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Microbial diversity and activity in the Nematostella vectensis holobiont: insights from 16S rRNA gene sequencing, isolate genomes, and a pilot-scale survey of gene expression.

Har JY, Helbig T, Lim JH, Fernando SC, Reitzel AM, Penn K, Thompson JR - Front Microbiol (2015)

Bottom Line: Molecular phylogenetic analysis of 16S rRNA gene clone libraries revealed four ribotypes associated with N. vectensis at multiple locations and times.A pilot metatranscriptome of laboratory-raised N. vectensis was compared to the isolate genomes and indicated expression of ORFs from L. thiooxidans with predicted functions of motility, nutrient scavenging (Fe and P), polyhydroxyalkanoate synthesis for carbon storage, and selective permeability (porins).We hypothesize that such activities may mediate acclimation and persistence of bacteria in a N. vectensis holobiont defined by both internal and external gradients of chemicals and nutrients in a dynamic coastal habitat.

View Article: PubMed Central - PubMed

Affiliation: Department of Civil and Environmental Engineering, Massachusetts Institute of Technology Cambridge, MA, USA.

ABSTRACT
We have characterized the molecular and genomic diversity of the microbiota of the starlet sea anemone Nematostella vectensis, a cnidarian model for comparative developmental and functional biology and a year-round inhabitant of temperate salt marshes. Molecular phylogenetic analysis of 16S rRNA gene clone libraries revealed four ribotypes associated with N. vectensis at multiple locations and times. These associates include two novel ribotypes within the ε-Proteobacterial order Campylobacterales and the Spirochetes, respectively, each sharing <85% identity with cultivated strains, and two γ-Proteobacterial ribotypes sharing >99% 16S rRNA identity with Endozoicomonas elysicola and Pseudomonas oleovorans, respectively. Species-specific PCR revealed that these populations persisted in N. vectensis asexually propagated under laboratory conditions. cDNA indicated expression of the Campylobacterales and Endozoicomonas 16S rRNA in anemones from Sippewissett Marsh, MA. A collection of bacteria from laboratory raised N. vectensis was dominated by isolates from P. oleovorans and Rhizobium radiobacter. Isolates from field-collected anemones revealed an association with Limnobacter and Stappia isolates. Genomic DNA sequencing was carried out on 10 cultured bacterial isolates representing field- and laboratory-associates, i.e., Limnobacter spp., Stappia spp., P. oleovorans and R. radiobacter. Genomes contained multiple genes identified as virulence (host-association) factors while S. stellulata and L. thiooxidans genomes revealed pathways for mixotrophic sulfur oxidation. A pilot metatranscriptome of laboratory-raised N. vectensis was compared to the isolate genomes and indicated expression of ORFs from L. thiooxidans with predicted functions of motility, nutrient scavenging (Fe and P), polyhydroxyalkanoate synthesis for carbon storage, and selective permeability (porins). We hypothesize that such activities may mediate acclimation and persistence of bacteria in a N. vectensis holobiont defined by both internal and external gradients of chemicals and nutrients in a dynamic coastal habitat.

No MeSH data available.


Related in: MedlinePlus

Neighbor-joining phylogeny of 16S rRNA gene sequences from clones and isolates analyzed in this study with the most closely related reference sequences from the Silva database with >95% rRNA identity. Symbols at branch termini denote origin of sequence from isolate (circle), cloned 16S rRNA from gDNA (square) or from cDNA (triangle). Symbol colors correspond to sample origin: Salt marsh collected anemones (green), laboratory-acclimated anemones (blue) or from both field and lab-acclimated anemones (red). Chart to the right of tree specifies the specific sample and date of sequence origin. Phylum is indicated at the far right and corresponds to the legend on the figure. Highlighted sequence clusters correspond to taxa discussed in this study (1) Pseudomonas oleovorans, (2) Endozoicomonas spp., (3) Limnobacter spp., (4) Stappia spp., (5) Rhizobium radiobacter, (6) uncultured Campylobacter lineage (note: the origin of the “termite group” sequence FJ202415 is the coral Orbicella (Montastrea) faveolata), (7) uncultured OTUs with highest sequence identity to a coral-derived Spirochete.
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Figure 3: Neighbor-joining phylogeny of 16S rRNA gene sequences from clones and isolates analyzed in this study with the most closely related reference sequences from the Silva database with >95% rRNA identity. Symbols at branch termini denote origin of sequence from isolate (circle), cloned 16S rRNA from gDNA (square) or from cDNA (triangle). Symbol colors correspond to sample origin: Salt marsh collected anemones (green), laboratory-acclimated anemones (blue) or from both field and lab-acclimated anemones (red). Chart to the right of tree specifies the specific sample and date of sequence origin. Phylum is indicated at the far right and corresponds to the legend on the figure. Highlighted sequence clusters correspond to taxa discussed in this study (1) Pseudomonas oleovorans, (2) Endozoicomonas spp., (3) Limnobacter spp., (4) Stappia spp., (5) Rhizobium radiobacter, (6) uncultured Campylobacter lineage (note: the origin of the “termite group” sequence FJ202415 is the coral Orbicella (Montastrea) faveolata), (7) uncultured OTUs with highest sequence identity to a coral-derived Spirochete.

