Uncultivated Microbial Eukaryotic Diversity: A Method to Link ssu rRNA Gene Sequences with Morphology
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{"title"=>"Uncultivated microbial eukaryotic diversity: A method to link ssu rRNA gene sequences with morphology", "type"=>"journal", "authors"=>[{"first_name"=>"Marissa B.", "last_name"=>"Hirst", "scopus_author_id"=>"54420056000"}, {"first_name"=>"Kelley N.", "last_name"=>"Kita", "scopus_author_id"=>"54420382800"}, {"first_name"=>"Scott C.", "last_name"=>"Dawson", "scopus_author_id"=>"55819393900"}], "year"=>2011, "source"=>"PLoS ONE", "identifiers"=>{"isbn"=>"1932-6203 (Electronic)\\r1932-6203 (Linking)", "sgr"=>"82955194832", "pui"=>"363053931", "pmid"=>"22174774", "issn"=>"19326203", "scopus"=>"2-s2.0-82955194832", "doi"=>"10.1371/journal.pone.0028158"}, "id"=>"7673359d-cc74-3635-afb3-fb7b5071ab1c", "abstract"=>"Protists have traditionally been identified by cultivation and classified taxonomically based on their cellular morphologies and behavior. In the past decade, however, many novel protist taxa have been identified using cultivation independent ssu rRNA sequence surveys. New rRNA \"phylotypes\" from uncultivated eukaryotes have no connection to the wealth of prior morphological descriptions of protists. To link phylogenetically informative sequences with taxonomically informative morphological descriptions, we demonstrate several methods for combining whole cell rRNA-targeted fluorescent in situ hybridization (FISH) with cytoskeletal or organellar immunostaining. Either eukaryote or ciliate-specific ssu rRNA probes were combined with an anti-alpha-tubulin antibody or phalloidin, a common actin stain, to define cytoskeletal features of uncultivated protists in several environmental samples. The eukaryote ssu rRNA probe was also combined with Mitotracker(R) or a hydrogenosomal-specific anti-Hsp70 antibody to localize mitochondria and hydrogenosomes, respectively, in uncultivated protists from different environments. Using rRNA probes in combination with immunostaining, we linked ssu rRNA phylotypes with microtubule structure to describe flagellate and ciliate morphology in three diverse environments, and linked Naegleria spp. to their amoeboid morphology using actin staining in hay infusion samples. We also linked uncultivated ciliates to morphologically similar Colpoda-like ciliates using tubulin immunostaining with a ciliate-specific rRNA probe. Combining rRNA-targeted FISH with cytoskeletal immunostaining or stains targeting specific organelles provides a fast, efficient, high throughput method for linking genetic sequences with morphological features in uncultivated protists. When linked to phylotype, morphological descriptions of protists can both complement and vet the increasing number of sequences from uncultivated protists, including those of novel lineages, identified in diverse environments.", "link"=>"http://www.mendeley.com/research/uncultivated-microbial-eukaryotic-diversity-method-link-ssu-rrna-gene-sequences-morphology", "reader_count"=>48, "reader_count_by_academic_status"=>{"Unspecified"=>2, "Professor > Associate Professor"=>3, "Student > Doctoral Student"=>6, "Researcher"=>12, "Student > Ph. D. Student"=>17, "Student > Master"=>3, "Other"=>2, "Student > Bachelor"=>2, "Professor"=>1}, "reader_count_by_user_role"=>{"Unspecified"=>2, "Professor > Associate Professor"=>3, "Student > Doctoral Student"=>6, "Researcher"=>12, "Student > Ph. D. Student"=>17, "Student > Master"=>3, "Other"=>2, "Student > Bachelor"=>2, "Professor"=>1}, "reader_count_by_subject_area"=>{"Unspecified"=>5, "Environmental Science"=>5, "Biochemistry, Genetics and Molecular Biology"=>1, "Agricultural and Biological Sciences"=>35, "Design"=>1, "Earth and Planetary Sciences"=>1}, "reader_count_by_subdiscipline"=>{"Design"=>{"Design"=>1}, "Earth and Planetary Sciences"=>{"Earth and Planetary Sciences"=>1}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>35}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>1}, "Unspecified"=>{"Unspecified"=>5}, "Environmental Science"=>{"Environmental Science"=>5}}, "reader_count_by_country"=>{"Canada"=>1, "United States"=>2, "Norway"=>2, "Brazil"=>2, "Chile"=>1, "Germany"=>3}, "group_count"=>2}

