scholarly journals A refined set of rRNA-targeted oligonucleotide probes for in situ detection and quantification of ammonia-oxidizing bacteria

2020 ◽  
Author(s):  
Michael Lukumbuzya ◽  
Jannie Munk Kristensen ◽  
Katharina Kitzinger ◽  
Andreas Pommerening-Röser ◽  
Per Halkjær Nielsen ◽  
...  
Keyword(s):  
16S Rrna ◽  
Rrna Gene ◽  
Ammonia Oxidizing Bacteria ◽  
Oligonucleotide Probes ◽  
Cross Hybridization ◽  
Approaches To Study ◽  
In Situ Detection ◽  
Probe Set ◽  
Weak Target

AbstractAmmonia-oxidizing bacteria (AOB) of the betaproteobacterial genera Nitrosomonas and Nitrosospira are key nitrifying microorganisms in many natural and engineered ecosystems. Since many AOB remain uncultured, fluorescence in situ hybridization (FISH) with rRNA-targeted oligonucleotide probes has been one of the most widely used approaches to study the community composition, abundance, and other features of AOB directly in environmental samples. However, the established and widely used AOB-specific 16S rRNA-targeted FISH probes were designed up to two decades ago, based on much smaller rRNA gene sequence datasets than available today. Several of these probes cover their target AOB lineages incompletely and suffer from a weak target specificity, which causes cross-hybridization of probes that should detect different AOB lineages. Here, a set of new highly specific 16S rRNA-targeted oligonucleotide probes was developed and experimentally evaluated that complements the existing probes and enables the specific detection and differentiation of the known, major phylogenetic clusters of betaproteobacterial AOB. The new probes were successfully applied to visualize and quantify AOB in activated sludge and biofilm samples from seven pilot- and full-scale wastewater treatment systems. Based on its improved target group coverage and specificity, the refined probe set will facilitate future in situ analyses of AOB.

scholarly journals A refined set of rRNA-targeted oligonucleotide probes for in situ detection and quantification of ammonia-oxidizing bacteria

2020 ◽  
Vol 186 ◽  
pp. 116372
Author(s):  
Michael Lukumbuzya ◽  
Jannie Munk Kristensen ◽  
Katharina Kitzinger ◽  
Andreas Pommerening-Röser ◽  
Per Halkjær Nielsen ◽  
...  
Keyword(s):  
Ammonia Oxidizing Bacteria ◽  
Oligonucleotide Probes ◽  
In Situ Detection ◽  
Detection And Quantification

scholarly journals Phylogenetic Characterization and In Situ Detection of a Cytophaga-Flexibacter-Bacteroides Phylogroup Bacterium in Tuber borchii Vittad. Ectomycorrhizal Mycelium

2000 ◽  
Vol 66 (11) ◽  
pp. 5035-5042 ◽  
Author(s):  
Elena Barbieri ◽  
Lucia Potenza ◽  
Ismaela Rossi ◽  
Davide Sisti ◽  
Giovanna Giomaro ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rdna ◽  
Direct Sequencing ◽  
Phylogenetic Analyses ◽  
Mycorrhizal Fungus ◽  
Rrna Gene ◽  
Small Subunit Rrna ◽  
Tuber Borchii ◽  
In Situ Detection

