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Microbial dioxygenase gene population shifts during polycyclic aromatic hydrocarbon biodegradation.

ABSTRACT: The degradation of polycyclic aromatic hydrocarbons (PAHs) by bacteria has been widely studied. While many pure cultures have been isolated and characterized for their ability to grow on PAHs, limited information is available on the diversity of microbes involved in PAH degradation in the environment. We have designed generic PCR primers targeting the gene fragment encoding the Rieske iron sulfur center common to all PAH dioxygenase enzymes. These Rieske primers were employed to track dioxygenase gene population shifts in soil enrichment cultures following exposure to naphthalene, phenanthrene, or pyrene. PAH degradation was monitored by gas chromatograph with flame ionization detection. DNA was extracted from the enrichment cultures following PAH degradation. 16S rRNA and Rieske gene fragments were PCR amplified from DNA extracted from each enrichment culture and an unamended treatment. The PCR products were cloned and sequenced. Molecular monitoring of the enrichment cultures before and after PAH degradation using denaturing gradient gel electrophoresis and 16S rRNA gene libraries suggests that specific phylotypes of bacteria were associated with the degradation of each PAH. Sequencing of the cloned Rieske gene fragments showed that different suites of genes were present in soil microbe populations under each enrichment culture condition. Many of the Rieske gene fragment sequences fell into clades which are distinct from the reference dioxygenase gene sequences used to design the PCR primers. The ability to profile not only the bacterial community but also the dioxygenases which they encode provides a powerful tool for both assessing bioremediation potential in the environment and for the discovery of novel dioxygenase genes.

SUBMITTER: Ni Chadhain SM

PROVIDER: S-EPMC1489606 | biostudies-literature | 2006 Jun

REPOSITORIES: biostudies-literature

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Microbial dioxygenase gene population shifts during polycyclic aromatic hydrocarbon biodegradation.

Ní Chadhain Sinéad M SM Norman R Sean RS Pesce Karen V KV Kukor Jerome J JJ Zylstra Gerben J GJ

Applied and environmental microbiology 20060601 6

The degradation of polycyclic aromatic hydrocarbons (PAHs) by bacteria has been widely studied. While many pure cultures have been isolated and characterized for their ability to grow on PAHs, limited information is available on the diversity of microbes involved in PAH degradation in the environment. We have designed generic PCR primers targeting the gene fragment encoding the Rieske iron sulfur center common to all PAH dioxygenase enzymes. These Rieske primers were employed to track dioxygenas ...[more]

PMID: 16751518

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Project description:BackgroundPolycyclic aromatic hydrocarbon (PAH) contamination has been a worldwide environmental issue because of its impact on ecosystems and human health. Biodegradation plays an important role in PAH removal in natural environments. To date, many PAH-degrading strains and degradation genes have been reported. However, a comprehensive PAH-degrading gene database is still lacking, hindering a deep understanding of PAH degraders in the era of big data. Furthermore, the relationships between the PAH-catabolic genotype and phenotype remain unclear.ResultsHere, we established a bacterial PAH-degrading gene database and explored PAH biodegradation capability via a genome-function relationship approach. The investigation of functional genes in the experimentally verified PAH degraders indicated that genes encoding hydratase-aldolase could serve as a biomarker for preliminarily identifying potential degraders. Additionally, a genome-centric interpretation of PAH-degrading genes was performed in the public genome database, demonstrating that they were ubiquitous in Proteobacteria and Actinobacteria. Meanwhile, the global phylogenetic distribution was generally consistent with the culture-based evidence. Notably, a few strains affiliated with the genera without any previously known PAH degraders (Hyphomonas, Hoeflea, Henriciella, Saccharomonospora, Sciscionella, Tepidiphilus, and Xenophilus) also bore a complete PAH-catabolic gene cluster, implying their potential of PAH biodegradation. Moreover, a random forest analysis was applied to predict the PAH-degrading trait in the complete genome database, revealing 28 newly predicted PAH degraders, of which nine strains encoded a complete PAH-catabolic pathway.ConclusionsOur results established a comprehensive PAH-degrading gene database and a genome-function relationship approach, which revealed several potential novel PAH-degrader lineages. Importantly, this genome-centric and function-oriented approach can overcome the bottleneck of conventional cultivation-based biodegradation research and substantially expand our current knowledge on the potential degraders of environmental pollutants.

Project description:The characterization of native polycyclic aromatic hydrocarbon (PAH)-degrading bacteria is significant for understanding the PAH degradation process in the natural environment and developing effective remediation technologies. Most previous investigations of PAH-degrading bacteria in environmental samples employ pahAc, which encodes the ?-subunit of PAH ring-hydroxylating dioxygenase, as a functional marker gene. However, the poor phylogenetic resolution and nonspecificity of pahAc result in a misestimation of PAH-degrading bacteria. Here, we propose a PAH hydratase-aldolase-encoding gene, pahE, as a superior biomarker for PAH-degrading bacteria. Comparative phylogenetic analysis of the key enzymes involved in the upper pathway of PAH degradation indicated that pahE evolved dependently from a common ancestor. A phylogenetic tree constructed based on PahE is largely congruent with PahAc-based phylogenies, except for the dispersion of several clades of other non-PAH-degrading aromatic hydrocarbon dioxygenases present in the PahAc tree. Analysis of pure strains by PCR confirmed that pahE can specifically distinguish PAH-degrading bacteria, while pahAc cannot. Illumina sequencing of pahE and pahAc amplicons showed more genotypes and higher specificity and resolution for pahE Novel reads were also discovered among the pahE amplicons, suggesting the presence of novel PAH-degrading populations. These results suggest that pahE is a more powerful biomarker for exploring the ecological role and degradation potential of PAH-degrading bacteria in ecosystems, which is significant to the bioremediation of PAH pollution and environmental microbial ecology.IMPORTANCE PAH contamination has become a worldwide environmental issue because of the potential toxic effects on natural ecosystems and human health. Biotransformation and biodegradation are considered the main natural elimination forms of PAHs from contaminated sites. Therefore, the knowledge of the degradation potential of the microbial community in contaminated sites is crucial for PAH pollution bioremediation. However, the nonspecificity of pahAc as a functional marker of PAH-degrading bacteria has resulted neither in a reliable prediction of PAH degradation potential nor an accurate assessment of degradation. Here, we introduced pahE encoding the PAH hydratase-aldolase as a new and better functional marker gene of PAH-degrading bacteria. This study provides a powerful molecular tool to more effectively explore the ecological role and degradation potential of PAH-degrading bacteria in ecosystems, which is significant to the bioremediation of PAH pollution.

Dataset Information

Microbial dioxygenase gene population shifts during polycyclic aromatic hydrocarbon biodegradation.

Publications

Microbial dioxygenase gene population shifts during polycyclic aromatic hydrocarbon biodegradation.

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