Development and application of metagenomic tools for characterization of toluene degradation in hydrocarbon polluted sediments

A metagenomic procedure was developed to assess microbial potential for toluene degradation within the sediment of a polluted aquifer at the tar-oil-contaminated site of Flingern, Germany. We targeted genes encoding the α-subunit of the toluene-4-monooxygenase (tmoA genes) to find new toluene degradation genes and pathways. Good quality and high molecular weight environmental DNA was extracted and cloned in BAC and fosmid vectors in Escherichia coli. The BAC library was transferred to other hosts, Cupriavidus metallidurans (Proteobacteria) and Edaphobacter aggregans (Acidobacteria). The use of an acidobacterial strain for the first time as an alternative host implied further characterization of its interaction with broad host range (BHR) plasmids (pMOL98, pKT230, and RP4). New ecological insight regarding host range of these plasmids was gained, which opened up new perspectives of developing BHR vectors for metagenomic applications. Exploitation of the acidobacterial-hosted BAC library led to the isolation of an as-yet unsequenced fragment from an Acidobacterium source that contains genes involved in aerobic and anaerobic toluene degradation. The functional screening of the BAC library in C. metallidurans CH34 led to the capture of 3 novel toluene monoxygenase-encoding operons located on IncP1 plasmids. The genetic screening using an innovative high throughput hybridization procedure allowed recovery of 16 significantly divergent tmoA-like genes. Their source fragments belonged exclusively to Proteobacteria and more specifically to Acinetobacter species. This unique metagenomic study applied to the Flingern groundwater sediment has opened future horizons for more applications.