Diversity and Evolution of Aromatic Degradation Pathway Enzymes in an Activated Sludge

Abstract

Metagenomics has emerged as an alternative approach to conventional microbial screening that allows exhaustive screening of microbial genomes in their natural environments. We adopted a functional metagenomic approach to screen for catabolic pathway genes for aromatic compounds. A fosmid library was created in Escherichia coli using an environmental DNA from an activated sludge used to treat industrial wastewater contaminated with aromatic compounds as a source of metagenome. By screening the library for extradiol dioxygenases (EDOs), we identified 91 positive clones, of which 38 were subjected to sequence analysis. Forty-three genes encoding EDOs were identified, including enzymes belonging to novel subfamilies. In particular, EDOs belonging to the I.2.G subfamily were overrepresented (20 clones). Enzymatic analysis revealed that the I.2.G EDOs were Mn(II)-dependent and had the strongest affinity for catechol reported thus far, which should enable host organisms to utilize the limited carbon sources and may be the basis for their dominance. As for the organization of catabolic pathway genes, unlike to known aromatic-degrading bacteria with complete pathways, the majority of the clones contained only subsets of the genes with novel arrangements. A circular plasmid-like 36.7-kb DNA form, designated pSKYE1, was assembled from the sequences of the clones carrying I.2.G EDOs, which possessed only a small set of genes for phenol degradation. Taking the dominance of this gene set (20 out of 38) into account, pSKYE1 may play a role in detoxification in the environment.