scholarly journals Feasibility of Transferring Fluorescent In Situ Hybridization Probes to an 18S rRNA Gene Phylochip and Mapping of Signal Intensities

2008 ◽  
Vol 74 (9) ◽  
pp. 2814-2821 ◽  
Author(s):  
Katja Metfies ◽  
Linda K. Medlin
Keyword(s):  
In Situ Hybridization ◽  
Secondary Structure ◽  
16S Rrna ◽  
Fluorescent In Situ Hybridization ◽  
18S Rrna ◽  
18S Rrna Gene ◽  
Rrna Gene ◽  
Rrna Molecule ◽  
The 16S Rrna Gene

ABSTRACT DNA microarray technology offers the possibility to analyze microbial communities without cultivation, thus benefiting biodiversity studies. We developed a DNA phylochip to assess phytoplankton diversity and transferred 18S rRNA probes from dot blot or fluorescent in situ hybridization (FISH) analyses to a microarray format. Similar studies with 16S rRNA probes have been done determined that in order to achieve a signal on the microarray, the 16S rRNA molecule had to be fragmented, or PCR amplicons had to be <150 bp in length to minimize the formation of a secondary structure in the molecule so that the probe could bind to the target site. We found different results with the 18S rRNA molecule. Four out of 12 FISH probes exhibited false-negative signals on the microarray; eight exhibited strong but variable signals using full-length 18S RNA molecules. A systematic investigation of the probe's accessibility to the 18S rRNA gene was made using Prymenisum parvum as the target. Fourteen additional probes identical to this target covered the regions not tested with existing FISH probes. Probes with a binding site in the first 900 bp of the gene generated positive signals. Six out of nine probes binding in the last 900 bp of the gene produced no signal. Our results suggest that although secondary structure affected probe binding, the effect is not the same for the 18S rRNA gene and the 16S rRNA gene. For the 16S rRNA gene, the secondary structure is stronger in the first half of the molecule, whereas in the 18S rRNA gene, the last half of the molecule is critical. Probe-binding sites within 18S rRNA gene molecules are important for the probe design for DNA phylochips because signal intensity appears to be correlated with the secondary structure at the binding site in this molecule. If probes are designed from the first half of the 18S rRNA molecule, then full-length 18S rRNA molecules can be used in the hybridization on the chip, avoiding the fragmentation and the necessity for the short PCR amplicons that are associated with using the 16S rRNA molecule. Thus, the 18S rRNA molecule is a more attractive molecule for use in environmental studies where some level of quantification is desired. Target size was a minor problem, whereas for 16S rRNA molecules target size rather than probe site was important.

Ultrastructural and molecular characterization of endosymbionts of the reed beetle genusMacroplea(Chrysomelidae, Donaciinae), and proposal of “CandidatusMacropleicola appendiculatae” and “CandidatusMacropleicola muticae”

2009 ◽  
Vol 55 (11) ◽  
pp. 1250-1260 ◽  
Author(s):  
Gregor Kölsch ◽  
Corinna Matz-Grund ◽  
Bo V. Pedersen
Keyword(s):  
Phylogenetic Analysis ◽  
16S Rrna ◽  
Fluorescent In Situ Hybridization ◽  
Malpighian Tubules ◽  
Rrna Gene ◽  
Beetle Species ◽  
Bacterial Symbionts ◽  
The 16S Rrna Gene

Intracellular bacterial symbionts are known from various insect groups, particularly from those feeding on unbalanced diets, where the bacteria provide essential nutrients to the host. In the case of reed beetles (Coleoptera: Chrysomelidae, Donaciinae), however, the endosymbionts appear to be associated with specialized “glands” that secrete a material used for the beetles’ unusual water-tight cocoon. These glands were discovered over a century ago, but the bacteria they contain have yet to be characterized and placed in a phylogenetic context. Here, we describe the ultrastructure of two endosymbiotic species (“ Candidatus Macropleicola appendiculatae” and “ Candidatus Macropleicola muticae”) that reside in cells of the Malpighian tubules of the reed beetle species Macroplea appendiculata and Macroplea mutica , respectively. Fluorescent in situ hybridization using oligonucleotides targeting the 16S rRNA gene specific to Macroplea symbionts verified the localization of the symbionts in these organs. Phylogenetic analysis of 16S rRNA placed “Candidatus Macropleicola” in a clade of typically endosymbiotic Enterobacteriaceae (γ-proteobacteria). Finally, we discuss the evidence available for the hypothesis that the beetle larvae use a secretion produced by the bacteria for the formation of an underwater cocoon.