ABSTRACT Mycorrhizal ascomycetous fungi are obligate ectosymbionts that colonize the roots of gymnosperms and angiosperms. In this paper we describe a straightforward approach in which a combination of morphological and molecular methods was used to survey the presence of potentially endo- and epiphytic bacteria associated with the ascomycetous ectomycorrhizal fungus Tuber borchii Vittad. Universal eubacterial primers specific for the 5′ and 3′ ends of the 16S rRNA gene (16S rDNA) were used for PCR amplification, direct sequencing, and phylogenetic analyses. The 16S rDNA was amplified directly from four pure cultures of T. borchii Vittad. mycelium. A nearly full-length sequence of the gene coding for the prokaryotic small-subunit rRNA was obtained from each T. borchii mycelium studied. The 16S rDNA sequences were almost identical (98 to 99% similarity), and phylogenetic analysis placed them in a single unique rRNA branch belonging to theCytophaga-Flexibacter-Bacteroides (CFB) phylogroup which had not been described previously. In situ detection of the CFB bacterium in the hyphal tissue of the fungus T. borchii was carried out by using 16S rRNA-targeted oligonucleotide probes for the eubacterial domain and the Cytophaga-Flexibacter phylum, as well as a probe specifically designed for the detection of this mycelium-associated bacterium. Fluorescent in situ hybridization showed that all three of the probes used bound to the mycelium tissue. This study provides the first direct visual evidence of a not-yet-cultured CFB bacterium associated with a mycorrhizal fungus of the genusTuber.

scholarly journals Emended description of the species Lampropedia hyalina

2004 ◽  
Vol 54 (5) ◽  
pp. 1709-1715 ◽  
Author(s):  
Natuschka Lee ◽  
Carmela Maria Cellamare ◽  
Cristiano Bastianutti ◽  
Ramon Rosselló-Mora ◽  
Peter Kämpfer ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Distinct Group ◽  
23S Rrna ◽  
Molecular Characteristics ◽  
Rrna Gene ◽  
Oligonucleotide Probes ◽  
Metabolic Characteristics ◽  
Gene Similarity

Three Lampropedia hyalina strains from different habitats were compared by phenotypic, chemotaxonomic and molecular characteristics. All strains form coccoid cells and have been reported to grow as square tablets of eight to 64 cells. However, two of these strains (ATCC 11041T and ATCC 43383) have apparently lost this ability, and the third strain may temporarily lose this capacity under certain cultivation conditions. The three strains showed only minor differences in metabolic characteristics: the main significant physiological difference was the ability to accumulate polyphosphate under alternating anaerobic–aerobic conditions found for DSM 15336. The three strains showed high similarity in fatty acid composition and only slight differences in the G+C content (63–67 mol%) and DNA–DNA reassociation (90–95 % relatedness). Comparative 16S rRNA gene sequence analyses on these three strains and three Lampropedia hyalina 16S rRNA gene sequences deposited at NCBI showed that they are all very similar (>98·8 %) and that they form a distinct group among the ‘Betaproteobacteria’, showing between 94·6 and 93 % 16S rRNA gene similarity to members of various genera such as Acidovorax, Aquaspirillum, Brachymonas, Comamonas, Delftia and Xenophilus. Fluorescent in situ hybridization with oligonucleotide probes targeting betaproteobacteria on the 16S rRNA and 23S rRNA gene level further supported the conclusion that all investigated strains are members of the ‘Betaproteobacteria’. Two oligonucleotide probes were designed and successfully applied for culture-independent identification of Lampropedia hyalina by means of fluorescent in situ hybridization.

scholarly journals Quantification of Different Eubacteriumspp. in Human Fecal Samples with Species-Specific 16S rRNA-Targeted Oligonucleotide Probes

2000 ◽  
Vol 66 (1) ◽  
pp. 375-382 ◽  
Author(s):  
Andreas Schwiertz ◽  
Gwenaelle Le Blay ◽  
Michael Blaut
Keyword(s):  
16S Rrna ◽  
Intestinal Bacteria ◽  
Dry Weight ◽  
Oligonucleotide Probes ◽  
Cross Hybridization ◽  
Fecal Samples ◽  
Cell Counts ◽  
Species Specific ◽  
Type Strains

