PRISTINE ENVIRONMENTS HARBOR A NEW GROUP OF OLIGOTROPHIC 2,4-DICHLOROPHENOXYACETIC ACID-DEGRADING BACTERIA

2,4-Dichlorophenoxyacetic acid (2,4-D)-degrading bacteria were isolate d from pristine environments which had no history of 2,4-D exposure. B y using 2,4-D dye indicator medium or C-14-labeled 2,4-D medium, six s trains were isolated from eight enrichment cultures capable of degradi ng 2,4-D. Phylogenetic analyses based on 16S ribosomal DNA (rDNA) sequ encing and physiological properties revealed that one isolate from Haw aiian volcanic soil could be classified in the genus Variovorax (a mem ber of the beta subdivision of the class Proteobacteria) and that the other five isolates from Hawaiian volcanic soils, Saskatchewan forest soil, and Chilean forest soil have 16S rDNAs with high degrees of simi larity to those of the Bradyrhizobium group (a member of the alpha sub division of the class Proteobacteria). All the isolates grow slowly on either nutrient media (0.1x Bacto Peptone-tryptone-yeast extract-gluc ose [PTYG] or 0.1x Luria broth [LB] medium) or 2,4-D medium, with mean generation times of 16 to 30 h, which are significantly slower than p reviously known 2,4-D degraders. Nutrient-rich media such as full-stre ngth PTYG and LB medium did not allow their growth. PCR amplification using internal consensus sequences of tfdA (a gene encoding an enzyme for the first step of 2,4-D mineralization, found in pJP4 of Alcaligen es eutrophus JMP134 and some other 2,4-D-degrading bacteria) as primer s and Southern hybridization,vith pJP4-tfdA as a probe revealed that t he isolate belonging to the genus Variovorax carried the tfdA gene. Th is gene was transmissible to A. eutrophus JMP228 carrying a plasmid wi th a mutant tfdA gene. The other five isolates did not appear to carry tfdA, and 2, 4-D-specific alpha-ketoglutarate-dependent dioxygenase a ctivity could not be detected in cell lysates. These results indicate that 2, l-D-degrading bacteria in pristine environments are slow-growi ng bacteria and that most of their phylogenies and catabolic genes dif fer from those of 2,4-D degraders typically isolated from agricultural soils or contaminated environments.