Development of in situ hybridisation using 16S rRNA gene to monitor black-lip pearl oyster,Pinctada margaritifera, larvae in plankton samples

ABSTRACT The geochemistry and microbiology of a uranium-contaminated subsurface environment that had undergone two seasons of acetate addition to stimulate microbial U(VI) reduction was examined. There were distinct horizontal and vertical geochemical gradients that could be attributed in large part to the manner in which acetate was distributed in the aquifer, with more reduction of Fe(III) and sulfate occurring at greater depths and closer to the point of acetate injection. Clone libraries of 16S rRNA genes derived from sediments and groundwater indicated an enrichment of sulfate-reducing bacteria in the order Desulfobacterales in sediment and groundwater samples. These samples were collected nearest the injection gallery where microbially reducible Fe(III) oxides were highly depleted, groundwater sulfate concentrations were low, and increases in acid volatile sulfide were observed in the sediment. Further down-gradient, metal-reducing conditions were present as indicated by intermediate Fe(II)/Fe(total) ratios, lower acid volatile sulfide values, and increased abundance of 16S rRNA gene sequences belonging to the dissimilatory Fe(III)- and U(VI)-reducing family Geobacteraceae. Maximal Fe(III) and U(VI) reduction correlated with maximal recovery of Geobacteraceae 16S rRNA gene sequences in both groundwater and sediment; however, the sites at which these maxima occurred were spatially separated within the aquifer. The substantial microbial and geochemical heterogeneity at this site demonstrates that attempts should be made to deliver acetate in a more uniform manner and that closely spaced sampling intervals, horizontally and vertically, in both sediment and groundwater are necessary in order to obtain a more in-depth understanding of microbial processes and the relative contribution of attached and planktonic populations to in situ uranium bioremediation.

Download Full-text

Diversity and Seasonal Dynamics of Actinobacteria Populations in Four Lakes in Northeastern Germany

Applied and Environmental Microbiology ◽

10.1128/aem.72.5.3489-3497.2006 ◽

2006 ◽

Vol 72 (5) ◽

pp. 3489-3497 ◽

Cited By ~ 159

Author(s):

Martin Allgaier ◽

Hans-Peter Grossart

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Seasonal Dynamics ◽

Phylogenetic Diversity ◽

Rrna Gene ◽

Lake District ◽

Gene Sequences ◽

16S Rrna Gene Sequences ◽

Northeastern Germany

ABSTRACT The phylogenetic diversity and seasonal dynamics of freshwater Actinobacteria populations in four limnologically different lakes of the Mecklenburg-Brandenburg Lake District (northeastern Germany) were investigated. Fluorescence in situ hybridization was used to determine the seasonal abundances and dynamics of total Actinobacteria (probe HGC69a) and the three actinobacterial subclusters acI, acI-A, and acI-B (probes AcI-852, AcI-840-1, and AcI-840-2). Seasonal means of total Actinobacteria abundances in the epilimnia of the lakes varied from 13 to 36%, with maximum values of 30 to 58%, of all DAPI (4′,6′-diamidino-2-phenylindole)-stained cells. Around 80% of total Actinobacteria belonged to the acI cluster. The two subclusters acI-A and acI-B accounted for 60 to 91% of the acI cluster and showed seasonal means of 49% (acI-B) and 23% (acI-A) in relation to the acI cluster. Total Actinobacteria and members of the clusters acI and acI-B showed distinct seasonal changes in their absolute abundances, with maxima in late spring and fall/winter. In eight clone libraries constructed from the lakes, a total of 76 actinobacterial 16S rRNA gene sequences were identified from a total of 177 clones. The majority of the Actinobacteria sequences belonged to the acI and acIV cluster. Several new clusters and subclusters were found (acSTL, scB1-4, and acIVA-D). The majority of all obtained 16S rRNA gene sequences are distinct from those of already-cultured freshwater Actinobacteria.

Download Full-text

Nitrifying bacterial communities in an aquaculture wastewater treatment system using fluorescence in situ hybridization (FISH), 16S rRNA gene cloning, and phylogenetic analysis

Biotechnology and Bioengineering ◽

10.1002/bit.21270 ◽

2006 ◽

Vol 97 (4) ◽

pp. 985-990 ◽

Cited By ~ 19

Author(s):

Chanyarat Paungfoo ◽

Poonsuk Prasertsan ◽

Paul C. Burrell ◽

Nugul Intrasungkha ◽

Linda L. Blackall

Keyword(s):

Phylogenetic Analysis ◽

Wastewater Treatment ◽

In Situ Hybridization ◽

16S Rrna ◽

16S Rrna Gene ◽

Fluorescence In Situ Hybridization ◽

Bacterial Communities ◽

Rrna Gene ◽

Aquaculture Wastewater

Download Full-text

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.

