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Applied and Environmental Microbiology, September 2009, p. 5501-5506, Vol. 75, No. 17
0099-2240/09/$08.00+0     doi:10.1128/AEM.00121-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

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

Woo Jun Sul,1 Joonhong Park,1,2 John F. Quensen III,1 Jorge L. M. Rodrigues,1 Laurie Seliger,3 Tamara V. Tsoi,1 Gerben J. Zylstra,3 and James M. Tiedje1*

Center for Microbial Ecology, Michigan State University, 540 Plant and Soil Sciences Building, East Lansing, Michigan 48824,1 Department of Civil and Environmental Engineering, Yonsei University, Seoul 120-749, Republic of Korea,2 Biotechnology Center for Agriculture and the Environment, School of Environmental and Biological Sciences, Cook College, Rutgers University, 59 Dudley Road, New Brunswick, New Jersey 08901-85203

Received 19 January 2009/ Accepted 2 July 2009

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.

* Corresponding author. Mailing address: Center for Microbial Ecology, Michigan State University, 540 Plant and Soil Sciences Building, East Lansing, MI 48824. Phone: (517) 355-0271, ext. 287. Fax: (517) 353-2917. E-mail: tiedjej{at}msu.edu

{triangledown} Published ahead of print on 31 July 2009.

Applied and Environmental Microbiology, September 2009, p. 5501-5506, Vol. 75, No. 17
0099-2240/09/$08.00+0     doi:10.1128/AEM.00121-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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