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 Urbach, E.
Right arrow Articles by Giovannoni, S. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Urbach, E.
Right arrow Articles by Giovannoni, S. J.
Agricola
Right arrow Articles by Urbach, E.
Right arrow Articles by Giovannoni, S. J.

 Previous Article  |  Next Article 

Applied and Environmental Microbiology, March 1999, p. 1207-1213, Vol. 65, No. 3
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Immunochemical Detection and Isolation of DNA from Metabolically Active Bacteria

Ena Urbach,* Kevin L. Vergin, and Stephen J. Giovannoni

Department of Microbiology, Oregon State University, Corvallis, Oregon 97331

Received 26 October 1998/Accepted 18 December 1998

Most techniques used to assay the growth of microbes in natural communities provide no information on the relationship between microbial productivity and community structure. To identify actively growing bacteria, we adapted a technique from immunocytochemistry to detect and selectively isolate DNA from bacteria incorporating bromodeoxyuridine (BrdU), a thymidine analog. In addition, we developed an immunocytochemical protocol to visualize BrdU-labeled microbial cells. Cultured bacteria and natural populations of aquatic bacterioplankton were pulse-labeled with exogenously supplied BrdU. Incorporation of BrdU into microbial DNA was demonstrated in DNA dot blots probed with anti-BrdU monoclonal antibodies and either peroxidase- or Texas red-conjugated secondary antibodies. BrdU-containing DNA was physically separated from unlabeled DNA by using antibody-coated paramagnetic beads, and the identities of bacteria contributing to both purified, BrdU-containing fractions and unfractionated, starting-material DNAs were determined by length heterogeneity PCR (LH-PCR) analysis. BrdU-containing DNA purified from a mixture of DNAs from labeled and unlabeled cultures showed >90-fold enrichment for the labeled bacterial taxon. The LH-PCR profile for BrdU-containing DNA from a labeled, natural microbial community differed from the profile for the community as a whole, demonstrating that BrdU was incorporated by a taxonomic subset of the community. Immunocytochemical detection of cells with BrdU-labeled DNA was accomplished by in situ probing with anti-BrdU monoclonal antibodies and Texas red-labeled secondary antibodies. Using this suite of techniques, microbial cells incorporating BrdU into their newly synthesized DNA can be quantified and the identities of these actively growing cells can be compared to the composition of the microbial community as a whole. Since not all strains tested could incorporate BrdU, these methods may be most useful when used to gain an understanding of the activities of specific species in the context of their microbial community.

* Corresponding author. Mailing address: Department of Microbiology, 220 Nash Hall, Oregon State University, Corvallis, OR 97331. Phone: (541) 737-0717. Fax: (541) 737-0496. E-mail: urbache{at}bcc.orst.edu.

Applied and Environmental Microbiology, March 1999, p. 1207-1213, Vol. 65, No. 3
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


This article has been cited by other articles:

  • Arthurson, V. (2008). Proper Sanitization of Sewage Sludge: a Critical Issue for a Sustainable Society. Appl. Environ. Microbiol. 74: 5267-5275 [Full Text]  
  • Hamasaki, K., Taniguchi, A., Tada, Y., Long, R. A., Azam, F. (2007). Actively Growing Bacteria in the Inland Sea of Japan, Identified by Combined Bromodeoxyuridine Immunocapture and Denaturing Gradient Gel Electrophoresis. Appl. Environ. Microbiol. 73: 2787-2798 [Abstract] [Full Text]  
  • Schwartz, E. (2007). Characterization of Growing Microorganisms in Soil by Stable Isotope Probing with H218O. Appl. Environ. Microbiol. 73: 2541-2546 [Abstract] [Full Text]  
  • Edlund, A., Jansson, J. K. (2006). Changes in Active Bacterial Communities before and after Dredging of Highly Polluted Baltic Sea Sediments. Appl. Environ. Microbiol. 72: 6800-6807 [Abstract] [Full Text]  
  • Walters, S. P., Field, K. G. (2006). Persistence and growth of fecal bacteroidales assessed by bromodeoxyuridine immunocapture.. Appl. Environ. Microbiol. 72: 4532-4539 [Abstract] [Full Text]  
  • Pernthaler, J., Amann, R. (2005). Fate of Heterotrophic Microbes in Pelagic Habitats: Focus on Populations. Microbiol. Mol. Biol. Rev. 69: 440-461 [Abstract] [Full Text]  
  • Ueda, J., Saito, H., Watanabe, H., Evers, B. M. (2005). Novel and quantitative DNA dot-blotting method for assessment of in vivo proliferation. Am. J. Physiol. Gastrointest. Liver Physiol. 288: G842-G847 [Abstract] [Full Text]  
  • Artursson, V., Jansson, J. K. (2003). Use of Bromodeoxyuridine Immunocapture To Identify Active Bacteria Associated with Arbuscular Mycorrhizal Hyphae. Appl. Environ. Microbiol. 69: 6208-6215 [Abstract] [Full Text]  
  • Pernthaler, A., Pernthaler, J., Schattenhofer, M., Amann, R. (2002). Identification of DNA-Synthesizing Bacterial Cells in Coastal North Sea Plankton. Appl. Environ. Microbiol. 68: 5728-5736 [Abstract] [Full Text]  
  • Roling, W. F. M., van Breukelen, B. M., Braster, M., Lin, B., van Verseveld, H. W. (2001). Relationships between Microbial Community Structure and Hydrochemistry in a Landfill Leachate-Polluted Aquifer. Appl. Environ. Microbiol. 67: 4619-4629 [Abstract] [Full Text]  
  • Yin, B., Crowley, D., Sparovek, G., De Melo, W. J., Borneman, J. (2000). Bacterial Functional Redundancy along a Soil Reclamation Gradient. Appl. Environ. Microbiol. 66: 4361-4365 [Abstract] [Full Text]  
  • Borneman, J. (1999). Culture-Independent Identification of Microorganisms That Respond to Specified Stimuli. Appl. Environ. Microbiol. 65: 3398-3400 [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»