This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ritalahti, K. M.
Right arrow Articles by Löffler, F. E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ritalahti, K. M.
Right arrow Articles by Löffler, F. E.
Agricola
Right arrow Articles by Ritalahti, K. M.
Right arrow Articles by Löffler, F. E.

 Previous Article  |  Next Article 

Applied and Environmental Microbiology, April 2006, p. 2765-2774, Vol. 72, No. 4
0099-2240/06/$08.00+0     doi:10.1128/AEM.72.4.2765-2774.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Quantitative PCR Targeting 16S rRNA and Reductive Dehalogenase Genes Simultaneously Monitors Multiple Dehalococcoides Strains

Kirsti M. Ritalahti,1* Benjamin K. Amos,1 Youlboong Sung,1,{dagger} Qingzhong Wu,1 Stephen S. Koenigsberg,3,{ddagger} and Frank E. Löffler1,2

School of Civil and Environmental Engineering,1 School of Biology, Georgia Institute of Technology, Atlanta, Georgia 30332-0512,2 Regenesis Bioremediation Products, San Clemente, California 92672-62443

Received 18 October 2005/ Accepted 7 February 2006

The 16S rRNA gene provides insufficient information to infer the range of chloroorganic electron acceptors used by different Dehalococcoides organisms. To overcome this limitation and provide enhanced diagnostic tools for growth measurements, site assessment, and bioremediation monitoring, a quantitative real-time PCR (qPCR) approach targeting 16S rRNA genes and three Dehalococcoides reductive dehalogenase (RDase) genes with assigned function (i.e., tceA, bvcA, and vcrA) was designed and evaluated. qPCR standard curves generated for the RDase genes by use of genomic DNA from Dehalococcoides pure cultures correlated with standard curves obtained for both Bacteria- and Dehalococcoides-targeted 16S rRNA genes, suggesting that the RDase genes are useful targets for quantitative assessment of Dehalococcoides organisms. RDase gene probe/primer pairs were specific for the Dehalococcoides strains known to carry the diagnostic RDase gene sequences, and the qPCR method allowed the detection of as few as 1 to 20 and quantification of as few as 50 to 100 tceA, bvcA, or vcrA gene targets per PCR volume. The qPCR approach was applied to dechlorinating enrichment cultures, microcosms, and samples from a contaminated site. In characterized enrichment cultures where known Dehalococcoides strains were enumerated, the sum of the three RDase genes equaled the total Dehalococcoides cell numbers. In site samples and chloroethane-dechlorinating microcosms, the sum of the three RDase genes was much less than that predicted by Dehalococcoides-targeted qPCR, totaling 10 to 30% of the total Dehalococcoides cell numbers. Hence, a large number of Dehalococcoides spp. contain as-yet-unidentified RDase genes, indicating that our current understanding of the dechlorinating Dehalococcoides community is incomplete.

* Corresponding author. Mailing address: Georgia Institute of Technology, School of Civil and Environmental Engineering, 311 Ferst Drive, 3230 ES&T Building, Atlanta, GA 30332-0512. Phone: (404) 385-4558. Fax: (404) 894-8266. E-mail: krita{at}ce.gatech.edu.

{dagger} Present address: University of Oklahoma, Norman, OK 73019.

{ddagger} Present address: Environmental Strategies Consulting, LLC (a Quanta Technical Services company), Irvine, CA 92612.

Applied and Environmental Microbiology, April 2006, p. 2765-2774, Vol. 72, No. 4
0099-2240/06/$08.00+0     doi:10.1128/AEM.72.4.2765-2774.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Freitag, T. E., Prosser, J. I. (2009). Correlation of Methane Production and Functional Gene Transcriptional Activity in a Peat Soil. Appl. Environ. Microbiol. 75: 6679-6687 [Abstract] [Full Text]  
  • Thomas, S. H., Padilla-Crespo, E., Jardine, P. M., Sanford, R. A., Loffler, F. E. (2009). Diversity and Distribution of Anaeromyxobacter Strains in a Uranium-Contaminated Subsurface Environment with a Nonuniform Groundwater Flow. Appl. Environ. Microbiol. 75: 3679-3687 [Abstract] [Full Text]  
  • Grostern, A., Edwards, E. A. (2009). Characterization of a Dehalobacter Coculture That Dechlorinates 1,2-Dichloroethane to Ethene and Identification of the Putative Reductive Dehalogenase Gene. Appl. Environ. Microbiol. 75: 2684-2693 [Abstract] [Full Text]  
  • Zhang, H., DiBaise, J. K., Zuccolo, A., Kudrna, D., Braidotti, M., Yu, Y., Parameswaran, P., Crowell, M. D., Wing, R., Rittmann, B. E., Krajmalnik-Brown, R. (2009). Human gut microbiota in obesity and after gastric bypass. Proc. Natl. Acad. Sci. USA 106: 2365-2370 [Abstract] [Full Text]  
  • Behrens, S., Azizian, M. F., McMurdie, P. J., Sabalowsky, A., Dolan, M. E., Semprini, L., Spormann, A. M. (2008). Monitoring Abundance and Expression of "Dehalococcoides" Species Chloroethene-Reductive Dehalogenases in a Tetrachloroethene-Dechlorinating Flow Column. Appl. Environ. Microbiol. 74: 5695-5703 [Abstract] [Full Text]  
  • Lee, P. K. H., Macbeth, T. W., Sorenson, K. S. Jr., Deeb, R. A., Alvarez-Cohen, L. (2008). Quantifying Genes and Transcripts To Assess the In Situ Physiology of "Dehalococcoides" spp. in a Trichloroethene-Contaminated Groundwater Site. Appl. Environ. Microbiol. 74: 2728-2739 [Abstract] [Full Text]  
  • Amos, B. K., Sung, Y., Fletcher, K. E., Gentry, T. J., Wu, W.-M., Criddle, C. S., Zhou, J., Loffler, F. E. (2007). Detection and Quantification of Geobacter lovleyi Strain SZ: Implications for Bioremediation at Tetrachloroethene- and Uranium-Impacted Sites. Appl. Environ. Microbiol. 73: 6898-6904 [Abstract] [Full Text]  
  • Bedard, D. L., Ritalahti, K. M., Loffler, F. E. (2007). The Dehalococcoides Population in Sediment-Free Mixed Cultures Metabolically Dechlorinates the Commercial Polychlorinated Biphenyl Mixture Aroclor 1260. Appl. Environ. Microbiol. 73: 2513-2521 [Abstract] [Full Text]  
  • Grostern, A., Edwards, E. A. (2006). A 1,1,1-Trichloroethane-Degrading Anaerobic Mixed Microbial Culture Enhances Biotransformation of Mixtures of Chlorinated Ethenes and Ethanes. Appl. Environ. Microbiol. 72: 7849-7856 [Abstract] [Full Text]  
  • Holmes, V. F., He, J., Lee, P. K. H., Alvarez-Cohen, L. (2006). Discrimination of Multiple Dehalococcoides Strains in a Trichloroethene Enrichment by Quantification of Their Reductive Dehalogenase Genes. Appl. Environ. Microbiol. 72: 5877-5883 [Abstract] [Full Text]  


Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»