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Cryo-Electron Tomography Reveals the Complex Ultrastructural Organization of Multicellular Filamentous Chloroflexota (Chloroflexi) Bacteria

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Cryo-Electron Tomography Reveals the Complex Ultrastructural Organization of Multicellular Filamentous Chloroflexota (Chloroflexi) Bacteria Research Collection Journal Article Cryo-Electron Tomography Reveals the Complex Ultrastructural Organization of Multicellular Filamentous Chloroflexota (Chloroflexi) Bacteria Author(s): Gaisin, Vasil A.; Kooger, Romain; Grouzdev, Denis S.; Gorlenko, Vladimir M.; Pilhofer, Martin Publication Date: 2020-06-26 Permanent Link: https://doi.org/10.3929/ethz-b-000426885 Originally published in: Frontiers in Microbiology 11, http://doi.org/10.3389/fmicb.2020.01373 Rights / License: Creative Commons Attribution 4.0 International This page was generated automatically upon download from the ETH Zurich Research Collection. For more information please consult the Terms of use. ETH Library fmicb-11-01373 June 24, 2020 Time: 17:43 # 1 ORIGINAL RESEARCH published: 26 June 2020 doi: 10.3389/fmicb.2020.01373 Cryo-Electron Tomography Reveals the Complex Ultrastructural Organization of Multicellular Filamentous Chloroflexota (Chloroflexi) Bacteria Vasil A. Gaisin1,2*†, Romain Kooger3†, Denis S. Grouzdev1, Vladimir M. Gorlenko1 and Martin Pilhofer3 1 Research Center of Biotechnology of the Russian Academy of Sciences, Moscow, Russia, 2 Algatech, Institute of Microbiology of the Czech Academy of Sciences, Treboˇ n,ˇ Czechia, 3 Institute of Molecular Biology & Biophysics, Eidgenössische Technische Hochschule Zürich, Zurich, Switzerland The cell biology of Chloroflexota is poorly studied. We applied cryo-focused ion beam milling and cryo-electron tomography to study the ultrastructural organization Edited by: of thermophilic Roseiflexus castenholzii and Chloroflexus aggregans, and mesophilic Felipe Cava, “Ca. Viridilinea mediisalina.” These species represent the three main lineages within Umeå University, Sweden a group of multicellular filamentous anoxygenic phototrophic Chloroflexota bacteria Reviewed by: Donald A. Bryant, belonging to the Chloroflexales order. We found surprising structural complexity in Pennsylvania State University (PSU), the Chloroflexales. As with filamentous cyanobacteria, cells of C. aggregans and “Ca. United States Viridilinea mediisalina” share the outer membrane-like layers of their intricate multilayer Conrad Mullineaux, Queen Mary University of London, cell envelope. Additionally, cells of R. castenholzii and “Ca. Viridilinea mediisalina” are United Kingdom connected by septal channels that resemble cyanobacterial septal junctions. All three *Correspondence: strains possess long pili anchored close to cell-to-cell junctions, a morphological feature Vasil A. Gaisin [email protected] comparable to that observed in cyanobacteria. The cytoplasm of the Chloroflexales †These authors have contributed bacteria is crowded with intracellular organelles such as different types of storage equally to this work granules, membrane vesicles, chlorosomes, gas vesicles, chemoreceptor-like arrays, and cytoplasmic filaments. We observed a higher level of complexity in the mesophilic Specialty section: This article was submitted to strain compared to the thermophilic strains with regards to the composition of Microbial Physiology and Metabolism, intracellular bodies and the organization of the cell envelope. The ultrastructural details a section of the journal that we describe in these Chloroflexales bacteria will motivate further cell biological Frontiers in Microbiology studies, given that the function and evolution of the many discovered morphological Received: 01 April 2020 Accepted: 27 May 2020 traits remain enigmatic in this diverse and widespread bacterial group. Published: 26 June 2020 Keywords: ultrastructural organization, Chloroflexi, Chloroflexota, cryo-electron tomography, filamentous Citation: bacteria, multicellular bacteria, cell envelope, intracellular organelles Gaisin VA, Kooger R, Grouzdev DS, Gorlenko VM and Pilhofer M (2020) Cryo-Electron INTRODUCTION Tomography Reveals the Complex Ultrastructural Organization of Multicellular Filamentous There are only two groups of filamentous multicellular phototrophic bacteria: a polyphyletic group Chloroflexota (Chloroflexi) Bacteria. of filamentous cyanobacteria and green non-sulfur bacteria, which belong to the Cyanobacteria and Front. Microbiol. 