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Soil type-dependent responses to phenanthrene as revealed by determining the diversity and abundance of polycyclic aromatic hydrocarbon ring-hydroxylating dioxygenase genes by using a novel PCR detection system.

ABSTRACT: A novel PCR primer system that targets a wide range of polycyclic aromatic hydrocarbon ring-hydroxylating dioxygenase (PAH-RHD(alpha)) genes of both Gram-positive and Gram-negative bacteria was developed and used to study their abundance and diversity in two different soils in response to phenanthrene spiking. The specificities and target ranges of the primers predicted in silico were confirmed experimentally by cloning and sequencing of PAH-RHD(alpha) gene amplicons from soil DNA. Cloning and sequencing showed the dominance of phnAc genes in the contaminated Luvisol. In contrast, high diversity of PAH-RHD(alpha) genes of Gram-positive and Gram-negative bacteria was observed in the phenanthrene-spiked Cambisol. Quantitative real-time PCR based on the same primers revealed that 63 days after phenanthrene spiking, PAH-RHD(alpha) genes were 1 order of magnitude more abundant in the Luvisol than in the Cambisol, while they were not detected in both control soils. In conclusion, sequence analysis of the amplicons obtained confirmed the specificity of the novel primer system and revealed a soil type-dependent response of PAH-RHD(alpha) gene-carrying soil bacteria to phenanthrene spiking.

SUBMITTER: Ding GC

PROVIDER: S-EPMC2901735 | biostudies-literature | 2010 Jul

REPOSITORIES: biostudies-literature

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Soil type-dependent responses to phenanthrene as revealed by determining the diversity and abundance of polycyclic aromatic hydrocarbon ring-hydroxylating dioxygenase genes by using a novel PCR detection system.

Ding Guo-Chun GC Ding Guo-Chun GC Heuer Holger H Zühlke Sebastian S Spiteller Michael M Pronk Geertje Johanna GJ Heister Katja K Kögel-Knabner Ingrid I Smalla Kornelia K

Applied and environmental microbiology 20100521 14

A novel PCR primer system that targets a wide range of polycyclic aromatic hydrocarbon ring-hydroxylating dioxygenase (PAH-RHD(alpha)) genes of both Gram-positive and Gram-negative bacteria was developed and used to study their abundance and diversity in two different soils in response to phenanthrene spiking. The specificities and target ranges of the primers predicted in silico were confirmed experimentally by cloning and sequencing of PAH-RHD(alpha) gene amplicons from soil DNA. Cloning and s ...[more]

PMID: 20495045

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Project description:BackgroundPolycyclic aromatic hydrocarbons (PAHs), widespread pollutants in the marine environment, can produce adverse effects in marine organisms and can be transferred to humans through seafood. Our knowledge of PAH-degrading bacterial populations in the marine environment is still very limited, and mainly originates from studies of cultured bacteria. In this work, genes coding catabolic enzymes from PAH-biodegradation pathways were characterized in coastal sediments of Patagonia with different levels of PAH contamination.ResultsGenes encoding for the catalytic alpha subunit of aromatic ring-hydroxylating dioxygenases (ARHDs) were amplified from intertidal sediment samples using two different primer sets. Products were cloned and screened by restriction fragment length polymorphism analysis. Clones representing each restriction pattern were selected in each library for sequencing. A total of 500 clones were screened in 9 gene libraries, and 193 clones were sequenced. Libraries contained one to five different ARHD gene types, and this number was correlated with the number of PAHs found in the samples above the quantification limit (r = 0.834, p < 0.05). Overall, eight different ARHD gene types were detected in the sediments. In five of them, their deduced amino acid sequences formed deeply rooted branches with previously described ARHD peptide sequences, exhibiting less than 70% identity to them. They contain consensus sequences of the Rieske type [2Fe-2S] cluster binding site, suggesting that these gene fragments encode for ARHDs. On the other hand, three gene types were closely related to previously described ARHDs: archetypical nahAc-like genes, phnAc-like genes as identified in Alcaligenes faecalis AFK2, and phnA1-like genes from marine PAH-degraders from the genus Cycloclasticus.ConclusionThese results show the presence of hitherto unidentified ARHD genes in this sub-Antarctic marine environment exposed to anthropogenic contamination. This information can be used to study the geographical distribution and ecological significance of bacterial populations carrying these genes, and to design molecular assays to monitor the progress and effectiveness of remediation technologies.

Dataset Information

Soil type-dependent responses to phenanthrene as revealed by determining the diversity and abundance of polycyclic aromatic hydrocarbon ring-hydroxylating dioxygenase genes by using a novel PCR detection system.

Publications

Soil type-dependent responses to phenanthrene as revealed by determining the diversity and abundance of polycyclic aromatic hydrocarbon ring-hydroxylating dioxygenase genes by using a novel PCR detection system.

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