Rudolf Amann | Max Planck Institute for Marine Microbiology (original) (raw)

Papers by Rudolf Amann

Research paper thumbnail of Use of green fluorescent protein as a marker for ecological studies of activated sludge communities

FEMS Microbiology Letters, 1997

Research paper thumbnail of Flow Cytometric Analysis of the In Situ Accessibility of Escherichia coli 16S rRNA for Fluorescently Labeled Oligonucleotide Probes

cleotide probes (mostly 18-mers) used with whole fixed cells of Escherichia coli DSM 30083 T. Two... more cleotide probes (mostly 18-mers) used with whole fixed cells of Escherichia coli DSM 30083 T. Two overlapping sets of adjacent oligonucleotides, 171 in total, were designed to cover the full length of the 16S rRNA. The two sets are shifted by 5 to 13 nucleotides. The probes were labeled with carboxyfluorescein, and signal intensities of hybridized cells were quantified by

Research paper thumbnail of Allochromatium humboldtianum sp. nov.*, a novel marine Allochromatium species isolated from soft coastal sediments off Peru

International journal of systematic and evolutionary microbiology, Jan 29, 2015

A new purple sulfur bacterium, strain AX1YPE, was isolated from marine sediments sampled at 47 m ... more A new purple sulfur bacterium, strain AX1YPE, was isolated from marine sediments sampled at 47 m depth in Callao Bay, Peru. Strain AX1YPE grows anaerobically, synthesizes bacteriochlorophyll a and carotenoid pigments of the spirilloxanthin series. Cells are Gram-negative rods and actively motile by a polar flagellum. Strain AX1YPE is able to grow photolithoautotrophically with sulfide and thiosulfate as electron donors. This new phototrophic organism uses ammonium salt, N2, urea, and glutamate as nitrogen sources. Strain AX1YPE has a DNA base composition of 63.9 mol% G + C. Analysis of the 16S rRNA gene sequence indicates that strain AX1YPE clusters in a separate branch within the genus Allochromatium of the family Chromatiaceae. Strain AX1YPE showed 16S rRNA gene sequence similarities of 98.2% with A. vinosum and A. minutissimum, 98.1% with A. phaeobacterium, 97.3% with A. renukae, and 96.8% with A. warmingii. DNA-DNA hybridization (DDH) values to the closest relatives A. vinosum a...

Research paper thumbnail of Taxonomy in the age of genomics

Systematic and Applied Microbiology, 2015

Research paper thumbnail of Detection and identification of Candida species in experimentally infected tissue and human blood by rRNA-specific fluorescent in situ hybridization

Journal of clinical microbiology, 1997

Two 18S rRNA-targeted oligonucleotide probes specific for Candida albicans and Candida parapsilos... more Two 18S rRNA-targeted oligonucleotide probes specific for Candida albicans and Candida parapsilosis were used to detect and identify by fluorescent in situ hybridization these medically important Candida species in deep organs of mice after experimental systemic infection. The C. albicans-specific probe detected fungal cells in kidney, spleen, and brain sections of a mouse infected with C. albicans but not in a mouse infected with the closely related species C. parapsilosis. Conversely, the C. parapsilosis-specific probe detected fungal cells in the deep organs of a mouse infected with C. parapsilosis but not in the deep organs of a C. albicans-infected mouse. In addition, the C. albicans-specific probe was used to detect this species in human blood spiked with yeast cells by a lysis-filtration assay and subsequent fluorescent in situ hybridization. By this assay, as few as three yeast cells per 0.5 ml of blood were consistently detected. Our results demonstrate that fluorescent in ...

Research paper thumbnail of Fluorescence in situ hybridization (FISH) with rRNA-targeted oligonucleotide probes

Methods in Microbiology, 2001

Note: This is a one-page preview only. Click here to download preview. ... Enable JavaScript for ... more Note: This is a one-page preview only. Click here to download preview. ... Enable JavaScript for PDF Excerpt to view it inline. ... Amann, R., Snaidr, J., Wagner, M., Ludwig, W. and Schleifer, K.-H., 1996. In situ visualization of high genetic diversity in a natural microbial ...

Research paper thumbnail of Phylogenetic identification and in situ detection of individual microbial cells without cultivation

Microbiological reviews, 1995

The frequent discrepancy between direct microscopic counts and numbers of culturable bacteria fro... more The frequent discrepancy between direct microscopic counts and numbers of culturable bacteria from environmental samples is just one of several indications that we currently know only a minor part of the diversity of microorganisms in nature. A combination of direct retrieval of rRNA sequences and whole-cell oligonucleotide probing can be used to detect specific rRNA sequences of uncultured bacteria in natural samples and to microscopically identify individual cells. Studies have been performed with microbial assemblages of various complexities ranging from simple two-component bacterial endosymbiotic associations to multispecies enrichments containing magnetotactic bacteria to highly complex marine and soil communities. Phylogenetic analysis of the retrieved rRNA sequence of an uncultured microorganism reveals its closest culturable relatives and may, together with information on the physicochemical conditions of its natural habitat, facilitate more directed cultivation attempts. F...

