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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

Species-specific PCR based detection of microbial populations in genomic DNA extracted from samples collected in June 2009 from Sippewissett Marsh or the laboratory-acclimated N. vectensis population. Lanes 1–6 correspond to (1) cloned 16S rRNA sequence as positive control, (2) negative control consisting of non-target 16S rRNA DNA, (3) N. vectensis from Sippewissett Marsh, (4) Sippewissett Marsh sediment, (5) Seawater from Sippewissett Marsh at sampling site, (6) laboratory-adapted anemones, and (7) No template control (master mix + water only), respectively. Positive controls were 105 copies of target 16S rRNA and samples were 10 ng DNA.
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Figure 4: Species-specific PCR based detection of microbial populations in genomic DNA extracted from samples collected in June 2009 from Sippewissett Marsh or the laboratory-acclimated N. vectensis population. Lanes 1–6 correspond to (1) cloned 16S rRNA sequence as positive control, (2) negative control consisting of non-target 16S rRNA DNA, (3) N. vectensis from Sippewissett Marsh, (4) Sippewissett Marsh sediment, (5) Seawater from Sippewissett Marsh at sampling site, (6) laboratory-adapted anemones, and (7) No template control (master mix + water only), respectively. Positive controls were 105 copies of target 16S rRNA and samples were 10 ng DNA.

Mentions: To determine whether the four OTU's associated with N. vectensis in multiple clone libraries during Summer/Fall 2008 (i.e., the Campylobacterales and Spirochete OTUs, and the Endozoicomonas elysicola-, and Pseudomonas oleovorans-like OTUs) remained associated with both laboratory-reared and Sippewissett Marsh N. vectensis collected in June 2009 (representing a timespan of 9–12 months) as well as to screen for their presence in the surrounding marsh habitat (sediment and water), specific PCR assays were designed for each OTU. These analyses confirmed that the Campylobacterales and Spirochete OTUs and Endozoicomonas elysicola remained associated with both laboratory-reared and field-collected anemones in June 2009 (Figure 4), however Pseudomonas oleovorans amplicons were not recovered from the field-collected anemone DNA. Marsh water DNA yielded the expected sized amplicons for all four-sequence types, although PCR inhibition of amplification was apparent by the reproducible faint band intensity of 16S rRNA amplicon from universal eubacterial primers, relative to other environments. In contrast, surface sediments did not appear to be associated with any of these four OTUs, and a positive signal for the universal 16S rRNA amplicon indicated PCR inhibition was not a confounding factor in the sediment analysis (Figure 4). Non-detection of Campylobacterales, Endozoicomonas elysicola, and Pseudomonas oleovorans amplicons in the sediment sample (June 2009) is consistent with their absence from the clone library prepared from surface sediments collected in November 2008 (data not shown). This suggests that the association of these ribotypes with N. vectensis may be more specific than ingestion of detritus or attachment of the surrounding sediment to the anemone surface. However, the absence of a Spirochete OTU-specific PCR amplicon from marsh sediment was surprising, and may be due to a low concentration of this population's DNA in the sediment in June 2009, in contrast to November 2008 when a single sequence was observed in the sediment clone library.


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)

Species-specific PCR based detection of microbial populations in genomic DNA extracted from samples collected in June 2009 from Sippewissett Marsh or the laboratory-acclimated N. vectensis population. Lanes 1–6 correspond to (1) cloned 16S rRNA sequence as positive control, (2) negative control consisting of non-target 16S rRNA DNA, (3) N. vectensis from Sippewissett Marsh, (4) Sippewissett Marsh sediment, (5) Seawater from Sippewissett Marsh at sampling site, (6) laboratory-adapted anemones, and (7) No template control (master mix + water only), respectively. Positive controls were 105 copies of target 16S rRNA and samples were 10 ng DNA.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 4: Species-specific PCR based detection of microbial populations in genomic DNA extracted from samples collected in June 2009 from Sippewissett Marsh or the laboratory-acclimated N. vectensis population. Lanes 1–6 correspond to (1) cloned 16S rRNA sequence as positive control, (2) negative control consisting of non-target 16S rRNA DNA, (3) N. vectensis from Sippewissett Marsh, (4) Sippewissett Marsh sediment, (5) Seawater from Sippewissett Marsh at sampling site, (6) laboratory-adapted anemones, and (7) No template control (master mix + water only), respectively. Positive controls were 105 copies of target 16S rRNA and samples were 10 ng DNA.
Mentions: To determine whether the four OTU's associated with N. vectensis in multiple clone libraries during Summer/Fall 2008 (i.e., the Campylobacterales and Spirochete OTUs, and the Endozoicomonas elysicola-, and Pseudomonas oleovorans-like OTUs) remained associated with both laboratory-reared and Sippewissett Marsh N. vectensis collected in June 2009 (representing a timespan of 9–12 months) as well as to screen for their presence in the surrounding marsh habitat (sediment and water), specific PCR assays were designed for each OTU. These analyses confirmed that the Campylobacterales and Spirochete OTUs and Endozoicomonas elysicola remained associated with both laboratory-reared and field-collected anemones in June 2009 (Figure 4), however Pseudomonas oleovorans amplicons were not recovered from the field-collected anemone DNA. Marsh water DNA yielded the expected sized amplicons for all four-sequence types, although PCR inhibition of amplification was apparent by the reproducible faint band intensity of 16S rRNA amplicon from universal eubacterial primers, relative to other environments. In contrast, surface sediments did not appear to be associated with any of these four OTUs, and a positive signal for the universal 16S rRNA amplicon indicated PCR inhibition was not a confounding factor in the sediment analysis (Figure 4). Non-detection of Campylobacterales, Endozoicomonas elysicola, and Pseudomonas oleovorans amplicons in the sediment sample (June 2009) is consistent with their absence from the clone library prepared from surface sediments collected in November 2008 (data not shown). This suggests that the association of these ribotypes with N. vectensis may be more specific than ingestion of detritus or attachment of the surrounding sediment to the anemone surface. However, the absence of a Spirochete OTU-specific PCR amplicon from marsh sediment was surprising, and may be due to a low concentration of this population's DNA in the sediment in June 2009, in contrast to November 2008 when a single sequence was observed in the sediment clone library.

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