Endosymbiotic Bacteria in the Parasitic Ciliate Ichthyophthirius multifiliis

ABSTRACT Endosymbiotic bacteria were identified in the parasitic ciliate Ichthyophthirius multifiliis, a common pathogen of freshwater fish. PCR amplification of DNA prepared from two isolates of I. multifiliis, using primers that bind conserved sequences in bacterial 16S rRNA genes, generated an ∼1,460-bp DNA product, which was cloned and sequenced. Sequence analysis demonstrated that 16S rRNA gene sequences from three classes of bacteria were present in the PCR product. These included Alphaproteobacteria (Rickettsiales), Sphingobacteria, and Flavobacterium columnare. DAPI (4′,6-diamidino-2-phenylindole) staining showed endosymbionts dispersed throughout the cytoplasm of trophonts and, in most, but not all theronts. Endosymbionts were observed by transmission electron microscopy in the cytoplasm, surrounded by a prominent, electron-translucent halo characteristic of Rickettsia. Fluorescence in situ hybridization demonstrated that bacteria from the Rickettsiales and Sphingobacteriales classes are endosymbionts of I. multifiliis, found in the cytoplasm, but not in the macronucleus or micronucleus. In contrast, F. columnare was not detected by fluorescence in situ hybridization. It likely adheres to I. multifiliis through association with cilia. The role that endosymbiotic bacteria play in the life history of I. multifiliis is not known.

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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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α-Proteobacterial Symbionts of Marine Bryozoans in the Genus Watersipora

Applied and Environmental Microbiology ◽

10.1128/aem.00604-06 ◽

2006 ◽

Vol 73 (1) ◽

pp. 303-311 ◽

Cited By ~ 33

Author(s):

Christine M. Anderson ◽

Margo G. Haygood

Keyword(s):

Phylogenetic Analysis ◽

In Situ Hybridization ◽

16S Rrna ◽

Fluorescence In Situ Hybridization ◽

16S Rrna Genes ◽

Rrna Genes ◽

Bacterial Symbionts ◽

California Coast ◽

Specific Fluorescence

ABSTRACT Bacterial symbionts that resembled mollicutes were discovered in the marine bryozoan Watersipora arcuata in the 1980s. In this study, we used PCR and sequencing of 16S rRNA genes, specific fluorescence in situ hybridization, and phylogenetic analysis to determine that the bacterial symbionts of “W. subtorquata” and “W. arcuata” from several locations along the California coast are actually closely related α-Proteobacteria, not mollicutes. We propose the names “Candidatus Endowatersipora palomitas” and “Candidatus Endowatersipora rubus” for the symbionts of “W. subtorquata” and “W. arcuata,” respectively.

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

Applied and Environmental Microbiology ◽

10.1128/aem.71.10.6308-6318.2005 ◽

2005 ◽

Vol 71 (10) ◽

pp. 6308-6318 ◽

Cited By ~ 172

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.

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Cultivation-Independent Examination of Horizontal Transfer and Host Range of an IncP-1 Plasmid among Gram-Positive and Gram-Negative Bacteria Indigenous to the Barley Rhizosphere

Applied and Environmental Microbiology ◽

10.1128/aem.00013-06 ◽

2006 ◽

Vol 72 (10) ◽

pp. 6687-6692 ◽

Cited By ~ 67

Author(s):

Sanin Musovic ◽

Gunnar Oregaard ◽

Niels Kroer ◽

Søren J. Sørensen

Keyword(s):

16S Rrna ◽

Host Range ◽

16S Rrna Genes ◽

Rrna Genes ◽

Rrna Gene ◽

Gram Positive ◽

Gram Negative ◽

Indigenous Bacteria ◽

Transfer Frequency

ABSTRACTThe host range and transfer frequency of an IncP-1 plasmid (pKJK10) among indigenous bacteria in the barley rhizosphere was investigated. A new flow cytometry-based cultivation-independent method for enumeration and sorting of transconjugants for subsequent 16S rRNA gene classification was used. Indigenous transconjugant rhizosphere bacteria were collected by fluorescence-activated cell sorting and identified by cloning and sequencing of 16S rRNA genes from the sorted cells. The host range of the pKJK10 plasmid was exceptionally broad, as it included not only bacteria belonging to the alpha, beta, and gamma subclasses of theProteobacteria, but alsoArthrobactersp., a gram-positive member of theActinobacteria. The transfer frequency (transconjugants per donor) from thePseudomonas putidadonor to the indigenous bacteria was 7.03 × 10−2± 3.84 × 10−2. This is the first direct documentation of conjugal transfer between gram-negative donor and gram-positive recipient bacteria in situ.

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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

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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.

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Detection of WWE2-relatedLentisphaeraeby 16S rRNA gene sequencing and fluorescence in situ hybridization in landfill leachate

Canadian Journal of Microbiology ◽

10.1139/w10-065 ◽

2010 ◽

Vol 56 (10) ◽

pp. 846-852 ◽

Cited By ~ 6

Author(s):

Rim Driss Limam ◽

Théodore Bouchez ◽

Rakia Chouari ◽

Tianlun Li ◽

Insaf Barkallah ◽

...

Keyword(s):

In Situ Hybridization ◽

16S Rrna ◽

16S Rrna Gene ◽

Fluorescence In Situ Hybridization ◽

Landfill Leachate ◽

16S Rrna Gene Sequencing ◽

Rrna Gene ◽

Rrna Gene Sequencing ◽

Victivallis Vadensis

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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