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. I n addition, we developed an immunocytochemical protocol to visualize BrdU-l abeled microbial cells. Cultured bacteria and natural populations of aquati c bacterioplankton were pulse-labeled with exogenously supplied BrdU. Incor poration of BrdU into microbial DNA was demonstrated in DNA dot blots probe d with anti-BrdU monoclonal antibodies and either peroxidase- or Texas red- conjugated secondary antibodies. BrdU-containing DNA was physically separat ed from unlabeled DNA by using antibody-coated paramagnetic beads, and the identities of bacteria contributing to both purified, BrdU-containing fract ions and unfractionated, starting-material DNAs were determined by length h eterogeneity PCR (LH-PCR) analysis. BrdU-containing DNA purified from a mix ture of DNAs from labeled and unlabeled cultures showed >90-fold enrichment for the labeled bacterial taxon. The LH-PCR profile for BrdU-containing DN A from a labeled, natural microbial community differed from the profile for the community as a whole, demonstrating that BrdU was incorporated by a ta xonomic subset of the community. Immunocytochemical detection of cells with BrdU-labeled DNA was accomplished by in situ probing with anti-BrdU monocl onal antibodies and Texas red-labeled secondary antibodies. Using this suit e of techniques, microbial cells incorporating BrdU into their newly synthe sized DNA can be quantified and the identities of these actively growing ce lls can be compared to the composition of the microbial community as a whol e. Since not all strains tested could incorporate BrdU, these methods may b e most useful when used to gain an understanding of the activities of speci fic species in the context of their microbial community.