Affiliation:
1. Department of Microbiology, Oregon State University, Corvallis, Oregon 97331
Abstract
ABSTRACT
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.
Publisher
American Society for Microbiology
Subject
Ecology,Applied Microbiology and Biotechnology,Food Science,Biotechnology
Reference32 articles.
1. Ausubel
F. A.
Brent
R.
Kingston
R. E.
Moore
D. D.
Smith
J. A.
Seidman
J. G.
Struhl
K.
Current protocols in molecular biology.
1988
John Wiley and Sons
New York N.Y
2. A species-specific bacterial productivity method using immunomagnetic separation and radiotracer experiments.;Bard D. G.;J. Microbiol. Methods,1997
3. Bacterial growth rate in the sea: direct analysis by thymidine autoradiography.;Brock T. D.;Science,1967
4. Relation between presence-absence of a visible nucleoid and metabolic activity in bacterioplankton cells.;Choi J. W.;Limnol. Oceanogr.,1996
5. Tolerance to bromodeoxyuridine in a thymidine-requiring strain of Bacillus subtilis.;Coote J. G.;J. Gen. Microbiol.,1986
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