Combination of 16S rRNA-targeted oligonucleotide probes with flow cytometry for analyzing mixed microbial populations

Amann, Rudolf; Binder, Brian J.; Olson, Robert J.; Chisholm, Sallie W.; Devereux, Richard; Stahl, David A.

doi:10.1128/aem.56.6.1919-1925.1990

Cited by 3,641 publications

(1,375 citation statements)

References 30 publications

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“…Samples were stained with the 16S rRNA probe SRB385 (CGGCGTCGCTGCGTCAGG) labelled with cyanine 3 to image sulphate-reducing bacteria (SRB). This oligoprobe targets SRB of the y-proteobacteria (Amann et al, 1990). Photosynthetic organisms and carbonate minerals were visualized by fluorescence.…”

Section: Methodsmentioning

confidence: 99%

Organomineralization processes in freshwater stromatolites: a living example from eastern Patagonia

Pacton

Hunger

Martinuzzi

et al. 2015

The Depositional Record

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Living stromatolites have been mostly described within shallow marine and (hyper)saline lacustrine environments. Southernmost South America lacks detailed investigations of these (organo)sedimentary buildups, particularly in regions experiencing extreme and variable environmental conditions. Here, we report and describe living freshwater stromatolites in the Maquinchao region, north-western Patagonia, Argentina. Fossil stromatolites characterized by globular and cauliflower shapes are also present in a continuous palaeoshoreline of a former lake at an altitude of 830 m, whereas their living counterparts only occur in the calm waters of sheltered or meandering sections of the Maquinchao River. The living stromatolites and their host waters have been sampled and studied using various chemical and microscopic techniques to better constrain the environmental versus biological factors controlling their development. Our results indicate that today stromatolites only proliferate in freshwater when Ca 2+ levels are high. A microscopic inspection of the living stromatolite mat indicates stronger photosynthetic activity in the upper green layer associated with crypto/ microcrystalline calcite (nanoglobules) compared to the lower beige-white biofilm. This biofilm contains more low-Mg calcite (rhombohedra) precipitates, which can form millimetre-sized aggregates in the underlying anoxic layer. Although sulphate-reducing bacteria are living in the entire mat, they appear more abundant and widely distributed in the lower beige-white layer and are always associated with Mg calcite. Low salinity and low-turbidity water along with microbial (photosynthetic and heterotrophic) activity are the most important factors promoting low-Mg calcite precipitation in the Maquinchao Basin. These conditions are very different from those proposed for recently described lacustrine stromatolites at high altitude in the subtropical and tropical Andes as well as in Chilean Patagonia. Hence, all these observations in modern freshwater stromatolites show the importance of geomicrobiological studies in identifying proxies of the hydrological conditions prevailing during their formation.

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Section: Methodsmentioning

confidence: 99%

Organomineralization processes in freshwater stromatolites: a living example from eastern Patagonia

Pacton

Hunger

Martinuzzi

et al. 2015

The Depositional Record

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“…The fluorescent tagged oligonucleotide probes used in this study were purchased from Thermo Electron Corporation GmbH, Ulm, Germany. These were EUB-338-mix [an equimolar mixture of EUB-338 (Amann et al 1990), EUB-338-II and EUB-338-III (Daims et al, 1999)], LGC-354-mix [an equimolar mixture of LGC-354-a, LGC-354-b and LGC-354-c (Meier et al, 1999)], Rhi-1247 (Ludwig et al, 1998) and EUK-516 (Amann et al, 1990) (Table 3). All of them were labelled either with FITC, Cy3 or Cy5.…”

Section: Fluorescence In Situ Hybridizationmentioning

confidence: 99%

Detection and identification of bacteria intimately associated with fungi of the orderSebacinales

Sharma

Schmid

Rothballer

et al. 2008

Cellular Microbiology

149

137

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Because of their beneficial impact on plants, the highly diverse mycorrhizal fungi grouped in the order Sebacinales lay claim to high ecological and agricultural significance. Here, we describe for the first time associations of Sebacinoid members with bacteria. Using quantitative PCR, denaturating gradient gel electrophoresis and fluorescence in situ hybridization, we detected an intimate association between Piriformospora indica and Rhizobium radiobacter, an alpha-Proteobacterium. The stability of the association, vertical transmission of the bacteria during asexual fungal reproduction and fungal plant colonization was monitored using R. radiobacter-specific primers. Treatment of mycelium or fungal protoplasts with antibiotics highly efficient against the free bacteria failed to cure the fungus. Barley seedlings dip-inoculated with R. radiobacter showed growth promotion and systemic resistance to the powdery mildew fungus Blumeria graminis comparable to P. indica inoculation. By screening additional isolates of the Sebacina vermifera complex, three species-specific associations with bacteria from the genera Paenibacillus, Acinetobacter and Rhodococcus were found. These findings suggest that Sebacinales species regularly undergo complex interactions involving host plants and bacteria reminiscent of other ectomycorrhizal and endomycorrhizal associations.

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“…Hybridisations were performed overnight. The bacterial probe EUB338 [31] served as a positive control, and its complement non-EUB338 [32] as the negative control. The specificities of the Pana134 and Pnasa1254 probes were increased by adding formamide 20% v ⁄ v, which is a duplex-destabilising agent, to the hybridisation buffer.…”

Section: Validationmentioning

confidence: 99%

Development of 16S rRNA-based probes for the identification of Gram-positive anaerobic cocci isolated from human clinical specimens

Wildeboer-Veloo

Harmsen

Welling

et al. 2007

Clinical Microbiology and Infection

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Fluorescent probes targeted at 16S rRNA were designed for Peptostreptococcus anaerobius and Peptostreptococcus stomatis (Pana134), Parvimonas micra (Pamic1435), Finegoldia magna (Fmag1250), Peptoniphilus asaccharolyticus (Pnasa1254), Peptoniphilus ivorii (Pnivo731), Peptoniphilus harei (Pnhar1466), Anaerococcus vaginalis (Avag1280) and Anaerococcus lactolyticus (Alac1438), based on the 16S rRNA sequences of reference strains and 88 randomly chosen clinical isolates. These strains were also used for validation of the probes. Application of the probes to an additional group of 100 clinical isolates revealed that 87% of Gram-positive anaerobic cocci (GPAC) could be identified with this set of probes. The 16S rRNAs of 13 clinical isolates that could not be identified were sequenced. Most of these isolates were GPAC that were not targeted by the probes. No clinical isolates of Pn. asaccharolyticus were encountered. Near full-length sequences were obtained from 71 of 101 (n = 88 + 13) sequenced clinical isolates. Of these, 25 showed <98% similarity with the homologues of the closest established species. The Fmag1250, Pamic1435, Pnhar1466, Pana134, Pnasa1254 and Pnivo731 probes allowed reliable identification and hybridised with all corresponding isolates. The Avag1280 and Alac1438 probes failed to hybridise with two isolates and one isolate, respectively, because of intra-species variation. However, overall, the set of probes yielded fast and reliable identification for the majority of clinical isolates.

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Combination of 16S rRNA-targeted oligonucleotide probes with flow cytometry for analyzing mixed microbial populations

Cited by 3,641 publications

References 30 publications

Organomineralization processes in freshwater stromatolites: a living example from eastern Patagonia

Organomineralization processes in freshwater stromatolites: a living example from eastern Patagonia

Detection and identification of bacteria intimately associated with fungi of the orderSebacinales

Development of 16S rRNA-based probes for the identification of Gram-positive anaerobic cocci isolated from human clinical specimens

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