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At least 19 records
All sourcesNASA NTRSDOE OSTI
DOE OSTI2026

Sampling Microbial Dynamics in the Salish Sea Estuary: Evaluating Methods to Capture Cyanobacteria and Cyanophage

Introduction: Picocyanobacteria from the genera Prochlorococcus and Synechococcus thrive across the globe in aquatic environments, have relatively small genomes, and have growth dynamics regulated by both viral interactions and abiotic conditions, making them excellent model organisms for exploring host-pathogencoevolution. Methods: We developed and refined methods to sample and sequence cyanobacteria, cyanophages, and measured features of their abiotic environment. Results: The protocol described herein can successfully discriminate large-cell eukaryotic organisms, but size fractionation of picocyanobacteria appears to be affected by the presence of free DNA, multicellular structures, and abundant tycheposons. Our preferred final protocol from this exploratory effort included a combination of in-line and single vacuum flask filtrations, which reduced filtration processing time by over threefold in some cases compared to other tested methods, such as a fully in-line sequence or in-site filtrations. We successfully extracted an average of approximately 400–1200 ng for all filter fractions, with some variations between kits. Discussion: The protocol described herein can successfully discriminate large-cell eukaryotic organisms, but size fractionation of picocyanobacteria appears to be affected by the presence of free DNA, multicellular structures, and abundant tycheposons.

Salish Sea↗
DOE OSTI2025

Systematic Mapping of Bacterial CRISPRa Systems for Synergistic Gene Activation Reveals Antagonistic Effects

CRISPR gene activation (CRISPRa) tools have shown great promise for bacterial strain engineering but often require customization for each intended application. Our goal is to create generalizable CRISPRa tools that can overcome previous limitations of gene activation in bacteria. In eukaryotic cells, multiple activators can be combined for synergistic gene activation. To identify potential effectors for synergistic activation in bacteria, we systematically characterized bacterial activator proteins with a set of engineered synthetic promoters. We found that optimal target sites for different activators could vary by up to 200 bases in the region upstream of the transcription start site (TSS). These optimal target sites qualitatively matched previous reports for each activator, but the precise targeting rules varied between different promoters. By characterizing targeting rules in the same promoter context, we were able to test activator combinations with each effector positioned at its optimal target site. We did not find any activator combinations that produced synergistic activation, and we found that many combinations were antagonistic. Furthermore, this systematic investigation highlights fundamental mechanistic differences between bacterial and eukaryotic transcriptional activation systems and suggests that alternative strategies will be necessary for strong bacterial gene activation at arbitrary endogenous targets.

CRISPR activation↗
DOE OSTI2024

Non-photosynthetic lineages sibling to Cyanobacteria associate with eukaryotes in the open ocean

Margulisbacteria are elusive uncultivated bacteria that have illuminated evolutionary transitions in the progenitor of Cyanobacteria, the latter being a critically important phylum that underpins oxygenic photosynthesis. The non-photosynthetic Margulisbacteria were discovered in a sulfidic spring and later in other habitats. Currently, this candidate phylum partitions into the Riflemargulisbacteria, primarily from sediments and groundwater, the Termititenax from insect gut microbiomes, and the Marinamargulisbacteria, from marine samples. We found that Marinamargulisbacteria amplicons were unusually distributed in size-fractionated samples from the sunlit photic and dark twilight zones of the ocean. Further, sequencing of wild marine protists rendered genomic information for distinct marinamargulisbacterial clades co-associated with uncultivated, non-photosynthetic Stramenopila and Opisthokonta protists. Phylogenomic analyses combining these data and available metagenome-assembled genomes (MAGs) and single-amplified genomes (SAGs) from sorted bacteria revealed new Marinamargulisbacteria lineages. The lineages delineate by their environment, forming clades comprising freshwater, marine pelagic, or sediment/hypoxic taxa. In conclusion, the remarkable diversity of Margulisbacteria indicates success in colonizing various habitats, potentially in a conserved strategy involving eukaryotic cells.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Emergent actin flows explain distinct modes of gliding motility

