TEM as an Important Tool to Study Aquatic Microorganisms and their Relationships with Ecological Processes

Silva, Thiago Pereira da; Melo, Rossana C. N.

doi:10.5772/61804

Cited by 6 publications

(9 citation statements)

References 60 publications

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“…Over the past years, our research group has been studying the ultrastructure of bacteria and cyanobacteria by TEM both in situ and in cultures ( Silva et al, 2014 , 2016 ; Noyma et al, 2015 ; Gamalier et al, 2017 ). Our EM methodology includes prompt aldehyde fixation while the cells are still in suspension and before any subsequent centrifugation procedure, which is important to optimal cell preservation and to capture specific biological events in response to varied stimuli.…”

Section: Resultsmentioning

confidence: 99%

The Cyanobacterium Cylindrospermopsis raciborskii (CYRF-01) Responds to Environmental Stresses with Increased Vesiculation Detected at Single-Cell Resolution

et al. 2018

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Secretion of membrane-limited vesicles, collectively termed extracellular vesicles (EVs), is an important biological process of both eukaryotic and prokaryotic cells. This process has been observed in bacteria, but remains to be better characterized at high resolution in cyanobacteria. In the present work, we address the release of EVs by Cylindrospermopsis raciborskii (CYRF-01), a filamentous bloom-forming cyanobacterium, exposed to environmental stressors. First, non-axenic cultures of C. raciborskii (CYRF-01) were exposed to ultraviolet radiation (UVA + UVB) over a 6 h period, which is known to induce structural damage to this species. Second, C. raciborskii was co-cultured in interaction with another cyanobacterium species, Microcystis aeruginosa (MIRF-01), over a 24 h period. After the incubation times, cell density and viability were analyzed, and samples were processed for transmission electron microscopy (TEM). Our ultrastructural analyses revealed that C. raciborskii constitutively releases EVs from the outer membrane during its normal growth and amplifies such ability in response to environmental stressors. Both situations induced significant formation of outer membrane vesicles (OMVs) by C. raciborskii compared to control cells. Quantitative TEM revealed an increase of 48% (UV) and 60% (interaction) in the OMV numbers compared to control groups. Considering all groups, the OMVs ranged in size from 20 to 300 nm in diameter, with most OMVs showing diameters between 20 and 140 nm. Additionally, we detected that OMV formation is accompanied by phosphatidylserine exposure, a molecular event also observed in EV-secreting eukaryotic cells. Altogether, we identified for the first time that C. raciborskii has the competence to secrete OMVs and that under different stress situations the genesis of these vesicles is increased. The amplified ability of cyanobacteria to release OMVs may be associated with adaptive responses to changes in environmental conditions and interspecies cell communication.

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Section: Resultsmentioning

confidence: 99%

The Cyanobacterium Cylindrospermopsis raciborskii (CYRF-01) Responds to Environmental Stresses with Increased Vesiculation Detected at Single-Cell Resolution

et al. 2018

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“…The morphology of bacterial cells was studied in electron micrographs randomly taken at high resolution (30,000–100,000 x magnification) to enable visualization of the entire cell profile and structural organization of the cell envelope. For bacterial classification as gram‐negative or gram‐positive in control cultures, 134 cells were analysed and the cell envelope composition determined as before (Silva et al, 2016). For quantitative analyses of OMVs (numbers and diameters) and VLPs (numbers in the cytoplasm), a total of 278 gram‐negative bacteria showing the entire cell profile were analysed ( n = 38 cells from natural ecosystems; n = 120 cells from untreated control group; and n = 120 cells from VLP‐treated group) using the software Image J 1.41 (National Institutes of Health, Bethesda, MD, USA).…”

Section: Methodsmentioning

confidence: 99%

Enhanced ability of freshwater bacteria to secrete extracellular vesicles upon interaction with virus

