Correlative SIP-FISH-Raman-SEM-NanoSIMS links identity, morphology, biochemistry, and physiology of environmental microbes

Schaible, George; Hatzenpichler, Roland S.; Cliff, John B.; Kohtz, Anthony

doi:10.1038/s43705-022-00134-3

Title: Correlative SIP-FISH-Raman-SEM-NanoSIMS links identity, morphology, biochemistry, and physiology of environmental microbes

Journal Article · 30 June 2022 · ISME Communications

DOI:https://doi.org/10.1038/s43705-022-00134-3· OSTI ID:1883223

Schaible, George ^[1]; Hatzenpichler, Roland S. ^[2];

^[3]; Kohtz, Anthony ^[1]

Montana State University
MONTANA STATE UNIVERSITY
BATTELLE (PACIFIC NW LAB)

Microscopic and spectroscopic techniques are commonly applied to study microbial cells but are typically used on separate samples, resulting in population-level datasets that are integrated across different cells with little spatial resolution. To address this shortcoming, we developed a workflow that correlates several microscopic and spectroscopic techniques to generate an in depth analysis of individual cells. By combining stable isotope probing (SIP), fluorescence in situ hybridization (FISH), scanning electron microscopy (SEM), confocal Raman microspectroscopy (Raman), and nano-scale secondary ion mass spectrometry (NanoSIMS), we illustrate how individual cells can be thoroughly interrogated to obtain information about their taxonomic identity, structure, physiology, and metabolic activity. Analysis of an artificial community demonstrated that our correlative approach was able to resolve the activity of single cells using heavy water SIP in conjunction with Raman and/or NanoSIMS and establish their taxonomy and morphology using FISH and SEM. We then applied this workflow to a sample of yet uncultured multicellular magnetotactic bacteria. In addition to establishing their identity and activity, backscatter electron microscopy (BSE), NanoSIMS, and energy-dispersive X-ray spectroscopy (EDS) were employed to characterize the magnetosomes within the cells. By integrating these techniques, we demonstrate a cohesive approach to thoroughly study environmental microbes at single cell resolution.

Cite

Export

Save

Research Organization:: Pacific Northwest National Laboratory (PNNL), Richland, WA (United States)

Sponsoring Organization:: USDOE

DOE Contract Number:: AC05-76RL01830

OSTI ID:: 1883223

Report Number(s):: PNNL-SA-172171

Journal Information:: ISME Communications, Vol. 2

Country of Publication:: United States

Language:: English

References (62)

