Q-197. Meta-Cleavage Pathways Expressed in Metagenomic Libraries from Petroleum Hydrocarbon Contaminated Soil

J. Josefiova1, H. Junca2, D. H. Pieper2, M. V. Brennerova1;
1Inst. of Microbiology, Prague, CZECH REPUBLIC, 2AG Biodegradation, Helmholtz-Zentrum für Infektionsforschung GmbH, Braunschweig, GERMANY.

Cloning of soil metagenome was applied to isolate genes involved in metabolism of petroleum hydrocarbons and to describe catabolic gene diversity. Fosmid libraries were constructed from soil DNA from a heavily polluted area under venting, air sparging and nutrient amendment treatment. Clones with catechol meta-cleavage activity were selected and screened for Extradiol Dioxygenase type I (EXDO) content by polymerase chain reaction with primers, designed to target different evolutionary branches inside this gene family, and with primers for genes encoding the α-subunit of Rieske non-heme iron oxygenases of the benzene/toluene/isopropylbenzene/biphenyl oxygenase branch (BTIB-RHDO). Majority of activity-selected clones were containing sequences affiliated to members of the known EXDO type I family, indicating the environmental importance of this family in the degradation of pollutants in ecosystems under strong selection. The main groups found were represented by 2,3-dihydroxybiphenyl dioxygenases, catechol 2,3-dioxygenases that are classified in a branch composed by putative enzymes found in genome projects of β- and γ- proteobacteria, and to a lesser extent, clones hosting sequences affiliated to dihydroxynaphthalene dioxygenases. Functionality of the identified meta-cleavage genes was confirmed in selected clones by transposon-insertion mutagenesis and partial purificaton and N-terminal sequencing. Substrate specificity assay showed complementary activities of the subgroups detected. There was a correlation between the presence of an EXDO gene and the detection of BTIB_RHDO in the same metagenomic insert, indicating the predominance of clusters similar to those involved in the biodegradation of toluene/biphenyl in Burkholderiales and Pseudomonadales. Despite the well known discrepancy of culture dependent and culture independent approaches, the predominant genes and pathways selected in this case were not absolutely unknown or dissimilar to the ones described in biodegradation pathways of cultured bacteria.
This work was supported by project BIOTOOL of the EC (GOCE-003998) and by the Ministry of Education, Youth and Sports of the Czech Republic - Grant No. 1M06011