During host infection, Toxoplasma gondii and related unicellular parasites move using gliding, which differs fundamentally from other known mechanisms of eukaryotic cell motility. Gliding is thought to be powered by a thin layer of flowing filamentous (F)-actin sandwiched between the plasma membrane and a myosin-covered inner membrane complex. How this surface actin layer drives the various gliding modes observed in experiments—helical, circular, twirling and patch, pendulum or rolling—is unclear. Here we suggest that F-actin flows arise through self-organization and develop a continuum model of emergent F-actin flow within the confines provided by Toxoplasma geometry. In the presence of F-actin turnover, our model predicts the emergence of a steady-state mode in which actin transport is largely directed rearward. Removing F-actin turnover leads to actin patches that recirculate up and down the cell, which we observe experimentally for drug-stabilized actin bundles in live Toxoplasma gondii parasites. These distinct self-organized actin states can account for observed gliding modes, illustrating how different forms of gliding motility can emerge as an intrinsic consequence of the self-organizing properties of F-actin flow in a confined geometry.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Is stochastic thermodynamics the key to understanding the energy costs of computation?

The relationship between the thermodynamic and computational properties of physical systems has been a major theoretical interest since at least the 19th century. It has also become of increasing practical importance over the last half-century as the energetic cost of digital devices has exploded. Importantly, real-world computers obey multiple physical constraints on how they work, which affects their thermodynamic properties. Moreover, many of these constraints apply to both naturally occurring computers, like brains or Eukaryotic cells, and digital systems. Most obviously, all such systems must finish their computation quickly, using as few degrees of freedom as possible. This means that they operate far from thermal equilibrium. Furthermore, many computers, both digital and biological, are modular, hierarchical systems with strong constraints on the connectivity among their subsystems. Yet another example is that to simplify their design, digital computers are required to be periodic processes governed by a global clock. None of these constraints were considered in 20th-century analyses of the thermodynamics of computation. The new field of stochastic thermodynamics provides formal tools for analyzing systems subject to all of these constraints. We argue here that these tools may help us understand at a far deeper level just how the fundamental thermodynamic properties of physical systems are related to the computation they perform.

computation↗
DOE OSTI2026

Comparative mitogenomics of kingdom Fungi – evolutionary insights and metagenomic applications

Mitochondria are essential components of eukaryotic cells, responsible for ATP production through oxidative phosphorylation. Despite their biological importance, unique challenges have hindered the adoption of automated mitochondrial genome (mitogenome) annotation methods, obstructing mitochondrial comparative genomics in a broad evolutionary context. Using Fungi as a study system and a Joint Genome Institute (JGI) annotated high-quality reference set, we observed broad patterns of mitochondrial evolution across the kingdom. We found that the median fungal mitogenome size is 58 kb and identified exceptionally large examples over 1 Mb in Pezizomycetes. All 14 expected oxidative phosphorylation protein-coding genes, plus rps3, were generally conserved. We found evidence of major evolutionary transitions within the Ascomycota, including the transfer of mitochondrially encoded atp8 and atp9 to the nuclear genomes across the Pezizomycotina and shifts in mitogenome tRNA patterns across the kingdom. We found substantial concordance between mitochondrial and nuclear evolution, enabling us to document 3131 total fungal mitogenomes from JGI-derived metagenomic datasets. We also identified 6467 total undeclared mitogenomes embedded in Genbank fungal nuclear assemblies. We provide interactive tools for mitogenome analysis through the JGI MycoCosm platform. Collectively, this work generated nearly 10 000 new fungal mitogenome annotations, providing a foundation and resources for future exploration of comparative fungal mitogenomics.

Ahrendt, Steven R. [USDOE Joint Genome Institute (↗
DOE OSTI2025

Structural dissection of ergosterol metabolism reveals a pathway optimized for membrane phase separation

Sterols are among the most abundant lipids in eukaryotic cells yet are synthesized through notoriously long metabolic pathways. It has been proposed that the molecular evolution of such pathways must have required each step to increase the capacity of its product to condense and order phospholipids. Here, we carry out a systematic analysis of the ergosterol pathway that leverages the yeast vacuole’s capacity to phase separate into ordered membrane domains. In the post-synthetic steps specific to ergosterol biosynthesis, we find that successive modifications act to oscillate ordering capacity, settling on a level that supports phase separation while retaining fluidity of the resulting domains. Simulations carried out with each intermediate showed how conformers in the sterol’s alkyl tail are capable of modulating long-range ordering of phospholipids, which could underlie changes in phase behavior. Our results indicate that the complexity of sterol metabolism could have resulted from the need to balance lipid interactions required for membrane organization.

