Investigation of a metagenomic library for aerobic toluene degradation genes
(2012) 14th International Symposium on Microbial Ecology (ISME14) — Location: Copenhagen, Danemark (19.August.2012)
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- Authors
Agathos, Spiros N. 
- Abstract
- A metagenomic study on sediment from a former gasworks site located in Düsseldorf- Flingern was conducted to assess the tar oil hydrocarbon degradation potential at that site. A sediment sample was collected from 6.70 m to 7.10 m below surface in the anoxic contaminant plume. This plume consisted mainly of benzene, toluene, ethylene, xylene and polyaromatic hydrocarbons. The first step was to extract enough high quality, high molecular weight DNA despite low cell density within the sediment. The extraction method used direct lysis of bacterial cells within the sediment followed by a phenol-chloroform extraction. The DNA was then purified using a centrifugal concentrator coupled with Q-Sepharose treatment. This protocol resulted in pure DNA of appropriate size range to construct a metagenomic library in the pCC1FOS vector with the CopyControlTM fosmid library system (Epicentre). The recombinant E.coli clones were robotically picked in duplicate onto nylon membranes at Libragen, France. The macroarrays contained 53,760 fosmid clones with an average insert size of 40 kb representing 537 E.coli genome equivalents. They were screened by colony blot hybridization for toluene mono-oxygenase. We used the primer set TMOA-F/TMOA-R to amplify the tmoA gene from the same DNA that was used for library construction to generate a digoxygenin-labeled-probe for downstream library screening. A total of 200 clones exhibited chemiluminescence after hybridization with the digoxigenin-labeled tmoA probe (in duplicate), indicating the presence of a toluene mono-oxygenase cluster and therefore a possible toluene degradation pathway. Assuming an equal abundance of genomes within the library, about 40 % of the screened genomes contained a tmoA gene. Only 34 clones out of these 200 positive tmoA clones identified by hybridization gave a tmoA PCR product. This result suggests that the remaining 166 clones contain mismatches with the TMOA-F/TMOAR primer set. These clones may contain a tmoA gene that is divergent from previously described sequences. Sequencing of these clones is expected to considerably enrich the catalogue of known genes involved in toluene degradation
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Citations
Bouhajja, E., George, I., Liles, M., & Agathos, S. N. (2012). Investigation of a metagenomic library for aerobic toluene degradation genes. 14th International Symposium on Microbial Ecology (ISME14), Copenhagen, Danemark. https://hdl.handle.net/2078.5/214842