Characterization of Novel Polycyclic Aromatic Hydrocarbon Dioxygenases from the Bacterial Metagenomic DNA of a Contaminated Soil

Abstract

f Ring-hydroxylating dioxygenases (RHDs) play a crucial role in the biodegradation of a range of aromatic hydrocarbons found on polluted sites, including polycyclic aromatic hydrocarbons (PAHs). Current knowledge on RHDs comes essentially from studies on culturable bacterial strains, while compelling evidence indicates that pollutant removal is mostly achieved by uncultured species. In this study, a combination of DNA-SIP labeling and metagenomic sequence analysis was implemented to investigate the metabolic potential of main PAH degraders on a polluted site. Following in situ labeling using [ 13 C]phenanthrene, the labeled metagenomic DNA was isolated from soil and subjected to shotgun sequencing. Most annotated sequences were predicted to belong to Betaproteobacteria, especially Rhodocyclaceae and Burkholderiales, which is consistent with previous findings showing that main PAH degraders on this site were affiliated to these taxa. Based on metagenomic data, four RHD gene sets were amplified and cloned from soil DNA. For each set, PCR yielded multiple amplicons with sequences differing by up to 321 nucleotides (17%), reflecting the great genetic diversity prevailing in soil. RHDs were successfully overexpressed in Escherichia coli, but full activity required the coexpression of two electron carrier genes, also cloned from soil DNA. Remarkably, two RHDs exhibited much higher activity when associated with electron carriers from a sphingomonad. The four RHDs showed markedly different preferences for two-and three-ring PAHs but were poorly active on four-ring PAHs. Three RHDs preferentially hydroxylated phenanthrene on the C-1 and C-2 positions rather than on the C-3 and C-4 positions, suggesting that degradation occurred through an alternate pathway.

Understanding and Designing the Strategies for the Microbe-Mediated Remediation of Environmental Contaminants Using Omics Approaches

“…Environmental biologists deem microbial bioremediation to be one of the potent tools to eliminate environmental contaminants (Bell et al, 2014 ; Chemerys et al, 2014 ; Roling, 2015 ). A number of review articles have recently been published and provide a much detailed account about the role of multi-omics (metagenomics, proteomics, metatranscriptomics, metabolomics, and fluxomics) approaches in microbial bioremediation.…”

Metagenomics — A Technological Drift in Bioremediation

“…Environmental scientists consider metagenomic bioremediation as one of the potential tools to remove contaminants from the environment [13][14][15]. As cited earlier, recent multiple studies have reported metagenomic approaches in bioremediation.…”

Challenges of unculturable bacteria: environmental perspectives

“…Metagenomic analysis showed a great abundance of aromaticring-hydroxylating oxygenase (RHO) genes, which were potentially responsible for polycyclic aromatic hydrocarbon (PAH) biodegradation, in uncultured microorganisms in chronically-polluted coastal sediments (Loviso et al 2015). Exploitation of metagenomic data has proved to be beneficial for the enzymatic characterization of RHOs from yet-to-becultured microorganisms in polluted soils (Singleton et al 2012;Martin et al 2013;Chemerys et al 2014) and marine environments (Musumeci et al 2019) providing a better understanding of the catabolic versatility of the vast majority of uncultured microbes. Dong and collegues used shotgun metagenomics to discover uncultured bacterial and archaeal phyla capable of the anaerobic degradation of aliphatic and aromatic hydrocarbons from deep-sea sediments.…”