Scopus | Further Information

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/704890"], "description"=>"<p>The evolutionary relationships of the rDNA sequences from the hay infusion were determined by bootstrap analysis using RAxML and are presented in A (only bootstrap values ≥50% are shown above the branches). Accession numbers follow the species name and sequences identified in this study are represented by the name “hay” followed by the accession number (A). The eukaryotic rRNA-targeted FISH (C) overlaid with the phalloidin (actin) stain (D) links phylotype with amoeboflagellate morphology in the hay infusion enrichment (B–E). The image overlay (also see 3D stack in <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028158#pone.0028158.s004\" target=\"_blank\">Video S3</a>) shows the eukaryote ssu rRNA probe (green), phalloidin stain (red), and DAPI nucleic acid stain (blue) (E). Scale bar = 10 µm.</p>", "links"=>[], "tags"=>["infusion", "enrichment"], "article_id"=>375258, "categories"=>["Microbiology", "Biochemistry", "Genetics", "Biological Sciences", "Ecology", "Evolutionary Biology"], "users"=>["Marissa B. Hirst", "Kelley N. Kita", "Scott C. Dawson"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0028158.g003", "stats"=>{"downloads"=>1, "page_views"=>6, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Amoebae_found_in_the_hay_infusion_enrichment_are_closely_related_to_Naegleria_spp_/375258", "title"=>"Amoebae found in the hay infusion enrichment are closely related to <i>Naegleria</i> spp.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2011-12-08 01:27:38"}
  • {"files"=>["https://ndownloader.figshare.com/files/357731", "https://ndownloader.figshare.com/files/357786", "https://ndownloader.figshare.com/files/357860", "https://ndownloader.figshare.com/files/357951", "https://ndownloader.figshare.com/files/357990"], "description"=>"<div><p>Protists have traditionally been identified by cultivation and classified taxonomically based on their cellular morphologies and behavior. In the past decade, however, many novel protist taxa have been identified using cultivation independent ssu rRNA sequence surveys. New rRNA “phylotypes” from uncultivated eukaryotes have no connection to the wealth of prior morphological descriptions of protists. To link phylogenetically informative sequences with taxonomically informative morphological descriptions, we demonstrate several methods for combining whole cell rRNA-targeted fluorescent <em>in situ</em> hybridization (FISH) with cytoskeletal or organellar immunostaining. Either eukaryote or ciliate-specific ssu rRNA probes were combined with an anti-α-tubulin antibody or phalloidin, a common actin stain, to define cytoskeletal features of uncultivated protists in several environmental samples. The eukaryote ssu rRNA probe was also combined with Mitotracker® or a hydrogenosomal-specific anti-Hsp70 antibody to localize mitochondria and hydrogenosomes, respectively, in uncultivated protists from different environments. Using rRNA probes in combination with immunostaining, we linked ssu rRNA phylotypes with microtubule structure to describe flagellate and ciliate morphology in three diverse environments, and linked <em>Naegleria</em> spp. to their amoeboid morphology using actin staining in hay infusion samples. We also linked uncultivated ciliates to morphologically similar <em>Colpoda</em>-like ciliates using tubulin immunostaining with a ciliate-specific rRNA probe. Combining rRNA-targeted FISH with cytoskeletal immunostaining or stains targeting specific organelles provides a fast, efficient, high throughput method for linking genetic sequences with morphological features in uncultivated protists. When linked to phylotype, morphological descriptions of protists can both complement and vet the increasing number of sequences from uncultivated protists, including those of novel lineages, identified in diverse environments.