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The architecture of sporangia and zoospores of Actinoplanes missouriensis was analyzed at a high resolution using quick-freeze deep-etch replica electron microscopy. This analysis revealed that (i) sporangia were surrounded by at least 2 membranous layers with smooth surfaces, (ii) zoospores were enclosed by a fibrillar layer, and (iii) flagella were generated in a restricted area on the zoospore surface. [ABSTRACT FROM AUTHOR]

Escherichia coli produces extracellular vesicles called outer membrane vesicles (OMVs) by releasing a part of its outer membrane. We previously reported that the combined deletion of nlpI and mlaE, related to envelope structure and phospholipid accumulation in the outer leaflet of the outer membrane, respectively, resulted in the synergistic increase of OMV production. In this study, the analysis of ΔmlaEΔnlpI cells using quick-freeze, deep-etch electron microscopy (QFDE-EM) revealed that plasmolysis occurred at the tip of the long axis in cells and that OMVs formed from this tip. Plasmolysis was also observed in the single-gene knockout mutants ΔnlpI and ΔmlaE. This study has demonstrated that plasmolysis was induced in the hypervesiculating mutant E. coli cells. Furthermore, intracellular vesicles and multilamellar OMV were observed in the ΔmlaEΔnlpI cells. Meanwhile, the secretion of recombinant green fluorescent protein (GFP) expressed in the cytosol of the ΔmlaEΔnlpI cells was more than 100 times higher than that of WT and ΔnlpI, and about 50 times higher than that of ΔmlaE in the OMV fraction, suggesting that cytosolic components were incorporated into outer-inner membrane vesicles (OIMVs) and released into the extracellular space. Additionally, QFDE-EM analysis revealed that ΔmlaEΔnlpI sacculi contained many holes noticeably larger than the mean radius of the peptidoglycan (PG) pores in wild-type (WT) E. coli. These results suggest that in ΔmlaEΔnlpI cells, cytoplasmic membrane materials protrude into the periplasmic space through the peptidoglycan holes and are released as OIMVs.

Escherichia coli produces extracellular vesicles called outer membrane vesicles (OMVs) by releasing a part of its outer membrane. We previously reported that the combined deletion of nlpI and mlaE , related to envelope structure and phospholipid accumulation in the outer leaflet of the outer membrane, respectively, resulted in the synergistic increase of OMV production. In this study, the analysis of Δ mlaE Δ nlpI cells using quick-freeze, deep-etch electron microscopy (QFDE-EM) revealed that plasmolysis occurred at the tip of the long axis in cells and that OMVs formed from this tip. Plasmolysis was also observed in the single-gene knockout mutants Δ nlpI and Δ mlaE. This study has demonstrated that plasmolysis was induced in the hypervesiculating mutant E. coli cells. Furthermore, intracellular vesicles and multilamellar OMV were observed in the Δ mlaE Δ nlpI cells. Meanwhile, the secretion of recombinant green fluorescent protein (GFP) expressed in the cytosol of the Δ mlaE Δ nlpI cells was more than 100 times higher than that of WT and Δ nlpI , and about 50 times higher than that of Δ mlaE in the OMV fraction, suggesting that cytosolic components were incorporated into outer-inner membrane vesicles (OIMVs) and released into the extracellular space. Additionally, QFDE-EM analysis revealed that Δ mlaE Δ nlpI sacculi contained many holes noticeably larger than the mean radius of the peptidoglycan (PG) pores in wild-type (WT) E. coli. These results suggest that in Δ mlaE Δ nlpI cells, cytoplasmic membrane materials protrude into the periplasmic space through the peptidoglycan holes and are released as OIMVs. [ABSTRACT FROM AUTHOR]

Nagilactone E, an antifungal agent derived from the root bark of Podocarpus nagi, inhibits 1,3-β glucan synthesis; however, its inhibitory activity is weak. Anethole, the principal component of anise oil, enhances the antifungal activity of nagilactone E. We aimed to determine the combinatorial effect and underlying mechanisms of action of nagilactone E and anethole against the budding yeast Saccharomyces cerevisiae. Analyses using gene-deficient strains showed that the multidrug efflux pump PDR5 is associated with nagilactone E resistance; its transcription was gradually restricted in cells treated with the drug combination for a prolonged duration but not in nagilactone-E-treated cells. Green-fluorescent-protein-tagged Pdr5p was intensively expressed and localized on the plasma membrane of nagilactone-E-treated cells but not in drug-combination-treated cells. Quick-freeze deep-etch electron microscopy revealed the smoothening of intertwined fiber structures on the cell surface of drug-combination-treated cells and spheroplasts, indicating a decline in cell wall components and loss of cell wall strength. Anethole enhanced the antifungal activity of nagilactone E by enabling its retention within cells, thereby accelerating cell wall damage. The combination of nagilactone E and anethole can be employed in clinical settings as an antifungal, as well as a food preservative to restrict food spoilage.

