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).