Unexpected Diversity and Complexity of the Guerrero Negro Hypersaline Microbial Mat

ABSTRACT We applied nucleic acid-based molecular methods, combined with estimates of biomass (ATP), pigments, and microelectrode measurements of chemical gradients, to map microbial diversity vertically on a millimeter scale in a hypersaline microbial mat from Guerrero Negro, Baja California Sur, Mexico. To identify the constituents of the mat, small-subunit rRNA genes were amplified by PCR from community genomic DNA extracted from layers, cloned, and sequenced. Bacteria dominated the mat and displayed unexpected and unprecedented diversity. The majority (1,336) of the 1,586 bacterial 16S rRNA sequences generated were unique, representing 752 species (≥97% rRNA sequence identity) in 42 of the main bacterial phyla, including 15 novel candidate phyla. The diversity of the mat samples differentiated according to the chemical milieu defined by concentrations of O2 and H2S. Bacteria of the phylum Chloroflexi formed the majority of the biomass by percentage of bulk rRNA and of clones in rRNA gene libraries. This result contradicts the general belief that cyanobacteria dominate these communities. Although cyanobacteria constituted a large fraction of the biomass in the upper few millimeters (>80% of the total rRNA and photosynthetic pigments), Chloroflexi sequences were conspicuous throughout the mat. Filamentous Chloroflexi bacteria were identified by fluorescence in situ hybridization within the polysaccharide sheaths of the prominent cyanobacterium Microcoleus chthonoplastes, in addition to free living in the mat. The biological complexity of the mat far exceeds that observed in other polysaccharide-rich microbial ecosystems, such as the human and mouse distal guts, and suggests that positive feedbacks exist between chemical complexity and biological diversity.

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Abundant and Diverse Fungal Microbiota in the Murine Intestine

Applied and Environmental Microbiology ◽

10.1128/aem.72.1.793-801.2006 ◽

2006 ◽

Vol 72 (1) ◽

pp. 793-801 ◽

Cited By ~ 113

Author(s):

Alexandra J Scupham ◽

Laura L. Presley ◽

Bo Wei ◽

Elizabeth Bent ◽

Natasha Griffith ◽

...

Keyword(s):

Small Subunit ◽

Rrna Genes ◽

Rrna Gene ◽

Small Subunit Rrna ◽

Culture Independent ◽

Specific Pathogen ◽

Health And Disease ◽

Oligonucleotide Fingerprinting ◽

Rrna Sequences

ABSTRACT Enteric microbiota play a variety of roles in intestinal health and disease. While bacteria in the intestine have been broadly characterized, little is known about the abundance or diversity of enteric fungi. This study utilized a culture-independent method termed oligonucleotide fingerprinting of rRNA genes (OFRG) to describe the compositions of fungal and bacterial rRNA genes from small and large intestines (tissue and luminal contents) of restricted-flora and specific-pathogen-free mice. OFRG analysis identified rRNA genes from all four major fungal phyla: Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota. The largest assemblages of fungal rRNA sequences were related to the genera Acremonium, Monilinia, Fusarium, Cryptococcus/Filobasidium, Scleroderma, Catenomyces, Spizellomyces, Neocallimastix, Powellomyces, Entophlyctis, Mortierella, and Smittium and the order Mucorales. The majority of bacterial rRNA gene clones were affiliated with the taxa Bacteroidetes, Firmicutes, Acinetobacter, and Lactobacillus. Sequence-selective PCR analyses also detected several of these bacterial and fungal rRNA genes in the mouse chow. Fluorescence in situ hybridization analysis with a fungal small-subunit rRNA probe revealed morphologically diverse microorganisms resident in the mucus biofilm adjacent to the cecal and proximal colonic epithelium. Hybridizing organisms comprised about 2% of the DAPI (4′,6-diamidino-2-phenylindole, dihydrochloride)-positive organisms in the mucus biofilm, but their abundance in fecal material may be much lower. These data indicate that diverse fungal taxa are present in the intestinal microbial community. Their abundance suggests that they may play significant roles in enteric microbial functions.

