Biology subjects

Stryhanyuk, H.

Publications and source records attributed to Stryhanyuk, H..

6 recordsLinked to original sources

bioRxiv2024

Stable Isotope Probing-nanoFTIR for Quantitation of Cellular Metabolism and Observation of Growth-dependent Spectral Features

This study utilizes nanoscale Fourier transform infrared spectroscopy (nanoFTIR) to perform stable isotope probing (SIP) on individual bacteria cells cultured in the presence of 13C-labelled glucose. SIP-nanoFTIR simultaneously quantifies single-cell metabolism through infrared spectroscopy and acquires cellular morphological information via atomic force microscopy. The redshift of the amide I peak corresponds to the isotopic enrichment of newly synthesized proteins. These observations of single-cell translational activity are comparable to those of conventional methods, examining bulk cell numbers. Observing cells cultured under conditions of limited carbon, SIP-nanoFTIR is used to identify environmentally-induced changes in metabolic heterogeneity and cellular morphology. Individuals outcompeting their neighboring cells will likely play a disproportionately large role in shaping population dynamics during adverse conditions or environmental fluctuations. Additionally, SIP-nanoFTIR enables the spectroscopic differentiation of specific cellular growth phases. During cellular replication, subcellular isotope distribution becomes more homogenous, which is reflected in the spectroscopic features dependent on the extent of 13C-13C mode coupling or to specific isotopic symmetries within protein secondary structures. As SIP-nanoFTIR captures single-cell metabolism, environmentally-induced cellular processes and subcellular isotope localization, this technique offers widespread applications across a variety of disciplines including microbial ecology, biophysics, biopharmaceuticals, medicinal science and cancer research.

biophysics↗

bioRxiv2024

Stable isotopes and nanoSIMS single-cell imaging reveals soil plastisphere colonizers able to assimilate sulfamethoxazole

The presence and accumulation of both plastics and antibiotics in soils may lead to the colonization, selection and propagation of bacteria with certain metabolic traits e.g. antibiotic resistance, in plastisphere. However, the impact of plastic-antibiotic tandem on the soil ecosystem functioning, particularly on microbial function and metabolism remains currently unexplored. Herein, we investigated the competence of soil bacteria to colonize plastics and to mineralize/degrade 13C-labelled sulfamethoxazole (SMX). Using single cell imaging, isotope tracers, soil respiration and SMX mineralization bulk measurements we show that microbial colonization of polystyrene (PE) and polyethylene (PS) surfaces takes place within the first 30 days of incubation. Morphologically diverse, microorganisms were colonizing both plastic types, with a preference for PE substrate. Nano-scale Secondary Ion Mass Spectrometry measurements show that 13C enrichment was highest at 130 days with values up to 1.29 atom %, similar to those of the 13CO2 pool (up to 1.26 atom%, or 22.55 {per thousand}). Our results provide direct evidence demonstrating, at single cell level, the capacity of bacterial colonizers of plastics to assimilate 13C from 13C-SMX. These findings expand our knowledge on the role of plastisphere microbiota in the ecological functioning of soils impacted by anthropogenic stressors.

microbiology↗

bioRxiv2021

Subcellular architecture and metabolic connection in the planktonic photosymbiosis between Collodaria (radiolarians) and their microalgae

Photosymbiosis is widespread and ecologically important in the oceanic plankton but remains poorly studied. Here, we used multimodal subcellular imaging to investigate the photosymbiosis between colonial Collodaria and their microalga dinoflagellate (Brandtodinium) collected in surface seawaters. We showed that this symbiosis is a very dynamic system whereby symbionts interact with different host cells via extracellular vesicles within the "greenhouse-like" colony. 3D electron microscopy revealed that the volume of the photosynthetic apparatus (plastid and pyrenoid) of the microalgae increased in symbiosis compared to free-living while the mitochondria volume was similar. Stable isotope probing coupled with NanoSIMS showed that carbon and nitrogen were assimilated and stored in the symbiotic microalga in starch granules and purine crystals, respectively. Nitrogen was also allocated to the algal nucleus (nucleolus). After 3 hours, low 13C and 15N transfer was detected in the host Golgi. Metal mapping revealed that intracellular iron concentration was similar in free-living and symbiotic microalgae (ca 40 ppm) and two-fold higher in the host, whereas copper concentration increased in symbiotic microalgae (up to 6900 ppm) and was detected in the host cell and extracellular vesicles. Sulfur mapping also pinpointed the importance of this nutrient for the algal metabolism. This study, which revealed subcellular changes of the morphology and nutrient homeostasis in symbiotic microalgae, improves our understanding on the metabolism of this widespread and abundant oceanic symbiosis and paves the way for more studies to investigate the metabolites exchanged.

ecology↗

bioRxiv2021

High-Resolution Chemical Mapping and Microbial Identification of Rhizosphere using Correlative Microscopy