Next-generation physiology approaches to study microbiome function at single cell level Hatzenpichler, Roland; Krukenberg, Viola; Spietz, Rachel L. Nature Reviews Microbiology, Vol. 18, Issue 4 https://doi.org/10.1038/s41579-020-0323-1	journal	February 2020
The 2018 correlative microscopy techniques roadmap Ando, Toshio; Bhamidimarri, Satya Prathyusha; Brending, Niklas Journal of Physics D: Applied Physics, Vol. 51, Issue 44 https://doi.org/10.1088/1361-6463/aad055	journal	August 2018
Advances in Correlative Single-Molecule Localization Microscopy and Electron Microscopy Endesfelder, Ulrike NanoBioImaging, Vol. 1, Issue 1 https://doi.org/10.2478/nbi-2014-0002	journal	January 2015
Individual microtubules viewed by immunofluorescence and electron microscopy in the same PtK2 cell Osborn, M.; Webster, Re; Weber, K. Journal of Cell Biology, Vol. 77, Issue 3 https://doi.org/10.1083/jcb.77.3.R27	journal	June 1978
Visualization of the same PtK2 cytoskeletons by both immunofluorescence and low power electron microscopy Webster, Robert E.; Osborn, Mary; Weber, Klaus Experimental Cell Research, Vol. 117, Issue 1 https://doi.org/10.1016/0014-4827(78)90426-3	journal	November 1978
Correlative Light- and Electron Microscopy with chemical tags Perkovic, Mario; Kunz, Michael; Endesfelder, Ulrike Journal of Structural Biology, Vol. 186, Issue 2 https://doi.org/10.1016/j.jsb.2014.03.018	journal	May 2014
Correlative fluorescence microscopy, transmission electron microscopy and secondary ion mass spectrometry (CLEM-SIMS) for cellular imaging Lange, Felix; Agüi-Gonzalez, Paola; Riedel, Dietmar PLOS ONE, Vol. 16, Issue 5 https://doi.org/10.1371/journal.pone.0240768	journal	May 2021
ColorEM: analytical electron microscopy for element-guided identification and imaging of the building blocks of life Pirozzi, Nicole M.; Hoogenboom, Jacob P.; Giepmans, Ben N. G. Histochemistry and Cell Biology, Vol. 150, Issue 5 https://doi.org/10.1007/s00418-018-1707-4	journal	August 2018
Correlation of fluorescence microscopy, electron microscopy, and NanoSIMS stable isotope imaging on a single tissue section Loussert-Fonta, Céline; Toullec, Gaëlle; Paraecattil, Arun Aby Communications Biology, Vol. 3, Issue 1 https://doi.org/10.1038/s42003-020-1095-x	journal	July 2020
Super-Resolution Correlative Light and Electron Microscopy (SR-CLEM) Reveals Novel Ultrastructural Insights Into Dendritic Cell Podosomes Joosten, Ben; Willemse, Marieke; Fransen, Jack Frontiers in Immunology, Vol. 9 https://doi.org/10.3389/fimmu.2018.01908	journal	August 2018
Correlative Electron and Fluorescence Microscopy of Magnetotactic Bacteria in Liquid: Toward In Vivo Imaging Woehl, Taylor J.; Kashyap, Sanjay; Firlar, Emre Scientific Reports, Vol. 4, Issue 1 https://doi.org/10.1038/srep06854	journal	October 2014
Juxtaposed membranes underpin cellular adhesion and display unilateral cell division of multicellular magnetotactic prokaryotes Qian, Xin‐Xin; Santini, Claire‐Lise; Kosta, Artemis Environmental Microbiology, Vol. 22, Issue 4 https://doi.org/10.1111/1462-2920.14710	journal	April 2020
Subgroup Characteristics of Marine Methane-Oxidizing ANME-2 Archaea and Their Syntrophic Partners as Revealed by Integrated Multimodal Analytical Microscopy McGlynn, Shawn E.; Chadwick, Grayson L.; O'Neill, Ariel Applied and Environmental Microbiology, Vol. 84, Issue 11 https://doi.org/10.1128/AEM.00399-18	journal	April 2018