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Redescriptions of three trachelocercid ciliates (Protista, Ciliophora, Karyorelictea), with notes on their phylogeny based on small subunit rRNA gene sequences

INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY ◽

10.1099/ijs.0.053959-0 ◽

2013 ◽

Vol 63 (Pt_9) ◽

pp. 3506-3514 ◽

Cited By ~ 9

Author(s):

Ying Yan ◽

Yuan Xu ◽

Zhenzhen Yi ◽

Alan Warren

Keyword(s):

Sequence Data ◽

Phylogenetic Analyses ◽

Small Subunit ◽

Rrna Genes ◽

Rrna Gene ◽

Ssu Rrna ◽

Small Subunit Rrna ◽

Small Subunit Rrna Gene ◽

Ssu Rrna Gene Sequence ◽

First Time

Three trachelocercid ciliates, Kovalevaia sulcata (Kovaleva, 1966) Foissner, 1997, Trachelocerca sagitta (Müller, 1786) Ehrenberg, 1840 and Trachelocerca ditis (Wright, 1982) Foissner, 1996, isolated from two coastal habitats at Qingdao, China, were investigated using live observation and silver impregnation methods. Data on their infraciliature and morphology are supplied. The small subunit rRNA (SSU rRNA) genes of K. sulcata and Trachelocerca sagitta were sequenced for the first time. Phylogenetic analyses based on SSU rRNA gene sequence data indicate that both organisms, and the previously sequenced Trachelocerca ditis, are located within the trachelocercid assemblage and that K. sulcata is sister to an unidentified taxon forming a clade that is basal to the core trachelocercids.

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Regulation of Plasmodium yoelii Oocyst Development by Strain- and Stage-Specific Small-Subunit rRNA

mBio ◽

10.1128/mbio.00117-15 ◽

2015 ◽

Vol 6 (2) ◽

Cited By ~ 4

Author(s):

Yanwei Qi ◽

Feng Zhu ◽

Richard T. Eastman ◽

Young Fu ◽

Martine Zilversmit ◽

...

Keyword(s):

Developmental Stages ◽

Small Subunit ◽

Plasmodium Yoelii ◽

Rrna Genes ◽

Parasite Development ◽

Rrna Gene ◽

Small Subunit Rrna ◽

Malaria Parasites ◽

Content Type ◽

Small Subunit Rrna Gene

ABSTRACT One unique feature of malaria parasites is the differential transcription of structurally distinct rRNA (rRNA) genes at different developmental stages: the A-type genes are transcribed mainly in asexual stages, whereas the S-type genes are expressed mostly in sexual or mosquito stages. Conclusive functional evidence of different rRNAs in regulating stage-specific parasite development, however, is still absent. Here we performed genetic crosses of Plasmodium yoelii parasites with one parent having an oocyst development defect (ODD) phenotype and another producing normal oocysts to identify the gene(s) contributing to the ODD. The parent with ODD—characterized as having small oocysts and lacking infective sporozoites—was obtained after introduction of a plasmid with a green fluorescent protein gene into the parasite genome and subsequent passages in mice. Quantitative trait locus analysis of genome-wide microsatellite genotypes of 48 progeny from the crosses linked an ~200-kb segment on chromosome 6 containing one of the S-type genes (D-type small subunit rRNA gene [D-ssu]) to the ODD. Fine mapping of the plasmid integration site, gene expression pattern, and gene knockout experiments demonstrated that disruption of the D-ssu gene caused the ODD phenotype. Interestingly, introduction of the D-ssu gene into the same parasite strain (self), but not into a different subspecies, significantly affected or completely ablated oocyst development, suggesting a stage- and subspecies (strain)-specific regulation of oocyst development by D-ssu. This study demonstrates that P. yoelii D-ssu is essential for normal oocyst and sporozoite development and that variation in the D-ssu sequence can have dramatic effects on parasite development. IMPORTANCE Malaria parasites are the only known organisms that express structurally distinct rRNA genes at different developmental stages. The differential expression of these genes suggests that they play unique roles during the complex life cycle of the parasites. Conclusive functional proof of different rRNAs in regulating parasite development, however, is still absent or controversial. Here we functionally demonstrate for the first time that a stage-specifically expressed D-type small-subunit rRNA gene (D-ssu) is essential for oocyst development of the malaria parasite Plasmodium yoelii in the mosquito. This study also shows that variations in D-ssu sequence and/or the timing of transcription may have profound effects on parasite oocyst development. The results show that in addition to protein translation, rRNAs of malaria parasites also regulate parasite development and differentiation in a strain-specific manner, which can be explored for controlling parasite transmission.

