scholarly journals In Situ Detection of Novel Bacterial Endosymbionts of Acanthamoeba spp. Phylogenetically Related to Members of the Order Rickettsiales

1999 ◽  
Vol 65 (1) ◽  
pp. 206-212 ◽  
Author(s):  
Thomas R. Fritsche ◽  
Matthias Horn ◽  
Seyedreza Seyedirashti ◽  
Romesh K. Gautom ◽  
Karl-Heinz Schleifer ◽  
...  
Keyword(s):  
16S Rdna ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Rrna Gene ◽  
Oligonucleotide Probes ◽  
Gram Negative ◽  
Rdna Sequences ◽  
16S Rdna Sequences ◽  
Bacterial Endosymbionts

ABSTRACT Acanthamoebae are ubiquitous soil and water bactivores which may serve as amplification vehicles for a variety of pathogenic facultative bacteria and as hosts to other, presently uncultured bacterial endosymbionts. The spectrum of uncultured endosymbionts includes gram-negative rods and gram-variable cocci, the latter recently shown to be members of the Chlamydiales. We report here the isolation from corneal scrapings of two Acanthamoebastrains that harbor gram-negative rod endosymbionts that could not be cultured by standard techniques. These bacteria were phylogenetically characterized following amplification and sequencing of the near-full-length 16S rRNA gene. We used two fluorescently labelled oligonucleotide probes targeting signature regions within the retrieved sequences to detect these organisms in situ. Phylogenetic analyses demonstrated that they displayed 99.6% sequence similarity and formed an independent and well-separated lineage within theRickettsiales branch of the alpha subdivision of theProteobacteria. Nearest relatives included members of the genus Rickettsia, with sequence similarities of approximately 85 to 86%, suggesting that these symbionts are representatives of a new genus and, perhaps, family. Distance matrix, parsimony, and maximum-likelihood tree-generating methods all consistently supported deep branching of the 16S rDNA sequences within the Rickettsiales. The oligonucleotide probes displayed at least three mismatches to all other available 16S rDNA sequences, and they both readily permitted the unambiguous detection of rod-shaped bacteria within intact acanthamoebae by confocal laser-scanning microscopy. Considering the long-standing relationship of mostRickettsiales with arthropods, the finding of a related lineage of endosymbionts in protozoan hosts was unexpected and may have implications for the preadaptation and/or recruitment of rickettsia-like bacteria to metazoan hosts.

scholarly journals Development of rRNA-Targeted PCR and In Situ Hybridization with Fluorescently Labelled Oligonucleotides for Detection of Yersinia Species

1998 ◽  
Vol 36 (9) ◽  
pp. 2557-2564 ◽  
Author(s):  
Karlheinz Trebesius ◽  
Dag Harmsen ◽  
Alexander Rakin ◽  
Jochen Schmelz ◽  
Jürgen Heesemann
Keyword(s):  
In Situ Hybridization ◽  
Laser Scanning Microscopy ◽  
Mouse Tissue ◽  
23S Rrna ◽  
Confocal Laser ◽  
Rrna Gene ◽  
Oligonucleotide Probes ◽  
Detection Techniques ◽  
Rdna Sequences

In this report, we present details of two rapid molecular detection techniques based on 16S and 23S rRNA sequence data to identify and differentiate Yersinia species from clinical and environmental sources. Near-full-length 16S rRNA gene (rDNA) sequences for three different Yersinia species and partial 23S rDNA sequences for three Y. pestis and three Y. pseudotuberculosis strains were determined. While 16S rDNA sequences of Y. pestis and Y. pseudotuberculosis were found to be identical, one base difference was identified within a highly variable region of 23S rDNA. The rDNA sequences were used to develop primers and fluorescently tagged oligonucleotide probes suitable for differential detection ofYersinia species by PCR and in situ hybridization, respectively. As few as 102 Yersinia cells per ml could be detected by PCR with a seminested approach. Amplification with a subgenus-specific primer pair followed by a second PCR allowed differentiation of Y. enterocolitica biogroup 1B from biogroups 2 to 5 or from other pathogenic Yersinia species. Moreover, a set of oligonucleotide probes suitable for rapid (3-h) in situ detection and differentiation of the three pathogenicYersinia species (in particular Y. pestis andY. pseudotuberculosis) was developed. The applicability of this technique was demonstrated by detection of Y. pestisand Y. pseudotuberculosis in spiked throat and stool samples, respectively. These probes were also capable of identifyingY. enterocolitica within cryosections of experimentally infected mouse tissue by the use of confocal laser scanning microscopy.

