Uptake kinetics of 2,4-dichlorophenoxyacetate by Delftia acidovorans MC1 and derivative strains: Complex characteristics in response to pH and growth substrate

The present investigation showed that active processes were involved in the uptake of 2,4-dichlorophenoxyacetate (2,4-D) by Delftia acidovorans MC1. With 2,4-D-grown cells, uptake at pH 6.8 was highly affine and showed a complex pattern-forming intermediary plateau at 20-100 microM 2,4-D. The kinetics became increasingly sigmoidal with raising of the pH to 7.5 and 8.5, and complexity disappeared. The apparent maximum was obtained at around 400 microM 2,4-D at either pH, and amounted to 15-20 nmol/min*mg protein. Higher substrate concentrations resulted in significant inhibition. With cells grown on (RS)-2-(2,4-dichlorophenoxy)propionate, 2,4-D uptake increased significantly and reached 45 nmol/min*mg, hinting at induction of a specific carrier(s). The kinetic characteristics made it apparent that several proteins contribute to 2,4-D uptake in MC1. An open reading frame was detected which has similarity to genes encoding major facilitator superfamily (MFS) transporters. Mutant strains that lacked this gene showed altered kinetics with decreased affinity to 2,4-D at pH 6.8. A mutant with complete deficiency in phenoxyalkanoate utilization showed an almost linear uptake pattern hinting at sole diffusion. Cloning of tfdK encoding a specific transporter for 2,4-D resulted in an increased uptake rate and, above all, higher affinity at slightly alkaline conditions due to hyperbolic kinetics. The presence of carbonylcyanide m-chlorophenylhydrazone led to the subsequent strong inhibition of 2,4-D uptake, suggesting proton symport as the likely active mechanism.