Study of diversity and function of the marine key group Vis6 with cultivation-independent methods

Phytoplankton in the ocean represents about 0.2% of all photosynthetic biomass on Earth, but is responsible for half of the global CO2 fixation. A large proportion of this produced organic matter is funneled through the microbial loop towards higher trophic levels by heterotrophic microorganisms. These have consequently a great impact on the global carbon cycle, the CO2 concentration in the atmosphere and ultimately our climate. Phytoplankton blooms are short-termed increases in phytoplankton biomass, followed by secondary blooms of heterotrophic bacteria. Taxonomic and ecological studies at the long term ecological research station Helgoland Roads, North Sea, revealed recurring patterns of bacteria on taxonomic and functional level. Members of the phylum Bacteroidetes and in particular of the class Flavobacteriia are among the major responders to phytoplankton blooms. Analyses with both cultivation and cultivation-independent techniques, based on genome sequencing and gene annotations, can reveal the role of individual groups in the degradation of phytoplankton derived organic matter. The focus of this work was the development of a culture-independent pipeline for the targeted access and the subsequent functional genome characterization of bacterial species from the environment. This pipeline combines fluorescence in situ hybridization (FISH) with fluorescence activated cell sorting (FACS) and subsequent genome sequencing. A taxon-specific FISH probe hybridizes to the target cells which are subsequently enriched by cell sorting based on the fluorescence signal. Finally, the DNA of the sorted cells is amplified and sequenced. The development of the pipeline involved an optimization of the recently developed hybridization chain reaction (HCR)-FISH method and the assessment of various cell fixatives concerning their influence on HCR-FISH signal intensity and the quality of genome sequencing and assembly. The developed pipeline was successfully applied on a planktonic seawater sample targeting the so far uncharacterized clade Vis6. This member of the Cryomorphaceae within the Flavobacteriia is recurring during spring phytoplankton blooms off the coast of Helgoland. The description of Vis6 as a novel candidate genus Candidatus Abditibacter with three species was enabled by the combination of the sequences retrieved from targeted sorting pipeline with additional data of North Sea metagenomes and abundance data based on the 16S rRNA identity of Vis6. The growth of Candidatus Abditibacter spp. is likely based on the utilization of phytoplankton-derived polysaccharides and proteins. In the future, the developed pipeline has the potential to be used on low abundance species from highly complex samples, where previous attempts with metagenomics have been unsuccessful so far

A pipeline for targeted metagenomics of environmental bacteria.

BackgroundMetagenomics and single cell genomics provide a window into the genetic repertoire of yet uncultivated microorganisms, but both methods are usually taxonomically untargeted. The combination of fluorescence in situ hybridization (FISH) and fluorescence activated cell sorting (FACS) has the potential to enrich taxonomically well-defined clades for genomic analyses.MethodsCells hybridized with a taxon-specific FISH probe are enriched based on their fluorescence signal via flow cytometric cell sorting. A recently developed FISH procedure, the hybridization chain reaction (HCR)-FISH, provides the high signal intensities required for flow cytometric sorting while maintaining the integrity of the cellular DNA for subsequent genome sequencing. Sorted cells are subjected to shotgun sequencing, resulting in targeted metagenomes of low diversity.ResultsPure cultures of different taxonomic groups were used to (1) adapt and optimize the HCR-FISH protocol and (2) assess the effects of various cell fixation methods on both the signal intensity for cell sorting and the quality of subsequent genome amplification and sequencing. Best results were obtained for ethanol-fixed cells in terms of both HCR-FISH signal intensity and genome assembly quality. Our newly developed pipeline was successfully applied to a marine plankton sample from the North Sea yielding good quality metagenome assembled genomes from a yet uncultivated flavobacterial clade.ConclusionsWith the developed pipeline, targeted metagenomes at various taxonomic levels can be efficiently retrieved from environmental samples. The resulting metagenome assembled genomes allow for the description of yet uncharacterized microbial clades. Video abstract

Combining real-time fluorescence spectroscopy and flow cytometry to reveal new insights in DOC and cell characterization of drinking water

