Faculty Opinions recommendation of Fluorescence in situ hybridization of 16S rRNA gene clones (Clone-FISH) for probe validation and screening of clone libraries.

We collected samples of anaerobic landfill leachate from municipal solid waste landfill (Vert-le-Grand, France) and constructed 16S rRNA clone libraries using primers targeting Planctomycetes and relatives (Pla46F and 1390R). Analyses of 16S rRNA gene sequences resulted in the abundant representation of WWE2-related Lentisphaerae, members of the phylum Lentisphaerae, in the clone library (98% of the retrieved sequences). Although the sequences that are phylogenetically affiliated with the cultured isolate Victivallis vadensis were identified (WWE2 subgroup II), the majority of the sequences were affiliated with an uncultured Lentisphaerae lineage (WWE2 subgroup I). We designed oligonucleotides probes targeting the specific 16S rRNA gene regions of those 2 subgroups. Fluorescence in situ hybridization confirmed the abundance of the uncultivated WWE2 subgroup I in our leachate samples.

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Molecular analyses of the diversity in marine bacterioplankton assemblages along the coastline of the northeastern Gulf of Mexico

Canadian Journal of Microbiology ◽

10.1139/w10-069 ◽

2010 ◽

Vol 56 (10) ◽

pp. 853-863 ◽

Cited By ~ 4

Author(s):

Ola A. Olapade

Keyword(s):

In Situ Hybridization ◽

16S Rrna ◽

16S Rrna Gene ◽

Gulf Of Mexico ◽

Fluorescence In Situ Hybridization ◽

Community Diversity ◽

Rrna Gene ◽

Marine Bacterioplankton ◽

Northeastern Gulf Of Mexico

Bacterial community diversity in marine bacterioplankton assemblages were examined in 3 coastal locations along the northeastern Gulf of Mexico (GOM) using 16S rRNA gene libraries and fluorescence in situ hybridization approaches. The majority of the sequences (30%–60%) were similar to the 16S rRNA gene sequences of unknown bacteria; however, the operational taxonomic units from members of the Cyanobacteria, Proteobacteria, and Bacteroidetes were also present at the 3 GOM sites. Overall, sequence diversity was more similar between the Gulf sites of Carrabelle and Ochlockonee than between either of the Gulf sites and Apalachicola Bay. Fluorescence in situ hybridization analyses revealed the quantitative predominance of members of the Alphaproteobacteria subclass and the Cytophaga – Flavobacterium cluster within the bacterioplankton assemblages. In general, the study further reveals the presence of many bacterial taxa that have been previously found to be dominant in coastal marine environments. Differences observed in the representation of the various bacterial phylogenetic groups among the GOM coastal sites could be partly attributed to dynamic variations in several site-specific conditions, including intermittent tidal events, nutrient availability, and anthropogenic influences.

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Distinct relationships between fluorescence in situ hybridization and 16S rRNA gene- and amplicon-based sequencing data of bacterioplankton lineages

Systematic and Applied Microbiology ◽

10.1016/j.syapm.2019.06.005 ◽

2019 ◽

Vol 42 (5) ◽

pp. 126000 ◽

Cited By ~ 1

Author(s):

Insa Bakenhus ◽

Bernd Wemheuer ◽

Pinar Akyol ◽

Helge-Ansgar Giebel ◽

Leon Dlugosch ◽

...

Keyword(s):

In Situ Hybridization ◽

16S Rrna ◽

16S Rrna Gene ◽

Fluorescence In Situ Hybridization ◽

Rrna Gene ◽

Sequencing Data

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Evidence of Multiple Treponema Phylotypes Involved in Bovine Digital Dermatitis as Shown by 16S rRNA Gene Analysis and Fluorescence In Situ Hybridization

Journal of Clinical Microbiology ◽

10.1128/jcm.00670-08 ◽

2008 ◽

Vol 46 (9) ◽

pp. 3012-3020 ◽

Cited By ~ 75

Author(s):

K. Klitgaard ◽

M. Boye ◽

N. Capion ◽

T. K. Jensen

Keyword(s):

In Situ Hybridization ◽

16S Rrna ◽

16S Rrna Gene ◽

Fluorescence In Situ Hybridization ◽

Gene Analysis ◽

Rrna Gene ◽

Digital Dermatitis ◽

16S Rrna Gene Analysis

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Change in Bacterial Community Structure during In Situ Biostimulation of Subsurface Sediment Cocontaminated with Uranium and Nitrate

Applied and Environmental Microbiology ◽

10.1128/aem.70.8.4911-4920.2004 ◽

2004 ◽

Vol 70 (8) ◽

pp. 4911-4920 ◽

Cited By ~ 200

Author(s):

Nadia N. North ◽

Sherry L. Dollhopf ◽

Lainie Petrie ◽

Jonathan D. Istok ◽

David L. Balkwill ◽

...

