Application of Pseudomurein Endoisopeptidase to Fluorescence In Situ Hybridization of Methanogens within the Family Methanobacteriaceae

ABSTRACT In situ detection of methanogens within the family Methanobacteriaceae is sometimes known to be unsuccessful due to the difficulty in permeability of oligonucleotide probes. Pseudomurein endoisopeptidase (Pei), a lytic enzyme that specifically acts on their cell walls, was applied prior to 16S rRNA-targeting fluorescence in situ hybridization (FISH). For this purpose, pure cultured methanogens within this family, Methanobacterium bryantii, Methanobrevibacter ruminantium, Methanosphaera stadtmanae, and Methanothermobacter thermautotrophicus together with a Methanothermobacter thermautotrophicus-containing syntrophic acetate-oxidizing coculture, endosymbiotic Methanobrevibacter methanogens within an anaerobic ciliate, and an upflow anaerobic sludge blanket (UASB) granule were examined. Even without the Pei treatment, Methanobacterium bryantii and Methanothermobacter thermautotrophicus cells are relatively well hybridized with oligonucleotide probes. However, almost none of the cells of Methanobrevibacter ruminantium, Methanosphaera stadtmanae, cocultured Methanothermobacter thermautotrophicus, and the endosymbiotic methanogens and the cells within UASB granule were hybridized. Pei treatment was able to increase the probe hybridization ratio in every specimen, particularly in the specimen that had shown little hybridization. Interestingly, the hybridizing signal intensity of Methanothermobacter thermautotrophicus cells in coculture with an acetate-oxidizing H2-producing syntroph was significantly improved by Pei pretreatment, whereas the probe was well hybridized with the cells of pure culture of the same strain. We found that the difference is attributed to the differences in cell wall thicknesses between the two culture conditions. These results indicate that Pei treatment is effective for FISH analysis of methanogens that show impermeability to the probe.

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Detection of fatty acid beta-oxidizing syntrophic bacteria by fluorescence in situ hybridization

Water Science & Technology ◽

10.2166/wst.2006.483 ◽

2006 ◽

Vol 54 (2) ◽

pp. 33-39 ◽

Cited By ~ 6

Author(s):

R.J. Menes ◽

D. Travers

Keyword(s):

In Situ Hybridization ◽

Fatty Acid ◽

Fluorescence In Situ Hybridization ◽

Upflow Anaerobic Sludge Blanket ◽

Anaerobic Sludge ◽

Enrichment Cultures ◽

The Family ◽

Syntrophic Bacteria ◽

Anaerobic Sludge Blanket

A new 16S rRNA-targeted oligonucleotide probe, specific for the cluster of fatty acid β-oxidizing syntrophic bacteria of the family Syntrophomonadaceae was designed for fluorescence in situ hybridization. This probe was evaluated with target as well as non-target cultures. Moreover this probe was assessed with butyrate and oleate degrading enrichment cultures and methanogenic sludges from full-scale plants. The results showed that the probe revealed the presence of fatty acid β-oxidizing syntrophic bacteria in some of the samples analyzed. However, cell quantification was possible only in enrichment cultures and in a flocculent sludge from a reactor that treats lipid-rich wastewaters, but not in methanogenic granular sludges from upflow anaerobic sludge blanket reactors.

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Microstructure of Anaerobic Granules Bioaugmented with Desulfitobacterium frappieri PCP-1

Applied and Environmental Microbiology ◽

10.1128/aem.68.8.4035-4043.2002 ◽

2002 ◽

Vol 68 (8) ◽

pp. 4035-4043 ◽

Cited By ~ 21

Author(s):

M. Lanthier ◽

B. Tartakovsky ◽

R. Villemur ◽

G. DeLuca ◽

S. R. Guiot

Keyword(s):

Growth Rate ◽

In Situ Hybridization ◽

Outer Layer ◽

Specific Growth ◽

Anaerobic Sludge ◽

Oligonucleotide Probes ◽

Reactor Operation ◽

Mathematical Simulations ◽

Successful Colonization

ABSTRACT Oligonucleotide probes were used to study the structure of anaerobic granular biofilm originating from a pentachlorophenol-fed upflow anaerobic sludge bed reactor augmented with Desulfitobacterium frappieri PCP-1. Fluorescence in situ hybridization demonstrated successful colonization of anaerobic granules by strain PCP-1. Scattered microcolonies of strain PCP-1 were detected on the biofilm surface after 3 weeks of reactor operation, and a dense outer layer of strain PCP-1 was observed after 9 weeks. Hybridization with probes specific for Eubacteria and Archaea probes showed that Eubacteria predominantly colonized the outer layer, while Archaea were observed in the granule interior. Mathematical simulations showed a distribution similar to that observed experimentally when using a specific growth rate of 2.2 day−1 and a low bacterial diffusion of 10−7 dm2 day−1. Also, the simulations showed that strain PCP-1 proliferation in the outer biofilm layer provided excellent protection of the biofilm from pentachlorophenol toxicity.