Novel prosthecate bacteria from the candidate phylum Acetothermia Hao, Liping; McIlroy, Simon Jon; Kirkegaard, Rasmus Hansen The ISME Journal, Vol. 12, Issue 9 https://doi.org/10.1038/s41396-018-0187-9	journal	June 2018
Three-Dimensional Mapping of Soil Chemical Characteristics at Micrometric Scale by Combining 2D SEM-EDX Data and 3D X-Ray CT Images Hapca, Simona; Baveye, Philippe C.; Wilson, Clare PLOS ONE, Vol. 10, Issue 9 https://doi.org/10.1371/journal.pone.0137205	journal	September 2015
Correlative Imaging Reveals Holistic View of Soil Microenvironments Schlüter, Steffen; Eickhorst, Thilo; Mueller, Carsten W. Environmental Science & Technology, Vol. 53, Issue 2 https://doi.org/10.1021/acs.est.8b05245	journal	December 2018
Spatially resolved correlative microscopy and microbial identification reveal dynamic depth‐ and mineral‐dependent anabolic activity in salt marsh sediment Marlow, Jeffrey; Spietz, Rachel; Kim, Keun‐Young Environmental Microbiology, Vol. 23, Issue 8 https://doi.org/10.1111/1462-2920.15667	journal	August 2021
Tracking microbial interactions with NanoSIMS Musat, Niculina; Musat, Florin; Weber, Peter Kilian Current Opinion in Biotechnology, Vol. 41 https://doi.org/10.1016/j.copbio.2016.06.007	journal	October 2016
Raman-FISH: combining stable-isotope Raman spectroscopy and fluorescence in situ hybridization for the single cell analysis of identity and function Huang, Wei E.; Stoecker, Kilian; Griffiths, Robert Environmental Microbiology, Vol. 9, Issue 8 https://doi.org/10.1111/j.1462-2920.2007.01352.x	journal	August 2007
Proposal to reclassify the proteobacterial classes Deltaproteobacteria and Oligoflexia, and the phylum Thermodesulfobacteria into four phyla reflecting major functional capabilities Waite, David W.; Chuvochina, Maria; Pelikan, Claus International Journal of Systematic and Evolutionary Microbiology, Vol. 70, Issue 11 https://doi.org/10.1099/ijsem.0.004213	journal	November 2020
Structure, Behavior, Ecology and Diversity of Multicellular Magnetotactic Prokaryotes Keim, Carolina N.; Lopes Martins, Juliana; Lins de Barros, Henrique Magnetoreception and Magnetosomes in Bacteria, p. 103-132 https://doi.org/10.1007/7171_040	book	January 2006
‘Candidatus Magnetoglobus multicellularis’, a multicellular, magnetotactic prokaryote from a hypersaline environment Abreu, Fernanda; Martins, Juliana Lopes; Silveira, Thaís Souza International Journal of Systematic and Evolutionary Microbiology, Vol. 57, Issue 6 https://doi.org/10.1099/ijs.0.64857-0	journal	June 2007
Cell Adhesion, Multicellular Morphology, and Magnetosome Distribution in the Multicellular Magnetotactic Prokaryote Candidatus Magnetoglobus multicellularis Abreu, Fernanda; Silva, Karen Tavares; Leão, Pedro Microscopy and Microanalysis, Vol. 19, Issue 3 https://doi.org/10.1017/S1431927613000329	journal	April 2013
Magnetotactic Bacteria and Magnetosomes Faivre, Damien; Schüler, Dirk Chemical Reviews, Vol. 108, Issue 11 https://doi.org/10.1021/cr078258w	journal	November 2008
Formation and function of bacterial organelles Greening, Chris; Lithgow, Trevor Nature Reviews Microbiology, Vol. 18, Issue 12 https://doi.org/10.1038/s41579-020-0413-0	journal	July 2020
Magnetosome biogenesis in magnetotactic bacteria Uebe, René; Schüler, Dirk Nature Reviews Microbiology, Vol. 14, Issue 10 https://doi.org/10.1038/nrmicro.2016.99	journal	September 2016
Multicellular photo-magnetotactic bacteria: Multicellular photo-magnetotactic bacteria Shapiro, Orr H.; Hatzenpichler, Roland; Buckley, Daniel H. Environmental Microbiology Reports, Vol. 3, Issue 2 https://doi.org/10.1111/j.1758-2229.2010.00215.x	journal	September 2010