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Single-cell genomics unveils a canonical origin of the diverse mitochondrial genomes of euglenozoans

BMC Biology ◽

10.1186/s12915-021-01035-y ◽

2021 ◽

Vol 19 (1) ◽

Author(s):

Kristína Záhonová ◽

Gordon Lax ◽

Savar D. Sinha ◽

Guy Leonard ◽

Thomas A. Richards ◽

...

Keyword(s):

Single Cell ◽

Small Subunit ◽

Rrna Genes ◽

Mitochondrial Genomes ◽

Trna Genes ◽

Rrna Gene ◽

Heterotrophic Flagellates ◽

Small Subunit Rrna ◽

Single Chromosome ◽

Linear Chromosomes

Abstract Background The supergroup Euglenozoa unites heterotrophic flagellates from three major clades, kinetoplastids, diplonemids, and euglenids, each of which exhibits extremely divergent mitochondrial characteristics. Mitochondrial genomes (mtDNAs) of euglenids comprise multiple linear chromosomes carrying single genes, whereas mitochondrial chromosomes are circular non-catenated in diplonemids, but circular and catenated in kinetoplastids. In diplonemids and kinetoplastids, mitochondrial mRNAs require extensive and diverse editing and/or trans-splicing to produce mature transcripts. All known euglenozoan mtDNAs exhibit extremely short mitochondrial small (rns) and large (rnl) subunit rRNA genes, and absence of tRNA genes. How these features evolved from an ancestral bacteria-like circular mitochondrial genome remains unanswered. Results We sequenced and assembled 20 euglenozoan single-cell amplified genomes (SAGs). In our phylogenetic and phylogenomic analyses, three SAGs were placed within kinetoplastids, 14 within diplonemids, one (EU2) within euglenids, and two SAGs with nearly identical small subunit rRNA gene (18S) sequences (EU17/18) branched as either a basal lineage of euglenids, or as a sister to all euglenozoans. Near-complete mitochondrial genomes were identified in EU2 and EU17/18. Surprisingly, both EU2 and EU17/18 mitochondrial contigs contained multiple genes and one tRNA gene. Furthermore, EU17/18 mtDNA possessed several features unique among euglenozoans including full-length rns and rnl genes, six mitoribosomal genes, and nad11, all likely on a single chromosome. Conclusions Our data strongly suggest that EU17/18 is an early-branching euglenozoan with numerous ancestral mitochondrial features. Collectively these data contribute to untangling the early evolution of euglenozoan mitochondria.

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Kinetic Bias in Estimates of Coastal Picoplankton Community Structure Obtained by Measurements of Small-Subunit rRNA Gene PCR Amplicon Length Heterogeneity

Applied and Environmental Microbiology ◽

10.1128/aem.64.11.4522-4529.1998 ◽

1998 ◽

Vol 64 (11) ◽

pp. 4522-4529 ◽

Cited By ~ 267

Author(s):

Marcelino Suzuki ◽

Michael S. Rappé ◽

Stephen J. Giovannoni

Keyword(s):

Clone Library ◽

Fluorescence Emission ◽

Small Subunit ◽

Ssu Rdna ◽

Rrna Genes ◽

Rrna Gene ◽

Length Variation ◽

Small Subunit Rrna ◽

Gene Frequencies ◽

Rdna Sequences

ABSTRACT Marine bacterioplankton diversity was examined by quantifying natural length variation in the 5′ domain of small-subunit (SSU) rRNA genes (rDNA) amplified by PCR from a DNA sample from the Oregon coast. This new technique, length heterogeneity analysis by PCR (LH-PCR), determines the relative proportions of amplicons originating from different organisms by measuring the fluorescence emission of a labeled primer used in the amplification reaction. Relationships between the sizes of amplicons and gene phylogeny were predicted by an analysis of 366 SSU rDNA sequences from cultivated marine bacteria and from bacterial genes cloned directly from environmental samples. LH-PCR was used to compare the distribution of bacterioplankton SSU rDNAs from a coastal water sample with that of an SSU rDNA clone library prepared from the same sample and also to examine the distribution of genes in the PCR products from which the clone library was prepared. The analysis revealed that the relative frequencies of genes amplified from natural communities are highly reproducible for replicate sets of PCRs but that a bias possibly caused by the reannealing kinetics of product molecules can skew gene frequencies when PCR product concentrations exceed threshold values.