ABSTRACTSpecies-specific 16S rRNA-targeted, Cy3 (indocarbocyanine)-labeled oligonucleotide probes were designed and validated to quantify differentEubacteriumspecies in human fecal samples. Probes were directed atEubacterium barkeri,E. biforme,E. contortum,E. cylindroides(two probes),E. dolichum,E. hadrum,E. lentum,E. limosum,E. moniliforme, andE. ventriosum. The specificity of the probes was tested with the type strains and a range of common intestinal bacteria. With one exception, none of the probes showed cross-hybridization under stringent conditions. The species-specific probes were applied to fecal samples obtained from 12 healthy volunteers.E. biforme,E. cylindroides,E. hadrum,E. lentum, andE. ventriosumcould be determined. All otherEubacteriumspecies for which probes had been designed were under the detection limit of 107cells g (dry weight) of feces−1. The cell counts obtained are essentially in accordance with the literature data, which are based on colony counts. This shows that whole-cell in situ hybridization with species-specific probes is a valuable tool for the enumeration ofEubacteriumspecies in feces.

scholarly journals Microbiological and Geochemical Heterogeneity in an In Situ Uranium Bioremediation Field Site

2005 ◽  
Vol 71 (10) ◽  
pp. 6308-6318 ◽  
Author(s):  
Helen A. Vrionis ◽  
Robert T. Anderson ◽  
Irene Ortiz-Bernad ◽  
Kathleen R. O'Neill ◽  
Charles T. Resch ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Acid Volatile Sulfide ◽  
Rrna Gene ◽  
Gene Sequences ◽  
16S Rrna Gene Sequences ◽  
Geochemical Heterogeneity

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.

Rapid detection of ammonia-oxidizing bacteria in activated sludge based on 16S-rRNA gene by using PCR and fluorometry

2002 ◽  
Vol 7 (5) ◽  
pp. 323-326
Author(s):  
Motohiko Hikuma ◽  
Masanori Nakajima ◽  
Toshiaki Hirai ◽  
Hiroshi Matsuoka
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Activated Sludge ◽  
Rapid Detection ◽  
Rrna Gene ◽  
Ammonia Oxidizing Bacteria

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

2006 ◽  
Vol 72 (5) ◽  
pp. 3489-3497 ◽  
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.

scholarly journals In Situ Characterization ofNitrospira-Like Nitrite-Oxidizing Bacteria Active in Wastewater Treatment Plants

2001 ◽  
Vol 67 (11) ◽  
pp. 5273-5284 ◽  
Author(s):  
Holger Daims ◽  
Jeppe L. Nielsen ◽  
Per H. Nielsen ◽  
Karl-Heinz Schleifer ◽  
Michael Wagner
Keyword(s):  
Wastewater Treatment ◽  
16S Rrna ◽  
Laser Scanning ◽  
Wastewater Treatment Plants ◽  
Carbon Sources ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Rrna Gene ◽  
Phylogenetic Affiliation

ABSTRACT Uncultivated Nitrospira-like bacteria in different biofilm and activated-sludge samples were investigated by cultivation-independent molecular approaches. Initially, the phylogenetic affiliation of Nitrospira-like bacteria in a nitrifying biofilm was determined by 16S rRNA gene sequence analysis. Subsequently, a phylogenetic consensus tree of theNitrospira phylum including all publicly available sequences was constructed. This analysis revealed that the genusNitrospira consists of at least four distinct sublineages. Based on these data, two 16S rRNA-directed oligonucleotide probes specific for the phylum and genus Nitrospira, respectively, were developed and evaluated for suitability for fluorescence in situ hybridization (FISH). The probes were used to investigate the in situ architecture of cell aggregates ofNitrospira-like nitrite oxidizers in wastewater treatment plants by FISH, confocal laser scanning microscopy, and computer-aided three-dimensional visualization. Cavities and a network of cell-free channels inside the Nitrospiramicrocolonies were detected that were water permeable, as demonstrated by fluorescein staining. The uptake of different carbon sources byNitrospira-like bacteria within their natural habitat under different incubation conditions was studied by combined FISH and microautoradiography. Under aerobic conditions, theNitrospira-like bacteria in bioreactor samples took up inorganic carbon (as HCO3 − or as CO2) and pyruvate but not acetate, butyrate, and propionate, suggesting that these bacteria can grow mixotrophically in the presence of pyruvate. In contrast, no uptake by theNitrospira-like bacteria of any of the carbon sources tested was observed under anoxic or anaerobic conditions.

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