During the past decades, several stand-alone and combinatory methods have been developed to investigate the chemistry (i.e. mapping of elemental, isotopic and molecular composition) and the role of microbes in soil and rhizosphere. However, none of these approaches are currently capable of characterizing soil-root-microbe interactions simultaneously in their spatial arrangement. Here we present a novel approach that allows chemical and microbial identification of the rhizosphere at micro-to nano-meter spatial resolution. Our approach includes i) a resin embedding and sectioning method suitable for simultaneous correlative characterization of Zea mays rhizosphere, ii) an analytical work flow that allows up to six instruments/techniques to be used correlatively, and iii) data and image correlation. Hydrophilic, immunohistochemistry compatible, low viscosity LR white resin was used to embed the rhizosphere sample. We employed waterjet cutting and avoided polishing the surface to prevent smearing of the sample surface at nanoscale. Embedding quality was analyzed by Helium Ion Microscopy (HIM). Bacteria in the embedded soil was identified by Catalyzed Reporter Deposition-Fluorescence In Situ Hybridization (CARD-FISH) to avoid interferences from high levels of auto fluorescence emitted by soil particles and organic matter. Chemical mapping of the rhizosphere was done by Scanning Electron Microscopy (SEM) with Energy-dispersive X-ray analysis (SEM-EDX), Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS), nano-focused Secondary Ion mass Spectrometry (nanoSIMS), and confocal Raman spectroscopy ({micro}-Raman). High-resolution correlative characterization by six different techniques followed by image registration shows that this method can meet the demanding requirements of multiple characterization techniques to chemically map the rhizosphere and identify spatial organization of bacteria. Finally, we presented individual and correlative workflows for imaging and image registration to analyze data. We hope this method will be a platform to combine various 2D analytics for an ample understanding of the rhizosphere processes and their ecological significance. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/429689v2_ufig1.gif" ALT="Figure 1"> View larger version (130K): org.highwire.dtl.DTLVardef@16c8234org.highwire.dtl.DTLVardef@d84266org.highwire.dtl.DTLVardef@1ef6001org.highwire.dtl.DTLVardef@87f51c_HPS_FORMAT_FIGEXP M_FIG C_FIG

plant biology↗

bioRxiv2020

Cytoklepty in the plankton: a host strategy to optimize the bioenergetic machinery of endosymbiotic algae

Endosymbioses have shaped the evolutionary trajectory of life and remain widespread and ecologically important. Investigating modern oceanic photosymbioses can illuminate how algal endosymbionts are energetically exploited by their heterotrophic hosts, and inform on putative initial steps of plastid acquisition in eukaryotes. By combining 3D subcellular imaging with photophysiology, carbon flux imaging and transcriptomics, we show that cell division of algal endosymbionts (Phaeocystis) is blocked within hosts (Acantharia), and that their cellular architecture and bioenergetic machinery are radically altered. Transcriptional evidence indicates that a nutrient-independent mechanism prevents symbiont cell division and decouples nuclear and plastid division. As endosymbiont plastids proliferate, the volume of the photosynthetic machinery volume increases 100-fold in correlation with expansion of a reticular mitochondrial network in close proximity to plastids. Photosynthetic efficiency tends to increase with cell size and photon propagation modeling indicates that the networked mitochondrial architecture enhances light capture. This is accompanied by 150-fold higher carbon uptake and upregulation of genes involved in photosynthesis and carbon fixation, which, in conjunction with a ca.15-fold size increase of pyrenoids demonstrates enhanced primary production in symbiosis. NanoSIMS analysis revealed major carbon allocation to plastids and transfer to the host cell. Invagination of the symbiosome into endosymbionts to optimize metabolic exchanges is strong evidence that the algal metamorphosis is irreversible. Hosts therefore trigger and unambiguously benefit from major bioenergetic remodeling of symbiotic microalgae with important consequences for the oceanic carbon cycle. Unlike other photosymbioses, this interaction represents a so-called cytoklepty, which is a putative initial step towards plastid acquisition.

physiology↗

bioRxiv2017

Conductive particles enable syntrophic acetate oxidation between Geobacter and Methanosarcina from coastal sediments

Coastal sediments are rich in conductive minerals, which could impact microbial processes for which acetate is a central intermediate. In the methanogenic zone, acetate is consumed by methanogens and/or syntrophic acetate oxidizing (SAO) consortia. SAO consortia live under extreme thermodynamic pressure and their survival depends on successful partnership. Here we demonstrate that conductive minerals facilitate a SAO partnership between Geobacter and Methanosarcina from the coastal sediments of the Bothnian Bay, Baltic Sea. Bothnian methanogenic sediments showed a high apparent isotopic fractionation (c 1.07) characteristic of CO2-reductive methanogenesis. The native community was represented by electrogens such as Geobacter and methanogens like Methanosarcina. Upon the addition of conductive particles (activated carbon and magnetite) methanogenesis from acetate increased four fold. Geobacter (96% related to G. psychrophilus) and Methanosarcina (99% related to M. subterranea) dominated the conductive particle-spiked SAO communities. Using NanoSIMS we demonstrated that during SAO, Geobacter incorporated 82% of the labeled acetate as compared to only 18% by Methanosarcina. At the same time Geobacter converted 27% of the 13C-acetate to 13CO2 as detected by IRMS. Indigenous soluble shuttles were not involved in SAO, since spiking fresh cultures with spent-media filtrate had no effect on methanogenic rates. Our results demonstrate that Geobacter oxidizes acetate to CO2 while transferring electrons extracellularly via conductive particles to Methanosarcina, which utilizes them for CO2 reduction to methane. In natural environments, mediation of SAO by conductive particles between electrogens and methanogens could impact the iron and methane cycles.\n\nSignificanceAcetate oxidizing bacteria are known to thrive in mutualistic consortia in which H2 or formate is shuttled to a methane-producing Archaea partner. Here we discovered that they could instead transfer electrons via conductive minerals. Mineral-SAO (syntrophic acetate oxidation) could be a vital pathway for CO2-reductive methanogenesis in the environment, especially in sediments rich in conductive minerals. Mineral-SAO is therefore of potential importance for both iron and methane cycles in sediments and soils. Additionally, our observations imply that agricultural runoff or amendments with conductive chars could trigger a significant increase in methane emissions.

microbiology↗