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Flow sorting of marine bacterioplankton after fluorescence in situ hybridization

MPS-Authors
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Sekar,  R.
Department of Biogeochemistry, Max Planck Institute for Marine Microbiology, Max Planck Society;

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Fuchs,  B. M.
Department of Molecular Ecology, Max Planck Institute for Marine Microbiology, Max Planck Society;

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Amann,  R.
Department of Molecular Ecology, Max Planck Institute for Marine Microbiology, Max Planck Society;

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Pernthaler,  J.
Department of Molecular Ecology, Max Planck Institute for Marine Microbiology, Max Planck Society;

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Citation

Sekar, R., Fuchs, B. M., Amann, R., & Pernthaler, J. (2004). Flow sorting of marine bacterioplankton after fluorescence in situ hybridization. Applied and Environmental Microbiology, 70(10), 6210-6219.


Cite as: http://hdl.handle.net/21.11116/0000-0001-D0F8-C
Abstract
We describe an approach to sort cells from coastal North Sea bacterioplankton by flow cytometry after in situ hybridization with rRNA-targeted horseradish peroxidase-labeled oligonucleotide probes and catalyzed fluorescent reporter deposition (CARD-FISH). In a sample from spring 2003 >90% of the cells were detected by CARD-FISH with a bacterial probe (EUB338). Approximately 30% of the microbial assemblage was affiliated with the Cytophaga-Flavobacterium lineage of the Bacteroidetes (CFB group) (probe CF319a), and almost 10% was targeted by a probe for the beta-proteobacteria (probe BET42a). A protocol was optimized to detach cells hybridized with EUB338, BET42a, and CF319a from membrane filters (recovery rate, 70%) and to sort the cells by flow cytometry. The purity of sorted cells was >95%. 16S rRNA gene clone libraries were constructed from hybridized and sorted cells (S-EUB, S-BET, and S-CF libraries) and from unhybridized and unsorted cells (UNHYB library). Sequences related to the CFB group were significantly more frequent in the S-CF library (66%) than in the UNHYB library (13%). No enrichment of beta-proteobacterial sequence types was found in the S-BET library, but novel sequences related to Nitrosospira were found exclusively in this library. These bacteria, together with members of marine clade OM43, represented >90% of the beta-proteobacteria in the water sample, as determined by CARD-FISH with specific probes. This illustrates that a combination of CARD-FISH and flow sorting might be a powerful approach to study the diversity and potentially the activity and the genomes of different bacterial populations in aquatic habitats.