A Complex Endomembrane System in the Archaeon Ignicoccus hospitalis Tapped by Nanoarchaeum equitans

Heimerl, Thomas; Flechsler, Jennifer; Pickl, Carolin; Heinz, Veronika; Salecker, Benjamin; Zweck, Josef; Wanner, Gerhard; Geimer, Stefan; Samson, Rachel Y.; Bell, Stephen D.; Huber, Harald; Wirth, Reinhard; Wurch, Louie L.; Podar, Mircea; Rachel, Reinhard

doi:10.3389/fmicb.2017.01072

Cited by 64 publications

(63 citation statements)

References 60 publications

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“…For instance, surface proteins homologous to hemolysine [8] could initiate such contacts as observed previously for acidophilic Micrarchaeota using cryo-electron tomography [5,17]. More recently, fusion of cell membranes of N. equitans and its host Ignicoccus hospitalis was observed by transmission electron microscopy [18].…”

Section: Introductionmentioning

confidence: 54%

An archaeal symbiont-host association from the deep terrestrial subsurface

et al. 2019

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DPANN archaea have reduced metabolic capacities and are diverse and abundant in deep aquifer ecosystems, yet little is known about their interactions with other microorganisms that reside there. Here, we provide evidence for an archaeal host-symbiont association from a deep aquifer system at the Colorado Plateau (Utah, USA). The symbiont, Candidatus Huberiarchaeum crystalense, and its host, Ca. Altiarchaeum hamiconexum, show a highly significant co-occurrence pattern over 65 metagenome samples collected over six years. The physical association of the two organisms was confirmed with genome-informed fluorescence in situ hybridization depicting small cocci of Ca. H. crystalense attached to Ca. A. hamiconexum cells. Based on genomic information, Ca. H. crystalense potentially scavenges vitamins, sugars, nucleotides, and reduced redoxequivalents from its host and thus has a similar metabolism as Nanoarchaeum equitans. These results provide insight into host-symbiont interactions among members of two uncultivated archaeal phyla that thrive in a deep subsurface aquifer.Recently, subsurface fluids discharged by a cold, CO 2 -driven geyser at the Colorado Plateau, Utah (Crystal Geyser) revealed the presence of multiple novel archaeal organisms, including the first representative of the Candidatus phylum Huberarchaeota [8], which we show to be part of the

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Section: Introductionmentioning

confidence: 54%

An archaeal symbiont-host association from the deep terrestrial subsurface

et al. 2019

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“…FS is generally performed in acetone containing up to several percent of OsO 4 and uranyl acetate (UA) aiming to stabilize sample structure and increase electron scattering. Recent studies on archaeal models indicated that OsO 4 can be excluded without loss of structure preservation and SEM contrast (Heimerl et al, 2017). The omission of OsO 4 also allows to preserve antigenicity after epoxy-embedding (Parthasarathy, 1995;Rachel et al, 2010).…”

Section: Introductionmentioning

confidence: 99%

FIB‐SEM imaging properties of Drosophila melanogaster tissues embedded in Lowicryl HM20