Science & Technology - Other Topics↗
DOE OSTI2024

Time-series metagenomics reveals changing protistan ecology of a temperate dimictic lake

Abstract Background Protists, single-celled eukaryotic organisms, are critical to food web ecology, contributing to primary productivity and connecting small bacteria and archaea to higher trophic levels. Lake Mendota is a large, eutrophic natural lake that is a Long-Term Ecological Research site and among the world’s best-studied freshwater systems. Metagenomic samples have been collected and shotgun sequenced from Lake Mendota for the last 20 years. Here, we analyze this comprehensive time series to infer changes to the structure and function of the protistan community and to hypothesize about their interactions with bacteria. Results Based on small subunit rRNA genes extracted from the metagenomes and metagenome-assembled genomes of microeukaryotes, we identify shifts in the eukaryotic phytoplankton community over time, which we predict to be a consequence of reduced zooplankton grazing pressures after the invasion of a invasive predator (the spiny water flea) to the lake. The metagenomic data also reveal the presence of the spiny water flea and the zebra mussel, a second invasive species to Lake Mendota, prior to their visual identification during routine monitoring. Furthermore, we use species co-occurrence and co-abundance analysis to connect the protistan community with bacterial taxa. Correlation analysis suggests that protists and bacteria may interact or respond similarly to environmental conditions. Cryptophytes declined in the second decade of the timeseries, while many alveolate groups (e.g., ciliates and dinoflagellates) and diatoms increased in abundance, changes that have implications for food web efficiency in Lake Mendota. Conclusions We demonstrate that metagenomic sequence-based community analysis can complement existing efforts to monitor protists in Lake Mendota based on microscopy-based count surveys. We observed patterns of seasonal abundance in microeukaryotes in Lake Mendota that corroborated expectations from other systems, including high abundance of cryptophytes in winter and diatoms in fall and spring, but with much higher resolution than previous surveys. Our study identified long-term changes in the abundance of eukaryotic microbes and provided context for the known establishment of an invasive species that catalyzes a trophic cascade involving protists. Our findings are important for decoding potential long-term consequences of human interventions, including invasive species introduction.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

The DYRKP1 kinase regulates cell wall degradation in Chlamydomonas by inducing matrix metalloproteinase expression

Abstract The cell wall of plants and algae is an important cell structure that protects cells from changes in the external physical and chemical environment. This extracellular matrix, composed of polysaccharides and glycoproteins, must be constantly remodeled throughout the life cycle. However, compared to matrix polysaccharides, little is known about the mechanisms regulating the formation and degradation of matrix glycoproteins. We report here that a plant kinase belonging to the dual-specificity tyrosine phosphorylation-regulated kinase (DYRKP1) family present in all eukaryotes regulates cell wall degradation after mitosis of Chlamydomonas reinhardtii by inducing the expression of matrix metalloproteinases. Without DYRKP1, daughter cells cannot disassemble parental cell walls and remain trapped inside for more than 10 days. On the other hand, the dual-specificity tyrosine phosphorylation-regulated kinase complementation lines show normal degradation of the parental cell wall. Transcriptomic and proteomic analyses indicate a marked downregulation of MMP gene expression and accumulation, respectively, in the dyrkp1 mutants. The mutants deficient in matrix metalloproteinases retain palmelloid structures for a longer time than the background strain, like dyrkp1 mutants. Our findings show that dual-specificity tyrosine phosphorylation-regulated kinase, by ensuring timely MMP expression, enables the successful execution of the cell cycle. Altogether, this study provides insight into the life cycle regulation in plants and algae.

