Novel Focused Ion Beam Liftouts for Spatial Characterization of Spherical Biominerals With Transmission Electron Microscopy.

Focused ion beam (FIB) is frequently used to prepare electron- and X-ray-beam-transparent thin sections of samples, called lamellae. Typically, lamellae are prepared from only a subregion of a sample. In this paper, we present a novel approach for FIB lamella preparation of microscopic samples, wherein the entire cross-section of the whole sample can be investigated. The approach was demonstrated using spherical, porous, and often hollow microprecipitates of biologically precipitated calcium carbonate. The microprecipitate morphology made these biogenic samples more fragile and challenging than materials commonly investigated using FIB lamellae. Our method enables the appropriate orientation of the lamellae required for further electron/X-ray analyses after attachment to the transmission electron microscopy (TEM) grid post and facilitates more secure adhesion onto the grid post. We present evidence of autofluorescence in bacterially precipitated vaterite using this lamella preparation method coupled with TEM selected area diffraction. This innovative approach allows studying biomineralization at the micro to nano scales, which can provide novel insights into bacterial responses to microenvironmental conditions.

A previously uncharacterized divisome-associated lipoprotein, DalA, is needed for normal cell division in Rhodobacterales.

The bacterial cell envelope is a key subcellular compartment with important roles in antibiotic resistance, nutrient acquisition, and cell morphology. We seek to gain a better understanding of proteins that contribute to the function of the cell envelope in Alphaproteobacteria. Using Rhodobacter sphaeroides, we show that a previously uncharacterized protein, RSP_1200, is an outer membrane (OM) lipoprotein that non-covalently binds peptidoglycan (PG). Using a fluorescently tagged version of this protein, we find that RSP_1200 undergoes a dynamic repositioning during the cell cycle and is enriched at the septum during cell division. We show that the position of RSP_1200 mirrors the location of FtsZ rings, leading us to propose that RSP_1200 is a newly identified component of the R. sphaeroides' divisome. Additional support for this hypothesis includes the co-precipitation of RSP_1200 with FtsZ, the Pal protein, and several predicted PG L,D-transpeptidases. We also find that a ∆RSP_1200 mutation leads to defects in cell division, sensitivity to PG-active antibiotics, and results in the formation of OM protrusions at the septum during cell division. Based on these results, we propose to name RSP_1200 DalA (for division-associated lipoprotein A) and postulate that DalA serves as a scaffold to position or modulate the activity of PG transpeptidases that are needed to form envelope invaginations during cell division. We find that DalA homologs are present in members of the Rhodobacterales order within Alphaproteobacteria. Therefore, we propose that further analysis of this and related proteins will increase our understanding of the macromolecular machinery and proteins that participate in cell division in Gram-negative bacteria. IMPORTANCE Multi-protein complexes of the bacterial cell envelope orchestrate key processes like growth, division, biofilm formation, antimicrobial resistance, and production of valuable compounds. The subunits of these protein complexes are well studied in some bacteria, and differences in their composition and function are linked to variations in cell envelope composition, shape, and proliferation. However, some envelope protein complex subunits have no known homologs across the bacterial phylogeny. We find that Rhodobacter sphaeroides RSP_1200 is a newly identified lipoprotein (DalA) and that loss of this protein causes defects in cell division and changes the sensitivity to compounds, affecting cell envelope synthesis and function. We find that DalA forms a complex with proteins needed for cell division, binds the cell envelope polymer peptidoglycan, and colocalizes with enzymes involved in the assembly of this macromolecule. The analysis of DalA provides new information on the cell division machinery in this and possibly other Alphaproteobacteria.

Resolving protein-mineral interfacial interactions during in vitro mineralization by atom probe tomography.

Organic macromolecules exert remarkable control over the nucleation and growth of inorganic crystallites during (bio)mineralization, as exemplified during enamel formation where the protein amelogenin regulates the formation of hydroxyapatite (HAP). However, it is poorly understood how fundamental processes at the organic-inorganic interface, such as protein adsorption and/or incorporation into minerals, regulates nucleation and crystal growth due to technical challenges in observing and characterizing mineral-bound organics at high-resolution. Here, atom probe tomography techniques were developed and applied to characterize amelogenin-mineralized HAP particles in vitro, revealing distinct organic-inorganic interfacial structures and processes at the nanoscale. Specifically, visualization of amelogenin across the mineralized particulate demonstrates protein can become entrapped during HAP crystal aggregation and fusion. Identification of protein signatures and structural interpretations were further supported by standards analyses, i.e., defined HAP surfaces with and without amelogenin adsorbed. These findings represent a significant advance in the characterization of interfacial structures and, more so, interpretation of fundamental organic-inorganic processes and mechanisms influencing crystal growth. Ultimately, this approach can be broadly applied to inform how potentially unique and diverse organic-inorganic interactions at different stages regulates the growth and evolution of various biominerals.