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The Polymorphisms of Oligonucleotide Probes in Wheat Cultivars Determined by ND-FISH

Molecules ◽

10.3390/molecules24061126 ◽

2019 ◽

Vol 24 (6) ◽

pp. 1126 ◽

Cited By ~ 2

Author(s):

Tianheng Ren ◽

Maojie He ◽

Zixin Sun ◽

Feiquan Tan ◽

Peigao Luo ◽

...

Keyword(s):

In Situ Hybridization ◽

Fluorescence In Situ Hybridization ◽

Wheat Genome ◽

Oligonucleotide Probes ◽

Wheat Cultivars ◽

Fish Signal

Non-denaturing fluorescence in situ hybridization (ND-FISH) has been used to distinguish wheat chromosomes and to detect alien chromosomes in the wheat genome. In this study, five different oligonucleotide probes were used with ND-FISH to examine 21 wheat cultivars and lines. These oligonucleotide probes distinguished 42 wheat chromosomes and also detected rye chromatin in the wheat genome. Moreover, the signal patterns of the oligonucleotide probes Oligo-pTa535-1 and Oligo-pSc119.2-1 showed high polymorphism in the wheat chromosomes. A total of 17.6% of the A group chromosomes, 25.9% of the B group chromosomes and 8.9% of the D group chromosomes showed obvious mutations when they were compared to the standard ND-FISH signal patterns, and most of them were Oligo-pSc119.2-1 mutants. The results suggested that these polymorphisms could be induced by the crossing of wheat cultivars. The results provided more information for the further application of oligonucleotide probes and ND-FISH.

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Quantification of Target Molecules Needed To Detect Microorganisms by Fluorescence In Situ Hybridization (FISH) and Catalyzed Reporter Deposition-FISH

Applied and Environmental Microbiology ◽

10.1128/aem.00208-08 ◽

2008 ◽

Vol 74 (16) ◽

pp. 5068-5077 ◽

Cited By ~ 87

Author(s):

Tatsuhiko Hoshino ◽

L. Safak Yilmaz ◽

Daniel R. Noguera ◽

Holger Daims ◽

Michael Wagner

Keyword(s):

In Situ Hybridization ◽

16S Rrna ◽

Activated Sludge ◽

Fluorescence In Situ Hybridization ◽

Oligonucleotide Probes ◽

Pure Cultures ◽

Technical Modifications ◽

Catalyzed Reporter Deposition ◽

Card Fish

ABSTRACT Fluorescence in situ hybridization (FISH) with rRNA-targeted oligonucleotide probes is a method that is widely used to detect and quantify microorganisms in environmental samples and medical specimens by fluorescence microscopy. Difficulties with FISH arise if the rRNA content of the probe target organisms is low, causing dim fluorescence signals that are not detectable against the background fluorescence. This limitation is ameliorated by technical modifications such as catalyzed reporter deposition (CARD)-FISH, but the minimal numbers of rRNA copies needed to obtain a visible signal of a microbial cell after FISH or CARD-FISH have not been determined previously. In this study, a novel competitive FISH approach was developed and used to determine, based on a thermodynamic model of probe competition, the numbers of 16S rRNA copies per cell required to detect bacteria by FISH and CARD-FISH with oligonucleotide probes in mixed pure cultures and in activated sludge. The detection limits of conventional FISH with Cy3-labeled probe EUB338-I were found to be 370 ± 45 16S rRNA molecules per cell for Escherichia coli hybridized on glass microscope slides and 1,400 ± 170 16S rRNA copies per E. coli cell in activated sludge. For CARD-FISH the values ranged from 8.9 ± 1.5 to 14 ± 2 and from 36 ± 6 to 54 ± 7 16S rRNA molecules per cell, respectively, indicating that the sensitivity of CARD-FISH was 26- to 41-fold higher than that of conventional FISH. These results suggest that optimized FISH protocols using oligonucleotide probes could be suitable for more recent applications of FISH (for example, to detect mRNA in situ in microbial cells).

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Composition of the bacterial sludge in occluded biliary stents characterized by fluorescence in situ hybridization with Rna-based oligonucleotide probes

Gastroenterology ◽

10.1016/s0016-5085(03)81237-4 ◽

2003 ◽

Vol 124 (4) ◽

pp. A246

Author(s):

Alexander Swidsinski ◽

Petra Schlien ◽

Sonja Swidsinski ◽

Guido Schachschal ◽

Vera Loening Baucke ◽

...

