Several metagenomic techniques were applied to analyze the microbial communities found in contaminated sites. A high-throughput metagenomic method called substrate-induced gene expression (SIGEX) was used to screen for genes that were upregulated by xenobiotic pollutants within a metagenomic library. SIGEX uses a promoterless green fluorescent protein (GFP) as a reporter for the transcription of metagenomic DNA that has been cloned in a plasmid library. Through propagation of metagenomic libraries in liquid culture, we used flow cytometry and fluorescence-activated cell-sorting to sort and
analyze rare clones with desired expression characteristics. Using microbial DNA isolated from a polycyclic aromatic-contaminated site, clones that were inducible by a variety of aromatic hydrocarbons were recovered using SIGEX. These inducible elements were examined for sequence similarity to known genes, and were found to contain different types of aromatic-metabolizing oxygenases and efflux pumps, along with their respective regulatory genes. Most often, the sequences were those of partial operons containing various nahG (salicylate oxygenase) family genes, along with their respective
upstream nahR regulators. Next-generation sequencing was used to map these small plasmid-based clones (representing relatively small fragments of the metagenome, on the order of 1 to 5 kilobases) to larger contigs (up to 61 kilobases) derived from the de novo assembly of 125 gigabases of shotgun-sequenced metagenomic DNA. These contigs were annotated using a variety of gene and protein prediction tools and were found to contain entire operons related to aromatic hydrocarbon metabolism; this enabled a more complete functional and taxonomic assignment of the sequences recovered through SIGEX
analysis. To assess the presence of relevant gene classes that were not recovered with SIGEX, genes with the capacity for xenobiotic metabolism were screened in silico within the assembled metagenomic reads using the biodegradation gene database and MG-RAST annotation. The taxonomic relationships between these functional genes were evaluated. As a direct application of this work, we show that SIGEX can be used to aid in the discovery and design of novel whole-cell bioreporters for the detection of xenobiotics.