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.

scholarly journals Phenotypic characterization of Astragalus glycyphyllos symbionts and their phylogeny based on the 16S rDNA sequences and RFLP of 16S rRNA gene

2014 ◽  
Vol 105 (6) ◽  
pp. 1033-1048 ◽  
Author(s):  
Sebastian Gnat ◽  
Magdalena Wójcik ◽  
Sylwia Wdowiak-Wróbel ◽  
Michał Kalita ◽  
Aneta Ptaszyńska ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
16S Rdna ◽  
Phenotypic Characterization ◽  
Rrna Gene ◽  
Rdna Sequences ◽  
16S Rdna Sequences ◽  
Astragalus Glycyphyllos Symbionts

Phylogenetic placement of unculturedCeanothusmicrosymbionts using 16S rRNA gene sequences

1999 ◽  
Vol 77 (9) ◽  
pp. 1208-1213 ◽  
Author(s):  
Nancy J Ritchie ◽  
David D Myrold
Keyword(s):  
16S Rdna ◽  
Phylogenetic Trees ◽  
Full Length ◽  
Likelihood Method ◽  
Rrna Gene ◽  
Consensus Sequences ◽  
Rdna Sequences ◽  
16S Rdna Sequences ◽  
Polymerase Chain ◽  
Ceanothus Americanus

Full-length 16S rDNA sequences were amplified directly from the nodules of Ceanothus americanus L. and Ceanothus thyrsiflorus Eschsch. using the polymerase chain reaction. Sequences were determined using an automated sequencer, compared against those in GenBank, and assembled into consensus sequences. The sequences were aligned with other full-length Frankia 16S rDNA sequences available from the data base. Phylogenetic trees were obtained using three different algorithms: neighbor joining, parsimony, and the maximum-likelihood method. All three methods showed that these Ceanothus L. microsymbionts were most closely related to the microsymbiont associated with Dryas drummondii Richardson ex Hook. Lvs. rather than Frankia isolated from the Elaeagnaceae.Key words: Frankia, Ceanothus, 16S rDNA.

scholarly journals Isolation, Characterization, and In Situ Detection of a Novel Chemolithoautotrophic Sulfur-Oxidizing Bacterium in Wastewater Biofilms Growing under Microaerophilic Conditions

2004 ◽  
Vol 70 (5) ◽  
pp. 3122-3129 ◽  
Author(s):  
Tsukasa Ito ◽  
Kenichi Sugita ◽  
Satoshi Okabe
Keyword(s):  
Laser Scanning ◽  
Elemental Sulfur ◽  
Sulfur Cycle ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Rrna Gene ◽  
Effective Measure ◽  
Sulfur Oxidizing ◽  
Microaerophilic Conditions

ABSTRACT We successfully isolated a novel aerobic chemolithotrophic sulfur-oxidizing bacterium, designated strain SO07, from wastewater biofilms growing under microaerophilic conditions. For isolation, the use of elemental sulfur (S0), which is the most abundant sulfur pool in the wastewater biofilms, as the electron donor was an effective measure to establish an enrichment culture of strain SO07 and further isolation. 16S rRNA gene sequence analysis revealed that newly isolated strain SO07 was affiliated with members of the genus Halothiobacillus, but it was only distantly related to previously isolated species (89% identity). Strain SO07 oxidized elemental sulfur, thiosulfate, and sulfide to sulfate under oxic conditions. Strain SO07 could not grow on nitrate. Organic carbons, including acetate, propionate, and formate, could not serve as carbon and energy sources. Unlike other aerobic sulfur-oxidizing bacteria, this bacterium was sensitive to NaCl; growth in medium containing more than 150 mM was negligible. In situ hybridization combined with confocal laser scanning microscopy revealed that a number of rod-shaped cells hybridized with a probe specific for strain SO07 were mainly present in the oxic biofilm strata (ca. 0 to 100 μm) and that they often coexisted with sulfate-reducing bacteria in this zone. These results demonstrated that strain SO07 was one of the important sulfur-oxidizing populations involved in the sulfur cycle occurring in the wastewater biofilm and was primarily responsible for the oxidation of H2S and S0 to SO4 2− under oxic conditions.