Unexpected diversity in populations of the many-celled magnetotactic prokaryote Simmons, Sheri L.; Edwards, Katrina J. Environmental Microbiology, Vol. 9, Issue 1 https://doi.org/10.1111/j.1462-2920.2006.01129.x	journal	January 2007
Microscale sulfur cycling in the phototrophic pink berry consortia of the Sippewissett Salt Marsh: Sulfur cycling in pink berries of the Sippewissett Marsh Wilbanks, Elizabeth G.; Jaekel, Ulrike; Salman, Verena Environmental Microbiology, Vol. 16, Issue 11 https://doi.org/10.1111/1462-2920.12388	journal	February 2014
The Green Berry Consortia of the Sippewissett Salt Marsh: Millimeter-Sized Aggregates of Diazotrophic Unicellular Cyanobacteria Wilbanks, Elizabeth G.; Salman-Carvalho, Verena; Jaekel, Ulrike Frontiers in Microbiology, Vol. 8 https://doi.org/10.3389/fmicb.2017.01623	journal	September 2017
Cable bacteria associated with long-distance electron transport in New England salt marsh sediment Larsen, Steffen; Nielsen, Lars Peter; Schramm, Andreas Environmental Microbiology Reports, Vol. 7, Issue 2 https://doi.org/10.1111/1758-2229.12216	journal	December 2014
Calcite-accumulating large sulfur bacteria of the genus Achromatium in Sippewissett Salt Marsh Salman, Verena; Yang, Tingting; Berben, Tom The ISME Journal, Vol. 9, Issue 11 https://doi.org/10.1038/ismej.2015.62	journal	April 2015
Seasonal Succession and Spatial Patterns of Synechococcus Microdiversity in a Salt Marsh Estuary Revealed through 16S rRNA Gene Oligotyping Mackey, Katherine R. M.; Hunter-Cevera, Kristen; Britten, Gregory L. Frontiers in Microbiology, Vol. 8 https://doi.org/10.3389/fmicb.2017.01496	journal	August 2017
Salt marsh sediment diversity: a test of the variability of the rare biosphere among environmental replicates Bowen, Jennifer L.; Morrison, Hilary G.; Hobbie, John E. The ISME Journal, Vol. 6, Issue 11 https://doi.org/10.1038/ismej.2012.47	journal	June 2012
Mirrored stainless steel substrate provides improved signal for Raman spectroscopy of tissue and cells Lewis, Aaran T.; Gaifulina, Riana; Isabelle, Martin Journal of Raman Spectroscopy, Vol. 48, Issue 1 https://doi.org/10.1002/jrs.4980	journal	July 2016
Sub-Micrometer-Scale Mapping of Magnetite Crystals and Sulfur Globules in Magnetotactic Bacteria Using Confocal Raman Micro-Spectrometry Eder, Stephan H. K.; Gigler, Alexander M.; Hanzlik, Marianne PLoS ONE, Vol. 9, Issue 9 https://doi.org/10.1371/journal.pone.0107356	journal	September 2014
Double Labeling of Oligonucleotide Probes for Fluorescence In Situ Hybridization (DOPE-FISH) Improves Signal Intensity and Increases rRNA Accessibility Stoecker, K.; Dorninger, C.; Daims, H. Applied and Environmental Microbiology, Vol. 76, Issue 3 https://doi.org/10.1128/AEM.02456-09	journal	December 2009
The Domain-specific Probe EUB338 is Insufficient for the Detection of all Bacteria: Development and Evaluation of a more Comprehensive Probe Set Daims, Holger; Brühl, Andreas; Amann, Rudolf Systematic and Applied Microbiology, Vol. 22, Issue 3 https://doi.org/10.1016/S0723-2020(99)80053-8	journal	September 1999
In Situ Accessibility of Small-Subunit rRNA of Members of the Domains Bacteria, Archaea, and Eucarya to Cy3-Labeled Oligonucleotide Probes Behrens, S.; Ruhland, C.; Inacio, J. Applied and Environmental Microbiology, Vol. 69, Issue 3 https://doi.org/10.1128/AEM.69.3.1748-1758.2003	journal	March 2003
