Flow Sorting of Marine Bacterioplankton after Fluorescence In Situ Hybridization

@article{citeulike:984447, 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.}, author = {Sekar, Raju and Fuchs, Bernhard M. and Amann, Rudolf and Pernthaler, Jakob }, citeulike-article-id = {984447}, doi = {10.1128/AEM.70.10.6210}, journal = {Appl. Environ. Microbiol.}, keywords = {fcm, fish}, month = {October}, number = {10}, pages = {6210--6219}, posted-at = {2008-05-05 16:14:23}, priority = {2}, title = {Flow Sorting of Marine Bacterioplankton after Fluorescence In Situ Hybridization}, url = {http://dx.doi.org/10.1128/AEM.70.10.6210}, volume = {70}, year = {2004} }

TY - JOUR ID - citeulike:984447 L3 - citeulike-article-id:984447 TI - Flow Sorting of Marine Bacterioplankton after Fluorescence In Situ Hybridization JF - Appl. Environ. Microbiol. VL - 70 IS - 10 SP - 6210 EP - 6219 N2 - 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. KW - fcm KW - fish AU - Sekar, Raju AU - Fuchs, Bernhard AU - Amann, Rudolf AU - Pernthaler, Jakob PY - 2004/10/01/ UR - http://dx.doi.org/10.1128/AEM.70.10.6210 ER -