Silva

Soares

Resende

et al. 2022

Environmental Microbiology

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The ability of freshwater bacteria to secrete extracellular vesicles (EVs) upon interaction with viruses remains to be established. Here, we investigated for the first time if freshwater virus-infected bacteria release EVs in both natural ecosystems and virus-like particles (VLPs)-enriched cultures. We performed a systematic study using transmission electron microscopy to visualize viruses and EVs at high resolution and single-cell imaging analyses to quantitate nascent EVs at the surface of gram-negative bacteria. First, by analysing freshwater samples from a tropical ecosystem (Negro River/Amazon Basin/Brazil), we captured bacteriophages-infected bacteria releasing EVs from their outer membrane. Next, VLPs isolated from these samples and inoculated in bacterial cultures not only impacted bacteria growth and viability but also led them to a significant release of EVs ($300% increase in numbers/cell section) compared to controls. The numbers of both budding and free EVs and EVs per linear micrometre of cell envelope were significantly higher in infected bacteria. Our findings identify a yet-not recognized capability of freshwater bacteria in generating EVs (overvesiculation) in response to viral infection. Since viruses are abundant members of aquatic ecosystems and bacteria are natural hosts for them, such interaction is an interesting event for microbial communities to be explored in freshwater ecosystems.

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“…Bacterial cells visualized by EM demonstrate high ultrastructural variability, but the causes of this diversity are unknown (22–34). Distinct reactions to environmental stress can be a reason for such heterogeneity, as shown for aquatic microorganisms (35). However, direct links between physiological state, stress factors, and the bacterial ultrastructure have not been demonstrated.…”

Section: Introductionmentioning

confidence: 99%

Exposure to stressors and antimicrobials induces cell-autonomous ultrastructural heterogeneity of an intracellular bacterial pathogen

Schulte

Hensel

Miśkiewicz

2020

Preprint

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Despite being clonal, bacterial pathogens show a remarkable physiological heterogeneity during infection of host and within host cells. This diversity is reflected by distinct ultrastructural morphotypes in transmission electron microscopy (TEM). Gram-negative bacteria visualized at high resolution by TEM show a rather simple composition of cytoplasm with a centrally located nucleoid and large number of ribosomes. The cytoplasm is separated from the external environment by inner and outer membranes. In this study, we show that individual cells of Salmonella enterica serovar Typhimurium (STM) are ultrastructural divergent in standard culture conditions, as well as during their intracellular lifestyle in mammalian host cells. STM can basically be discriminated into two morphotypes based on the criterion of cytoplasmic density. We identified environmental conditions which affect cytoplasmic densities. Using chemical treatments and defined mutant strains, we were able to link the occurrence of an electron-dense type to oxidative stress and other noxes. Furthermore, ultrastructural analyses of STM during infection and fluorescence reporter analyses for cell viability were combined in a correlative light and electron microscopy approach. We provide evidence that two newly characterized ultrastructural types with lucent or dense cytoplasm represent viable cells. Moreover, the presence of electron-dense types is stress related and can be experimentally induced only when amino acids are available in the environment. This study sheds more light on diversities between individual bacteria in populations and possible physiological meanings like a stress response to explain the diversities discussed.ImportanceBacterial pathogens show a remarkable resilience to adverse conditions during infection. Although being genetically identical, a clonal population may contain dead, dormant, slowly as well as rapidly proliferating cells. The physiological state of individual cells in a population may be analyzed by fluorescent probes or reporters. In contrast, reliable markers to interrogate single cells regarding viability, response to environmental cues, and exposure to antimicrobial compounds are sparse for ultrastructural approaches. For intracellular Salmonella enterica we observed distinct ultrastructural morphotypes. Using defined experimental conditions, these morphotypes were linked to reactions of bacteria to stressors or antimicrobials. The parameters defined here provide criteria for the interpretation of bacterial heterogeneity on the ultrastructural level.

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TEM as an Important Tool to Study Aquatic Microorganisms and their Relationships with Ecological Processes

Cited by 6 publications

References 60 publications

The Cyanobacterium Cylindrospermopsis raciborskii (CYRF-01) Responds to Environmental Stresses with Increased Vesiculation Detected at Single-Cell Resolution

The Cyanobacterium Cylindrospermopsis raciborskii (CYRF-01) Responds to Environmental Stresses with Increased Vesiculation Detected at Single-Cell Resolution

Enhanced ability of freshwater bacteria to secrete extracellular vesicles upon interaction with virus

Exposure to stressors and antimicrobials induces cell-autonomous ultrastructural heterogeneity of an intracellular bacterial pathogen

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