The tfdR gene product can successfully take over the role of the insertion element-inactivated TfdT protein as a transcriptional activator of the tfdCDEF gene cluster, which encodes chlorocatechol degradation in Ralstonia eutropha JMP134(pJP4)

Abstract

The tfdT gene is located upstream of and transcribed divergently from the tfdCDEF chlorocatechol-degradative operon on plasmid pJP4 of Ralstonia eutropha (formerly Alcaligenes eutrophus) JMP134. It is 684 bp long and encodes a 25-kDa protein. On the basis of its predicted amino acid sequence, the TfdT protein could be classified as a LysR-type transcriptional regulator. It has the highest degree of similarity with the proteins TcbR, ClcR, and TfdR, which are involved in the regulation of chloroaromatic breakdown. Despite this homology, the TfdT protein failed to activate the expression of its presumed target operon, tfdCDEF. This failure could be attributed to the inability of TfdT to bind the tfdC promoter region, an absolute requirement for transcriptional activation. Sequence analysis downstream of the tfdT gene revealed the presence of an insertion element-like element. We postulate that this element disrupted the tfdT open reading frame, leading to a premature termination and the production of a truncated, disfunctional TfdT protein. As an alternative to the inactivated TfdT protein, we propose that the product of the tfdR gene (or its identical twin, tfdS), located elsewhere on plasmid pJP4, can successfully take over the regulation of tfdCDEF expression. The TfdR protein was capable of binding to the tfdC promoter region and activated tfdCDEF gene expression by a factor of 80 to 100 when provided in cis as a tfdR-tfdCDEF hybrid regulon. Although to a lesser extent, induction of tfdCDEF expression was also observed when no functional TfdR protein was provided, implying cross-activation by chromosomally encoded regulatory elements in R. eutropha JMP134(pJP4).

Importance of DifferenttfdGenes for Degradation of Chloroaromatics byRalstonia eutrophaJMP134

“…Expression of catechol and chlorocatechol operons usually requires LysR-type transcriptional activators and inducer muconate (catechol operons) or 2-chloromuconate (chlorocatechol operons) (28). In strain JMP134, the tfdR gene product is responsible for expression of the tfd operons (25) and it is activated by chloromuconates (14). It has been proposed that, even in the absence of tfdR, low-level expression of tfdCDEF occurs, implying a cross-activation by chromosomally encoded regulatory elements (14).…”

HbpR, a New Member of the XylR/DmpR Subclass within the NtrC Family of Bacterial Transcriptional Activators, Regulates Expression of 2-Hydroxybiphenyl Metabolism in Pseudomonas azelaica HBP1

“…DNA amplification by PCR was performed with Taq DNA polymerase (Gibco BRL, Life Technologies, Inc., Gaithersburg, Md.) as described elsewhere (37).…”

Steroid degradation in Comamonas testosteroni

“…It is an activator which induces 3,17-hsd, 3-hsd, ksi, TesA2 to ORF18-encoded enzyme, TesA1 to TesG, SteA to ORF6-encoded enzyme, the meta-cleavage enzyme and the ORFs in the downstream DNA region [17,67] Homology search for putative steroid degradation genes in the genomic sequence data on the web using deduced amino acid sequences of tesB to ORF33 revealed the presence of putative steroid degradation gene clusters similar to tesB to ORF33 in bacteria of different genera. Among a number of candidate bacteria, ten strains of seven genera were selected for comparison (C. testosteroni CNB-1 [29], C. testosteroni KF1 (draft sequence, accession: NZ_AAUJ02000001) [70,71], Cupriavidu necator (formally R. eutropha) JMP134 (accession: NC_007347) [72,73], Ralstonia eutropha H16 (accession: NC_008314) [74], Burkholderia cenocepacia J2315 (accession: NC_011001) [75], Burkholderia sp. 383 (accession: NC_007511) [76], Cu.…”