scholarly journals Tannins from senescent Rhizophora mangle mangrove leaves have a distinctive effect on prokaryotic and eukaryotic communities in a Distichlis spicata salt marsh soil

2020 ◽  
Vol 96 (9) ◽  
Author(s):  
Qiu-Fang Zhang ◽  
Hendrikus J Laanbroek
Keyword(s):  
Salt Marsh ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Condensed Tannins ◽  
18S Rrna ◽  
18S Rrna Gene ◽  
Rrna Gene ◽  
Marsh Soil ◽  
Salt Marsh Soil ◽  
The 16S Rrna Gene

ABSTRACT Due to climate warming, tannin-rich Rhizophora mangle migrates into tannin-poor salt marshes, where the tannins interfere with the biogeochemistry in the soil. Changes in biogeochemistry are likely associated with changes in microbial communities. This was studied in microcosms filled with salt marsh soil and amended with leaf powder, crude condensed tannins, purified condensed tannins (PCT), all from senescent R. mangle leaves, or with tannic acid. Size and composition of the microbial communities were determined by denaturing gradient gel electrophoresis, high-throughput sequencing and real-time PCR based on the 16S and 18S rRNA genes. Compared with the control, the 16S rRNA gene abundance was lowered by PCT, while the 18S rRNA gene abundance was enhanced by all treatments. The treatments also affected the composition of the 16S rRNA and 18S rRNA gene assemblies, but the effects on the 18S rRNA gene were greater. The composition of the 18S rRNA gene, but not of the 16S rRNA gene, was significantly correlated with the mineralization of carbon, nitrogen and phosphorus. Distinctive microbial groups emerged during the different treatments. This study revealed that migration of mangroves may affect both the prokaryotic and the eukaryotic communities in salt marsh soils, but that the effects on the eukaryotes will likely be greater.

scholarly journals 16S rRNA gene and 18S rRNA gene diversity in microbial mat communities in meltwater ponds on the McMurdo Ice Shelf, Antarctica

Polar Biology ◽  
2021 ◽  
Author(s):  
Eleanor E. Jackson ◽  
Ian Hawes ◽  
Anne D. Jungblut
Keyword(s):  
16S Rrna ◽  
18S Rrna ◽  
High Throughput Sequencing ◽  
Microbial Mats ◽  
Gene Diversity ◽  
Salinity Gradient ◽  
18S Rrna Gene ◽  
Rrna Gene ◽  
Ice Shelf ◽  
The Impact

AbstractThe undulating ice of the McMurdo Ice Shelf, Southern Victoria Land, supports one of the largest networks of ice-based, multiyear meltwater pond habitats in Antarctica, where microbial mats are abundant and contribute most of the biomass and biodiversity. We used 16S rRNA and 18S rRNA gene high-throughput sequencing to compare variance of the community structure in microbial mats within and between ponds with different salinities and pH. Proteobacteria and Cyanobacteria were the most abundant phyla, and composition at OTU level was highly specific for the meltwater ponds with strong community sorting along the salinity gradient. Our study provides the first detailed evaluation of eukaryote communities for the McMurdo Ice Shelf using the 18S rRNA gene. They were dominated by Ochrophyta, Chlorophyta and Ciliophora, consistent with previous microscopic analyses, but many OTUs belonging to less well-described heterotrophic protists from Antarctic ice shelves were also identified including Amoebozoa, Rhizaria and Labyrinthulea. Comparison of 16S and 18S rRNA gene communities showed that the Eukaryotes had lower richness and greater similarity between ponds in comparison with Bacteria and Archaea communities on the McMurdo Ice shelf. While there was a weak correlation between community dissimilarity and geographic distance, the congruity of microbial assemblages within ponds, especially for Bacteria and Archaea, implies strong habitat filtering in ice shelf meltwater pond ecosystems, especially due to salinity. These findings help to understand processes that are important in sustaining biodiversity and the impact of climate change on ice-based aquatic habitats in Antarctica.

scholarly journals Combined Microautoradiography–16S rRNA Probe Technique for Determination of Radioisotope Uptake by Specific Microbial Cell Types In Situ

