DNA-Stable Isotope Probing Integrated with Metagenomics for Retrieval of Biphenyl Dioxygenase Genes from Polychlorinated Biphenyl-Contaminated River Sediment

Stable isotope probing with ¹³C]biphenyl was used to explore the genetic properties of indigenous bacteria able to grow on biphenyl in PCB-contaminated River Raisin sediment. A bacterial 16S rRNA gene clone library generated from ¹³C]DNA after a 14-day incubation with ¹³C]biphenyl revealed the dominant organisms to be members of the genera Achromobacter and Pseudomonas. A library built from PCR amplification of genes for aromatic-ring-hydroxylating dioxygenases from the ¹³C]DNA fraction revealed two sequence groups similar to bphA (encoding biphenyl dioxygenase) of Comamonas testosteroni strain B-356 and of Rhodococcus sp. RHA1. A library of 1,568 cosmid clones was produced from the ¹³C]DNA fraction. A 31.8-kb cosmid clone, detected by aromatic dioxygenase primers, contained genes of biphenyl dioxygenase subunits bphAE, while the rest of the clone''s sequence was similar to that of an unknown member of the Gammaproteobacteria. A discrepancy in G+C content near the bphAE genes implies their recent acquisition, possibly by horizontal transfer. The biphenyl dioxygenase from the cosmid clone oxidized biphenyl and unsubstituted and para-only-substituted rings of polychlorinated biphenyl (PCB) congeners. A DNA-stable isotope probing-based cosmid library enabled the retrieval of functional genes from an uncultivated organism capable of PCB metabolism and suggest dispersed dioxygenase gene organization in nature.Commercially used polychlorinated biphenyls (PCBs), which are mixtures of more than 60 individual chlorinated biphenyl congeners, are among the most persistent anthropogenic chemical pollutants that threaten natural ecosystems and human health (1). Numerous biphenyl-degrading microorganisms have been isolated and studied, especially for the range of PCB congeners that they degrade. Research has been primarily focused on the biodegradative pathways and the biphenyl dioxygenases responsible for initial PCB oxidation by isolated bacteria (14, 27). Knowledge, however, is limited concerning the indigenous microbial populations that metabolize PCBs in the environment. Stable isotope probing (SIP) coupled with metagenomics is one approach to more directly explore which organisms and genetic information may be involved in PCB degradation in PCB-contaminated sites.SIP was developed to separate and concentrate the nucleic acids or fatty acids of microbial populations that metabolize and, hence, assimilate the isotopically labeled substrates into new cell material (4, 5, 28). Recently, the active PCB degraders in a biofilm community on PCB droplets were revealed as Burkholderia species by using DNA-SIP (32). In another DNA-SIP study, 75 different genera that acquired carbon from ¹³C]biphenyl were found in the PCB-contaminated root zone of a pine tree (22). In addition, that heavy ¹³C]DNA fraction revealed new dioxygenase sequences and possible PCB degradation pathways from GeoChip (16) results and from PCR-amplified sequences obtained by using primers targeting aromatic-ring-hydroxylating dioxygenase (ARHD) genes (22).A major hurdle in using DNA-SIP for metagenomic analyses (9) is the very small amount of heavy DNA that is produced and, hence, recovered, making library construction difficult. Two studies have shown the feasibility of DNA-SIP for metagenomic analyses for C-1 compound-utilizing communities, but they first increased the amount of the heavy DNA fraction by multiple-displacement amplification (6, 10) or enriched the community by growth in sediment slurries. (18).In this study, we used ¹³C]biphenyl to probe for potential PCB-degrading populations in a PCB-contaminated river sediment and to recover genes potentially involved in the critical first step of PCB degradation, the dioxygenase attack. We found a 31.8-kb cosmid clone that contained a biphenyl dioxygenase sequence (bphAE) and demonstrated its activity on PCBs.