Immunochemical detection and isolation of DNA from metabolically active bacteria - PubMed (original) (raw)

Immunochemical detection and isolation of DNA from metabolically active bacteria

E Urbach et al. Appl Environ Microbiol. 1999 Mar.

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.

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Figures

FIG. 1

FIG. 1

Growth kinetics for bacterial cultures with and without BrdU. Arrows indicate times at which cultures were supplemented with BrdU and TdR (+BrdU) or TdR alone (−BrdU). Cultures were harvested at the final time points and used to prepare DNA for dot blots.

FIG. 2

FIG. 2

Immunochemical detection of BrdU in DNA dot blots. (A) DNA from cultured bacteria grown in the presence (+) or absence (−) of BrdU, probed with anti-BrdU monoclonal antibodies and peroxidase-conjugated secondary antibodies. (B) DNA from Cronemiller Lake bacterioplankton, incubated with or without BrdU, probed with anti-BrdU monoclonal antibodies and Texas red-conjugated secondary antibodies.

FIG. 3

FIG. 3

Immunocytochemical detection of BrdU-labeled Alteromonas sp. strain C250.5-4. The panels on the left show fluorescence from Texas red-conjugated secondary antibodies bound to anti-BrdU monoclonal antibodies; the panels on the right show DAPI fluorescence. The upper panels show BrdU-labeled cultures; the lower panels show unlabeled cultures.

FIG. 4

FIG. 4

Immunochemical purification of BrdU-containing DNA, analyzed by LH-PCR. (A) Experimental mixture containing DNA from a BrdU-labeled Alteromonas cultures and an unlabeled Roseobacter culture. (B) DNA immunochemically purified from the mixture by using anti-BrdU monoclonal antibodies and paramagnetic beads. (C) BrdU-labeled Alteromonas DNA. (D) Unlabeled Roseobacter DNA. The electropherogram for unlabeled Alteromonas DNA was identical to that shown in panel C.

FIG. 5

FIG. 5

LH-PCR analysis of active microbial taxa in Cronemiller Lake. (A) BrdU-labeled lake water DNA. (B) Active fraction isolated from labeled lake water DNA by using anti-BrdU monoclonal antibodies and paramagnetic beads. (C) Control (unlabeled lake water DNA). (D) Control fraction isolated from unlabeled lake water DNA by using anti-BrdU monoclonal antibodies and paramagnetic beads. Dashed lines indicate the positions of the major peaks at 317 and 319 nucleotides.

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