Mentions: A total of 393 non-chimeric Bacterial and chloroplast 16S rRNA gene sequences (E. coli positions 27–805) were obtained from N. vectensis from Mahone Bay, Nova Scotia (MB), Clinton Harbor, Connecticut (CT) and Sippewissett Marsh, Massachusetts (MA-I, MA-II) (Figure 2A, Table 1). An additional 39 16S rRNA gene sequences were recovered from cDNA libraries prepared from N. vectensis total RNA (Sippewissett Marsh, MA-IV), 82 Bacterial 16S rRNA genes were recovered from laboratory-reared N. vectensis and 66 16S rRNA sequences were obtained from Sippewissett Marsh surface sediments (Table 1). Archaeal 16S rRNA genes were not recovered by amplification of N. vectensis DNA (20 ng) with the Archaeal primer pair 21F to 958R. Three to ten bacterial phyla were recovered from samples of N. vectensis consisting of representatives from Cytophaga-Flexibacter-Bacteroides (CFB), Chloroflexi, Cyanobacteria, Deferribacteres, Firmicutes, OD1, Planctomycetes, Proteobacteria, Spirochetes, Tenericutes and Verrucomicrobia (Figures 2A, 3). Operational taxonomic units (OTUs) were defined as clusters of 16S rRNA sequences sharing >99% identity (i.e., a ribotype).


Microbial diversity and activity in the Nematostella vectensis holobiont: insights from 16S rRNA gene sequencing, isolate genomes, and a pilot-scale survey of gene expression.

Har JY, Helbig T, Lim JH, Fernando SC, Reitzel AM, Penn K, Thompson JR - Front Microbiol (2015)

Neighbor-joining phylogeny of 16S rRNA gene sequences from clones and isolates analyzed in this study with the most closely related reference sequences from the Silva database with >95% rRNA identity. Symbols at branch termini denote origin of sequence from isolate (circle), cloned 16S rRNA from gDNA (square) or from cDNA (triangle). Symbol colors correspond to sample origin: Salt marsh collected anemones (green), laboratory-acclimated anemones (blue) or from both field and lab-acclimated anemones (red). Chart to the right of tree specifies the specific sample and date of sequence origin. Phylum is indicated at the far right and corresponds to the legend on the figure. Highlighted sequence clusters correspond to taxa discussed in this study (1) Pseudomonas oleovorans, (2) Endozoicomonas spp., (3) Limnobacter spp., (4) Stappia spp., (5) Rhizobium radiobacter, (6) uncultured Campylobacter lineage (note: the origin of the “termite group” sequence FJ202415 is the coral Orbicella (Montastrea) faveolata), (7) uncultured OTUs with highest sequence identity to a coral-derived Spirochete.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4557100&req=5