Kim, Minjae (ORCID:0000000223561295)↗
DOE OSTI2025

Engineered Endosymbionts that Modulate Primary Macrophage Function and Attenuate Tumor Growth by Shifting the Tumor Microenvironment

Modulating gene expression in macrophages can be used to improve tissue regeneration and redirect tumor microenvironments (TMEs) toward positive therapeutic outcomes. We have developed Bacillus subtilis as an engineered endosymbiont (EES) capable of residing inside the eukaryotic host cell cytoplasm and controlling the fate of macrophages. Secretion of mammalian transcription factors (TFs) from B. subtilis that expresses listeriolysin O (LLO; allowing the EES to escape destruction by the macrophage) modulated expression of surface markers, cytokines, and chemokines, indicating functional changes in a macrophage/monocyte cell line. The engineered B. subtilis LLO TF strains were evaluated in murine bone marrow-derived macrophages (BMDMs) by flow cytometry, chemokine/cytokine profiling, metabolic assays, and RNA-Seq delivery of TFs by the EES shifted BMDM gene expression, production of cytokine and chemokines, and metabolic patterns, indicating that the TF strains could guide primary macrophage function. Thereafter, the ability of the TF strains to alter the TME was characterized in vivo in an orthotopic murine model of triple-negative breast cancer to assess therapeutic effects. The TF strains altered the TME by shifting immune cell composition and attenuating tumor growth. Additionally, multiple doses of the TF strains were well-tolerated by the mice. The use of B. subtilis LLO TF strains as EES showed promise as a unique cancer immunotherapy by directing the immune function intracellularly. The uses of EES could be expanded to modulate other mammalian cells over a range of biomedical applications.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2024

Exploiting bacterial effector proteins to uncover evolutionarily conserved antiviral host machinery

Arboviruses are a diverse group of insect-transmitted pathogens that pose global public health challenges. Identifying evolutionarily conserved host factors that combat arbovirus replication in disparate eukaryotic hosts is important as they may tip the balance between productive and abortive viral replication, and thus determine virus host range. Here, we exploit naturally abortive arbovirus infections that we identified in lepidopteran cells and use bacterial effector proteins to uncover host factors restricting arbovirus replication. Bacterial effectors are proteins secreted by pathogenic bacteria into eukaryotic hosts cells that can inhibit antimicrobial defenses. Since bacteria and viruses can encounter common host defenses, we hypothesized that some bacterial effectors may inhibit host factors that restrict arbovirus replication in lepidopteran cells. Thus, we used bacterial effectors as molecular tools to identify host factors that restrict four distinct arboviruses in lepidopteran cells. By screening 210 effectors encoded by seven different bacterial pathogens, we identify several effectors that individually rescue the replication of all four arboviruses. We show that these effectors encode diverse enzymatic activities that are required to break arbovirus restriction. We further characterize Shigella flexneri-encoded IpaH4 as an E3 ubiquitin ligase that directly ubiquitinates two evolutionarily conserved proteins, SHOC2 and PSMC1, promoting their degradation in insect and human cells. We show that depletion of either SHOC2 or PSMC1 in insect or human cells promotes arbovirus replication, indicating that these are ancient virus restriction factors conserved across invertebrate and vertebrate hosts. Collectively, our study reveals a novel pathogen-guided approach to identify conserved antimicrobial machinery, new effector functions, and conserved roles for SHOC2 and PSMC1 in virus restriction.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Towards chloroplastic nanofactories: formation of proteinaceous scaffolds for metabolic engineering

The evolution of eukaryotic lipid-bound organelles allows for specialized metabolism to occur within spatially distinct metabolic landscapes within the same cell. However, this strategy of compartmentalization is not unique to eukaryotic organisms. Many bacteria, spread across 45 different phyla, contain loci that encode for specialized bacterial microcompartments (BMCs) (Sutter et al., 2021). The formation of a BMC involves self-assembly from three families of shell proteins to form the outer shell membrane, in addition to the encapsulation of an enzymatic core packaged within the lumen of a BMC (Kerfeld et al., 2018). These proteinaceous organelles provide a competitive growth advantage by enabling organisms to process inaccessible substrates by sequestering metabolic intermediates (i.e. aldehydes) that would otherwise be toxic within the cytoplasm. While BMCs perform specific metabolic functions in their native organism, synthetic, empty BMC shells can form without the requirement of native cargo inside (Doron and Kerfeld, 2024). This provides a transferable and tunable platform of protein scaffolding for guiding metabolic engineering within a host organism of choice (Raba and Kerfeld, 2022).

