title: Identification of novel toluene monooxygenase genes in a hydrocarbon-polluted sediment using sequence- and function-based screening of metagenomic libraries.
abstract: The microbial potential for toluene degradation within sediments from a tar oil-contaminated site in Flingern, Germany, was assessed using a metagenomic approach. High molecular weight environmental DNA from contaminated sediments was extracted, purified, and cloned into fosmid and BAC vectors and transformed into Escherichia coli. The fosmid library was screened by hybridization with a PCR amplicon of the α-subunit of the toluene 4-monooxygenase gene to identify genes and pathways encoding toluene degradation. Fourteen clones were recovered from the fosmid library, among which 13 were highly divergent from known tmoA genes and several had the closest relatives among Acinetobacter species. The BAC library was transferred to the heterologous hosts Cupriavidus metallidurans (phylum Proteobacteria) and Edaphobacter aggregans (phylum Acidobacteria). The resulting libraries were screened for expression of toluene degradation in the non-degradative hosts. From expression in C. metallidurans, three novel toluene monooxygenase-encoding operons were identified that were located on IncP1 plasmids. The E. aggregans-hosted BAC library led to the isolation of a cloned genetic locus putatively derived from an Acidobacteria taxon that contained genes involved in aerobic and anaerobic toluene degradation. These data suggest the important role of plasmids in the spread of toluene degradative capacity and indicate putative novel tmoA genes present in this hydrocarbon-polluted environment.
authors: Earth and Life Institute, Laboratoire de Génie Biologique, Université catholique de Louvain, Place Croix du Sud 2, boite L7.05.19, 1348, Louvain-la-Neuve, Belgium.
authors: E
authors: E
authors: Bouhajja
authors: Department of Biological Sciences, Auburn University, 101 Rouse Life Science Building, Auburn, Alabama, 36849, USA.
authors: M
authors: M
authors: McGuire
authors: Department of Biological Sciences, Auburn University, 101 Rouse Life Science Building, Auburn, Alabama, 36849, USA.
authors: M R
authors: MR
authors: Liles
authors: Earth and Life Institute, Biodiversity Research Centre, Université catholique de Louvain, Place Croix du Sud 4-5, Bte L.7.07.04, 1348, Louvain-la-Neuve, Belgium.
authors: G
authors: G
authors: Bataille
authors: Earth and Life Institute, Laboratoire de Génie Biologique, Université catholique de Louvain, Place Croix du Sud 2, boite L7.05.19, 1348, Louvain-la-Neuve, Belgium.|School of Life Sciences and Biotechnology, Yachay Tech University, San Miguel de Urcuquí, Ecuador.
authors: S N
authors: SN
authors: Agathos
authors: Laboratoire d'Ecologie des Systèmes Aquatiques, Université libre de Bruxelles, Campus de la Plaine CP 221, Boulevard du Triomphe, 1050, Brussels, Belgium. igeorge@ulb.ac.be.
authors: I F
authors: IF
authors: George
mesh_terms: D003001:Cloning, Molecular; D004783:Environmental Microbiology; D004785:Environmental Pollutants / Q000378:metabolism*; D005820:Genetic Testing; D005858:Germany; D056186:Metagenomics; D006899:Mixed Function Oxygenases / Q000032:analysis* / Q000235:genetics*; D009693:Nucleic Acid Hybridization; D017422:Sequence Analysis, DNA; D014050:Toluene / Q000378:metabolism*
chemical_list: D004785:Environmental Pollutants; D014050:Toluene; D006899:Mixed Function Oxygenases
keywords: Biodegradation; Catabolic gene diversity; Expression in heterologous hosts; Macroarray; Metagenomics; Toluene 4-monooxygenase