Immunochemical Detection and Isolation of DNA from Metabolically Active Bacteria

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.

Combined bromodeoxyuridine immunocapture and terminal‐restriction fragment length polymorphism analysis highlights differences in the active soil bacterial metagenome due to Glomus mosseae inoculation or plant species

“…The approach adopted in the present study has several advantages over other approaches available to date to study microbial community structure and function, such as techniques to measure growth rates and productivity for particular taxa within natural communities (DeLong et al, 1989;Kerkhof and Ward, 1993). Taxon-specific productivity estimates can also be obtained from incorporation of radiolabelled tracer molecules into natural populations, followed by immunocapture of cells binding to specific antibodies (Bard and Ward, 1997;Urbach et al, 1999), or by combination with group-specific fluorescent oligonucleotide probes [STAR-FISH (Ouverney and Fuhrman, 1999) or MAR-FISH (Kindaichi et al, 2004)]. These approaches provide information about group-specific growth rates for specific microorganisms of interest, but they cannot directly provide information on active members of entire microbial communities.…”

“…This method relies on incorporation of the thymidine analogue, bromodeoxyuridine (BrdU), into growing cells during DNA replication. The newly synthesized DNA can then be isolated by BrdU immunocapture (Borneman, 1999;Urbach et al ., 1999). This approach permits identification of specific populations that grow in response to specified stimuli.…”

Active bacterial community structure along vertical redox gradients in Baltic Sea sediment

“…DNA from metabolically active bacteria (i.e. DNAreplicating bacteria) was extracted by employing the BrdUimmunocapture approach (Urbach et al, 1999;Artursson and Jansson, 2003;. This method relies on incorporation of BrdU as a thymidine analogue into growing cells during DNA replication.…”

“…For this purpose, we used two different approaches for studying metabolically active and potentially growing bacteria in the sediments, i.e. amplification of 16S rRNA genes from bromodeoxyuridine (BrdU)-labelled DNA (Urbach et al, 1999;Artursson and Jansson, 2003;Warnecke et al, 2005) and from cDNA obtained by reverse transcription polymerase chain reaction (rt-PCR) of RNA (Teske et al, 1996). Bromodeoxyuridine is incorporated into the DNA of growing cells as a thymidine analogue and BrdU-immunocapture was previously demonstrated as a simple approach for detection of growing (i.e.…”

Microbial growth in Arctic tundra soil at −2°C

“…In addition to synthesizing new membrane components, microbes were also replicating, as indicated by our second line of evidence: incorporation of BrdU into DNA. BrdU is a thymidine analogue that can be taken up and incorporated by many microorganisms (Urbach et al, 1999), making it a useful tracer for DNA replication. Although widely used in mammalian systems, this technique is relatively new to microbial ecology (e.g.…”