Optimizing fluorescent in situ hybridization with rRNA-targeted oligonucleotide probes for flow cytometric identification of microorganisms Wallner, Günter; Amann, Rudolf; Beisker, Wolfgang Cytometry, Vol. 14, Issue 2 https://doi.org/10.1002/cyto.990140205	journal	January 1993
Phenotypic heterogeneity in metabolic traits among single cells of a rare bacterial species in its natural environment quantified with a combination of flow cell sorting and NanoSIMS Zimmermann, Matthias; Escrig, Stéphane; Hübschmann, Thomas Frontiers in Microbiology, Vol. 06 https://doi.org/10.3389/fmicb.2015.00243	journal	April 2015
A pipeline for targeted metagenomics of environmental bacteria Grieb, Anissa; Bowers, Robert M.; Oggerin, Monike Microbiome, Vol. 8, Issue 1 https://doi.org/10.1186/s40168-020-0790-7	journal	February 2020
NanoSIMS sample preparation decreases isotope enrichment: magnitude, variability and implications for single‐cell rates of microbial activity Meyer, Nicolette R.; Fortney, Julian L.; Dekas, Anne E. Environmental Microbiology, Vol. 23, Issue 1 https://doi.org/10.1111/1462-2920.15264	journal	October 2020
The effect of FISH and CARD-FISH on the isotopic composition of 13C- and 15N-labeled Pseudomonas putida cells measured by nanoSIMS Musat, Niculina; Stryhanyuk, Hryhoriy; Bombach, Petra Systematic and Applied Microbiology, Vol. 37, Issue 4 https://doi.org/10.1016/j.syapm.2014.02.002	journal	June 2014
Single-cell identification in microbial communities by improved fluorescence in situ hybridization techniques Amann, Rudolf; Fuchs, Bernhard M. Nature Reviews Microbiology, Vol. 6, Issue 5 https://doi.org/10.1038/nrmicro1888	journal	May 2008
Raman microspectroscopy for microbiology Lee, Kang Soo; Landry, Zachary; Pereira, Fátima C. Nature Reviews Methods Primers, Vol. 1, Issue 1 https://doi.org/10.1038/s43586-021-00075-6	journal	November 2021
Single cell stable isotope probing in microbiology using Raman microspectroscopy Wang, Yun; Huang, Wei E.; Cui, Li Current Opinion in Biotechnology, Vol. 41 https://doi.org/10.1016/j.copbio.2016.04.018	journal	October 2016
Advancements in the application of NanoSIMS and Raman microspectroscopy to investigate the activity of microbial cells in soils Eichorst, Stephanie A.; Strasser, Florian; Woyke, Tanja FEMS Microbiology Ecology, Vol. 91, Issue 10 https://doi.org/10.1093/femsec/fiv106	journal	August 2015
Influence of Carbon Sources on Quantification of Deuterium Incorporation in Heterotrophic Bacteria: A Raman-Stable Isotope Labeling Approach Matanfack, Georgette Azemtsop; Taubert, Martin; Guo, Shuxia Analytical Chemistry, Vol. 92, Issue 16 https://doi.org/10.1021/acs.analchem.0c02443	journal	July 2020
Single‐cell determination of iron content in magnetotactic bacteria: implications for the iron biogeochemical cycle Amor, Matthieu; Tharaud, Mickaël; Gélabert, Alexandre Environmental Microbiology, Vol. 22, Issue 3 https://doi.org/10.1111/1462-2920.14708	journal	June 2019
Magnetic iron-sulphur crystals from a magnetotactic microorganism Farina, Marcos; Esquivel, Darci Motta S.; de Barros, Henrique G. P. Lins Nature, Vol. 343, Issue 6255 https://doi.org/10.1038/343256a0	journal	January 1990
Ultrastructure, tactic behaviour and potential for sulfate reduction of a novel multicellular magnetotactic prokaryote from North Sea sediments Wenter, Roland; Wanner, Gerhard; Schüler, Dirk Environmental Microbiology, Vol. 11, Issue 6 https://doi.org/10.1111/j.1462-2920.2009.01877.x	journal	June 2009