A lichen is a slow-growing niche-constructing organism that forms a thallus via scripted symbiotic/mutualist relationships between fungi, algae, and bacteria. Here we use quick-freeze deep-etch electron microscopy (QFDEEM), in conjunction with light microscopy, to document the structural manifestations of hyphal differentiation during the formation of three lichen tissues that localize between the algal layer and the surface of the thallus: the outer cortex of foliose lobes; the outer layer of fruticose stems; and the enwrapping layer of asexual propagules called soredia that protrude from squamulose podetia and foliose lobes. Our observations document features of outer-layer architecture and the role played by extracellular matrices (ECM). They also lead us to propose the medullary stem-cell hypothesis for lichen organization wherein totipotent medullary hyphae produce lateral branches that undergo specific differentiation pathways in specific domains of the thallus. • Lichen medullary hyphae grow by bifurcation and differentiate by branching. • Outer cortex of lichen lobe conglutinated by hyphal sub-branches and extracellular matrix fibrils. • Crystals of secondary products coat hyphal walls in lichen propagules. • Medullary hyphae display characteristics of stem cells. [ABSTRACT FROM AUTHOR]

A lichen is a slow-growing niche-constructing organism that forms a thallus via scripted symbiotic/mutualist relationships between fungi, algae, and bacteria. Here we use quick-freeze deep-etch electron microscopy (QFDEEM) and light microscopy to probe a hallmark lichen construction wherein clusters of algae and hyphae, inter-connected by wall-to-wall junctions, form stable consortia that we call green modules. These assemble in the pseudo-meristem and then localize to the algal layer of the thallus. In the foliose lobes of Candelaria concolor , the cells in each module are enveloped in a continuous 10-nm-thick film of hydrophobin proteins, which binds to wall and matrix surfaces on its hydrophilic side and faces air or water on its hydrophobic side. We document patterned relationships between modules and associated cords of hyphae destined for the outer layers, and propose ways that these relationships could form the structural foundation for water and air regulation within foliose lobes. [ABSTRACT FROM AUTHOR]

A lichen is a slow-growing niche-constructing organism that form a thallus via scripted symbiotic/mutualist relationships between fungi, algae, and bacteria. In preceding papers we have presented quick-freeze deep-etch EM (QFDEEM) images of the algal and outer layers of squamulose, foliose and fruticose lichens. Here we examine the remaining layers, one occupied by medullary fungal hyphae, and the others including a stunning variety of bacterial biofilms and novel extracellular matrix (ECM) materials. We document that the medullary compartment is filled with an ECM wherein fungal hyphae and secondary metabolites, many crystalline, are suspended in a ground substance that we call fog. We propose that fog is a liquid-glass-like mixture of secondary metabolites (synthesized by the fungi), polyols (synthesized by the algae), and polyol-sequestered water. Bacteria are described in several contexts. In the outer cortical layers of foliose Candelaria and Physcia lobes, they form patchy biofilm islands atop the fungal walls and the polysaccharide-based ECM. In the inner surface of Cladonia podetia they form long heterospecific biofilms at the lichen/external water boundary and at the fog boundary, reinforcing the intrinsic fog/water phase separation and preventing the fog from leaking out. In the outer layer of Usnea fibrils and the inner cortex of foliose lobes, they join fungi and extracellular materials to form surface boundaries. Hence lichenized bacteria not only participate in metabolic exchange but also serve architectural roles in lichen construction and maintenance. • Medullary space contains viscous liquid called fog, posited to contain polyols. • Bacterial biofilms associate with fungal walls and with one another. • Biofilms include distinct bacterial species in matrix continuity. • Biofilm matrices participate in forming outer surface boundaries of lichens. [ABSTRACT FROM AUTHOR]

