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Applied and Environmental Microbiology, November 2002, p. 5728-5736, Vol. 68, No. 11
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.11.5728-5736.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Identification of DNA-Synthesizing Bacterial Cells in Coastal North Sea Plankton

Annelie Pernthaler, Jakob Pernthaler,* Martha Schattenhofer, and Rudolf Amann

Max Planck Institute for Marine Microbiology, Bremen, Germany

Received 18 April 2002/ Accepted 30 July 2002

We describe a method for microscopic identification of DNA-synthesizing cells in bacterioplankton samples. After incubation with the halogenated thymidine analogue bromodeoxyuridine (BrdU), environmental bacteria were identified by fluorescence in situ hybridization (FISH) with horseradish peroxidase (HRP)-linked oligonucleotide probes. Tyramide signal amplification was used to preserve the FISH staining during the subsequent immunocytochemical detection of BrdU incorporation. DNA-synthesizing cells were visualized by means of an HRP-labeled antibody Fab fragment and a second tyramide signal amplification step. We applied our protocol to samples of prefiltered (pore size, 1.2 µm) North Sea surface water collected during early autumn. After 4 h of incubation, BrdU incorporation was detected in 3% of all bacterial cells. Within 20 h the detectable DNA-synthesizing fraction increased to >14%. During this period, the cell numbers of members of the Roseobacter lineage remained constant, but the fraction of BrdU-incorporating Roseobacter sp. cells doubled, from 24 to 42%. In Alteromonas sp. high BrdU labeling rates after 4 to 8 h were followed by a 10-fold increase in abundance. Rapid BrdU incorporation was also observed in members of the SAR86 lineage. After 4 h of incubation, cells affiliated with this clade constituted 8% of the total bacteria but almost 50% of the visibly DNA-synthesizing bacterial fraction. Thus, this clade might be an important contributor to total bacterioplankton activity in coastal North Sea water during periods of low phytoplankton primary production. The small size and low ribosome content of SAR86 cells are probably not indications of inactivity or dormancy.

* Corresponding author. Mailing address: Max-Planck-Institut für Marine Mikrobiologie, Celsiusstraße 1, D-28359 Bremen, Germany. Phone: 49 421 2028940. Fax: 49 421 2028580. E-mail: jperntha{at}mpi-bremen.de.

Applied and Environmental Microbiology, November 2002, p. 5728-5736, Vol. 68, No. 11
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.11.5728-5736.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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