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Characterization of microbial associations with methanotrophic archaea and sulfate-reducing bacteria through statistical comparison of nested Magneto-FISH enrichments

Trembath-Reichert, Elizabeth and Case, David H. and Orphan, Victoria J. (2016) Characterization of microbial associations with methanotrophic archaea and sulfate-reducing bacteria through statistical comparison of nested Magneto-FISH enrichments. PeerJ, 4 . Art. No. e1913. ISSN 2167-8359. PMCID PMC4841229. http://resolver.caltech.edu/CaltechAUTHORS:20160502-081644901

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Abstract

Methane seep systems along continental margins host diverse and dynamic microbial assemblages, sustained in large part through the microbially mediated process of sulfate-coupled Anaerobic Oxidation of Methane (AOM). This methanotrophic metabolism has been linked to consortia of anaerobic methane-oxidizing archaea (ANME) and sulfate-reducing bacteria (SRB). These two groups are the focus of numerous studies; however, less is known about the wide diversity of other seep associated microorganisms. We selected a hierarchical set of FISH probes targeting a range of Deltaproteobacteria diversity. Using the Magneto-FISH enrichment technique, we then magnetically captured CARD-FISH hybridized cells and their physically associated microorganisms from a methane seep sediment incubation. DNA from nested Magneto-FISH experiments was analyzed using Illumina tag 16S rRNA gene sequencing (iTag). Enrichment success and potential bias with iTag was evaluated in the context of full-length 16S rRNA gene clone libraries, CARD-FISH, functional gene clone libraries, and iTag mock communities. We determined commonly used Earth Microbiome Project (EMP) iTAG primers introduced bias in some common methane seep microbial taxa that reduced the ability to directly compare OTU relative abundances within a sample, but comparison of relative abundances between samples (in nearly all cases) and whole community-based analyses were robust. The iTag dataset was subjected to statistical co-occurrence measures of the most abundant OTUs to determine which taxa in this dataset were most correlated across all samples. Many non-canonical microbial partnerships were statistically significant in our co-occurrence network analysis, most of which were not recovered with conventional clone library sequencing, demonstrating the utility of combining Magneto-FISH and iTag sequencing methods for hypothesis generation of associations within complex microbial communities. Network analysis pointed to many co-occurrences containing putatively heterotrophic, candidate phyla such as OD1, Atribacteria, MBG-B, and Hyd24-12 and the potential for complex sulfur cycling involving Epsilon-, Delta-, and Gammaproteobacteria in methane seep ecosystems.


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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4841229/PubMed CentralArticle
ORCID:
AuthorORCID
Orphan, Victoria J.0000-0002-5374-6178
Additional Information:© 2016 Trembath-Reichert et al. Distributed under Creative Commons CC-BY 4.0. Submitted 12 January 2016; Accepted 18 March 2016; Published 18 April 2016. We thank the crew of the R/V Atlantis and DSV JASON II, as well as Abigail Green-Saxena and Joshua Steele for assistance with optimization of the Magneto-FISH protocol and Stephanie Connon for assistance with sequencing. We also are grateful to Katherine Dawson, Emil Ruff, and two anonymous reviewers for providing comments on this manuscript. Funding: This research is funded by the Department of Energy, Office of Science, Office of Biological and Environmental Research (DE-SC0003940) and the Gordon and Betty Moore Foundation through Grant GBMF 3780 (both to VJO). ETR was supported by a NIH/NRSA Training Grant (5 T32 GM07616). Funding for DHC was provided in part by an NSF Graduate Research Fellowship. Samples were collected with funding from the National Science Foundation (BIO-OCE 0825791; to VJO). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Author Contributions: Elizabeth Trembath-Reichert conceived and designed the experiments, performed the experiments, analyzed the data, wrote the paper, prepared figures and/or tables, reviewed drafts of the paper. David H. Case conceived and designed the experiments, performed the experiments, reviewed drafts of the paper. Victoria J. Orphan contributed reagents/materials/analysis tools, designed the experiments, reviewed drafts of the paper. Data availability: The following information was supplied regarding data availability: GenBank sequences: AprA ( KT280505– KT280517), DsrA ( KT280518– KT280533), McrA ( KT280534– KT280581), Archaeal 16S rRNA ( KT280582– KT280632), Bacterial 16S rRNA ( KT280633– KT280909), SoxB ( KT280910– KT280928) SRA: accession SAMN03879962, BioSample: SAMN03879962, Sample name: PC47 (5133–5137) mixed slurry. The authors declare there are no competing interests.
Funders:
Funding AgencyGrant Number
Department of Energy (DOE)DE-SC0003940
Gordon and Betty Moore FoundationGBMF 3780
NIH5 T32 GM07616
NSF Graduate Research FellowshipUNSPECIFIED
NSFOCE 0825791
Subject Keywords:Magneto-FISH, Co-occurrence network, Earth microbiome project, iTag sequencing, Anaerobic oxidation of methane, Microbial association, Microbial ecology
PubMed Central ID:PMC4841229
Record Number:CaltechAUTHORS:20160502-081644901
Persistent URL:http://resolver.caltech.edu/CaltechAUTHORS:20160502-081644901
Official Citation:Trembath-Reichert E, Case DH, Orphan VJ. (2016) Characterization of microbial associations with methanotrophic archaea and sulfate-reducing bacteria through statistical comparison of nested Magneto-FISH enrichments. PeerJ 4:e1913 https://doi.org/10.7717/peerj.1913
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:66572
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:02 May 2016 16:11
Last Modified:02 May 2016 16:11

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