Research paper thumbnail of Graphical representation of ribosomal RNA probe accessibility data using ARB software package

BMC bioinformatics, 2005

Taxon specific hybridization probes in combination with a variety of commonly used hybridization ... more Taxon specific hybridization probes in combination with a variety of commonly used hybridization formats nowadays are standard tools in microbial identification. A frequently applied technology, fluorescence in situ hybridization (FISH), besides single cell identification, allows the localization and functional studies of the microbial community composition. Careful in silico design and evaluation of potential oligonucleotide probe targets is therefore crucial for performing successful hybridization experiments. The PROBE Design tools of the ARB software package take into consideration several criteria such as number, position and quality of diagnostic sequence differences while designing oligonucleotide probes. Additionally, new visualization tools were developed to enable the user to easily examine further sequence associated criteria such as higher order structure, conservation, G+C content, transition-transversion profiles and in situ target accessibility patterns. The different...

Research paper thumbnail of 16S rRNA-targeted oligonucleotide probes for the in situ detection of members of the phylum Cytophaga-Flavobacterium-Bacteroides

Systematic and applied microbiology, 2000

Bacteria of the Cytophaga-Flavobacterium-Bacteroides phylum (CFB-phylum) are numerically importan... more Bacteria of the Cytophaga-Flavobacterium-Bacteroides phylum (CFB-phylum) are numerically important members of many microbial communities. A suite of five 16S rRNA-targeted oligonucleotide probes for members of this group is described which was designed to dominantly target bacteria of the CFB-phylum that are found in particular habitats. For this we initially performed a literature survey-for the sources and sites of isolation of hitherto described members of the CFB-phylum. Probe CFB286 is mostly complementary to the 16S rRNA of species originally isolated from freshwater habitats, however, detects some marine and soil isolates and is the only probe which includes some food isolates. Probe CFB563 detects marine as well as animal-associated isolates. Probe CFB719, which also detects some environmental isolates, and probe CFB972 are mostly targeting animal-associated isolates. All probes are complementary to a variety of human-associated species within the CFB-phylum which, in the da...

Research paper thumbnail of Improved sensitivity of whole-cell hybridization by the combination of horseradish peroxidase-labeled oligonucleotides and tyramide signal amplification

Applied and environmental microbiology, 1997

The substrate fluorescein-tyramide was combined with oligonucleotide probes directly labeled with... more The substrate fluorescein-tyramide was combined with oligonucleotide probes directly labeled with horseradish peroxidase to improve the sensitivity of in situ hybridization of whole fixed bacterial cells. Flow cytometry and quantitative microscopy of cells hybridized by this technique showed 10- to 20-fold signal amplifications relative to fluorescein-monolabeled probes. The application of the new technique to the detection of natural bacterial communities resulted in very bright signals; however, the number of detected cells was significantly lower than that detected with fluorescently monolabeled, rRNA-targeted oligonucleotide probes.

Research paper thumbnail of Analysis of Broad-scale Differences in Microbial Community Composition of Two Pristine Forest Soils

Systematic and Applied Microbiology, 1998

Broad-scale differences in soil microbial community composition were analyzed in two contrasting ... more Broad-scale differences in soil microbial community composition were analyzed in two contrasting soils using DNA reassociation and % G+C profiles for analysis on the community-level, and filter-and whole cell hybridization techniques for a coarse-level characterization of larger phylogenetic groups of bacteria. Reassociation analysis of DNA from bacterial fractions extracted from the organic soil Seim and the mineral soil Hau revealed similar complexity of the communities with 5700 and 4900 different bacterial genomes (g soil [dry wt])-l, respectively. Thermal denaturation studies showed wide % G+C distributions in DNA from bacteria of both soils. Differences in the median % G+C with 55 to 61 % for the bacterial community in soil Seim and 61 to 66% for that in soil Hau indicated a higher proportion of bacteria with a high DNA G+C content in soil Hau. In situ hybridization with fluorescent (Cy3-labeled) probes targeting larger phylogenetic groups showed minor differences between both soils, and between direct detection of bacteria in dispersed soil slurries and in bacterial fractions extracted from soils though about 90% of the total bacteria were lost during extraction. In dispersed slurries of both soils, only probes ALFI b, SRB385, and PLA46 hybridized to cells accounting for more than 1 % of the DAPIstained cells, while numbers obtained after hybridization with probes ARCH915, BET42a, GAM42a, HGC69a, and CF319a were below the detection limit set at <1 %. These results were confirmed by in situ hybridization with horseradish peroxidase (HRP)-labeled probes and subsequent Cy3-tyramide signal amplification. In contrast, dot blot hybridization with probe HGC69a indicated significant amounts of Gram-positive bacteria with a high DNA G+C content in both soils. These could subsequently be visualized in non-dispersed soil slurries by in situ hybridization with HRP-Iabeled probe HGC69a and Cy3-tyramide signal amplification. Filamentous Gram-positive bacteria with a high DNA G+C content, likely actinomycetes, which are present in soil Hau in significant numbers are obviously destroyed by procedures used for soil dispersion.