Download Full-text

Phylogenetic Analysis and In Situ Identification of Bacteria Community Composition in an Acidic Sphagnum Peat Bog

Applied and Environmental Microbiology ◽

10.1128/aem.72.3.2110-2117.2006 ◽

2006 ◽

Vol 72 (3) ◽

pp. 2110-2117 ◽

Cited By ~ 213

Author(s):

Svetlana N. Dedysh ◽

Timofei A. Pankratov ◽

Svetlana E. Belova ◽

Irina S. Kulichevskaya ◽

Werner Liesack

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Community Composition ◽

Significant Proportion ◽

Peat Bog ◽

Rrna Gene ◽

Bacterial Phyla ◽

Cell Counts ◽

Novel Strategy

ABSTRACT The Bacteria community composition in an acidic Sphagnum peat bog (pH 3.9 to 4.5) was characterized by a combination of 16S rRNA gene clone library analysis, rRNA-targeted fluorescence in situ hybridization (FISH), and cultivation. Among 84 environmental 16S rRNA gene clones, a set of only 16 cloned sequences was closely related (≥95% similarity) to taxonomically described organisms. Main groups of clones were affiliated with the Acidobacteria (24 clones), Alphaproteobacteria (20), Verrucomicrobia (13), Actinobacteria (8), Deltaproteobacteria (4), Chloroflexi (3), and Planctomycetes (3). The proportion of cells that hybridized with oligonucleotide probes specific for members of the domains Bacteria (EUB338-mix) and Archaea (ARCH915 and ARC344) accounted for only 12 to 22% of the total cell counts. Up to 24% of the EUB338-positive cells could be assigned by FISH to specific bacterial phyla. Alphaproteobacteria and Planctomycetes were the most numerous bacterial groups (up to 1.3 × 107 and 1.1 × 107 cells g−1 peat, respectively). In contrast to conventional plating techniques, a novel biofilm-mediated enrichment approach allowed us to isolate some representatives of predominant Bacteria groups, such as Acidobacteria and Planctomycetes. This novel strategy has great potential to enable the isolation of a significant proportion of the peat bog bacterial diversity.

Download Full-text

Molecular Characterization of Potential Nitrogen Fixation by Anaerobic Methane-Oxidizing Archaea in the Methane Seep Sediments at the Number 8 Kumano Knoll in the Kumano Basin, Offshore of Japan

Applied and Environmental Microbiology ◽

10.1128/aem.01184-09 ◽

2009 ◽

Vol 75 (22) ◽

pp. 7153-7162 ◽

Cited By ~ 33

Author(s):

Junichi Miyazaki ◽

Ryosaku Higa ◽

Tomohiro Toki ◽

Juichiro Ashi ◽

Urumu Tsunogai ◽

...

Keyword(s):

Nitrogen Fixation ◽

16S Rrna ◽

16S Rrna Gene ◽

Gene Clusters ◽

Rrna Gene ◽

Methane Seep ◽

Core Sediments ◽

The Core ◽

Group 2

ABSTRACT The potential for microbial nitrogen fixation in the anoxic methane seep sediments in a mud volcano, the number 8 Kumano Knoll, was characterized by molecular phylogenetic analyses. A total of 111 of the nifH (a gene coding a nitrogen fixation enzyme, Fe protein) clones were obtained from different depths of the core sediments, and the phylogenetic analysis of the clones indicated the genetic diversity of nifH genes. The predominant group detected (methane seep group 2), representing 74% of clonal abundance, was phylogenetically related to the nifH sequences obtained from the Methanosarcina species but was most closely related to the nifH sequences potentially derived from the anoxic methanotrophic archaea (ANME-2 archaea). The recovery of the nif gene clusters including the nifH sequences of the methane seep group 2 and the subsequent reverse transcription-PCR detection of the nifD and nifH genes strongly suggested that the genetic components of the gene clusters would be operative for the in situ assimilation of molecular nitrogen (N2) by the host microorganisms. DNA-based quantitative PCR of the archaeal 16S rRNA gene, the group-specific mcrA (a gene encoding the methyl-coenzyme M reductase α subunit) gene, and the nifD and nifH genes demonstrated the similar distribution patterns of the archaeal 16S rRNA gene, the mcrA groups c-d and e, and the nifD and nifH genes through the core sediments. These results supported the idea that the anoxic methanotrophic archaea ANME-2c could be the microorganisms hosting the nif gene clusters and could play an important role in not only the in situ carbon (methane) cycle but also the nitrogen cycle in subseafloor sediments.