</p> </div>", "links"=>[], "tags"=>["uncultivated", "microbial", "eukaryotic", "ssu", "rrna", "sequences", "morphology"], "article_id"=>130708, "categories"=>["Microbiology", "Biochemistry", "Genetics", "Biological Sciences", "Ecology", "Evolutionary Biology"], "users"=>["Marissa B. Hirst", "Kelley N. Kita", "Scott C. Dawson"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0028158.s001", "https://dx.doi.org/10.1371/journal.pone.0028158.s002", "https://dx.doi.org/10.1371/journal.pone.0028158.s003", "https://dx.doi.org/10.1371/journal.pone.0028158.s004", "https://dx.doi.org/10.1371/journal.pone.0028158.s005"], "stats"=>{"downloads"=>6, "page_views"=>16, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/Uncultivated_Microbial_Eukaryotic_Diversity_A_Method_to_Link_ssu_rRNA_Gene_Sequences_with_Morphology/130708", "title"=>"Uncultivated Microbial Eukaryotic Diversity: A Method to Link ssu rRNA Gene Sequences with Morphology", "pos_in_sequence"=>0, "defined_type"=>4, "published_date"=>"2011-12-08 00:11:48"}
  • {"files"=>["https://ndownloader.figshare.com/files/704984"], "description"=>"<p>Live hay infusion (A–E) was incubated with Mitotracker® Red CM-H<sub>2</sub>XRos (C), fixed, hybridized with the eukaryote ssu rRNA probe (B), and overlaid with an anti-α-tubulin antibody to stain cytoskeletal features of each cell (D). Tailed arrow marks ovoid mitochondria. The image overlay (E) shows the eukaryotic ssu rRNA probe (green), Mitotracker® Red CM-H<sub>2</sub>XRos (red), and anti-α-tubulin (blue). Scale bar = 10 µm. Fixed termite hindgut samples (F–J) were hybridized with the eukaryote ssu rRNA probe (G), overlaid with and anti-Hsp70 antibody to stain hydrogenosomes (H, arrowhead). The image overlay (J) shows the eukaryotic ssu rRNA probe (green), anti-Hsp70 antibody (red), and DAPI nucleic acid stain (blue). Scale bar = 25 µm. Also see 3D stacks of overlays in <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028158#pone.0028158.s005\" target=\"_blank\">Video S4</a>.</p>", "links"=>[], "tags"=>["environments", "links", "eukaryotic", "phylotypes", "subcellular", "localization", "mitochondria"], "article_id"=>375345, "categories"=>["Microbiology", "Biochemistry", "Genetics", "Biological Sciences", "Ecology", "Evolutionary Biology"], "users"=>["Marissa B. Hirst", "Kelley N. Kita", "Scott C. Dawson"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0028158.g004", "stats"=>{"downloads"=>4, "page_views"=>4, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_ImmunoFISH_in_two_environments_links_eukaryotic_phylotypes_with_the_subcellular_localization_of_their_mitochondria_or_hydrogenosomes_/375345", "title"=>"ImmunoFISH in two environments links eukaryotic phylotypes with the subcellular localization of their mitochondria or hydrogenosomes.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2011-12-08 01:29:05"}
  • {"files"=>["https://ndownloader.figshare.com/files/705061"], "description"=>"1<p><i>Trichomonas vaginalis</i> anti-Hsp70 antibody.</p>2<p>The eukb mix is composed of three probes used simultaneously.</p>3<p>For Putah Creek, 5 mM EDTA was added to the wash buffer.</p>4<p>Antibody dilution is shown in parentheses.</p>", "links"=>[], "tags"=>["conditions", "immunofish"], "article_id"=>375426, "categories"=>["Microbiology", "Biochemistry", "Genetics", "Biological Sciences", "Ecology", "Evolutionary Biology"], "users"=>["Marissa B. Hirst", "Kelley N. Kita", "Scott C. Dawson"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0028158.t001", "stats"=>{"downloads"=>0, "page_views"=>1, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Specific_conditions_for_the_immunoFISH_protocol_for_each_environment_or_control_sample_/375426", "title"=>"Specific conditions for the immunoFISH protocol for each environment or control sample.", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2011-12-08 01:30:26"}