Research paper thumbnail of Optimizing fluorescent in situ hybridization with rRNA-targeted oligonucleotide probes for flow cytometric identification of microorganisms

Cytometry, 1993

A combination of fluorescent rRNA-targeted oligonucleotide probes ("phylogenetic stains&quot... more A combination of fluorescent rRNA-targeted oligonucleotide probes ("phylogenetic stains") and flow cytometry was used for a high resolution automated analysis of mixed microbial populations. Fixed cells of bacteria and yeasts were hybridized in suspension with fluorescein- or tetramethylrhodamine-labeled oligonucleotide probes complementary to group-specific regions of the 16S ribosomal RNA (rRNA) molecules. Quantifying probe-conferred cell fluorescence by flow cytometry, we could discriminate between target and nontarget cell populations. We critically examined changes of the hybridization conditions, kinetics of the hybridization, and posthybridization treatments. Intermediate probe concentrations, addition of detergent to the hybridization buffer, and a posthybridization washing step were found to increase the signal to noise ratio. We could demonstrate a linear correlation between growth rate and probe-conferred fluorescence of Escherichia coli and Pseudomonas cepacia ...

Research paper thumbnail of Diverse sulfate-reducing bacteria of the Desulfosarcina/Desulfococcus clade are the key alkane degraders at marine seeps

The ISME journal, 2014

Biogeochemical and microbiological data indicate that the anaerobic oxidation of non-methane hydr... more Biogeochemical and microbiological data indicate that the anaerobic oxidation of non-methane hydrocarbons by sulfate-reducing bacteria (SRB) has an important role in carbon and sulfur cycling at marine seeps. Yet, little is known about the bacterial hydrocarbon degraders active in situ. Here, we provide the link between previous biogeochemical measurements and the cultivation of degraders by direct identification of SRB responsible for butane and dodecane degradation in complex on-site microbiota. Two contrasting seep sediments from Mediterranean Amon mud volcano and Guaymas Basin (Gulf of California) were incubated with (13)C-labeled butane or dodecane under sulfate-reducing conditions and analyzed via complementary stable isotope probing (SIP) techniques. Using DNA- and rRNA-SIP, we identified four specialized clades of alkane oxidizers within Desulfobacteraceae to be distinctively active in oxidation of short- and long-chain alkanes. All clades belong to the Desulfosarcina/Desulf...

Research paper thumbnail of Past and future species definitions for Bacteria and Archaea

Systematic and Applied Microbiology, 2015

Species is the basic unit of biological diversity. However, among the different microbiological d... more Species is the basic unit of biological diversity. However, among the different microbiological disciplines there is an important degree of disagreement as to what this unit may be. In this minireview, we argue that the main point of disagreement is the definition (i.e. the way species are circumscribed by means of observable characters) rather than the concept (i.e. the idea of what a species may be as a unit of biodiversity, the meaning of the patterns of recurrence observed in nature, and the why of their existence). Taxonomists have defined species by means of genetic and expressed characters that ensure the members of the unit are monophyletic, and exhibit a large degree of genomic and phenotypic coherence. The new technologies allowing high-throughput data acquisition are expected to improve future classifications significantly and will lead to database-based taxonomy centered on portable and interactive data. Future species descriptions of Bacteria and Archaea should include a high quality genome sequence of at least the type strain as an obligatory requirement, just as today an almost full-length 16S rRNA gene sequence must be provided. Serious efforts are needed in order to re-evaluate the major guidelines for standard descriptions.

Research paper thumbnail of Microscale distribution of populations and acitivities of Nitrosospira and Nitrospira ssp. along a macroscale gradient in a nitrifying bioreactor: quantification by in situ hybridization and the use of microsensors

The change of activity and abundance of Nitrosospira and Nitrospira spp. along a bulk water gradi... more The change of activity and abundance of Nitrosospira and Nitrospira spp. along a bulk water gradient in a nitrifying fluidized bed reactor was analyzed by a combination of microsensor measurements and fluorescence in situ hybridization. Nitrifying bacteria were immobilized in bacterial aggregates that remained in fixed positions within the reactor column due to the flow regimen. Nitrification occurred in a

Research paper thumbnail of Identification of single bacterial cells using digoxigenin-labelled, rRNA-targeted oligonucleotides

Journal of General Microbiology, 1991

Oligonucleotides were end-labelled with digoxigenin (DIG), chemically at the 5'-end or enzymicall... more Oligonucleotides were end-labelled with digoxigenin (DIG), chemically at the 5'-end or enzymically at the 3'-end. Following specific in situ hybridization of these probes to intracellular rRNA molecules, the hybrids were detected with anti-DIG Fab fragments labelled with fluorescent dyes. The antibody fragments penetrated through the bacterial cell periphery and specifically bound to their antigens. Probe-conferred and non-specific fluorescence per cell were quantified by flow cytometry and compared to values obtained with end-labelled fluorescent probes. The DIG reporter molecules could also be detected in whole fixed cells by antibodies labelled with either alkaline phosphatase or horseradish peroxidase. The penetration of the large antibody-enzyme complexes into the cells required lysozyme/EDTA treatment prior to the hybridization and has so far only been achieved for Gramnegative bacteria. This technique has the potential for significant signal amplification as compared to the fluorescently end-labelled oligonucleotides hitherto used for single cell identification in microbial ecology. Moreover, it can be used instead of fluorescent assays in natural samples showing autofluorescence.