Keyword(s):

In Situ Hybridization ◽

Fluorescence In Situ Hybridization ◽

Oligonucleotide Probes ◽

Biliary Stents

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Design and Evaluation of 16S rRNA-Targeted Oligonucleotide Probes for Fluorescence In Situ Hybridization

Gene Probes ◽

10.1385/1-59259-238-4:029 ◽

2003 ◽

pp. 029-042 ◽

Cited By ~ 13

Author(s):

Philip Hugenholtz ◽

Gene W Tyson ◽

Linda L Blackall

Keyword(s):

In Situ Hybridization ◽

16S Rrna ◽

Fluorescence In Situ Hybridization ◽

Oligonucleotide Probes

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Monitoring of microbial diversity by fluorescence in situ hybridization and fluorescence spectrometry

Water Science & Technology ◽

10.2166/wst.2003.0300 ◽

2003 ◽

Vol 47 (5) ◽

pp. 133-138 ◽

Cited By ~ 4

Author(s):

V. Ivanov ◽

S.T.-L. Tay ◽

J.-H. Tay

Keyword(s):

In Situ Hybridization ◽

Fluorescence In Situ Hybridization ◽

Environmental Samples ◽

Specific Binding ◽

Oligonucleotide Probes ◽

Simple Method ◽

Total Binding ◽

Unlabeled Probe ◽

Microbial Groups

The goal of the research was the development of a simple method to quantify microbial groups in environmental samples. Fluorescence intensity was measured in the sample before and after whole cell fluorescence in situ hybridization with rRNA-targeted, fluorochrome-labeled oligonucleotide probes. To determine specific and non-specific binding of different oligonucleotide probes the following approaches have been used: (1) incubation of the sample with probes at two different temperatures; (2) hybridization of labeled probe in the presence of unlabeled probe; (3) incubation of the sample with labeled specific probe or labeled nonsense probe. Specific binding (hybridization) of the probe was calculated as the difference between total binding and non-specific binding of the probe. Specific binding was 40-50% of total binding in the environmental samples tested. The ratio of the specific binding of different probes may be used to quantify the ratio of different microbial groups in the environmental samples. This quantification is suitable for the microbiological monitoring of microbial aggregates because it is a simple technique and the results can be measured by a portable fluorometer.

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Differential sensitivity of 16S rRNA targeted oligonucleotide probes used for fluorescence in situ hybridization is a result of ribosomal higher order structure

Canadian Journal of Microbiology ◽

10.1139/m96-136 ◽

1996 ◽

Vol 42 (10) ◽

pp. 1061-1071 ◽

Cited By ~ 39

Author(s):

Marc E. Frischer ◽

Peter J. Floriani ◽

Sandra A. Nierzwicki-Bauer

Keyword(s):

In Situ Hybridization ◽

16S Rrna ◽

Fluorescence In Situ Hybridization ◽

Differential Sensitivity ◽

Probe Design ◽

Oligonucleotide Probes ◽

Whole Cells ◽

Gene Probes ◽

Wide Range

The use of 16S rRNA targeted gene probes for the direct analysis of microbial communities has revolutionized the field of microbial ecology, yet a comprehensive approach for the design of such probes does not exist. The development of 16S rRNA targeted oligonucleotide probes for use with fluorescence in situ hybridization (FISH) procedures has been especially difficult as a result of the complex nature of the rRNA target molecule. In this study a systematic comparison of 16S rRNA targeted oligonucleotide gene probes was conducted to determine if target location influences the hybridization efficiency of oligonucleotide probes when used with in situ hybridization protocols for the detection of whole microbial cells. Five unique universal 12-mer oligonucleotide sequences, located at different regions of the 16S rRNA molecule, were identified by a computer-aided sequence analysis of over 1000 partial and complete 16S rRNA sequences. The complements of these oligomeric sequences were chemically synthesized for use as probes and end labeled with either [γ-32P] ATP or the fluorescent molecule tetramethylrhodamine-5/-6. Hybridization sensitivity for each of the probes was determined by hybridization to heat-denatured RNA immobilized on blots or to formaldehyde fixed whole cells. All of the probes hybridized with equal efficiency to denatured RNA. However, the probes exhibited a wide range of sensitivity (from none to very strong) when hybridized with whole cells using a previously developed FISH procedure. Differential hybridization efficiencies against whole cells could not be attributed to cell wall type, since the relative probe efficiency was preserved when either Gram-negative or -positive cells were used. These studies represent one of the first attempts to systematically define criteria for 16S rRNA targeted probe design for use against whole cells and establish target site location as a critical parameter in probe design.Key words: 16S rRNA, oligonucleotide probes, in situ hybridization.

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