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.

FIG. 1.

Microbial growth (Δ) and biodegradation of PAHs (O) over time. (A) Enrichment cultures with NP; (B) enrichment cultures with PH; (C) enrichment cultures with PY. Monitoring of NP cultures ended at 84 h due to rapid degradation of the compound in comparison to PH and PY. Data represent the averages and standard errors of triplicate data.

FIG. 2.

Denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rRNA gene fragments from PAH-amended enrichment cultures over time. The time of sampling (hours) is listed above the lanes. DGGE lanes are representative of triplicate data.

FIG. 3.

Ordination plots, by treatments and time, of bacterial communities generated by principle component analysis of bacterial species occurrence from 16S rRNA gene DGGE profiles. ⧫, unamended (UN); ▪, naphthalene amended (NP); •, phenanthrene amended (PH); ▴, pyrene amended (PY).

FIG. 4.

Phylogenetic distribution of the Rieske gene fragment families from the soil (S) and enrichment libraries. The dendrogram was constructed from a ClustalW alignment of the Rieske sequences by neighbor-joining analysis using Mega 3.0. Nodes supported by bootstrap values greater than 50 are indicated with a filled black circle. Major clone families are in boldface and have the number of clones observed indicated in parentheses. Reference sequences from GenBank include the accession number. The scale bar represents substitutions per nucleotide.

FIG. 5.

Rieske clone family accumulation curves showing the number of clones sequenced versus the number of Rieske families observed. Soil (⧫), naphthalene (▪), phenanthrene (▵), and pyrene (○) samples are shown.