Lichens are slow-growing niche-constructing organisms that form a thallus via scripted symbiotic/mutualist relationships between fungi, algae, and bacteria. Here we use quick-freeze deep-etch electron microscopy (QFDEEM) to document the ultrastructure of the eukaryotic organisms and the extracellular matrix materials that are found in four lichens. Previous studies have shown that each thallus in a complex lichen consists of a central medullary layer containing aligned fungal hyphae. These medullary hyphae send lateral branches to an upper layer, some making contact with resident algae, and usually to a lower layer. As the thallus ages, such "regular" branches transform into acellular versions called "struts" and "honeycombs." We conclude with a consideration of two materials that are synthesized and secreted by lichenized fungi: abundant secondary products/metabolites that often crystallize, and hydrophobin proteins that self-assemble into films. • Some lichenized hyphae differentiate into acellular struts and honeycombs • Algal pyrenoids are enlarged and contain plastoglobules • Fungal secondary products form crystals • Fungal hydrophobin proteins form extensive films [ABSTRACT FROM AUTHOR]

Lichens are slow-growing niche-constructing organisms that form a thallus via scripted symbiotic/mutualistic relationships between fungi, algae, and bacteria, and that are distributed across nearly all terrestrial ecosystems. Here we use quick-freeze deep-etch electron microscopy (QFDEEM) to probe the ultrastructure of a lichen-forming fungus, Cladonia grayi , and its partner alga, Asterochloris glomerata , grown separately (solo) in the laboratory. The solo fungus resembles its lichenized counterpart in: a) general cellular organization; b) the capacity to form lateral branches; c) the production of extracellular materials; and d) the generation of acellular hyphae that we call struts. It differs in carrying a fibrillar coat on its wall exterior and in often adopting two novel plasma-membrane configurations called pleated and pitted. The solo alga also resembles its lichenized counterpart in general cellular organization, but its algaenan-based wall carries a fibrillar coat not evident in the lichen, and its pyrenoid and plastoglobule endowments are less well developed. These findings are followed by four reports on the QFDEEM ultrastructure of four species of lichens, where we describe the differentiations that occur when the fungi and algae are living together in community with bacteria. • Solo fungal walls and solo algal walls carry fibrillar coats. • Solo fungal plasma membranes carry differentiations called pleats and pits. • Solo fungi secrete coarse fibers and plates that form a meshwork. • Some solo hyphal branches differentiate into acellular forms called struts. [ABSTRACT FROM AUTHOR]

A lichen is a slow-growing niche-constructing organism that forms a thallus via scripted symbiotic/mutualist relationships between fungi, algae, and bacteria. Here we use quick-freeze deep-etch electron microscopy (QFDEEM), in conjunction with light microscopy, to document the structural manifestations of hyphal differentiation during the formation of three lichen tissues that localize between the algal layer and the surface of the thallus: the outer cortex of foliose lobes; the outer layer of fruticose stems; and the enwrapping layer of asexual propagules called soredia that protrude from squamulose podetia and foliose lobes. Our observations document features of outer-layer architecture and the role played by extracellular matrices (ECM). They also lead us to propose the medullary stem-cell hypothesis for lichen organization wherein totipotent medullary hyphae produce lateral branches that undergo specific differentiation pathways in specific domains of the thallus. • Lichen medullary hyphae grow by bifurcation and differentiate by branching. • Outer cortex of lichen lobe conglutinated by hyphal sub-branches and extracellular matrix fibrils. • Crystals of secondary products coat hyphal walls in lichen propagules. • Medullary hyphae display characteristics of stem cells. [ABSTRACT FROM AUTHOR]

Nagilactone E, an antifungal agent derived from the root bark of Podocarpus nagi , inhibits 1,3-β glucan synthesis; however, its inhibitory activity is weak. Anethole, the principal component of anise oil, enhances the antifungal activity of nagilactone E. We aimed to determine the combinatorial effect and underlying mechanisms of action of nagilactone E and anethole against the budding yeast Saccharomyces cerevisiae . Analyses using gene-deficient strains showed that the multidrug efflux pump PDR5 is associated with nagilactone E resistance; its transcription was gradually restricted in cells treated with the drug combination for a prolonged duration but not in nagilactone-E-treated cells. Green-fluorescent-protein-tagged Pdr5p was intensively expressed and localized on the plasma membrane of nagilactone-E-treated cells but not in drug-combination-treated cells. Quick-freeze deep-etch electron microscopy revealed the smoothening of intertwined fiber structures on the cell surface of drug-combination-treated cells and spheroplasts, indicating a decline in cell wall components and loss of cell wall strength. Anethole enhanced the antifungal activity of nagilactone E by enabling its retention within cells, thereby accelerating cell wall damage. The combination of nagilactone E and anethole can be employed in clinical settings as an antifungal, as well as a food preservative to restrict food spoilage.