Comparison of the Specificities and Efficacies of Primers for Aromatic Dioxygenase Gene Analysis of Environmental Samples

ABSTRACTA betaproteobacterium within the familyRhodocyclaceaepreviously identified as a pyrene degrader via stable-isotope probing (SIP) of contaminated soil (designated pyrene group 1 or PG1) was cultivated as the dominant member of a mixed bacterial culture. A metagenomic library was constructed, and the largest contigs were analyzed for genes associated with polycyclic aromatic hydrocarbon (PAH) metabolism. Eight pairs of genes with similarity to the α- and β-subunits of ring-hydroxylating dioxygenases (RHDs) associated with aerobic bacterial PAH degradation were identified and linked to PG1 through PCR analyses of a simplified enrichment culture. In tandem with a ferredoxin and reductase found in close proximity to one pair of RHD genes, six of the RHDs were cloned and expressed inEscherichia coli. Each cloned RHD was tested for activity against nine PAHs ranging in size from two to five rings. Despite differences in their predicted protein sequences, each of the six RHDs was capable of transforming phenanthrene and pyrene. Three RHDs could additionally transform naphthalene and fluorene, and these genotypes were also associated with the ability of theE. coliconstructs to convert indole to indigo. Only one of the six cloned RHDs was capable of transforming anthracene and benz[a]anthracene. None of the tested RHDs were capable of significantly transforming fluoranthene, chrysene, or benzo[a]pyrene.

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confidence: 99%

Heterologous Expression of Polycyclic Aromatic Hydrocarbon Ring-Hydroxylating Dioxygenase Genes from a Novel Pyrene-Degrading Betaproteobacterium

Singleton

Aitken

2012

“…The identification of functionally important PAHdegrading bacterial populations in marine sediments is the primary step to advancing our understanding of the ecological mechanisms governing PAH biodegradation in this environmental matrix (9). The most common target used to study PAHdegrading bacterial populations is the gene encoding the alpha subunit of the dioxygenase that catalyzes the first step of aerobic degradation pathways (5). In previous studies (2, 9), we identified eight distinct variants of this functional gene in intertidal sediments of Patagonia using PCR clone libraries targeting PAH dioxygenase genes previously identified in Gram-negative bacteria.…”

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confidence: 99%

Abundance, Dynamics, and Biogeographic Distribution of Seven Polycyclic Aromatic Hydrocarbon Dioxygenase Gene Variants in Coastal Sediments of Patagonia

Marcos

Lozada

Marzio

et al. 2012

“…This underscores the broad diversity of oxygenase genes in the environment and the potential limitations and biases of PCR-based inquiries (8,14). Despite the high diversity among genes encoding enzymes involved in the attack of monoaromatic hydrocarbons, there seems to be a high degree of conservation in the biochemical pathways that enable aerobic destabilization of the aromatic ring (8,14).…”

Section: Discussionmentioning

confidence: 99%

“…Numerous studies have applied assays with molecular biomarker approaches to contaminated sites, with the objective of exploring the diversities of biodegradation genes and of the naturally occurring populations involved in metabolizing hydrocarbons (10)(11)(12)(13)(14)(15)(16). In addition, cultivation efforts have successfully isolated hydrocarbon-degrading organisms from habitats, including from soil, groundwater, marine waters, and deep sea sediments (12,(17)(18)(19)(20)(21)(22).…”

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confidence: 99%

“…In addition, cultivation efforts have successfully isolated hydrocarbon-degrading organisms from habitats, including from soil, groundwater, marine waters, and deep sea sediments (12,(17)(18)(19)(20)(21)(22). Despite the many studies successfully reporting new information on the genetic basis (e.g., dioxygenase) of hydrocarbon metabolism, much remains to be learned regarding both the identities of populations responsible for biodegradation and the diversity of biodegradation genes (4,10,14,16).…”

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confidence: 99%

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Benzene Degradation by a Variovorax Species within a Coal Tar-Contaminated Groundwater Microbial Community

Posman

DeRito

Madsen

2017

Investigations of environmental microbial communities are crucial for the discovery of populations capable of degrading hazardous compounds and may lead to improved bioremediation strategies. The goal of this study was to identify microorganisms responsible for aerobic benzene degradation in coal tar-contaminated groundwater. Benzene degradation was monitored in laboratory incubations of well waters using gas chromatography mass spectrometry (GC-MS). Stable isotope probing (SIP) experiments using [13C]benzene enabled us to obtain 13C-labled community DNA. From this, 16S rRNA clone libraries identified Gammaproteobacteria and Betaproteobacteria as the active benzene-metabolizing microbial populations. Subsequent cultivation experiments yielded nine bacterial isolates that grew in the presence of benzene; five were confirmed in laboratory cultures to grow on benzene. The isolated benzene-degrading organisms were genotypically similar (>97% 16S rRNA gene nucleotide identities) to the organisms identified in SIP experiments. One isolate, Variovorax MAK3, was further investigated for the expression of a putative aromatic ring-hydroxylating dioxygenase (RHD) hypothesized to be involved in benzene degradation. Microcosm experiments using Variovorax MAK3 revealed a 10-fold increase in RHD (Vapar_5383) expression, establishing a link between this gene and benzene degradation. Furthermore, the addition of Variovorax MAK3 to microcosms prepared from site waters accelerated community benzene degradation and correspondingly increased RHD gene expression. In microcosms using uninoculated groundwater, quantitative (q)PCR assays (with 16S rRNA and RDH genes) showed that Variovorax was present and responsive to added benzene. These data demonstrate how the convergence of cultivation-dependent and -independent techniques can boost understandings of active populations and functional genes in complex benzene-degrading microbial communities. IMPORTANCE Benzene is a human carcinogen whose presence in contaminated groundwater drives environmental cleanup efforts. Although the aerobic biodegradation of benzene has long been established, knowledge of the identity of the microorganisms in complex naturally occurring microbial communities responsible for benzene biodegradation has evaded scientific inquiry for many decades. Here, we applied a molecular biology technique known as stable isotope probing (SIP) to the microbial communities residing in contaminated groundwater samples to identify the community members active in benzene biodegradation. We complemented this approach by isolating and growing in the laboratory a bacterium representative of the bacteria found using SIP. Further characterization of the isolated bacterium enabled us to track the expression of a key gene that attacks benzene both in pure cultures of the bacterium and in the naturally occurring groundwater microbial community. This work advances information regarding the documentation of microbial processes, especially the populations and genes that contribute to bioremediation.