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)

The tfdT gene is located upstream of and transcribed divergently from the tfdCDEF chlorocatechol-degradative operon on plasmid pJP4 of Ralst onia eutropha (formerly Alcaligenes eutrophus) JMP134. It is 684 bp lo ng and encodes a 25-kDa protein, On the basis of its predicted amino a cid sequence, the TfdT protein could be classified as a LysR-type tran scriptional regulator, It has the highest degree of similarity with th e proteins TcbR, ClcR, and TfdR, which are involved in the regulation of chloroaromatic breakdown. Despite this homolog, the TfdT protein fa iled to activate the expression of its presumed target operon, tfdCDEF . This failure could be attributed to the inability of TfdT to bind th e tfdC promoter legion, an absolute requirement for transcriptional ac tivation, Sequence analysis downstream of the tfdT gene revealed the p resence of an insertion element-like element. We postulate that this e lement disrupted the tfdT open reading frame, leading to a premature t ermination and the production of a truncated, disfunctional TfdT prote in, As an alternative to the inactivated TfdT protein, we propose that the product of the tfdR gene (or its identical twin, tfdS), located e lsewhere on plasmid pJP4, can successfully take over the regulation of tfdCDEF expression. The TfdR protein was capable of binding to the tf dC promoter region and activated tfdCDEF gene expression by a factor o f 80 to 100 when provided in cis as a tfdR-tfdCDEF hybrid regulon, Alt hough to a lesser extent, induction of tfdCDEF expression was also obs erved when no functional TfdR protein was provided, implying cross-act ivation by chromosomally encoded regulatory elements in R. eutropha JM P134(pJP4).