monomer in solution. Overexpressed TsaR(His) bound specifically to three regions within the promoter between the divergently transcribed tsaR and tsaMBCD. The dissociation constant (K(D)) for the whole promoter region was about 0.9 micro M, and the interaction was a function of the concentration of the ligand TSA. A regulatory model for this LysR-type regulator is proposed on the basis of these data.

SUBMITTER: Tralau T

PROVIDER: S-EPMC154824 | biostudies-literature | 2003 Apr

REPOSITORIES: biostudies-literature

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Characterization of TsaR, an oxygen-sensitive LysR-type regulator for the degradation of p-toluenesulfonate in Comamonas testosteroni T-2.

Tralau Tewes T Mampel Jörg J Cook Alasdair M AM Ruff Jürgen J

Applied and environmental microbiology 20030401 4

TsaR is the putative LysR-type regulator of the tsa operon (tsaMBCD) which encodes the first steps in the degradation of p-toluenesulfonate (TSA) in Comamonas testosteroni T-2. Transposon mutagenesis was used to knock out tsaR. The resulting mutant lacked the ability to grow with TSA and p-toluenecarboxylate (TCA). Reintroduction of tsaR in trans on an expression vector reconstituted growth with TSA and TCA. The tsaR gene was cloned into Escherichia coli with a C-terminal His tag and overexpress ...[more]

PMID: 12676713

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Project description:Comamonas testosteroni T-2 uses a standard, if seldom examined, attack on an aromatic compound and oxygenates the side chain of p-toluenesulfonate (TS) (or p-toluenecarboxylate) to p-sulfobenzoate (or terephthalate) prior to complete oxidation. The expression of the first three catabolic enzymes in the pathway, the TS methyl-monooxygenase system (comprising reductase B and oxygenase M; TsaMB), p-sulfobenzyl alcohol dehydrogenase (TsaC), and p-sulfobenzaldehyde dehydrogenase (TsaD), is coregulated as regulatory unit R1 (H. R. Schlafli Oppenberg, G. Chen, T. Leisinger, and A. M. Cook, Microbiology [Reading] 141:1891-1899, 1995). The components of the oxygenase system were repurified, and the N-terminal amino acid sequences were confirmed and extended. An internal sequence of TsaM was obtained, and the identity of the [2Fe-2S] Rieske center was confirmed by electron paramagnetic resonance spectroscopy. We purified both dehydrogenases (TsaC and TsaD) and determined their molecular weights and N-terminal amino acid sequences. Oligonucleotides derived from the partial sequences of TsaM were used to identify cloned DNA from strain T-2, and about 6 kb of contiguous cloned DNA was sequenced. Regulatory unit R1 was presumed to represent a four-gene operon (tsaMBCD) which was regulated by the LysR-type regulator, TsaR, encoded by a deduced one-gene transcriptional unit. The genes for the inducible TS transport system were not at this locus. The oxygenase system was confirmed to be a class IA mononuclear iron oxygenase, and class IA can now be seen to have two evolutionary groups, the monooxygenases and the dioxygenases, though the divergence is limited to the oxygenase components. The alcohol dehydrogenase TsaC was confirmed to belong to the short-chain, zinc-independent dehydrogenases, and the aldehyde dehydrogenase TsaD was found to resemble several other aldehyde dehydrogenases. The operon and its putative regulator are compared with units of the TOL plasmid.

Dataset Information

Characterization of TsaR, an oxygen-sensitive LysR-type regulator for the degradation of p-toluenesulfonate in Comamonas testosteroni T-2.

Publications

Characterization of TsaR, an oxygen-sensitive LysR-type regulator for the degradation of p-toluenesulfonate in Comamonas testosteroni T-2.

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