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Applied and Environmental Microbiology, September 2006, p. 5877-5883, Vol. 72, No. 9
0099-2240/06/$08.00+0     doi:10.1128/AEM.00516-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Discrimination of Multiple Dehalococcoides Strains in a Trichloroethene Enrichment by Quantification of Their Reductive Dehalogenase Genes

Victor F. Holmes,1 Jianzhong He,1 Patrick K. H. Lee,1 and Lisa Alvarez-Cohen1,2*

Department of Civil and Environmental Engineering, University of California at Berkeley, Berkeley, California 94720,1 Earth Sciences Division, Lawrence Berkeley National Laboratory, 1 Cyclotron Road, Berkeley, California 947202

Received 3 March 2006/ Accepted 9 June 2006

While many anaerobic microbial communities are capable of reductively dechlorinating tetrachloroethene (PCE) and trichloroethene (TCE) to dichloroethene (DCE), vinyl chloride (VC), and finally ethene, the accumulation of the highly toxic intermediates, cis-DCE (cDCE) and VC, presents a challenge for bioremediation processes. Members of the genus Dehalococcoides are apparently solely responsible for dechlorination beyond DCE, but isolates of Dehalococcoides each metabolize only a subset of PCE dechlorination intermediates and the interactions among distinct Dehalococcoides strains that result in complete dechlorination are not well understood. Here we apply quantitative PCR to 16S rRNA and reductase gene sequences to discriminate and track Dehalococcoides strains in a TCE enrichment derived from soil taken from the Alameda Naval Air Station (ANAS) using a four-gene plasmid standard. This standard increased experimental accuracy such that 16S rRNA and summed reductase gene copy numbers matched to within 10%. The ANAS culture was found to contain only a single Dehalococcoides 16S rRNA gene sequence, matching that of D. ethenogenes 195, but both the vcrA and tceA reductive dehalogenase genes. Quantities of these two genes in the enrichment summed to the quantity of the Dehalococcoides 16S rRNA gene. Further, between ANAS subcultures enriched on TCE, cDCE, or VC, the relative copy number of the two dehalogenases shifted 14-fold, indicating that the genes are present in two different Dehalococcoides strains. Comparison of cell yields in VC-, cDCE-, and TCE-enriched subcultures suggests that the tceA-containing strain is responsible for nearly all of the TCE and cDCE metabolism in ANAS, whereas the vcrA-containing strain is responsible for all of the VC metabolism.

* Corresponding author. Mailing address: 726 Davis Hall, Department of Civil and Environmental Engineering, University of California at Berkeley, Berkeley, CA 94720-1710. Phone: (510) 643-5969. Fax: (510) 642-7483. E-mail: alvarez{at}ce.berkeley.edu.

Applied and Environmental Microbiology, September 2006, p. 5877-5883, Vol. 72, No. 9
0099-2240/06/$08.00+0     doi:10.1128/AEM.00516-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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