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

Abstract

ABSTRACT Stable isotope probing with [ 13 C]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 [ 13 C]DNA after a 14-day incubation with [ 13 C]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 [ 13 C]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 [ 13 C]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.