Research paper thumbnail of Niches of two polysaccharide-degrading Polaribacter isolates from the North Sea during a spring diatom bloom

The ISME Journal, 2014

Members of the flavobacterial genus Polaribacter thrive in response to North Sea spring phytoplan... more Members of the flavobacterial genus Polaribacter thrive in response to North Sea spring phytoplankton blooms. We analyzed two respective Polaribacter species by whole genome sequencing, comparative genomics, substrate tests and proteomics. Both can degrade algal polysaccharides but occupy distinct niches. The liquid culture isolate Polaribacter sp. strain Hel1_33_49 has a 3.0-Mbp genome with an overall peptidase:CAZyme ratio of 1.37, four putative polysaccharide utilization loci (PULs) and features proteorhodopsin, whereas the agar plate isolate Polaribacter sp. strain Hel1_85 has a 3.9-Mbp genome with an even peptidase:CAZyme ratio, eight PULs, a mannitol dehydrogenase for decomposing algal mannitol-capped polysaccharides but no proteorhodopsin. Unlike other sequenced Polaribacter species, both isolates have larger sulfatase-rich PULs, supporting earlier assumptions that Polaribacter take part in the decomposition of sulfated polysaccharides. Both strains grow on algal laminarin and the sulfated polysaccharide chondroitin sulfate. For strain Hel1_33_49, we identified by proteomics (i) a laminarin-induced PUL, (ii) chondroitin sulfate-induced CAZymes and (iii) a chondroitin-induced operon that likely enables chondroitin sulfate recognition. These and other data suggest that strain Hel1_33_49 is a planktonic flavobacterium feeding on proteins and a small subset of algal polysaccharides, while the more versatile strain Hel1_85 can decompose a broader spectrum of polysaccharides and likely associates with algae.The ISME Journal advance online publication, 5 December 2014; doi:10.1038/ismej.2014.225.

Research paper thumbnail of Obligate Intracellular Bacterial Parasites of Acanthamoebae Related toChlamydiaspp

The phylogeny of obligate intracellular coccoid parasites of acanthamoebae isolated from the nasa... more The phylogeny of obligate intracellular coccoid parasites of acanthamoebae isolated from the nasal mucosa of humans was analyzed by the rRNA approach. The primary structures of the 16S and 23S rRNA molecules ofonestrainweredeterminedinalmostfulllength.Insituhybridizationwithahorseradishperoxidase-labeled oligonucleotide probe targeted to a unique signature site undoubtedly correlated the retrieved 16S rRNA sequence to the respective intracellular parasite. This probe also hybridized with the second

Research paper thumbnail of Microbial Community Composition of Wadden Sea Sediments as Revealed by Fluorescence In Situ Hybridization

The microbial community composition of Wadden Sea sediments of the German North Sea coast was inv... more The microbial community composition of Wadden Sea sediments of the German North Sea coast was investigated by in situ hybridization with group-specific fluorescently labeled, rRNA-targeted oligonucleotides. A large fraction (up to 73%) of the DAPI (4*,6-diamidino-2-phenylindole)-stained cells hybridized with the bacterial probes. Nearly 45% of the total cells could be further identified as belonging to known phyla. Members of the

Research paper thumbnail of Development of a 16S rRNA-targeted probe set for Verrucomicrobia and its application for fluorescence in situ hybridization in a humic lake

Systematic and Applied Microbiology, 2010

Members of the highly diverse bacterial phylum Verrucomicrobia are globally distributed in variou... more Members of the highly diverse bacterial phylum Verrucomicrobia are globally distributed in various terrestrial and aquatic habitats. They are key players in soils, but little is known about their role in aquatic systems. Here, we report on the design and evaluation of a 16S rRNA-targeted probe set for the identification of Verrucomicrobia and of clades within this phylum. Subsequently, the probe set was applied to a study concerning the seasonal abundance of Verrucomicrobia in waters of the humic lake Große Fuchskuhle (Germany) by catalyzed reporter deposition fluorescence in situ hybridization. The lake hosted diverse Verrucomicrobia clades in all seasons. Either Spartobacteria (up to 19%) or Opitutus spp. (up to 7%) dominated the communities, whereas Prosthecobacter spp. were omnipresent in low numbers (o 1%). Verrucomicrobial abundance and community composition varied between the seasons, and between more and less humic basins, but were rather stable in oxic and seasonally anoxic waters.

