Immunochemical detection and isolation of DNA from metabolically active bacteria.
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.
Department of Microbiology, Oregon State University, Corvallis, Oregon 97331, USA. urbache@bcc.orst.edu
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Urbach
K L
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Vergin
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SJ
Giovannoni
D000911:Antibodies, Monoclonal / Q000276:immunology; D001419:Bacteria / Q000145:classification / Q000235:genetics / Q000254:growth & development* / Q000378:metabolism; D001973:Bromodeoxyuridine / Q000276:immunology / Q000378:metabolism*; D004269:DNA, Bacterial / Q000032:analysis / Q000096:biosynthesis / Q000302:isolation & purification*; D017753:Ecosystem; D005618:Fresh Water; D005798:Genes, Bacterial; D020459:Genes, rRNA; D015151:Immunoblotting; D007150:Immunohistochemistry / Q000379:methods*; D018189:Immunomagnetic Separation; D010802:Phylogeny; D016133:Polymerase Chain Reaction / Q000379:methods; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D014871:Water Microbiology
D000911:Antibodies, Monoclonal; D004269:DNA, Bacterial; D012336:RNA, Ribosomal, 16S; D001973:Bromodeoxyuridine