Dynamics of multigene expression during catabolic adaptation of Ralstonia eutropha JMP134 (pJP4) to the herbicide 2,4‐dichlorophenoxyacetate

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Abstract
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.