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Small Cells with Big Photosynthetic Productivities: Biotechnological Potential of the Picochlorum Genus

The Picochlorum genus is a distinctive eukaryotic green-algal clade that is the focus of several current biotechnological studies. It is capable of extremely rapid growth rates and has exceptional tolerances to high salinity, intense light, and elevated temperatures. Importantly, it has robust stability and high-biomass productivities in outdoor field trials in seawater. These features have propelled Picochlorum into the spotlight as a promising model for both fundamental and biotechnological research. Recently, several genetic tools, including genome editing, were developed for these algae, enabling insights into Picochlorum photophysiology and algal transformations for expanded capabilities. Here, we survey the Picochlorum genus, its genetic toolbox, recently characterized transformants, and discuss the commercial potential of Picochlorum as a salt-water photoautotrophic biocatalyst.

09 BIOMASS FUELS↗
DOE OSTI2026

Many paths, similar destinations: viruses and bacterial microcompartments form polyhedra inside cells

A large number of biological entities assemble into icosahedral structures, and these are ubiquitous throughout nature. Examples include eukaryotic and prokaryotic viral capsids and more recently discovered bacterial microcompartments. Viral capsids and bacterial microcompartments are both composed of pentameric and hexameric subunits; however, they differ in the type of cargo they encapsulate: nucleic acid or protein. Also, both depart from strict icosahedral symmetry: while this is less common in viruses, among bacterial microcompartments, diverse and heterogeneous polyhedra are common. We review shared principles and key distinctions between the self-directed assembly of various icosahedral architectures and their polyhedral variants in nature and explore the concept that there are multiple paths, influenced by their cargo, to arriving at similar protein cage morphologies.

bacterial microcompartments↗
DOE OSTI2026

Arabidopsis lipins mediate lipid droplet biogenesis to protect cells from lipotoxicity

Lipin proteins, a family of phosphatidic acid phosphatases (PAHs), are key regulators of lipid metabolism, storage, and homeostasis across eukaryotes. While Arabidopsis (Arabidopsis thaliana) lipins function in lipid biosynthesis and gene regulation, their roles in lipid droplet (LD) biogenesis and lipid homeostasis remain largely unknown. Here, we show that double knockout of two PAH genes (PAH1/2) results in impaired LD biogenesis, accelerated triacylglycerol (TAG) hydrolysis, and lipid imbalance. pah1/2 mutant leaves exhibited a marked reduction in TAG levels and a significant decrease in LD size, while the rates of TAG and diacylglycerol (DAG) synthesis remained largely unchanged. In seeds, PAH1/2 disruption minimally affected TAG content but significantly reduced LD size. Fatty acid feeding experiments demonstrated impaired LD formation and increased lipotoxicity in pah1/2 leaves and seedlings. Furthermore, knockout of PAH1/2 in mutants with enhanced fatty acid flux through phosphatidylcholine (PC) led to severe reductions in leaf TAG levels, despite increases in TAG synthesis rates, indicating accelerated TAG turnover. Phosphatidic acid, free fatty acids, and PC accumulated, leading to massive proliferation of endoplasmic reticulum membranes and severe growth and developmental defects. These findings demonstrate evolutionarily conserved roles for PAH1/2 in LD biogenesis, membrane lipid homeostasis, and cellular protection against lipotoxicity, particularly under conditions of elevated fatty acid flux.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

DNA double-strand break movement in heterochromatin depends on the histone acetyltransferase dGcn5

Abstract Cells employ diverse strategies to repair double-strand breaks (DSBs), a dangerous form of DNA damage that threatens genome integrity. Eukaryotic nuclei consist of different chromatin environments, each displaying distinct molecular and biophysical properties that can significantly influence the DSB-repair process. DSBs arising in the compact and silenced heterochromatin domains have been found to move to the heterochromatin periphery in mouse and Drosophila to prevent aberrant recombination events. However, it is poorly understood how chromatin components, such as histone post-translational modifications, contribute to these DSB movements within heterochromatin. Using irradiation as well as locus-specific DSB induction in Drosophila tissues and cultured cells, we find enrichment of histone H3 lysine 9 acetylation (H3K9ac) at DSBs in heterochromatin but not euchromatin. We find this increase is mediated by the histone acetyltransferase dGcn5, which rapidly localizes to heterochromatic DSBs. Moreover, we demonstrate that in the absence of dGcn5, heterochromatic DSBs display impaired recruitment of the SUMO E3 ligase Nse2/Qjt and fail to relocate to the heterochromatin periphery to complete repair. In summary, our results reveal a previously unidentified role for dGcn5 and H3K9ac in heterochromatic DSB repair and underscore the importance of differential chromatin responses at heterochromatic and euchromatic DSBs to promote safe repair.