1999 ◽  
Vol 65 (4) ◽  
pp. 1746-1752 ◽  
Author(s):  
Cleber C. Ouverney ◽  
Jed A. Fuhrman
Keyword(s):  
In Situ Hybridization ◽  
16S Rrna ◽  
Microbial Communities ◽  
Fluorescent In Situ Hybridization ◽  
Amino Acid Uptake ◽  
Cell Types ◽  
Black Box ◽  
Acid Uptake ◽  
Substrate Uptake

ABSTRACT We propose a novel method for studying the function of specific microbial groups in situ. Since natural microbial communities are dynamic both in composition and in activities, we argue that the microbial “black box” should not be regarded as homogeneous. Our technique breaks down this black box with group-specific fluorescent 16S rRNA probes and simultaneously determines 3H-substrate uptake by each of the subgroups present via microautoradiography (MAR). Total direct counting, fluorescent in situ hybridization, and MAR are combined on a single slide to determine (i) the percentages of different subgroups in a community, (ii) the percentage of total cells in a community that take up a radioactively labeled substance, and (iii) the distribution of uptake within each subgroup. The method was verified with pure cultures. In addition, in situ uptake by members of the α subdivision of the class Proteobacteria(α-Proteobacteria) and of the Cytophaga-Flavobacteriumgroup obtained off the California coast and labeled with fluorescent oligonucleotide probes for these subgroups showed that not only do these organisms account for a large portion of the picoplankton community in the sample examined (∼60% of the universal probe-labeled cells and ∼50% of the total direct counts), but they also are significant in the uptake of dissolved amino acids in situ. Nearly 90% of the total cells and 80% of the cells belonging to the α-Proteobacteria and Cytophaga-Flavobacterium groups were detectable as active organisms in amino acid uptake tests. We suggest a name for our triple-labeling technique, substrate-tracking autoradiographic fluorescent in situ hybridization (STARFISH), which should aid in the “dissection” of microbial communities by type and function.

scholarly journals Investigation of an Acetate-Fed Denitrifying Microbial Community by Stable Isotope Probing, Full-Cycle rRNA Analysis, and Fluorescent In Situ Hybridization-Microautoradiography

2005 ◽  
Vol 71 (12) ◽  
pp. 8683-8691 ◽  
Author(s):  
Maneesha P. Ginige ◽  
Jürg Keller ◽  
Linda L. Blackall
Keyword(s):  
In Situ Hybridization ◽  
Stable Isotope ◽  
Activated Sludge ◽  
Fluorescent In Situ Hybridization ◽  
Batch Reactor ◽  
External Source ◽  
Stable Isotope Probing ◽  
Rrna Gene ◽  
The Impact

ABSTRACT The acetate-utilizing microbial consortium in a full-scale activated sludge process was investigated without prior enrichment using stable isotope probing (SIP). [13C]acetate was used in SIP to label the DNA of the denitrifiers. The [13C]DNA fraction that was extracted was subjected to a full-cycle rRNA analysis. The dominant 16S rRNA gene phylotypes in the 13C library were closely related to the bacterial families Comamonadaceae and Rhodocyclaceae in the class Betaproteobacteria. Seven oligonucleotide probes for use in fluorescent in situ hybridization (FISH) were designed to specifically target these clones. Application of these probes to the sludge of a continuously fed denitrifying sequencing batch reactor (CFDSBR) operated for 16 days revealed that there was a significant positive correlation between the CFDSBR denitrification rate and the relative abundance of all probe-targeted bacteria in the CFDSBR community. FISH-microautoradiography demonstrated that the DEN581 and DEN124 probe-targeted cells that dominated the CFDSBR were capable of taking up [14C]acetate under anoxic conditions. Initially, DEN444 and DEN1454 probe-targeted bacteria also dominated the CFDSBR biomass, but eventually DEN581 and DEN124 probe-targeted bacteria were the dominant bacterial groups. All probe-targeted bacteria assessed in this study were denitrifiers capable of utilizing acetate as a source of carbon. The rapid increase in the number of organisms positively correlated with the immediate increase in denitrification rates observed by plant operators when acetate is used as an external source of carbon to enhance denitrification. We suggest that the impact of bacteria on activated sludge subjected to intermittent acetate supplementation should be assessed prior to the widespread use of acetate in the wastewater industry to enhance denitrification.

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

2007 ◽  
Vol 53 (1) ◽  
pp. 116-128 ◽  
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.