Characterization and phylogenetic identification of a species of spherical multicellular magnetotactic prokaryotes that produces both magnetite and greigite crystals Zhang, Rui; Chen, Yi-Ran; Du, Hai-Jian Research in Microbiology, Vol. 165, Issue 7 https://doi.org/10.1016/j.resmic.2014.07.012	journal	September 2014
Diversity and Characterization of Multicellular Magnetotactic Prokaryotes From Coral Reef Habitats of the Paracel Islands, South China Sea Teng, Zhaojie; Zhang, Yuyang; Zhang, Wenyan Frontiers in Microbiology, Vol. 9 https://doi.org/10.3389/fmicb.2018.02135	journal	September 2018
The transformation of mackinawite into greigite studied by Raman spectroscopy Bourdoiseau, Jacques-André; Jeannin, Marc; Rémazeilles, Céline Journal of Raman Spectroscopy, Vol. 42, Issue 3 https://doi.org/10.1002/jrs.2729	journal	March 2011
Iron sulfides from magnetotactic bacteria; structure, composition, and phase transitions Posfai, Mihaly; Buseck, Peter R.; Bazylinski, Dennis A. American Mineralogist, Vol. 83, Issue 11-12 Part 2 https://doi.org/10.2138/am-1998-11-1235	journal	December 1998
Imaging Intracellular Fluorescent Proteins at Nanometer Resolution Betzig, E.; Patterson, G. H.; Sougrat, R. Science, Vol. 313, Issue 5793 https://doi.org/10.1126/science.1127344	journal	September 2006
Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM) Rust, Michael J.; Bates, Mark; Zhuang, Xiaowei Nature Methods, Vol. 3, Issue 10 https://doi.org/10.1038/nmeth929	journal	August 2006
In situ visualization of newly synthesized proteins in environmental microbes using amino acid tagging and click chemistry : Visualizing new proteins in microbes by click chemistry Hatzenpichler, Roland; Scheller, Silvan; Tavormina, Patricia L. Environmental Microbiology, Vol. 16, Issue 8 https://doi.org/10.1111/1462-2920.12436	journal	April 2014
Individual cell DNA synthesis within natural marine bacterial assemblages as detected by ‘click’ chemistry Smriga, S.; Samo, Tj; Malfatti, F. Aquatic Microbial Ecology, Vol. 72, Issue 3 https://doi.org/10.3354/ame01698	journal	July 2014
d -Amino Acid Chemical Reporters Reveal Peptidoglycan Dynamics of an Intracellular Pathogen Siegrist, M. Sloan; Whiteside, Sarah; Jewett, John C. ACS Chemical Biology, Vol. 8, Issue 3 https://doi.org/10.1021/cb3004995	journal	January 2013
Cell organization and ultrastructure of a magnetotactic multicellular organism Keim, Carolina N.; Abreu, Fernanda; Lins, Ulysses Journal of Structural Biology, Vol. 145, Issue 3 https://doi.org/10.1016/j.jsb.2003.10.022	journal	March 2004

Similar Records

Multicellular magnetotactic bacteria are genetically heterogeneous consortia with metabolically differentiated cells

Journal Article · 2024 · PLoS Biology (Online) · OSTI ID:1883223

Schaible, George A.; Jay, Zackary J.; Cliff, John; +7 more

Journal Article · 2013 · Microscopy and Microanalysis · OSTI ID:1883223

Carpenter, Kevin J.; Weber, Peter K.; Davisson, M. Lee; +3 more

Advancements in the application of NanoSIMS and Raman microspectroscopy to investigate the activity of microbial cells in soils

Journal Article · 2015 · FEMS Microbiology Ecology (Online) · OSTI ID:1883223

Eichorst, Stephanie A.; Strasser, Florian; Woyke, Tanja; +4 more

Title: Correlative SIP-FISH-Raman-SEM-NanoSIMS links identity, morphology, biochemistry, and physiology of environmental microbes

Citation Formats

References (62)

Similar Records

Related Subjects