Figure 3: Neighbor-joining phylogeny of 16S rRNA gene sequences from clones and isolates analyzed in this study with the most closely related reference sequences from the Silva database with >95% rRNA identity. Symbols at branch termini denote origin of sequence from isolate (circle), cloned 16S rRNA from gDNA (square) or from cDNA (triangle). Symbol colors correspond to sample origin: Salt marsh collected anemones (green), laboratory-acclimated anemones (blue) or from both field and lab-acclimated anemones (red). Chart to the right of tree specifies the specific sample and date of sequence origin. Phylum is indicated at the far right and corresponds to the legend on the figure. Highlighted sequence clusters correspond to taxa discussed in this study (1) Pseudomonas oleovorans, (2) Endozoicomonas spp., (3) Limnobacter spp., (4) Stappia spp., (5) Rhizobium radiobacter, (6) uncultured Campylobacter lineage (note: the origin of the “termite group” sequence FJ202415 is the coral Orbicella (Montastrea) faveolata), (7) uncultured OTUs with highest sequence identity to a coral-derived Spirochete.
Mentions: A total of 393 non-chimeric Bacterial and chloroplast 16S rRNA gene sequences (E. coli positions 27–805) were obtained from N. vectensis from Mahone Bay, Nova Scotia (MB), Clinton Harbor, Connecticut (CT) and Sippewissett Marsh, Massachusetts (MA-I, MA-II) (Figure 2A, Table 1). An additional 39 16S rRNA gene sequences were recovered from cDNA libraries prepared from N. vectensis total RNA (Sippewissett Marsh, MA-IV), 82 Bacterial 16S rRNA genes were recovered from laboratory-reared N. vectensis and 66 16S rRNA sequences were obtained from Sippewissett Marsh surface sediments (Table 1). Archaeal 16S rRNA genes were not recovered by amplification of N. vectensis DNA (20 ng) with the Archaeal primer pair 21F to 958R. Three to ten bacterial phyla were recovered from samples of N. vectensis consisting of representatives from Cytophaga-Flexibacter-Bacteroides (CFB), Chloroflexi, Cyanobacteria, Deferribacteres, Firmicutes, OD1, Planctomycetes, Proteobacteria, Spirochetes, Tenericutes and Verrucomicrobia (Figures 2A, 3). Operational taxonomic units (OTUs) were defined as clusters of 16S rRNA sequences sharing >99% identity (i.e., a ribotype).

Bottom Line: Molecular phylogenetic analysis of 16S rRNA gene clone libraries revealed four ribotypes associated with N. vectensis at multiple locations and times.A pilot metatranscriptome of laboratory-raised N. vectensis was compared to the isolate genomes and indicated expression of ORFs from L. thiooxidans with predicted functions of motility, nutrient scavenging (Fe and P), polyhydroxyalkanoate synthesis for carbon storage, and selective permeability (porins).We hypothesize that such activities may mediate acclimation and persistence of bacteria in a N. vectensis holobiont defined by both internal and external gradients of chemicals and nutrients in a dynamic coastal habitat.

View Article: PubMed Central - PubMed

Affiliation: Department of Civil and Environmental Engineering, Massachusetts Institute of Technology Cambridge, MA, USA.

ABSTRACT
We have characterized the molecular and genomic diversity of the microbiota of the starlet sea anemone Nematostella vectensis, a cnidarian model for comparative developmental and functional biology and a year-round inhabitant of temperate salt marshes. Molecular phylogenetic analysis of 16S rRNA gene clone libraries revealed four ribotypes associated with N. vectensis at multiple locations and times. These associates include two novel ribotypes within the ε-Proteobacterial order Campylobacterales and the Spirochetes, respectively, each sharing <85% identity with cultivated strains, and two γ-Proteobacterial ribotypes sharing >99% 16S rRNA identity with Endozoicomonas elysicola and Pseudomonas oleovorans, respectively. Species-specific PCR revealed that these populations persisted in N. vectensis asexually propagated under laboratory conditions. cDNA indicated expression of the Campylobacterales and Endozoicomonas 16S rRNA in anemones from Sippewissett Marsh, MA. A collection of bacteria from laboratory raised N. vectensis was dominated by isolates from P. oleovorans and Rhizobium radiobacter. Isolates from field-collected anemones revealed an association with Limnobacter and Stappia isolates. Genomic DNA sequencing was carried out on 10 cultured bacterial isolates representing field- and laboratory-associates, i.e., Limnobacter spp., Stappia spp., P. oleovorans and R. radiobacter. Genomes contained multiple genes identified as virulence (host-association) factors while S. stellulata and L. thiooxidans genomes revealed pathways for mixotrophic sulfur oxidation. A pilot metatranscriptome of laboratory-raised N. vectensis was compared to the isolate genomes and indicated expression of ORFs from L. thiooxidans with predicted functions of motility, nutrient scavenging (Fe and P), polyhydroxyalkanoate synthesis for carbon storage, and selective permeability (porins). We hypothesize that such activities may mediate acclimation and persistence of bacteria in a N. vectensis holobiont defined by both internal and external gradients of chemicals and nutrients in a dynamic coastal habitat.

No MeSH data available.


Related in: MedlinePlus