Lupin kernel fiber consumption modifies fecal microbiota in healthy men as determined by rRNA gene fluorescent in situ hybridization

2006 ◽  
Vol 45 (6) ◽  
pp. 335-341 ◽  
Author(s):  
Stuart C. Smith ◽  
Rachel Choy ◽  
Stuart K. Johnson ◽  
Ramon S. Hall ◽  
Alida C.M. Wildeboer-Veloo ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Fluorescent In Situ Hybridization ◽  
Fecal Microbiota ◽  
Rrna Gene ◽  
Healthy Men ◽  
Fiber Consumption

Novel Nitrospira-like bacteria as dominant nitrite-oxidizers in biofilms from wastewater treatment plants: diversity and in situ physiology

2000 ◽  
Vol 41 (4-5) ◽  
pp. 85-90 ◽  
Author(s):  
H. Daims ◽  
P.H. Nielsen ◽  
J.L. Nielsen ◽  
S. Juretschko ◽  
M. Wagner
Keyword(s):  
Wastewater Treatment ◽  
In Situ Hybridization ◽  
16S Rrna ◽  
Fluorescent In Situ Hybridization ◽  
Wastewater Treatment Plants ◽  
Combined Application ◽  
Fundamental Understanding ◽  
Unculturable Bacteria ◽  
Rrna Sequences

The frequency and distribution of putatively nitrite-oxidizing, Nitrospira- like bacteria in nitrifying biofilms from two reactors receiving wastewater with different ammonia and salt concentrations were observed by fluorescent in situ hybridization. For this purpose, new 16S rRNA-directed oligonucleotide probes targeting the bacterial phylum Nitrospira and the three main lineages within this phylum were developed and evaluated. The diversity of Nitrospira-like bacteria in the reactors was additionally investigated by retrieval and comparative analysis of full 16S rRNA sequences from the biofilms. We found that, despite of the differences in the influent composition, Nitrospira-like bacteria form dominant populations in both reactors. In addition, first insights into the physiology of these still unculturable bacteria were obtained by the incubation of active biofilm samples with radioactively labeled substrates followed by the combined application of fluorescent in situ hybridization and microautoradiography. The results are discussed in consideration of the frequently observed dominance of Nitrospira-like bacteria in nitrifying bioreactors. Consequently, high priority should be assigned to future studies on the ecology and physiology of these organisms in order to increase our fundamental understanding of nitrogen cycling and to enable knowledge-driven future improvements of nitrifying wastewater treatment plants.

The structure and organization of the 16S ribosomal RNA gene from the archaebacterium Thermoplasma acidophilum

1989 ◽  
Vol 35 (1) ◽  
pp. 124-133 ◽  
Author(s):  
Heesoo K. Ree ◽  
Kaiming Cao ◽  
David L. Thurlow ◽  
Robert A. Zimmermann
Keyword(s):  
Secondary Structure ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Transcription Initiation ◽  
Single Copy ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Transcription Control ◽  
Thermoplasma Acidophilum ◽  
The 16S Rrna Gene

The complete nucleotide sequence of the 16S rRNA gene from Thermoplasma acidophilum, as well as its 5′ and 3′ flanking regions, were determined by the dideoxynucleotide chain termination method. The 16S rRNA gene encodes 1471 nucleotides. The primary and secondary structures of T. acidophilum 16S rRNA both exhibit typical archaebacterial features. The sequence appears to be more closely related to 16S rRNAs of the methanogen–halophile group than to those of the thermoacidophile group. Secondary-structure comparisons generally support this relationship, although there are several examples in which the single-stranded loops in particular helices of T. acidophilum 16S rRNA more strongly resemble their counterparts in the 16S rRNA of Sulfolobus solfataricus, a member of the thermoacidophile group. In contrast to the polycistronic rRNA operons found in most organisms, the three rRNA genes from T. acidophilum occur in only a single copy per genome and appear to be physically unlinked. Consistent with this, the 16S rRNA gene is flanked by putative promoter and terminator sequences that are comparable to the transcription control signals from other archaebacterial genes. The sequence TATATATA, which is very similar to the archaebacterial promoter consensus TTTAT/AATA, is located 18 bases before the probable site of transcription initiation, TGCACAT. There is a potential transcription termination site immediately downstream from the gene that consists of a relatively stable stem and loop structure followed by stretches of Tresidues.Key words: archaebacteria, thermoacidophile, rRNA sequence, rRNA secondary structure, promoter.

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