Biochemistry & Molecular Biology↗
DOE OSTI2024

Inhibition of the eukaryotic initiation factor-2α kinase PERK decreases risk of autoimmune diabetes in mice

Preventing the onset of autoimmune type 1 diabetes (T1D) is feasible through pharmacological interventions that target molecular stress–responsive mechanisms. Cellular stresses, such as nutrient deficiency, viral infection, or unfolded proteins, trigger the integrated stress response (ISR), which curtails protein synthesis by phosphorylating eukaryotic translation initiation factor-2α (eIF2α). In T1D, maladaptive unfolded protein response (UPR) in insulin-producing β cells renders these cells susceptible to autoimmunity. We found that inhibition of the eIF2α kinase PKR-like ER kinase (PERK), a common component of the UPR and ISR, reversed the mRNA translation block in stressed human islets and delayed the onset of diabetes, reduced islet inflammation, and preserved β cell mass in T1D-susceptible mice. Single-cell RNA-Seq of islets from PERK-inhibited mice showed reductions in the UPR and PERK signaling pathways and alterations in antigen-processing and presentation pathways in β cells. Spatial proteomics of islets from these mice showed an increase in the immune checkpoint protein programmed death-ligand 1 (PD-L1) in β cells. Golgi membrane protein 1, whose levels increased following PERK inhibition in human islets and EndoC-βH1 human β cells, interacted with and stabilized PD-L1. Collectively, our studies show that PERK activity enhances β cell immunogenicity and that inhibition of PERK may offer a strategy for preventing or delaying the development of T1D.

Research & Experimental Medicine↗
DOE OSTI2025

Investigation of the Cyanothece nitrogenase cluster in Synechocystis : a blueprint for engineering nitrogen-fixing photoautotrophs

ABSTRACT The nitrogenase gene cluster of unicellular diazotrophic cyanobacteria, such asCyanothece, is frequently selected by nature for nitrogen-fixing partnerships with eukaryotic phototrophs. The essential cluster components that confer an advantage in such partnerships remain underexplored. To use this cluster for the development of synthetic, phototrophic nitrogen-fixing systems, a thorough and systematic analysis of its constituent genes is necessary. An initial effort to assess the possibility of engineering this cluster into non-diazotrophic phototrophs led to the generation of aSynechocystis6803 strain with significant nitrogenase activity. In the current study, a refactoring approach was taken to determine the dispensability of the non-structural genes in the cluster and define a minimal gene set for constructing a functional nitrogenase for phototrophs. Using a bottom-up strategy, thenifgenes fromCyanothece51142 were re-organized to form new operons. The genes were then seamlessly removed to determine their essentiality in the nitrogen fixation process. We demonstrate that besides the structural genesnifHDK,nifBSUENPVZTXW, as well ashesAB, are important for optimal nitrogenase function in a phototroph. We also show that optimal expression of these genes is crucial for efficient nitrogenase activity. Our findings provide a solid foundation for generating synthetic systems that will facilitate solar-powered conversion of atmospheric nitrogen into nitrogen-rich compounds, a stride toward a greener world. IMPORTANCE Integrating nitrogen fixation genes into various photosynthetic organisms is an exciting strategy for converting atmospheric nitrogen into nitrogen-rich products in a green and energy-efficient way. In order to facilitate this process, it is essential that we understand the fundamentals of the functioning of a prokaryotic nitrogen-fixing machinery in a non-diazotrophic, photoautotrophic cell. This study examines a nitrogenase gene cluster that has been naturally selected on multiple occasions for a nitrogen-fixing partnership by eukaryotic photoautotrophs and provides a basic blueprint for designing a photosynthetic organism with nitrogen-fixing ability.

Microbiology↗