Research paper thumbnail of Use of green fluorescent protein as a marker for ecological studies of activated sludge communities

FEMS Microbiology Letters, 1997

Research paper thumbnail of Flow Cytometric Analysis of the In Situ Accessibility of Escherichia coli 16S rRNA for Fluorescently Labeled Oligonucleotide Probes

cleotide probes (mostly 18-mers) used with whole fixed cells of Escherichia coli DSM 30083 T. Two... more cleotide probes (mostly 18-mers) used with whole fixed cells of Escherichia coli DSM 30083 T. Two overlapping sets of adjacent oligonucleotides, 171 in total, were designed to cover the full length of the 16S rRNA. The two sets are shifted by 5 to 13 nucleotides. The probes were labeled with carboxyfluorescein, and signal intensities of hybridized cells were quantified by

Research paper thumbnail of Allochromatium humboldtianum sp. nov.*, a novel marine Allochromatium species isolated from soft coastal sediments off Peru

International journal of systematic and evolutionary microbiology, Jan 29, 2015

A new purple sulfur bacterium, strain AX1YPE, was isolated from marine sediments sampled at 47 m ... more A new purple sulfur bacterium, strain AX1YPE, was isolated from marine sediments sampled at 47 m depth in Callao Bay, Peru. Strain AX1YPE grows anaerobically, synthesizes bacteriochlorophyll a and carotenoid pigments of the spirilloxanthin series. Cells are Gram-negative rods and actively motile by a polar flagellum. Strain AX1YPE is able to grow photolithoautotrophically with sulfide and thiosulfate as electron donors. This new phototrophic organism uses ammonium salt, N2, urea, and glutamate as nitrogen sources. Strain AX1YPE has a DNA base composition of 63.9 mol% G + C. Analysis of the 16S rRNA gene sequence indicates that strain AX1YPE clusters in a separate branch within the genus Allochromatium of the family Chromatiaceae. Strain AX1YPE showed 16S rRNA gene sequence similarities of 98.2% with A. vinosum and A. minutissimum, 98.1% with A. phaeobacterium, 97.3% with A. renukae, and 96.8% with A. warmingii. DNA-DNA hybridization (DDH) values to the closest relatives A. vinosum a...

Research paper thumbnail of Taxonomy in the age of genomics

Systematic and Applied Microbiology, 2015

Research paper thumbnail of Detection and identification of Candida species in experimentally infected tissue and human blood by rRNA-specific fluorescent in situ hybridization

Journal of clinical microbiology, 1997

Two 18S rRNA-targeted oligonucleotide probes specific for Candida albicans and Candida parapsilos... more Two 18S rRNA-targeted oligonucleotide probes specific for Candida albicans and Candida parapsilosis were used to detect and identify by fluorescent in situ hybridization these medically important Candida species in deep organs of mice after experimental systemic infection. The C. albicans-specific probe detected fungal cells in kidney, spleen, and brain sections of a mouse infected with C. albicans but not in a mouse infected with the closely related species C. parapsilosis. Conversely, the C. parapsilosis-specific probe detected fungal cells in the deep organs of a mouse infected with C. parapsilosis but not in the deep organs of a C. albicans-infected mouse. In addition, the C. albicans-specific probe was used to detect this species in human blood spiked with yeast cells by a lysis-filtration assay and subsequent fluorescent in situ hybridization. By this assay, as few as three yeast cells per 0.5 ml of blood were consistently detected. Our results demonstrate that fluorescent in ...

Research paper thumbnail of Fluorescence in situ hybridization (FISH) with rRNA-targeted oligonucleotide probes

Methods in Microbiology, 2001

Note: This is a one-page preview only. Click here to download preview. ... Enable JavaScript for ... more Note: This is a one-page preview only. Click here to download preview. ... Enable JavaScript for PDF Excerpt to view it inline. ... Amann, R., Snaidr, J., Wagner, M., Ludwig, W. and Schleifer, K.-H., 1996. In situ visualization of high genetic diversity in a natural microbial ...

Research paper thumbnail of Phylogenetic identification and in situ detection of individual microbial cells without cultivation

Microbiological reviews, 1995

The frequent discrepancy between direct microscopic counts and numbers of culturable bacteria fro... more The frequent discrepancy between direct microscopic counts and numbers of culturable bacteria from environmental samples is just one of several indications that we currently know only a minor part of the diversity of microorganisms in nature. A combination of direct retrieval of rRNA sequences and whole-cell oligonucleotide probing can be used to detect specific rRNA sequences of uncultured bacteria in natural samples and to microscopically identify individual cells. Studies have been performed with microbial assemblages of various complexities ranging from simple two-component bacterial endosymbiotic associations to multispecies enrichments containing magnetotactic bacteria to highly complex marine and soil communities. Phylogenetic analysis of the retrieved rRNA sequence of an uncultured microorganism reveals its closest culturable relatives and may, together with information on the physicochemical conditions of its natural habitat, facilitate more directed cultivation attempts. F...

