Molecular characterization of a deletion/duplication rearrangement in tfd genes from Ralstonia eutropha JMP134(pJP4) that improves growth on 3-chlorobenzoic acid but abolishes growth on 2,4-dichlorophenoxyacetic acid

The Alcaligenes eutrophus JMP134 tfdA gene, encoding the enzyme responsible for the first step in 2,4-dichlorophenoxyacetic acid (2,4-D) biodegradation, was overexpressed in Escherichia coli, and several enzymatic properties of the partially purified gene product were examined. Although the tfdA-encoded enzyme is typically referred to as 2,4-D monooxygenase, we were unable to observe any reductant-dependent activity. Rather, we demonstrate that this enzyme is a ferrous ion-dependent dioxygenase that uses alpha-ketoglutarate as a cosubstrate. The alpha-ketoglutarate is converted to succinate concomitant with 2,4-D conversion to 2,4-dichlorophenol. By using [1-14C]alpha-ketoglutarate, we established that carbon dioxide is the second product derived from alpha-ketoglutarate. Finally, we verified the proposal that glyoxylate is the second product derived from 2,4-D.

The structure of a transposon specifying the biodegradation of chlorobenzoate contaminants is described. Tn5271 is a 17-kilobase (kb) transposon that resides in the plasmid or chromosome of Alcaligenes sp. strain BR60 and allows this organism to grow on 3- and 4-chlorobenzoate. The transposon is flanked by a directly repeated sequence of 3201 base pairs (bp), which in turn is flanked by 110-bp inverted repeats. The 3.2-kb repeated sequence, designated IS1071, exists in multiple copies in the genome of Alcaligenes sp. strain BR60 and is involved in recombination of the catabolic genes into the chromosome of this strain. Sequence analysis revealed that the inverted repeat of IS1071 and the derived amino acid sequence of the single open reading frame within IS1071 are related to the inverted repeats and transposase (TnpA) proteins of the class II (Tn3 family) transposable elements. The absence of a resolvase gene within IS1071 suggests that this element is capable of determining the first step in class II transposition only. This was confirmed by observations on the IS1071-dependent formation of stable cointegrates in a recombination-deficient Escherichia coli. These results support an evolutionary scheme in which the class II transposable elements descended from simple insertion sequences.

Ralstonia eutropha JMP134 carries a 22 kb DNA region on plasmid pJP4 necessary for the degradation of 2,4-D (2,4-dichlorophenoxyacetate). In this study, expression of the 2,4-D pathway genes (designated tfd ) upon exposure to different concentrations of 2,4-D was measured at a detailed timescale in chemostat-grown R. eutropha cultures. A sharp increase in mRNA levels for tfdA, tfdCDEF-B, tfdDIICIIEIIFII-BII and tfdK was detected between 2 and 13 min after exposure to 2,4-D. This response time was not dependent on the 2,4-D concentration. The genes tfdA, tfdCD and tfdDIICII were expressed immediately upon induction, whereas tfdB, tfdBII and tfdK mRNAs could be detected only around 10 min later. The number of tfd mRNA transcripts per cell was estimated to be around 200-500 during maximal expression, after which they decreased to between 1 and 30 depending on the 2,4-D concentration used for induction. Unlike the mRNAs, the specific activity of the 2,4-D pathway enzyme chlorocatechol 1,2-dioxygenase did not increase sharply but accumulated to a steady-state plateau, which was dependent on the 2, 4-D concentration in the medium. At 1 mM 2,4-D, several oscillations in mRNA levels were observed before steady-state expression was reached, which was caused by transient accumulation of the first pathway intermediate, 2,4-dichlorophenol, to toxic concentrations. Expression of tfdR and tfdS, the (identical) regulatory genes for the tfd pathway remained low and essentially unchanged during the entire adaptation phase.