11:1373. Chloroflexota (Chloroflexi) phyla, respectively. Despite green non-sulfur bacteria and filamentous doi: 10.3389/fmicb.2020.01373 cyanobacteria being phylogenetically distant lineages that have many differences in their biology, Frontiers in Microbiology| www.frontiersin.org 1 June 2020| Volume 11| Article 1373 fmicb-11-01373 June 24, 2020 Time: 17:43 # 2 Gaisin et al. Cryo-ET on Chloroflexi their phenotypes often share many significant similarities that (Pilhofer et al., 2010; Beck and Baumeister, 2016). Cryo-ET is are adapted to specific ecological niches. Their multicellular limited to the imaging of thin samples (<700 nm). Therefore, filaments often form a dense “fabric” of cyanobacterial mats imaging thicker bacteria has been dependent on the advances or biofilms (Bauld and Brock, 1973; Doemel and Brock, 1977; in sample thinning techniques such as cryo-focused ion beam Ley et al., 2006; Gaisin et al., 2015). In addition to their (cryo-FIB) milling (Marko et al., 2007). Here, we used cryo- the multicellular filamentous morphology, the cells contain ET and cryo-FIB milling followed by cryo-ET to study the (bacterio)chlorophylls that are essential to a phototrophic ultrastructural organization of the thermophilic Roseiflexus lifestyle. In addition, they employ comparable surface-dependent castenholzii, thermophilic Chloroflexus aggregans, and the “gliding” motility to migrate through a mat or form aggregates recently described mesophilic bacterium “Ca. Viridilinea (Richardson and Castenholz, 1987; Hanada, 2014). Thus, mediisalina” (Hanada et al., 1995, 2002; Gaisin et al., 2019a). We filamentous cyanobacteria and green non-sulfur bacteria possibly chose these species because each represents one of the three main present an example of convergent evolution that has led to lineages within the Chloroflexales order: bacteriochlorophyll resembling phenotypes in phylogenetically distant lineages. a-containing Roseiflexus-related members, bacteriochlorophyll Therefore, we postulated that studying the differences and a and c-containing Chloroflexus-related members, and a similarities between these two groups would help understand group of mesophilic bacteriochlorophyll a, c and d-containing the evolution of the morphological traits associated with members with gas vesicles. The results presented here allow multicellular phototrophic bacteria. To be able to compare the for a deeper understanding of the cell biology of Chloroflexales, morphologies between these two phyla, their ultrastructural as well as Chloroflexota bacteria in general. Particularly, these traits first need to be characterized. However, in contrast to data provide new information to the debate surrounding the cyanobacteria, the cell architecture of Chloroflexota bacteria is organization of the cell envelope in Chloroflexota (Sutcliffe, poorly understood. 2011; Cavalier-Smith and Chao, 2020), the mechanism of their Green non-sulfur bacteria is an outdated term used to motility and adherence (Fukushima et al., 2016; Fukushima, define phototrophic species of the Chloroflexota phylum. The 2016), and finally on the multicellular organization of the majority of phototrophic Chloroflexota bacteria belong to the phototrophic bacteria. Chloroflexales order (Grouzdev et al., 2018; Thiel et al., 2018). All Chloroflexales bacteria display a branchless filamentous multicellular morphology (Hanada, 2014). Their cells are colored MATERIALS AND METHODS because they contain bacteriochlorophylls and carotenoids. In all but one case, no noticeable ultrastructural differences have been Bacterial Cultures reported between cells of an individual multicellular filament, Culture of R. castenholzii DSM 13941 was grown in liquid leaving it unclear whether cell specialization occurs, as in the case medium consisting of the following components (g l−1): 0.5 of many cyanobacterial species. Oscillochloris chrysea, in which KH2PO4, 0.5 NH4Cl, 0.3 MgCl2·6H2O, 0.5 KCl, 0.5 NaCl, 0.5 the terminal cells have a morphology distinct from the rest of the Na2SO4, 0.2 Na2S·9H2O, 0.3 NaHCO3, 2.0 yeast extract, 3.0 filament, is the only exception (Gorlenko and Pivovarova, 1977; HEPES. The medium was completed by Wolfe’s vitamin (10 ml Garrity et al., 2001). In contrast, many filamentous cyanobacteria l−1) and trace element (10 ml l−1) solutions from ATCC. The pH possess specialized cells, such as vegetative cells, heterocysts of the medium was adjusted to 7.7. The culture was maintained and akinetes. In general, Chloroflexales bacteria seem to display in a glass tube with screw caps and rubber septa at a temperature a simpler organization than cyanobacteria, although this view of 50.5◦C under constant light (incandescent light bulb). might originate from the techniques that have been applied Culture of C. aggregans DSM 9485 was grown in liquid for their imaging. medium consisting of the following components (g l−1): 0.5 Progress in cryo-electron microscopy has enabled new KH2PO4, 0.5 NH4Cl, 0.3 MgCl2·6H2O, 0.5 KCl, 0.5 NaCl, 0.5 advances in the exploration of cyanobacterial cell biology, Na2SO4, 0.5 Na2S·9H2O, 0.3 NaHCO3, 1.5 yeast extract, 3.0 leading to the discovery of intricate macromolecular details HEPES. The medium was complemented with the 1000x trace (Dai et al., 2018; Rast et al., 2019; Weiss et al., 2019). As of element solution (1 ml l−1) and iron (III) citrate (0.006 g l−1) today, ultrastructural data on Chloroflexales bacteria are relatively
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