Porrati

Grewe

Seybert

et al. 2018

Journal of Microscopy

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Summary Lowicryl resins enable processing of biological material for electron microscopy at the lowest temperatures compatible with resin embedding. When combined with high‐pressure freezing and freeze‐substitution, Lowicryl embedding supports preservation of fine structural details and fluorescent markers. Here, we analysed the applicability of Lowicryl HM20 embedding for focused ion beam (FIB) scanning electron microscopy (SEM) tomography of Drosophila melanogaster embryonic and larval model systems. We show that the freeze‐substitution with per‐mill concentrations of uranyl acetate provided sufficient contrast and an image quality of SEM imaging in the range of similar samples analysed by transmission electron microscopy (TEM). Preservation of genetically encoded fluorescent proteins allowed correlative localization of regions of interest (ROI) within the embedded tissue block. TEM on sections cut from the block face enabled evaluation of structural preservation to allow ROI ranking and thus targeted, time‐efficient FIB‐SEM tomography data collection. The versatility of Lowicryl embedding opens new perspectives for designing hybrid SEM‐TEM workflows to comprehensively analyse biological structures. Lay Description Focused ion beam scanning electron microscopy is becoming a widely used technique for the three‐dimensional analysis of biological samples at fine structural details beyond levels feasible for light microscopy. To withstand the abrasion of material by the ion beam and the imaging by the scanning electron beam, biological samples have to be embedded into resins, most commonly these are very dense epoxy‐based plastics. However, dense resins generate electron scattering which interferes with the signal from the biological specimen. Furthermore, to improve the imaging contrast, epoxy embedding requires chemical treatments with e.g. heavy metals, which deteriorate the ultrastructure of the biological specimen. In this study we explored the applicability of an electron lucent resin, Lowicryl HM 20, for focused ion beam scanning electron microscopy. The Lowicryl embedding workflow operates at milder chemical treatments and lower temperatures, thus preserving the sub‐cellular and sub‐organellar organization, as well as fluorescent markers visible by light microscopy. Here we show that focus ion beam scanning electron microscopy of Lowicryl‐embedded fruit flies tissues provides reliable imaging revealing fine structural details. Our workflow benefited from use of transmission electron microscopy for the quality control of the ultrastructural preservation and fluorescent light microscopy for localization of regions of interest. The versatility of Lowicryl embedding opens up new perspectives for designing hybrid workflows combining fluorescent light, scanning, and transmission electron microscopy techniques to comprehensively analyze biological structures.

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“…Antibodies directed against the Ignicoccus A 1 ‐part of the ATP synthase (polyclonal anti‐A 1 antibodies were used in a dilution of 1:200), and antibodies directed against the acetyl‐CoA synthetase (ACS; polyclonal anti‐ACS antibodies were used in a dilution of 1:100) were raised in rabbits from the purified native complex (Heimerl et al, ; Küper, Meyer, Müller, Rachel, & Huber, ; Mayer et al, ). The monoclonal antibody against ß‐glucan chrysolaminarin was raised in mice and was purchased at Biosupplies (Australia).…”

Section: Methodsmentioning

confidence: 99%

“…For 3D reconstructions, we used serial ultrathin sections with a nominal thickness of 70 nm. Segmentation and 3D reconstruction of EM data was carried out with AMIRA (Thermo Fisher Scientific) as described previously (Heimerl et al, ).…”

Section: Methodsmentioning

confidence: 99%

2D and 3D immunogold localization on (epoxy) ultrathin sections with and without osmium tetroxide

Flechsler¹,

Heimerl

Pickl³

et al. 2020

Microscopy Res & Technique

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For nearly 50 years immunogold labeling on ultrathin sections has been successfully used for protein localization in laboratories worldwide. In theory and in practice, this method has undergone continual improvement over time. In this study, we carefully analyzed circulating protocols for postembedding labeling to find out if they are still valid under modern laboratory conditions, and in addition, we tested unconventional protocols. For this, we investigated immunolabeling of Epon-embedded cells, immunolabeling of cells treated with osmium, and the binding behavior of differently sized gold particles. Here we show that (in contrast to widespread belief) immunolabeling of Epon-embedded cells and of cells treated with osmium tetroxide is actually working. Furthermore, we established a "speed protocol" for immunolabeling by reducing antibody incubation times. Finally, we present our results on threedimensional immunogold labeling. K E Y W O R D S(serial) immunogold labeling, Archaea, Diatoms, Epon 812-osmium tetroxide

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A Complex Endomembrane System in the Archaeon Ignicoccus hospitalis Tapped by Nanoarchaeum equitans

Cited by 64 publications

References 60 publications

An archaeal symbiont-host association from the deep terrestrial subsurface

An archaeal symbiont-host association from the deep terrestrial subsurface

FIB‐SEM imaging properties of Drosophila melanogaster tissues embedded in Lowicryl HM20

2D and 3D immunogold localization on (epoxy) ultrathin sections with and without osmium tetroxide

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