Research paper thumbnail of Graphical representation of ribosomal RNA probe accessibility data using ARB software package

BMC bioinformatics, 2005

Taxon specific hybridization probes in combination with a variety of commonly used hybridization ... more Taxon specific hybridization probes in combination with a variety of commonly used hybridization formats nowadays are standard tools in microbial identification. A frequently applied technology, fluorescence in situ hybridization (FISH), besides single cell identification, allows the localization and functional studies of the microbial community composition. Careful in silico design and evaluation of potential oligonucleotide probe targets is therefore crucial for performing successful hybridization experiments. The PROBE Design tools of the ARB software package take into consideration several criteria such as number, position and quality of diagnostic sequence differences while designing oligonucleotide probes. Additionally, new visualization tools were developed to enable the user to easily examine further sequence associated criteria such as higher order structure, conservation, G+C content, transition-transversion profiles and in situ target accessibility patterns. The different...

Research paper thumbnail of 16S rRNA-targeted oligonucleotide probes for the in situ detection of members of the phylum Cytophaga-Flavobacterium-Bacteroides

Systematic and applied microbiology, 2000

Bacteria of the Cytophaga-Flavobacterium-Bacteroides phylum (CFB-phylum) are numerically importan... more Bacteria of the Cytophaga-Flavobacterium-Bacteroides phylum (CFB-phylum) are numerically important members of many microbial communities. A suite of five 16S rRNA-targeted oligonucleotide probes for members of this group is described which was designed to dominantly target bacteria of the CFB-phylum that are found in particular habitats. For this we initially performed a literature survey-for the sources and sites of isolation of hitherto described members of the CFB-phylum. Probe CFB286 is mostly complementary to the 16S rRNA of species originally isolated from freshwater habitats, however, detects some marine and soil isolates and is the only probe which includes some food isolates. Probe CFB563 detects marine as well as animal-associated isolates. Probe CFB719, which also detects some environmental isolates, and probe CFB972 are mostly targeting animal-associated isolates. All probes are complementary to a variety of human-associated species within the CFB-phylum which, in the da...

Research paper thumbnail of Improved sensitivity of whole-cell hybridization by the combination of horseradish peroxidase-labeled oligonucleotides and tyramide signal amplification

Applied and environmental microbiology, 1997

The substrate fluorescein-tyramide was combined with oligonucleotide probes directly labeled with... more The substrate fluorescein-tyramide was combined with oligonucleotide probes directly labeled with horseradish peroxidase to improve the sensitivity of in situ hybridization of whole fixed bacterial cells. Flow cytometry and quantitative microscopy of cells hybridized by this technique showed 10- to 20-fold signal amplifications relative to fluorescein-monolabeled probes. The application of the new technique to the detection of natural bacterial communities resulted in very bright signals; however, the number of detected cells was significantly lower than that detected with fluorescently monolabeled, rRNA-targeted oligonucleotide probes.

Research paper thumbnail of Analysis of Broad-scale Differences in Microbial Community Composition of Two Pristine Forest Soils

Systematic and Applied Microbiology, 1998

Broad-scale differences in soil microbial community composition were analyzed in two contrasting ... more Broad-scale differences in soil microbial community composition were analyzed in two contrasting soils using DNA reassociation and % G+C profiles for analysis on the community-level, and filter-and whole cell hybridization techniques for a coarse-level characterization of larger phylogenetic groups of bacteria. Reassociation analysis of DNA from bacterial fractions extracted from the organic soil Seim and the mineral soil Hau revealed similar complexity of the communities with 5700 and 4900 different bacterial genomes (g soil [dry wt])-l, respectively. Thermal denaturation studies showed wide % G+C distributions in DNA from bacteria of both soils. Differences in the median % G+C with 55 to 61 % for the bacterial community in soil Seim and 61 to 66% for that in soil Hau indicated a higher proportion of bacteria with a high DNA G+C content in soil Hau. In situ hybridization with fluorescent (Cy3-labeled) probes targeting larger phylogenetic groups showed minor differences between both soils, and between direct detection of bacteria in dispersed soil slurries and in bacterial fractions extracted from soils though about 90% of the total bacteria were lost during extraction. In dispersed slurries of both soils, only probes ALFI b, SRB385, and PLA46 hybridized to cells accounting for more than 1 % of the DAPIstained cells, while numbers obtained after hybridization with probes ARCH915, BET42a, GAM42a, HGC69a, and CF319a were below the detection limit set at <1 %. These results were confirmed by in situ hybridization with horseradish peroxidase (HRP)-labeled probes and subsequent Cy3-tyramide signal amplification. In contrast, dot blot hybridization with probe HGC69a indicated significant amounts of Gram-positive bacteria with a high DNA G+C content in both soils. These could subsequently be visualized in non-dispersed soil slurries by in situ hybridization with HRP-Iabeled probe HGC69a and Cy3-tyramide signal amplification. Filamentous Gram-positive bacteria with a high DNA G+C content, likely actinomycetes, which are present in soil Hau in significant numbers are obviously destroyed by procedures used for soil dispersion.