Sudden changes in drinking water quality can cause harmful consequences for end users. Thus, real-time monitoring of drinking water quality can allow early warning and provide crucial gains for securing safe water distribution. This study investigated the advantages of simultaneous real-time measuring of flow cytometry and fluorescence spectroscopy. A contamination event was investigated in a laboratory-scale analysis by spiking drinking water samples with organic nutrients. Flow cytometric data were analyzed by creating fingerprints based on differentiation into high and low nucleic acid cells (HNA/LNA). The detailed characterization of these data showed that an increase in HNA cells indicated an increase in the bacterial growth potential even before actual TCC increases. The fluorescence data was decomposed via the PARAFAC method to reveal seven fluorescent components. Three aromatic protein-like components were associated with the microbiological condition of the drinking water cells; namely, Components 4 (λEx = 279 nm, λEm = 351 nm), 6 (λEx = 279 nm, λEm = 332 nm), and 7 (λEx = 276 nm, λEm = 302 nm). Component 6 was identified as a possible organic variable for appropriate monitoring of TCC, whereas Components 4 and 7 were identified as organic compounds representing nutrients for organisms present in drinking water. Overall, combining both methods for real-time monitoring can be a powerful tool to guarantee drinking water quality

Erforschung von Diversität und Funktion der marinen Schlüsselgruppe Vis6 mit kultivierungsunabhängigen Methoden

Phytoplankton in the ocean represents about 0.2% of all photosynthetic biomass on Earth, but is responsible for half of the global CO2 fixation. A large proportion of this produced organic matter is funneled through the microbial loop towards higher trophic levels by heterotrophic microorganisms. These have consequently a great impact on the global carbon cycle, the CO2 concentration in the atmosphere and ultimately our climate. Phytoplankton blooms are short-termed increases in phytoplankton biomass, followed by secondary blooms of heterotrophic bacteria. Taxonomic and ecological studies at the long term ecological research station Helgoland Roads, North Sea, revealed recurring patterns of bacteria on taxonomic and functional level. Members of the phylum Bacteroidetes and in particular of the class Flavobacteriia are among the major responders to phytoplankton blooms. Analyses with both cultivation and cultivation-independent techniques, based on genome sequencing and gene annotations, can reveal the role of individual groups in the degradation of phytoplankton derived organic matter. The focus of this work was the development of a culture-independent pipeline for the targeted access and the subsequent functional genome characterization of bacterial species from the environment. This pipeline combines fluorescence in situ hybridization (FISH) with fluorescence activated cell sorting (FACS) and subsequent genome sequencing. A taxon-specific FISH probe hybridizes to the target cells which are subsequently enriched by cell sorting based on the fluorescence signal. Finally, the DNA of the sorted cells is amplified and sequenced. The development of the pipeline involved an optimization of the recently developed hybridization chain reaction (HCR)-FISH method and the assessment of various cell fixatives concerning their influence on HCR-FISH signal intensity and the quality of genome sequencing and assembly. The developed pipeline was successfully applied on a planktonic seawater sample targeting the so far uncharacterized clade Vis6. This member of the Cryomorphaceae within the Flavobacteriia is recurring during spring phytoplankton blooms off the coast of Helgoland. The description of Vis6 as a novel candidate genus Candidatus Abditibacter with three species was enabled by the combination of the sequences retrieved from targeted sorting pipeline with additional data of North Sea metagenomes and abundance data based on the 16S rRNA identity of Vis6. The growth of Candidatus Abditibacter spp. is likely based on the utilization of phytoplankton-derived polysaccharides and proteins. In the future, the developed pipeline has the potential to be used on low abundance species from highly complex samples, where previous attempts with metagenomics have been unsuccessful so far

A pipeline for targeted metagenomics of environmental bacteria.

BackgroundMetagenomics and single cell genomics provide a window into the genetic repertoire of yet uncultivated microorganisms, but both methods are usually taxonomically untargeted. The combination of fluorescence in situ hybridization (FISH) and fluorescence activated cell sorting (FACS) has the potential to enrich taxonomically well-defined clades for genomic analyses.MethodsCells hybridized with a taxon-specific FISH probe are enriched based on their fluorescence signal via flow cytometric cell sorting. A recently developed FISH procedure, the hybridization chain reaction (HCR)-FISH, provides the high signal intensities required for flow cytometric sorting while maintaining the integrity of the cellular DNA for subsequent genome sequencing. Sorted cells are subjected to shotgun sequencing, resulting in targeted metagenomes of low diversity.ResultsPure cultures of different taxonomic groups were used to (1) adapt and optimize the HCR-FISH protocol and (2) assess the effects of various cell fixation methods on both the signal intensity for cell sorting and the quality of subsequent genome amplification and sequencing. Best results were obtained for ethanol-fixed cells in terms of both HCR-FISH signal intensity and genome assembly quality. Our newly developed pipeline was successfully applied to a marine plankton sample from the North Sea yielding good quality metagenome assembled genomes from a yet uncultivated flavobacterial clade.ConclusionsWith the developed pipeline, targeted metagenomes at various taxonomic levels can be efficiently retrieved from environmental samples. The resulting metagenome assembled genomes allow for the description of yet uncharacterized microbial clades. Video abstract