  • {"files"=>["https://ndownloader.figshare.com/files/704619"], "description"=>"<p>Representative immunoFISH of protists in Putah Creek (A–D), the termite hindgut (E–H), and the hay infusion enrichment (I–L) are presented. Positive control of the protist <i>G. intestinalis</i> ATCC 50803 (M–P) and negative control of the bacterium <i>P. putida</i> F1 (Q–T) are also shown. Fixed samples were hybridized with the broad eukaryote ssu rRNA probe (B, F, J, N, R) and overlaid with an anti-α-tubulin antibody to stain cytoskeletal features of each cell (C, G, K, O, S). The image overlays (also 3D stack in <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028158#pone.0028158.s002\" target=\"_blank\">Video S1</a>) show the eukaryote ssu rRNA probe (green), anti-α-tubulin antibody (red), and DAPI nucleic acid stain (blue) (D, H, L, P, T). Scale bars = 10 µm with the exception of the hay infusion enrichment (I) with the scale bar = 25 µm.</p>", "links"=>[], "tags"=>["links", "phylotype", "cytoskeletal", "morphology", "protists"], "article_id"=>374977, "categories"=>["Microbiology", "Biochemistry", "Genetics", "Biological Sciences", "Ecology", "Evolutionary Biology"], "users"=>["Marissa B. Hirst", "Kelley N. Kita", "Scott C. Dawson"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0028158.g001", "stats"=>{"downloads"=>1, "page_views"=>5, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_ImmunoFISH_links_phylotype_with_cytoskeletal_morphology_in_protists_from_three_environments_/374977", "title"=>"ImmunoFISH links phylotype with cytoskeletal morphology in protists from three environments.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2011-12-08 01:22:57"}
  • {"files"=>["https://ndownloader.figshare.com/files/704779"], "description"=>"<p>Ciliate positive control <i>P. aurelia</i> (I–L) and the diplomonad negative control <i>G. intestinali</i>s ATCC 50803 (M–P) are also shown. Fixed samples were hybridized with the ciliate-specific ssu rRNA probe (B, F, J, N) and overlaid with an anti-α-tubulin antibody to stain cytoskeletal features of each cell (C, G, K, O). The image overlays (also see 3D stack in <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028158#pone.0028158.s003\" target=\"_blank\">Video S2</a>) show the ciliate-specific ssu rRNA probe (green), anti-α-tubulin antibody (red), and DAPI nucleic acid stain (blue) (D, H, L, P). The tailed arrow marks a bacterium, and the arrowhead marks an amoeba. Scale bars = 25 µm with the exception of the positive control <i>G. intestinalis</i> ATCC 50803 (M) with the scale bar = 10 µm.</p>", "links"=>[], "tags"=>["links", "ciliate-specific", "phylotypes", "cytoskeletal", "morphologies", "infusion"], "article_id"=>375135, "categories"=>["Microbiology", "Biochemistry", "Genetics", "Biological Sciences", "Ecology", "Evolutionary Biology"], "users"=>["Marissa B. Hirst", "Kelley N. Kita", "Scott C. Dawson"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0028158.g002", "stats"=>{"downloads"=>0, "page_views"=>1, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_ImmunoFISH_links_ciliate_specific_phylotypes_with_their_cytoskeletal_morphologies_in_the_hay_infusion_enrichment_/375135", "title"=>"ImmunoFISH links ciliate-specific phylotypes with their cytoskeletal morphologies in the hay infusion enrichment.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2011-12-08 01:25:35"}

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Relative Metric

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