Research paper thumbnail of Optimizing fluorescent in situ hybridization with rRNA-targeted oligonucleotide probes for flow cytometric identification of microorganisms

Cytometry, 1993

A combination of fluorescent rRNA-targeted oligonucleotide probes ("phylogenetic stains&quot... more A combination of fluorescent rRNA-targeted oligonucleotide probes ("phylogenetic stains") and flow cytometry was used for a high resolution automated analysis of mixed microbial populations. Fixed cells of bacteria and yeasts were hybridized in suspension with fluorescein- or tetramethylrhodamine-labeled oligonucleotide probes complementary to group-specific regions of the 16S ribosomal RNA (rRNA) molecules. Quantifying probe-conferred cell fluorescence by flow cytometry, we could discriminate between target and nontarget cell populations. We critically examined changes of the hybridization conditions, kinetics of the hybridization, and posthybridization treatments. Intermediate probe concentrations, addition of detergent to the hybridization buffer, and a posthybridization washing step were found to increase the signal to noise ratio. We could demonstrate a linear correlation between growth rate and probe-conferred fluorescence of Escherichia coli and Pseudomonas cepacia ...

Research paper thumbnail of Diverse sulfate-reducing bacteria of the Desulfosarcina/Desulfococcus clade are the key alkane degraders at marine seeps

The ISME journal, 2014

Biogeochemical and microbiological data indicate that the anaerobic oxidation of non-methane hydr... more Biogeochemical and microbiological data indicate that the anaerobic oxidation of non-methane hydrocarbons by sulfate-reducing bacteria (SRB) has an important role in carbon and sulfur cycling at marine seeps. Yet, little is known about the bacterial hydrocarbon degraders active in situ. Here, we provide the link between previous biogeochemical measurements and the cultivation of degraders by direct identification of SRB responsible for butane and dodecane degradation in complex on-site microbiota. Two contrasting seep sediments from Mediterranean Amon mud volcano and Guaymas Basin (Gulf of California) were incubated with (13)C-labeled butane or dodecane under sulfate-reducing conditions and analyzed via complementary stable isotope probing (SIP) techniques. Using DNA- and rRNA-SIP, we identified four specialized clades of alkane oxidizers within Desulfobacteraceae to be distinctively active in oxidation of short- and long-chain alkanes. All clades belong to the Desulfosarcina/Desulf...

Research paper thumbnail of Past and future species definitions for Bacteria and Archaea

Systematic and Applied Microbiology, 2015

Species is the basic unit of biological diversity. However, among the different microbiological d... more Species is the basic unit of biological diversity. However, among the different microbiological disciplines there is an important degree of disagreement as to what this unit may be. In this minireview, we argue that the main point of disagreement is the definition (i.e. the way species are circumscribed by means of observable characters) rather than the concept (i.e. the idea of what a species may be as a unit of biodiversity, the meaning of the patterns of recurrence observed in nature, and the why of their existence). Taxonomists have defined species by means of genetic and expressed characters that ensure the members of the unit are monophyletic, and exhibit a large degree of genomic and phenotypic coherence. The new technologies allowing high-throughput data acquisition are expected to improve future classifications significantly and will lead to database-based taxonomy centered on portable and interactive data. Future species descriptions of Bacteria and Archaea should include a high quality genome sequence of at least the type strain as an obligatory requirement, just as today an almost full-length 16S rRNA gene sequence must be provided. Serious efforts are needed in order to re-evaluate the major guidelines for standard descriptions.

Research paper thumbnail of Microscale distribution of populations and acitivities of Nitrosospira and Nitrospira ssp. along a macroscale gradient in a nitrifying bioreactor: quantification by in situ hybridization and the use of microsensors

The change of activity and abundance of Nitrosospira and Nitrospira spp. along a bulk water gradi... more The change of activity and abundance of Nitrosospira and Nitrospira spp. along a bulk water gradient in a nitrifying fluidized bed reactor was analyzed by a combination of microsensor measurements and fluorescence in situ hybridization. Nitrifying bacteria were immobilized in bacterial aggregates that remained in fixed positions within the reactor column due to the flow regimen. Nitrification occurred in a

Research paper thumbnail of Identification of single bacterial cells using digoxigenin-labelled, rRNA-targeted oligonucleotides

Journal of General Microbiology, 1991

Oligonucleotides were end-labelled with digoxigenin (DIG), chemically at the 5'-end or enzymicall... more Oligonucleotides were end-labelled with digoxigenin (DIG), chemically at the 5'-end or enzymically at the 3'-end. Following specific in situ hybridization of these probes to intracellular rRNA molecules, the hybrids were detected with anti-DIG Fab fragments labelled with fluorescent dyes. The antibody fragments penetrated through the bacterial cell periphery and specifically bound to their antigens. Probe-conferred and non-specific fluorescence per cell were quantified by flow cytometry and compared to values obtained with end-labelled fluorescent probes. The DIG reporter molecules could also be detected in whole fixed cells by antibodies labelled with either alkaline phosphatase or horseradish peroxidase. The penetration of the large antibody-enzyme complexes into the cells required lysozyme/EDTA treatment prior to the hybridization and has so far only been achieved for Gramnegative bacteria. This technique has the potential for significant signal amplification as compared to the fluorescently end-labelled oligonucleotides hitherto used for single cell identification in microbial ecology. Moreover, it can be used instead of fluorescent assays in natural samples showing autofluorescence.

