An Improved Protocol for Quantification of Freshwater Actinobacteria by Fluorescence In Situ Hybridization
| Autor: | Raju Sekar, Annelie Pernthaler, Thomas Posch, Jakob Pernthaler, Falk Warnecke, Rudolf Amann |
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| Rok vydání: | 2003 |
| Předmět: |
Cell Membrane Permeability
Gram-positive bacteria Colony Count Microbial Fresh Water Gram-Positive Bacteria Applied Microbiology and Biotechnology Actinobacteria Microbiology Marine bacteriophage Methods Picoplankton In Situ Hybridization Fluorescence Bacteria Ecology biology Serine Endopeptidases Bacterioplankton Plankton biology.organism_classification Limnohabitans Muramidase Proteobacteria Food Science Biotechnology |
| Zdroj: | Applied and Environmental Microbiology |
| ISSN: | 1098-5336 0099-2240 |
| Popis: | We tested a previously described protocol for fluorescence in situ hybridization of marine bacterioplankton with horseradish peroxidase-labeled rRNA-targeted oligonucleotide probes and catalyzed reporter deposition (CARD-FISH) in plankton samples from different lakes. The fraction of Bacteria detected by CARD-FISH was significantly lower than after FISH with fluorescently monolabeled probes. In particular, the abundances of aquatic Actinobacteria were significantly underestimated. We thus developed a combined fixation and permeabilization protocol for CARD-FISH of freshwater samples. Enzymatic pretreatment of fixed cells was optimized for the controlled digestion of gram-positive cell walls without causing overall cell loss. Incubations with high concentrations of lysozyme (10 mg ml 1 ) followed by achromopeptidase (60 U ml 1 ) successfully permeabilized cell walls of Actinobacteria for subsequent CARD-FISH both in enrichment cultures and environmental samples. Between 72 and >99% (mean, 86%) of all Bacteria could be visualized with the improved assay in surface waters of four lakes. For freshwater samples, our method is thus superior to the CARD-FISH protocol for marine Bacteria (mean, 55%) and to FISH with directly fluorochrome labeled probes (mean, 67%). Actinobacterial abundances in the studied systems, as detected by the optimized protocol, ranged from 32 to >55% (mean, 45%). Our findings confirm that members of this lineage are among the numerically most important Bacteria of freshwater picoplankton. The taxonomic composition of picoplankton communities profoundly differs in freshwater and marine systems. Cultivation-independent molecular approaches have demonstrated that bacteria from large phylogenetic lineages of typical freshwater microbes, e.g., the -subdivision of the proteobacteria, are virtually absent in the marine picoplankton (15, 23). For other bacterial lineages the evidence is less conclusive. Comparative analysis of 16S rDNA genes indicate that uncultured members of the class Actinobacteria are ubiquitous in lakes of various trophic state, size or geographic location (16, 36), but actinobacterial sequence types are also known from marine systems (31). |
| Databáze: | OpenAIRE |
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