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

Abstract

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 the 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; rather, 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 toward 2,3-dihydroxybiphenyl. This metagenomic analysis has substantially increased our knowledge of the existing biodiversity of Edos. Large amounts of several types of contaminants are released into the environment due to industrial activities and accidental spills. Many of these contaminants, such as aromatic hydrocarbons, have complex and stable chemical structures, which make them prevail in the environment for a long time, thus resulting in the contamination of ecosystems. Biological treatment of industrial wastewater and bioremediation of contaminated soils and water are therefore critical to prevent or combat this contamination.Standardized culturing techniques have been successfully applied for many years to isolate many different types of bacteria that are capable of utilizing a variety of aromatic hydrocarbons and to characterize their biodegradation pathways and the coding genes responsible for these capabilities, which have shown that bacteria can adapt to utilize a plethora of organic contaminants, including xenobiotics. The aerobic biodegradation of aromatics has been well documented and has been shown to follow two pathways, involving either intradiol or extradiol cleavage of the aromatic rings of di-or trihydroxylated intermediates. The intradiol dioxygenases that have been described to date appear to belong to the same superfamily, but extradiol dioxygenases (Edos) of three different phylogenetic origins have been reported (1, 2). Type I extradiol dioxygenases are more numerous and belong to the vicinal oxygen chelate superfa...

Complete Genome Sequence of a Bacterium Representing a Deep Uncultivated Lineage within the Gammaproteobacteria Associated with the Degradation of Polycyclic Aromatic Hydrocarbons

“…Two novel genes were identified likely to encode for the large subunit of ring-hydroxylating dioxygenases (RHD), enzymes which catalyze the initial reaction in the aerobic metabolism of many PAHs. RHD gene sequences highly similar to those predicted in TR3.2 (>93% amino acid identity) have been independently recovered in separate studies analyzing functional genes in microbial communities of petroleum-contaminated soils ( 14 , 15 ) and may have been derived from uncultivated bacteria closely related to TR3.2.…”

Functional soil metagenomics: elucidation of polycyclic aromatic hydrocarbon degradation potential following 12 years of in situ bioremediation

“…This number of positive clones corresponds to one active clone per 22Mb of sequence screened. This amount of clones was higher than previously reported in activity screening studies using 2,3‐dihydroxybiphenyl as substrate, where one clone per 155 Mb of screened DNA was observed (Terron‐Gonzalez et al ., ) and similar to values observed for catechol 2,3‐dioxygenase activities in contaminated environments (varying from one clone per 3.6Mb to 1 clone per 35 Mb of screened DNA (Suenaga et al ., ; Brennerova et al ., )) and reflects not only a high abundance of ring cleavage activities in the analysed soil but also high soil potential for the degradation of aromatic compounds.…”

“…Such enzymes had not been observed in catechol based screenings which has been attributed to their scarcity in the environment (Suenaga et al ., ). However, such enzymes have been identified, even though at a low extent, in metagenome based screenings recently (Terron‐Gonzalez et al ., ), and the high number observed in the contaminated environment analysed here suggest the importance of these enzymes in biodegradation. Twenty of these EXDOs showed highest similarity (>70%) to enzymes encoded in the I. cernigliae TR3.2 genome.…”

“…Only one specific amplification product each was obtained, using EXDO-3 and EXDO-4 primers targeting dihydroxynaphthalene dioxygenases and related enzymes in Proteobacteria. Of the previous reports on the functionbased screening of extradiol dioxygenases, only one applied a primer-based approach and could classify 72% of extradiol dioxygenase encoding genes (Brennerova et al, 2009), whereas others (Suenaga et al, 2007;Terron-Gonzalez et al, 2016) claimed the majority of genes identified to be only distantly related to previously observed ones. Clearly, the diversity of extradiol dioxygenases is too broad to be identified by approaches based on available sequence information (Vilchez-Vargas et al, 2010).…”

“…Despite the advantages, functional metagenomics dedicated to the analysis of aromatic degradation has only been applied in few studies, whereas metagenomic surveys, which can only cover organisms and genes present in high abundance in a given environment, are often attempted (Guazzaroni et al, 2013). The use of functional screening to retrieve aromatic catabolic activities has so far, to our knowledge, been restricted to the study of extradiol dioxygenases with high activity against catechol and thus, to the degradation of simple aromatic compounds such as benzene/toluene (Brennerova et al, 2009) and phenols (Suenaga et al, 2007) or to petroleum contaminated sites not stressed with a high level of PAH contamination (Terron-Gonzalez et al, 2016). These studies indicated screens for extradiol dioxygenases as promising tools to identify the diversity of such enzymes in contaminated environments.…”