Research paper thumbnail of Niches of two polysaccharide-degrading Polaribacter isolates from the North Sea during a spring diatom bloom

The ISME Journal, 2014

Members of the flavobacterial genus Polaribacter thrive in response to North Sea spring phytoplan... more Members of the flavobacterial genus Polaribacter thrive in response to North Sea spring phytoplankton blooms. We analyzed two respective Polaribacter species by whole genome sequencing, comparative genomics, substrate tests and proteomics. Both can degrade algal polysaccharides but occupy distinct niches. The liquid culture isolate Polaribacter sp. strain Hel1_33_49 has a 3.0-Mbp genome with an overall peptidase:CAZyme ratio of 1.37, four putative polysaccharide utilization loci (PULs) and features proteorhodopsin, whereas the agar plate isolate Polaribacter sp. strain Hel1_85 has a 3.9-Mbp genome with an even peptidase:CAZyme ratio, eight PULs, a mannitol dehydrogenase for decomposing algal mannitol-capped polysaccharides but no proteorhodopsin. Unlike other sequenced Polaribacter species, both isolates have larger sulfatase-rich PULs, supporting earlier assumptions that Polaribacter take part in the decomposition of sulfated polysaccharides. Both strains grow on algal laminarin and the sulfated polysaccharide chondroitin sulfate. For strain Hel1_33_49, we identified by proteomics (i) a laminarin-induced PUL, (ii) chondroitin sulfate-induced CAZymes and (iii) a chondroitin-induced operon that likely enables chondroitin sulfate recognition. These and other data suggest that strain Hel1_33_49 is a planktonic flavobacterium feeding on proteins and a small subset of algal polysaccharides, while the more versatile strain Hel1_85 can decompose a broader spectrum of polysaccharides and likely associates with algae.The ISME Journal advance online publication, 5 December 2014; doi:10.1038/ismej.2014.225.

Research paper thumbnail of Obligate Intracellular Bacterial Parasites of Acanthamoebae Related toChlamydiaspp

The phylogeny of obligate intracellular coccoid parasites of acanthamoebae isolated from the nasa... more The phylogeny of obligate intracellular coccoid parasites of acanthamoebae isolated from the nasal mucosa of humans was analyzed by the rRNA approach. The primary structures of the 16S and 23S rRNA molecules ofonestrainweredeterminedinalmostfulllength.Insituhybridizationwithahorseradishperoxidase-labeled oligonucleotide probe targeted to a unique signature site undoubtedly correlated the retrieved 16S rRNA sequence to the respective intracellular parasite. This probe also hybridized with the second

Research paper thumbnail of Microbial Community Composition of Wadden Sea Sediments as Revealed by Fluorescence In Situ Hybridization

The microbial community composition of Wadden Sea sediments of the German North Sea coast was inv... more The microbial community composition of Wadden Sea sediments of the German North Sea coast was investigated by in situ hybridization with group-specific fluorescently labeled, rRNA-targeted oligonucleotides. A large fraction (up to 73%) of the DAPI (4*,6-diamidino-2-phenylindole)-stained cells hybridized with the bacterial probes. Nearly 45% of the total cells could be further identified as belonging to known phyla. Members of the

Research paper thumbnail of Development of a 16S rRNA-targeted probe set for Verrucomicrobia and its application for fluorescence in situ hybridization in a humic lake

Systematic and Applied Microbiology, 2010

Members of the highly diverse bacterial phylum Verrucomicrobia are globally distributed in variou... more Members of the highly diverse bacterial phylum Verrucomicrobia are globally distributed in various terrestrial and aquatic habitats. They are key players in soils, but little is known about their role in aquatic systems. Here, we report on the design and evaluation of a 16S rRNA-targeted probe set for the identification of Verrucomicrobia and of clades within this phylum. Subsequently, the probe set was applied to a study concerning the seasonal abundance of Verrucomicrobia in waters of the humic lake Große Fuchskuhle (Germany) by catalyzed reporter deposition fluorescence in situ hybridization. The lake hosted diverse Verrucomicrobia clades in all seasons. Either Spartobacteria (up to 19%) or Opitutus spp. (up to 7%) dominated the communities, whereas Prosthecobacter spp. were omnipresent in low numbers (o 1%). Verrucomicrobial abundance and community composition varied between the seasons, and between more and less humic basins, but were rather stable in oxic and seasonally anoxic waters.