Characterization of Biphenyl Dioxygenase Sequences and Activities Encoded by the Metagenomes of Highly Polychlorobiphenyl-Contaminated Soils

Abstract

ABSTRACT Total extracted DNA from two heavily polychlorobiphenyl-contaminated soils was analyzed with respect to biphenyl dioxygenase sequences and activities. This was done by PCR amplification and cloning of a DNA segment encoding the active site of the enzyme. The translated sequences obtained fell into three similarity clusters (I to III). Sequence identities were high within but moderate or low between the clusters. Members of clusters I and II showed high sequence similarities with well-known biphenyl dioxygenases. Cluster III showed low (43%) sequence identity with a biphenyl dioxygenase from Rhodococcus jostii RHA1. Amplicons from the three clusters were used to reconstitute and express complete biphenyl dioxygenase operons. In most cases, the resulting hybrid dioxygenases were detected in cell extracts of the recombinant hosts. At least 83% of these enzymes were catalytically active. Several amino acid exchanges were identified that critically affected activity. Chlorobiphenyl turnover by the enzymes containing the prototype sequences of clusters I and II was characterized with 10 congeners that were major, minor, or not constituents of the contaminated soils. No direct correlations were observed between on-site concentrations and rates of productive dioxygenations of these chlorobiphenyls. The prototype enzymes displayed markedly different substrate and product ranges. The cluster II dioxygenase possessed a broader substrate spectrum toward the assayed congeners, whereas the cluster I enzyme was superior in the attack of ortho -chlorinated aromatic rings. These results demonstrate the feasibility of the applied approach to functionally characterize dioxygenase activities of soil metagenomes via amplification of incomplete genes.