scholarly journals Gram-Negative, Oxidase-Positive Bacteria in Rainwater and Wind Samples

Fine Focus ◽  
2016 ◽  
Vol 2 (2) ◽  
pp. 92-103
Author(s):  
Kathryn E. Preston ◽  
Jennifer Huddlestone
Keyword(s):  
Microbial Communities ◽  
16S Rdna ◽  
Opportunistic Infections ◽  
Novel Species ◽  
Human Pathogens ◽  
Gram Negative ◽  
Biological Particles ◽  
Rdna Sequences ◽  
16S Rdna Sequences

Gram-negative, rod-shaped, oxidase-positive bacteria, such as Aeromonas and Pseduomonas, are widespread in the environment. Aeromonas are emerging human pathogens associated with extraintestinal and opportunistic infections. Though there are various biological particles known in the atmosphere, these microbial communities are poorly characterized. Bacteria have the ability to remain suspended in the air for prolonged periods of time and can be transmitted through both airborne and droplet means. This study aimed to isolate Aeromonas and other similar bacteria from samples from the troposphere in order to learn more about the distribution of these organisms. Eleven precipitation and wind samples were aseptically collected in Abilene, Texas, and plated onto Aeromonas Blue Medium plates with and without ampicillin. The 16S rDNA sequences were amplified from 28 Gram-negative, oxidase-positive isolates. The analyzed sequences showed that none of the isolates belonged to the genus Aeromonas, but did include Pseudomonas, Sphingomonas, Massilia, Naxibacter, Paracoccus, Novosphingobium, and Mesorhizobium, giving clues to the distribution of these organisms. Furthermore, six isolates appeared to be novel species of bacteria, and several more were uncultured before this study.

Towards understanding the taxonomy of some of the filamentous bacteria causing bulking and foaming in activated sludge plants

1996 ◽  
Vol 34 (5-6) ◽  
pp. 137-144 ◽  
Author(s):  
Linda L. Blackall ◽  
Elizabeth M. Seviour ◽  
Debbie Bradford ◽  
Helen M. Stratton ◽  
Mitchell A. Cunningham ◽  
...  
Keyword(s):  
Activated Sludge ◽  
16S Rdna ◽  
Phylogenetic Relationship ◽  
Pure Culture ◽  
Gram Negative Bacteria ◽  
Filamentous Bacteria ◽  
Gram Negative ◽  
Rdna Sequences ◽  
16S Rdna Sequences ◽  
Microthrix Parvicella

Several filamentous bacteria obtained in pure culture from activated sludge plants were characterised using their 16S rDNA sequences to determine their phylogenetic relationship to other bacteria. “Microthrix parvicella” was shown to be an unusual actinomycete, while the Gram negative bacteria Type 0092, Type 0411 and Type 1863 all belong to the Flexibacter-Cytophaga-Bacteroides phylum, and Type 0803 is a member of the beta subclass of the Proteobacteria. The practical value of obtaining this information is discussed.

scholarly journals A Straightforward DOPE (Double Labeling of Oligonucleotide Probes)-FISH (FluorescenceIn SituHybridization) Method for Simultaneous Multicolor Detection of Six Microbial Populations

2012 ◽  
Vol 78 (15) ◽  
pp. 5138-5142 ◽  
Author(s):  
Faris Behnam ◽  
Andreas Vilcinskas ◽  
Michael Wagner ◽  
Kilian Stoecker
Keyword(s):  
Laser Scanning ◽  
Simultaneous Detection ◽  
Laser Scanning Microscopy ◽  
Clinical Samples ◽  
Confocal Laser ◽  
Oligonucleotide Probes ◽  
Double Labeling ◽  
Attractive Option ◽  
Higher Sensitivity

ABSTRACTFluorescencein situhybridization (FISH) with rRNA-targeted oligonucleotide probes is an essential tool for the cultivation-independent identification of microbes within environmental and clinical samples. However, one of the major constraints of conventional FISH is the very limited number of different target organisms that can be detected simultaneously with standard epifluorescence or confocal laser scanning microscopy. Recently, this limitation has been overcome via an elegant approach termed combinatorial labeling and spectral imaging FISH (CLASI-FISH) (23). This technique, however, suffers compared to conventional FISH from an inherent loss in sensitivity and potential probe binding biases caused by the competition of two differentially labeled oligonucleotide probes for the same target site. Here we demonstrate that the application of multicolored, double-labeled oligonucleotide probes enables the simultaneous detection of up to six microbial target populations in a straightforward and robust manner with higher sensitivity and less bias. Thus, this newly developed technique should be an attractive option for all researchers interested in applying conventional FISH methods for the study of microbial communities.