Download Full-text

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.

Download Full-text

Characterization of a Culturable Alphaproteobacterial Symbiont Common to Many Marine Sponges and Evidence for Vertical Transmission via Sponge Larvae

Applied and Environmental Microbiology ◽

10.1128/aem.72.5.3724-3732.2006 ◽

2006 ◽

Vol 72 (5) ◽

pp. 3724-3732 ◽

Cited By ~ 155

Author(s):

Julie J. Enticknap ◽

Michelle Kelly ◽

Olivier Peraud ◽

Russell T. Hill

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Denaturing Gradient Gel Electrophoresis ◽

Marine Sponges ◽

Rrna Gene ◽

Surrounding Water ◽

Axinella Corrugata ◽

Gradient Gel Electrophoresis ◽

Related Group

ABSTRACT A closely related group of alphaproteobacteria were found to be present in seven genera of marine sponges from several locations and were shown to be transferred between sponge generations through the larvae in one of these sponges. Isolates of the alphaproteobacterium were cultured from the sponges Axinella corrugata, Mycale laxissima, Monanchora unguifera, and Niphates digitalis from Key Largo, Florida; Didiscus oxeata and Monanchora unguifera from Discovery Bay, Jamaica; an Acanthostronglyophora sp. from Manado, Indonesia; and Microciona prolifera from the Cheasapeake Bay in Maryland. Isolates were very similar to each other on the basis of 16S rRNA gene sequence (>99% identity) and are closely related to Pseudovibrio denitrificans. The bacterium was never isolated from surrounding water samples and was cultured from larvae of M. laxissima, indicating that it is a vertically transmitted symbiont in this sponge. Denaturing gradient gel electrophoresis, 16S rRNA gene clone library analysis, and fluorescent in situ hybridization with probes specific to the alphaproteobacterium confirmed the presence of this bacterium in the M. laxissima larvae. The alphaproteobacterium was densely associated with the larvae rather than being evenly distributed throughout the mesohyl. This is the first report of the successful culture of a bacterial symbiont of a sponge that is transferred through the gametes.

Download Full-text

In-depth analysis of Bacillus anthracis 16S rRNA genes and transcripts reveals intra- and intergenomic diversity and facilitates anthrax detection

10.1101/2021.08.02.454851 ◽

2021 ◽

Author(s):

Peter Braun ◽

Fee Zimmermann ◽

Mathias C Walter ◽

Sonja Mantel ◽

Karin Aistleitner ◽

...

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Bacterial Species ◽

16S Rrna Genes ◽

Rrna Genes ◽

Close Relative ◽

Rrna Gene ◽

Copy Numbers ◽

Depth Analysis

Analysis of 16S ribosomal RNA (rRNA) genes provides a central means of taxonomic classification of bacterial species. Based on presumed sequence identity among species of the Bacillus cereus sensu lato group, the 16S rRNA genes of B. anthracis have been considered unsuitable for diagnosis of the anthrax pathogen. With the recent identification of a single nucleotide polymorphism in some 16S rRNA gene copies, specific identification of B. anthracis becomes feasible. Here, we designed and evaluated a set of in situ-, in vitro- and in silico-assays to assess the yet unknown 16S-state of B. anthracis from different perspectives. Using a combination of digital PCR, fluorescence in situ hybridization, long-read genome sequencing and bioinformatics we were able to detect and quantify a unique 16S rRNA gene allele of B. anthracis (16S-BA-allele). This allele was found in all available B. anthracis genomes and may facilitate differentiation of the pathogen from any close relative. Bioinformatics analysis of 959 B. anthracis genome data-sets inferred that abundances and genomic arrangements of the 16S-BA-allele and the entire rRNA operon copy-numbers differ considerably between strains. Expression ratios of 16S-BA-alleles were proportional to the respective genomic allele copy-numbers. The findings and experimental tools presented here provide detailed insights into the intra- and intergenomic diversity of 16S rRNA genes and may pave the way for improved identification of B. anthracis and other pathogens with diverse rRNA operons.

Download Full-text