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Study of Genetic Diversity of Eukaryotic Picoplankton in Different Oceanic Regions by Small-Subunit rRNA Gene Cloning and Sequencing

Applied and Environmental Microbiology ◽

10.1128/aem.67.7.2932-2941.2001 ◽

2001 ◽

Vol 67 (7) ◽

pp. 2932-2941 ◽

Cited By ~ 354

Author(s):

Beatriz Dı́ez ◽

Carlos PedrÃ¯Â¿Â½s-AliÃ¯Â¿Â½ ◽

Ramon Massana

Keyword(s):

Operational Taxonomic Unit ◽

Small Subunit ◽

Small Cells ◽

Rrna Genes ◽

Rrna Gene ◽

Polymorphism Analysis ◽

Small Subunit Rrna ◽

Phylogenetic Groups ◽

18S Rrna Genes ◽

A Minor

ABSTRACT Very small eukaryotic organisms (picoeukaryotes) are fundamental components of marine planktonic systems, often accounting for a significant fraction of the biomass and activity in a system. Their identity, however, has remained elusive, since the small cells lack morphological features for identification. We determined the diversity of marine picoeukaryotes by sequencing cloned 18S rRNA genes in five genetic libraries from North Atlantic, Southern Ocean, and Mediterranean Sea surface waters. Picoplankton were obtained by filter size fractionation, a step that excluded most large eukaryotes and recovered most picoeukaryotes. Genetic libraries of eukaryotic ribosomal DNA were screened by restriction fragment length polymorphism analysis, and at least one clone of each operational taxonomic unit (OTU) was partially sequenced. In general, the phylogenetic diversity in each library was rather great, and each library included many different OTUs and members of very distantly related phylogenetic groups. Of 225 eukaryotic clones, 126 were affiliated with algal classes, especially the Prasinophyceae, the Prymnesiophyceae, the Bacillariophyceae, and the Dinophyceae. A minor fraction (27 clones) was affiliated with clearly heterotrophic organisms, such as ciliates, the chrysomonad Paraphysomonas, cercomonads, and fungi. There were two relatively abundant novel lineages, novel stramenopiles (53 clones) and novel alveolates (19 clones). These lineages are very different from any organism that has been isolated, suggesting that there are previously unknown picoeukaryotes. Prasinophytes and novel stramenopile clones were very abundant in all of the libraries analyzed. These findings underscore the importance of attempts to grow the small eukaryotic plankton in pure culture.

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Phylogenetic Analysis of CryptosporidiumParasites Based on the Small-Subunit rRNA Gene Locus

Applied and Environmental Microbiology ◽

10.1128/aem.65.4.1578-1583.1999 ◽

1999 ◽

Vol 65 (4) ◽

pp. 1578-1583 ◽

Cited By ~ 439

Author(s):

Lihua Xiao ◽

Lillian Escalante ◽

Chunfu Yang ◽

Irshad Sulaiman ◽

Anannias A. Escalante ◽

...

Keyword(s):

Phylogenetic Analysis ◽

Distinct Species ◽

Small Subunit ◽

Biological Data ◽

Rrna Genes ◽

Recent Analysis ◽

Species Differentiation ◽

Rrna Gene ◽

Small Subunit Rrna ◽

Rock Hyrax

ABSTRACT Biological data support the hypothesis that there are multiple species in the genus Cryptosporidium, but a recent analysis of the available genetic data suggested that there is insufficient evidence for species differentiation. In order to resolve the controversy in the taxonomy of this parasite genus, we characterized the small-subunit rRNA genes of Cryptosporidium parvum,Cryptosporidium baileyi, Cryptosporidium muris, and Cryptosporidium serpentis and performed a phylogenetic analysis of the genus Cryptosporidium. Our study revealed that the genus Cryptosporidium contains the phylogenetically distinct species C. parvum, C. muris, C. baileyi, and C. serpentis, which is consistent with the biological characteristics and host specificity data. The Cryptosporidium species formed two clades, with C. parvum and C. baileyi belonging to one clade and C. muris and C. serpentisbelonging to the other clade. Within C. parvum, human genotype isolates and guinea pig isolates (known asCryptosporidium wrairi) each differed from bovine genotype isolates by the nucleotide sequence in four regions. A C. muris isolate from cattle was also different from parasites isolated from a rock hyrax and a Bactrian camel. Minor differences were also detected between C. serpentis isolates from snakes and lizards. Based on the genetic information, a species- and strain-specific PCR-restriction fragment length polymorphism diagnostic tool was developed.

