Bioremediation relies on the stability of enzymatic activities, particularly when bioavailable contaminant concentrations do not permit much renewal of microbial biomass. Starving Delftia acidovorans MC1 were found to lose specific degradation activity, while accumulating variants of the alpha-ketoglutarate-dependent dioxygenase RdpA, the enzyme initiating the degradation of (RS)-2-(2,4-dichlorophenoxy)propionate. These variants differed in their pI and originated from post-translational modification, since there is only one rdpA gene in the genome. It was tested if RdpA modification resulted from carbonylation by reactive oxygen species, known side products of dioxygenase reactions. Carbonylated amino acids in proteins of starved cells were specifically derivatized with 2,4-dinitrophenylhydrazine. Subsequent immunolabeling of the resulting hydrazones and mass spectrometry of tryptic digests confirmed different levels of carbonylation of RdpA.

Delftia acidovorans MC1071 can productively degrade R-2-(2,4-dichlorophenoxy)propionate (R-2,4-DP) but not 2,4-dichlorophenoxyacetate (2,4-D) herbicides. This work demonstrates adaptation of MC1071 to degrade 2,4-D in a model two-dimensional porous medium (referred to here as a micromodel). Adaptation for 2,4-D degradation in the 2 cm-long micromodel occurred within 35 days of exposure to 2,4-D, as documented by substrate removal. The amount of 2,4-D degradation in the adapted cultures in two replicate micromodels (~10 and 20 % over 142 days) was higher than a theoretical maximum (4 %) predicted using published numerical simulation methods, assuming instantaneous biodegradation and a transverse dispersion coefficient obtained for the same pore structure without biomass present. This suggests that the presence of biomass enhances substrate mixing. Additional evidence for adaptation was provided by operation without R-2,4-DP, where degradation of 2,4-D slowly decreased over 20 days, but was restored almost immediately when R-2,4-DP was again provided. Compared to suspended growth systems, the micromodel system retained the ability to degrade 2,4-D longer in the absence of R-2,4-DP, suggesting slower responses and greater resilience to fluctuations in substrates might be expected in the soil environment than in a chemostat.

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Microbial activities and the versatility gained through adaptation to xenobiotic compounds are the main biological forces to counteract environmental pollution.The current results present a new adaptive mechanism that is mediated through posttranslational modifications. Strains of Delftia acidovorans incapable of growing autochthonously on 2,4-dichlorophenoxyacetate (2,4-D) were cultivated in a chemostat on 2,4-D in the presence of (R)-2-(2,4-dichlorophenoxy)propionate. Long-term cultivation led to enhanced 2,4-D degradation, as demonstrated by improved values of the Michaelis-Menten constant K m for 2,4-D and the catalytic efficiency k cat /K m of the initial degradative key enzyme (R)-2-(2,4-dichlorophenoxy)propionate/ α-ketoglutarate-dependent dioxygenases (RdpA). Analyses of the rdpA gene did not reveal any mutations, indicating a nongenetic mechanism of adaptation. 2-DE of enzyme preparations, however, showed a series of RdpA forms varying in their pI. During adaptation increased numbers of RdpA variants were observed. Subsequent immunoassays of the RdpA variants showed a specific reaction with 2,4-dinitrophenylhydrazine (DNPH), characteristic of carbonylation modifications. Together these results indicate that posttranslational carbonylation modified the substrate specificity of RdpA. A model was implemented explaining the segregation of clones with improved degradative activity within the chemostat. The process described is capable of quickly responding to environmental conditions by reversibly adapting the degradative potential to various phenoxyalkanoate herbicides.Keywords: 2,4-Dichlorophenoxyacetate (2,4-D) / (R)-2-(2,4-Dichlorophenoxy)propionate / α-ketoglutarate-dependent dioxygenases (RdpA) / Individual-based modeling / Posttranslational carbonylationAdditional supporting information may be found in the online version of this article at the publisher's web-site