Functional Metagenomics of a Biostimulated Petroleum-Contaminated Soil Reveals an Extraordinary Diversity of Extradiol Dioxygenases

  • Laura Terrón-González, Guadalupe Martín-Cabello, Manuel Ferrer, Eduardo Santero
  • Applied and Environmental Microbiology, February 2016, ASM Journals
  • DOI: 10.1128/aem.03811-15

Diversity of extradiol dioxygenases found bu functional metagenomics

What is it about?

A metagenomic library of a petroleum-contaminated soil was constructed in a fosmid vector that allowed heterologous expression of metagenomic DNA. The library, consisting of 6.5 Gb of metagenomic DNA, was screened for extradiol dioxygenase (EDO) activity using catechol and 2,3-dihydroxybiphenyl as substrates. Fifty-eight independent clones encoding extradiol dioxygenase activity were identified. Forty-one different EDO encoding genes were identified. The population of EDO genes was not dominated by a particular gene or by highly similar genes, but the genes had an even distribution and high diversity. Phylogenetic analyses revealed that most of the genes could not be ascribed to previously defined subfamilies of EDOs. Rather, the EDO genes led to the definition of 10 new subfamilies of type I EDOs. Phylogenetic analysis of type II enzymes defined 7 families, 2 of which harbored the type II EDOs that were found in this work. Particularly striking was the diversity found in family I.3 EDOs; 15 out of the 17 sequences assigned to this family belonged to 7 newly defined subfamilies. A strong bias was found that depended on the substrate used for the screening: catechol mainly led to the detection of EDOs belonging to the I.2 family, while 2,3-dihydroxybiphenyl led to the detection of most other EDOs. Members of the I.2 family showed a clear substrate preference for monocyclic substrates, while those from the I.3 family showed a broader substrate range and high activity towards 2,3-dihydroxybiphenyl. This metagenomic analysis has substantially increased our knowledge of the existing biodiversity of EDOs.

Why is it important?

This work reveals an extraordinary diversity of extradiol dioxygenases, much higher than previously found in other metagenomic functional analyses. The main differences are that transcription of the environmental DNA in the metagenomic library is driven by a vector promoter subject to antitermination by the lambda phage antitermination system, thus facilitating metagenomic gene expression, and that two different indicator substrates have been used in the screening. The results show a strong bias in the type of extradiol dioxygenases identified, depending on the indicator used.


Professor Eduardo Santero
Universidad Pablo de Olavide

On the one hand, this work shows the extraordinary hidden diversity of enzymes encoded by uncultured bacteria, which can be uncovered by metagenomic functional analyses, On the other hand, it revealed the strong bias in the type of enzymes found by the metagenomic functional analyses, depending on the substrate used for the functional screening, which shows the limitations of the functional approach.

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The following have contributed to this page: Professor Eduardo Santero