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Effect of Primers Hybridizing to Different Evolutionarily Conserved Regions of the Small-Subunit rRNA Gene in PCR-Based Microbial Community Analyses and Genetic Profiling

Applied and Environmental Microbiology ◽

10.1128/aem.67.8.3557-3563.2001 ◽

2001 ◽

Vol 67 (8) ◽

pp. 3557-3563 ◽

Cited By ~ 193

Author(s):

Achim Schmalenberger ◽

Frank Schwieger ◽

Christoph C. Tebbe

Keyword(s):

Microbial Community ◽

Small Subunit ◽

Sscp Analysis ◽

Rrna Genes ◽

Rrna Gene ◽

Small Subunit Rrna ◽

Genetic Profiling ◽

Evolutionarily Conserved ◽

Pcr Products ◽

Pcr Targeting

ABSTRACT Genetic profiling techniques of microbial communities based on PCR-amplified signature genes, such as denaturing gradient gel electrophoresis or single-strand-conformation polymorphism (SSCP) analysis, are normally done with PCR products of less than 500-bp. The most common target for diversity analysis, the small-subunit rRNA genes, however, are larger, and thus, only partial sequences can be analyzed. Here, we compared the results obtained by PCR targeting different variable (V) regions (V2 and V3, V4 and V5, and V6 to V8) of the bacterial 16S rRNA gene with primers hybridizing to evolutionarily conserved flanking regions. SSCP analysis of single-stranded PCR products generated from 13 different bacterial species showed fewer bands with products containing V4-V5 (average, 1.7 bands per organism) than with V2-V3 (2.2 bands) and V6-V8 (2.3 bands). We found that the additional bands (>1 per organism) were caused by intraspecies operon heterogeneities or by more than one conformation of the same sequence. Community profiles, generated by PCR-SSCP from bacterial-cell consortia extracted from rhizospheres of field-grown maize (Zea mays), were analyzed by cloning and sequencing of the dominant bands. A total of 48 sequences could be attributed to 34 different strains from 10 taxonomical groups. Independent of the primer pairs, we found proteobacteria (α, β, and γ subgroups) and members of the genus Paenibacillus (low G+C gram-positive) to be the dominant organisms. Other groups, however, were only detected with single primer pairs. This study gives an example of how much the selection of different variable regions combined with different specificities of the flanking “universal” primers can affect a PCR-based microbial community analysis.

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Phenotypic effects of targeted mutations in the small subunit rRNA gene of Tetrahymena thermophila

Molecular and Cellular Biology ◽

10.1128/mcb.13.8.4814-4825.1993 ◽

1993 ◽

Vol 13 (8) ◽

pp. 4814-4825

Author(s):

R Sweeney ◽

L Chen ◽

M C Yao

Keyword(s):

Tertiary Structure ◽

Tetrahymena Thermophila ◽

Ribosomal Subunit ◽

Small Subunit ◽

Rrna Genes ◽

Rrna Gene ◽

Small Subunit Rrna ◽

Variable Regions ◽

Small Subunit Rrna Gene

Tetrahymena thermophila is an ideal organism with which to study functional aspects of the rRNAs in vivo since the somatic rRNA genes of T. thermophila can be totally replaced by cloned copies introduced via microinjection. In this study, we made small insertions into seven sites within the small subunit rRNA gene and observed their phenotypic effects on transformed cells. Two mutated genes coding for rRNA (rDNAs), both of which bear insertions in highly conserved sequences, failed to transform and are therefore believed to produce nonfunctional rRNAs. Three other altered rDNAs produce functional rRNAs that can substitute for most or all of the cellular rRNA. Two of these bear insertions in highly variable regions, and, surprisingly, the other has an insertion in a region that is well conserved for both sequence and secondary structure among eucaryotes. In addition, two other insertions appear to destabilize rRNAs that contain them. Our findings make predictions concerning the positions of some of these sites within the tertiary structure of the small ribosomal subunit and thus serve as an in vivo test of the existing tertiary structure models for the small subunit rRNA. Our results are in good agreement with expectations based on sequence comparison and in vitro work.

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