Several types of environmental bacteria that can aerobically degrade various aromatic compounds have been identified. The catabolic genes in these bacteria have generally been found to form operons, which promote efficient and complete degradation. However, little is known about the degradation pathways in bacteria that are difficult to culture in the laboratory. By functionally screening a metagenomic library created from activated sludge, we had earlier identified 91 fosmid clones carrying genes for extradiol dioxygenase (EDO), a key enzyme in the degradation of aromatic compounds. In this study, we analyzed 38 of these fosmids for the presence and organization of novel genes for aromatics degradation. Only two of the metagenomic clones contained complete degradation pathways similar to those found in known aromatic compound-utilizing bacteria. The rest of the clones contained only subsets of the pathway genes, with novel gene arrangements. A circular 36.7-kb DNA form was assembled from the sequences of clones carrying genes belonging to a novel EDO subfamily. This plasmid-like DNA form, designated pSKYE1, possessed genes for DNA replication and stable maintenance as well as a small set of genes for phenol degradation; the encoded enzymes, phenol hydroxylase and EDO, are capable of the detoxification of aromatic compounds. This gene set was found in 20 of the 38 analyzed clones, suggesting that this 'detoxification apparatus' may be widespread in the environment.
Title: A metagenomic approach to the identification of UDP-glucose 4-epimerase as a menadione resistance protein Mori T, Suenaga H, Miyazaki K Ref: Biosci Biotechnol Biochem, 72:1611, 2008 : PubMed
Quinones are potentially toxic agents that generate reactive oxygen species (ROS) upon reduction. We screened a metagenomic library for a menadione resistance gene to identify UDP-glucose 4-epimerase (UGE). Escherichia coli carrying the gene became resistant to various quinones, but not to ROS. Because UGE is involved in the biosynthesis of lipopolysaccharides, it may have contributed to forming a permeability barrier against lipophilic quinones.
Title: Functional screening of a metagenomic library for genes involved in microbial degradation of aromatic compounds Suenaga H, Ohnuki T, Miyazaki K Ref: Environ Microbiol, 9:2289, 2007 : PubMed
A metagenomic approach was taken to retrieve catabolic operons for aromatic compounds from activated sludge used to treat coke plant wastewater. Metagenomic DNA extracted from the sludge was cloned into fosmids and the resulting Escherichia coli library was screened for extradiol dioxygenases (EDOs) using catechol as a substrate, yielding 91 EDO-positive clones. Based on their substrate specificity for various catecholic compounds, 38 clones were subjected to sequence analysis. Each insert contained at least one EDO gene, and a total of 43 EDO genes were identified. More than half of these belonged to new EDO subfamilies: I.1.C (2 clones), I.2.G (20 clones), I.3.M (2 clones) and I.3.N (1 clone). The fact that novel I.2.G family genes were over-represented in these clones suggested that these genes play a specific role in environmental aromatic degradation. The I.2.G clones were further classified into six groups based on single-nucleotide polymorphisms (SNPs). Based on the combination of the SNPs, the evolutionary lineage of the genes was reconstructed; further, taking the activities of the clones into account, potential adaptive mutations were identified. The metagenomic approach was thus used to retrieve novel EDO genes as well as to gain insights into the gene evolution of EDOs.