scholarly journals Niche-Partitioning of ProchlorococcusPopulations in a Stratified Water Column in the Eastern North Atlantic Ocean

1999 ◽  
Vol 65 (6) ◽  
pp. 2585-2591 ◽  
Author(s):  
Nyree J. West ◽  
David J. Scanlan
Keyword(s):  
Water Column ◽  
16S Rdna ◽  
North Atlantic ◽  
North Atlantic Ocean ◽  
Niche Partitioning ◽  
Rdna Sequences ◽  
16S Rdna Sequences ◽  
Eastern North Atlantic ◽  
Eastern North Atlantic Ocean

ABSTRACT The in situ community structure of Prochlorococcuspopulations in the eastern North Atlantic Ocean was examined by analysis of Prochlorococcus 16S rDNA sequences with three independent approaches: cloning and sequencing, hybridization to specific oligonucleotide probes, and denaturing gradient gel electrophoresis (DGGE). The hybridization of high-light (HL) and low-light (LL) Prochlorococcus genotype-specific probes to two depth profiles of PCR-amplified 16S rDNA sequences revealed that in these two stratified water columns, an obvious niche-partitioning ofProchlorococcus genotypes occurred. In each water column a shift from the HL to the LL genotype was observed, a transition correlating with the depth of the surface mixed layer (SML). Only the HL genotype was found in the SML in each water column, whereas the LL genotype was distributed below the SML. The range of in situ irradiance to which each genotype was subjected within these distinct niches was consistent with growth irradiance studies of cultured HL- and LL-adapted Prochlorococcus strains. DGGE analysis and the sequencing of Prochlorococcus 16S rDNA clones were in full agreement with the genotype-specific oligonucleotide probe hybridization data. These observations of a partitioning ofProchlorococcus genotypes in a stratified water column provide a genetic basis for the dim and brightProchlorococcus populations observed in flow cytometric signatures in several oceanic provinces.

scholarly journals Phylogenetic Analysis of Nonthermophilic Members of the Kingdom Crenarchaeota and Their Diversity and Abundance in Soils

1998 ◽  
Vol 64 (11) ◽  
pp. 4333-4339 ◽  
Author(s):  
Daniel H. Buckley ◽  
Joseph R. Graber ◽  
Thomas M. Schmidt
Keyword(s):  
16S Rrna ◽  
16S Rdna ◽  
Phylogenetic Analyses ◽  
Pcr Primers ◽  
Soil Samples ◽  
Molecular Techniques ◽  
Rrna Gene ◽  
Rdna Sequences ◽  
16S Rdna Sequences ◽  
Long Term Ecological Research

ABSTRACT Within the last several years, molecular techniques have uncovered numerous 16S rRNA gene (rDNA) sequences which represent a unique and globally distributed lineage of the kingdom Crenarchaeotathat is phylogenetically distinct from currently characterized crenarchaeotal species. rDNA sequences of members of this novel crenarchaeotal group have been recovered from low- to moderate-temperature environments (−1.5 to 32°C), in contrast to the high-temperature environments (temperature, >80°C) required for growth of the currently recognized crenarchaeotal species. We determined the diversity and abundance of the nonthermophilic members of the Crenarchaeota in soil samples taken from cultivated and uncultivated fields located at the Kellogg Biological Station’s Long-Term Ecological Research site (Hickory Corners, Mich.). Clones were generated from 16S rDNA that was amplified by using broad-specificity archaeal PCR primers. Twelve crenarchaeotal sequences were identified, and the phylogenetic relationships between these sequences and previously described crenarchaeotal 16S rDNA sequences were determined. Phylogenetic analyses included nonthermophilic crenarchaeotal sequences found in public databases and revealed that the nonthermophilic Crenarchaeota group is composed of at least four distinct phylogenetic clusters. A 16S rRNA-targeted oligonucleotide probe specific for all known nonthermophilic crenarchaeotal sequences was designed and used to determine their abundance in soil samples. The nonthermophilicCrenarchaeota accounted for as much as 1.42% ± 0.42% of the 16S rRNA in the soils analyzed.

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