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Microbial Communities ◽

Quantitative Pcr ◽

Rrna Gene ◽

Gene Sequences ◽

16S Rrna Gene Sequences ◽

Clone Libraries ◽

Subsurface Sediments

ABSTRACT Previous studies have demonstrated that metal-reducing microorganisms can effectively promote the precipitation and removal of uranium from contaminated groundwater. Microbial communities were stimulated in the acidic subsurface by pH neutralization and addition of an electron donor to wells. In single-well push-pull tests at a number of treated sites, nitrate, Fe(III), and uranium were extensively reduced and electron donors (glucose, ethanol) were consumed. Examination of sediment chemistry in cores sampled immediately adjacent to treated wells 3.5 months after treatment revealed that sediment pH increased substantially (by 1 to 2 pH units) while nitrate was largely depleted. A large diversity of 16S rRNA gene sequences were retrieved from subsurface sediments, including species from the α, β, δ, and γ subdivisions of the class Proteobacteria, as well as low- and high-G+C gram-positive species. Following in situ biostimulation of microbial communities within contaminated sediments, sequences related to previously cultured metal-reducing δ-Proteobacteria increased from 5% to nearly 40% of the clone libraries. Quantitative PCR revealed that Geobacter-type 16S rRNA gene sequences increased in biostimulated sediments by 1 to 2 orders of magnitude at two of the four sites tested. Evidence from the quantitative PCR analysis corroborated information obtained from 16S rRNA gene clone libraries, indicating that members of the δ-Proteobacteria subdivision, including Anaeromyxobacter dehalogenans-related and Geobacter-related sequences, are important metal-reducing organisms in acidic subsurface sediments. This study provides the first cultivation-independent analysis of the change in metal-reducing microbial communities in subsurface sediments during an in situ bioremediation experiment.

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Heterogeneity between 16S ribosomal RNA gene copies borne by one Desulfitobacterium strain is caused by different 100-200 bp insertions in the 5´ region

Canadian Journal of Microbiology ◽

10.1139/w06-111 ◽

2007 ◽

Vol 53 (1) ◽

pp. 116-128 ◽

Cited By ~ 13

Author(s):

Richard Villemur ◽

Philippe Constant ◽

Annie Gauthier ◽

Martine Shareck ◽

Réjean Beaudet

Keyword(s):

In Situ Hybridization ◽

16S Rrna ◽

16S Rrna Gene ◽

Ribosomal Rna ◽

16S Ribosomal Rna ◽

16S Rrna Genes ◽

Rrna Genes ◽

Rrna Gene ◽

Gene Copies

Strains of Desulfitobacterium hafniense, such as strains PCP-1, DP7, TCE1, and TCP-A, have unusual long 16S ribosomal RNA (rRNA) genes due to an insertion of approximately 100 bp in the 5' region. In this report, we analyzed the 16S rRNA genes of different Desulfitobacterium strains to determine if such an insertion is a common feature of desulfitobacteria. We amplified this region by polymerase chain reaction (PCR) from eight Desulfitobacterium strains (D. hafniense strains PCP-1, DP7, TCP-A, TCE1, and DCB-2; D. dehalogenans; D. chlororespirans; and Desulfitobacterium sp. PCE1) and resolved each PCR product by denaturing gradient gel electrophoresis (DGGE). All strains had from two to seven DGGE- migrating bands, suggesting heterogeneity in their 16S rRNA gene copies. For each strain, the 5' region of the 16S rRNA genes was amplified and a clone library was derived. Clones corresponding to most PCR–DGGE migration bands were isolated. Sequencing of representative clones revealed that the heterogeneity was generated by insertions of 100–200 bp. An insertion was found in at least one copy of the 16S rRNA gene in all examined strains. In total, we found eight different types of insertions (INS1–INS8) that varied from 123 to 193 nt in length. Two-dimensional structural analyses of transcribed sequences predicted that all insertions would form an energetically stable loop. Reverse transcriptase – PCR experiments revealed that most of the observed insertions in the Desulfitobacterium strains were excised from the mature 16S rRNA transcripts. Insertions were not commonly found in bacterial 16S rRNA genes, and having a different insertion in several 16S rRNA gene copies borne by a single bacterial species was rarely observed. The function of these insertions is not known, but their occurrence can have an important impact in deriving 16S rRNA oligonucleotidic fluorescence in situ hybridization probes, as these insertions can be excised from 16S rRNA transcripts.Key words: Desulfitobacterium, 16S ribosomal RNA genes, heterogeneity, gene insertions, fluorescence in situ hybridization.

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Community Composition of Marine Bacterioplankton Determined by 16S rRNA Gene Clone Libraries and Fluorescence In Situ Hybridization

Applied and Environmental Microbiology ◽

10.1128/aem.66.12.5116-5122.2000 ◽

2000 ◽

Vol 66 (12) ◽

pp. 5116-5122 ◽

Cited By ~ 313

Author(s):

Matthew T. Cottrell ◽

David L. Kirchman

Keyword(s):

In Situ Hybridization ◽

16S Rrna ◽

Fluorescence In Situ Hybridization ◽

Community Composition ◽

16S Rrna Genes ◽

Rrna Genes ◽

Clone Libraries ◽

Marine Bacterioplankton ◽

Bacterioplankton Communities

ABSTRACT We determined the compositions of bacterioplankton communities in surface waters of coastal California using clone libraries of 16S rRNA genes and fluorescence in situ hybridization (FISH) in order to compare the community structures inferred from these two culture-independent approaches. The compositions of two clone libraries were quite similar to those of clone libraries of marine bacterioplankton examined by previous studies. Clones from γ-proteobacteria comprised ca. 28% of the libraries, while approximately 55% of the clones came from α-proteobacteria, which dominated the clone libraries. TheCytophaga-Flavobacter group and three others each comprised 10% or fewer of the clone libraries. The community composition determined by FISH differed substantially from the composition implied by the clone libraries. The Cytophaga-Flavobacter group dominated 8 of the 11 communities assayed by FISH, including the two communities assayed using clone libraries. On average only 10% of DAPI (4′,6′-diamidino-2-phenylindole)-stained bacteria were detected by FISH with a probe for α-proteobacteria, but 30% of DAPI-stained bacteria appeared to be in the Cytophaga-Flavobacter group as determined by FISH. α-Proteobacteria were greatly overrepresented in clone libraries compared to their relative abundance determined by FISH, while the Cytophaga-Flavobacter group was underrepresented in clone libraries. Our data show that theCytophaga-Flavobacter group can be a numerically dominant component of coastal marine bacterioplankton communities.

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