Obtaining genomes from uncultivated environmental microorganisms using FACS–based single-cell genomics

Rinke, Christian; Lee, Janey; Nath, Nandita; Goudeau, Danielle; Thompson, Brian; Poulton, Nicole; Dmitrieff, Elizabeth; Malmstrom, Rex R.; Stepanauskas, Ramunas; Woyke, Tanja

doi:10.1038/nprot.2014.067

Cited by 235 publications

(254 citation statements)

References 45 publications

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“…After successful tests, BONCAT-treated sediment-extracted consortia from samples #3730 and #7142 were analyzed. A BD Influx cell sorter was sterilized, and sheath fluid was prepared using 1× PBS, as recently described (65).…”

Section: Methodsmentioning

confidence: 99%

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Visualizing in situ translational activity for identifying and sorting slow-growing archaeal−bacterial consortia

Hatzenpichler

Connon

Goudeau

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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165

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To understand the biogeochemical roles of microorganisms in the environment, it is important to determine when and under which conditions they are metabolically active. Bioorthogonal noncanonical amino acid tagging (BONCAT) can reveal active cells by tracking the incorporation of synthetic amino acids into newly synthesized proteins. The phylogenetic identity of translationally active cells can be determined by combining BONCAT with rRNAtargeted fluorescence in situ hybridization (BONCAT-FISH). In theory, BONCAT-labeled cells could be isolated with fluorescenceactivated cell sorting (BONCAT-FACS) for subsequent genetic analyses. Here, in the first application, to our knowledge, of BONCAT-FISH and BONCAT-FACS within an environmental context, we probe the translational activity of microbial consortia catalyzing the anaerobic oxidation of methane (AOM), a dominant sink of methane in the ocean. These consortia, which typically are composed of anaerobic methane-oxidizing archaea (ANME) and sulfatereducing bacteria, have been difficult to study due to their slow in situ growth rates, and fundamental questions remain about their ecology and diversity of interactions occurring between ANME and associated partners. Our activity-correlated analyses of >16,400 microbial aggregates provide the first evidence, to our knowledge, that AOM consortia affiliated with all five major ANME clades are concurrently active under controlled conditions. Surprisingly, sorting of individual BONCAT-labeled consortia followed by whole-genome amplification and 16S rRNA gene sequencing revealed previously unrecognized interactions of ANME with members of the poorly understood phylum Verrucomicrobia. This finding, together with our observation that ANME-associated Verrucomicrobia are found in a variety of geographically distinct methane seep environments, suggests a broader range of symbiotic relationships within AOM consortia than previously thought.activity-based cell sorting | BONCAT | click chemistry | ecophysiology | single-cell microbiology

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Section: Methodsmentioning

confidence: 99%

“…Consortia sorted into individual wells of microtiter plates were lysed and their genomes amplified via multiple displacement amplification (MDA) following established protocols (65). For ethanol-fixed biomass from sediment #3730, 76 out of 168 sorted events resulted in an MDA product.…”

Section: Identification Of Microbial Partners Within Activity-sorted mentioning

confidence: 99%

Visualizing in situ translational activity for identifying and sorting slow-growing archaeal−bacterial consortia

Hatzenpichler

Connon

Goudeau

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

165

208

View full text Add to dashboard Cite

show abstract

“…Pyrotag sequencing was done using 16S rRNA gene primers for the V3-V6 (B343F/B1099rc; Liu et al, 2007) and V6-V9 (A934F/U1492R; Lane, 1991;Teske and Sorensen, 2008) regions for bacteria and archaea, respectively. Cell sorting and single-cell genomics were conducted at the Single Cell Genomics Center, Bigelow Laboratory for Ocean Sciences (East Boothbay, ME, USA) as described in Rinke et al (2014). Comparative genome analysis was performed using the phylogenomic platform EDGAR (Blom et al, 2009), which uses an automatically calculated Blast score ratio value cutoff for identifying conserved and unique gene sets among genome pairs based on bi-directional best blast hit analyses.…”

Section: Methodsmentioning

confidence: 99%

Comparative genomics reveals adaptations of a halotolerant thaumarchaeon in the interfaces of brine pools in the Red Sea

et al. 2014

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The bottom of the Red Sea harbors over 25 deep hypersaline anoxic basins that are geochemically distinct and characterized by vertical gradients of extreme physicochemical conditions. Because of strong changes in density, particulate and microbial debris get entrapped in the brine-seawater interface (BSI), resulting in increased dissolved organic carbon, reduced dissolved oxygen toward the brines and enhanced microbial activities in the BSI. These features coupled with the deep-sea prevalence of ammonia-oxidizing archaea (AOA) in the global ocean make the BSI a suitable environment for studying the osmotic adaptations and ecology of these important players in the marine nitrogen cycle. Using phylogenomic-based approaches, we show that the local archaeal community of five different BSI habitats (with up to 18.2% salinity) is composed mostly of a single, highly abundant Nitrosopumilus-like phylotype that is phylogenetically distinct from the bathypelagic thaumarchaea; ammonia-oxidizing bacteria were absent. The composite genome of this novel Nitrosopumilus-like subpopulation (RSA3) co-assembled from multiple single-cell amplified genomes (SAGs) from one such BSI habitat further revealed that it shares B54% of its predicted genomic inventory with sequenced Nitrosopumilus species. RSA3 also carries several, albeit variable gene sets that further illuminate the phylogenetic diversity and metabolic plasticity of this genus. Specifically, it encodes for a putative proline-glutamate 'switch' with a potential role in osmotolerance and indirect impact on carbon and energy flows. Metagenomic fragment recruitment analyses against the composite RSA3 genome, Nitrosopumilus maritimus, and SAGs of mesopelagic thaumarchaea also reiterate the divergence of the BSI genotypes from other AOA.

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“…Although metagenomics was very useful, it remained inefficient in handling natural genomic heterogeneity and cross-strain assemblies could not be avoided (Rinke, Lee et al 2014). Also, taxonomic assignments of metagenomic reads/contigs remain challenging.…”

Section: Accepted M Manuscriptmentioning

confidence: 99%

Contemporary molecular tools in microbial ecology and their application to advancing biotechnology

Rashid

Stingl

2015

Biotechnology Advances

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Obtaining genomes from uncultivated environmental microorganisms using FACS–based single-cell genomics

Cited by 235 publications

References 45 publications

Visualizing in situ translational activity for identifying and sorting slow-growing archaeal−bacterial consortia

Visualizing in situ translational activity for identifying and sorting slow-growing archaeal−bacterial consortia

Comparative genomics reveals adaptations of a halotolerant thaumarchaeon in the interfaces of brine pools in the Red Sea

Contemporary molecular tools in microbial ecology and their application to advancing biotechnology

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