Critical role for uricase and xanthine dehydrogenase in soybean nitrogen fixation and nodule development.

De novo purine biosynthesis is required for the incorporation of fixed nitrogen in ureide exporting nodules, as formed on soybean [Glycine max (L.) Merr.] roots. However, in many cases, the enzymes involved in this pathway have been deduced strictly from genome annotations with little direct genetic evidence, such as mutant studies, to confirm their biochemical function or importance to nodule development. While efforts to develop large mutant collections of soybean are underway, research on this plant is still hampered by the inability to obtain mutations in any specific gene of interest. Using a forward genetic approach, as well as CRISPR/Cas9 gene editing via Agrobacterium rhizogenes-mediated hairy root transformation, we identified and characterized the role of GmUOX (Uricase) and GmXDH (Xanthine Dehydrogenase) in nitrogen fixation and nodule development in soybean. The gmuox knockout soybean mutants displayed nitrogen deficiency chlorosis and early nodule senescence, as exemplified by the reduced nitrogenase (acetylene reduction) activity in nodules, the internal greenish-white internal appearance of nodules, and diminished leghemoglobin production. In addition, gmuox1 nodules showed collapsed infected cells with degraded cytoplasm, aggregated bacteroids with no discernable symbiosome membranes, and increased formation of poly-ß-hydroxybutyrate granules. Similarly, knockout gmxdh mutant nodules, generated with the CRISPR/Cas9 system, also exhibited early nodule senescence. These genetic studies confirm the critical role of the de novo purine metabolisms pathway not only in the incorporation of fixed nitrogen but also in the successful development of a functional, nitrogen-fixing nodule. Furthermore, these studies demonstrate the great utility of the CRISPR/Cas9 system for studying root-associated gene traits when coupled with hairy root transformation.

Changes in the C-terminal, N-terminal, and histidine regions of amelogenin reveal the role of oligomer quaternary structure on adsorption and hydroxyapatite mineralization.

Adsorption interactions between amelogenin and calcium phosphate minerals are believed to be important to amelogenin's function in enamel formation, however, the role of specific amino acid residues and domains within the protein in controlling adsorption is not well known. We synthesized "mechanistic probes" by systematically removing charged regions of amelogenin in order to elucidate their roles. The probes included amelogenin without the charged residues in the N-terminus (SEKR), without two, three, or eight histidines (H) in the central protein region (H2, H3, H8), or without the C-terminal residues (Delta). In-situ atomic force microscopy (AFM) adsorption studies onto hydroxyapatite (HAP) single crystals confirmed that the C-terminus was the dominant domain in promoting adsorption. We propose that subtle changes in protein-protein interactions for proteins with histidines and N-terminal residues removed resulted in changes in the oligomer quaternary size and structure that also affected protein adsorption. HAP mineralization studies revealed that the oligomer-HAP binding energy and protein layer thickness were factors in controlling the amorphous calcium phosphate (ACP) to HAP induction time. Our studies with mechanistic probes reveal the importance of the oligomer quaternary structure in controlling amelogenin adsorption and HAP mineralization.

Intramyocardial hemorrhage drives fatty degeneration of infarcted myocardium.

Sudden blockage of arteries supplying the heart muscle contributes to millions of heart attacks (myocardial infarction, MI) around the world. Although re-opening these arteries (reperfusion) saves MI patients from immediate death, approximately 50% of these patients go on to develop chronic heart failure (CHF) and die within a 5-year period; however, why some patients accelerate towards CHF while others do not remains unclear. Here we show, using large animal models of reperfused MI, that intramyocardial hemorrhage - the most damaging form of reperfusion injury (evident in nearly 40% of reperfused ST-elevation MI patients) - drives delayed infarct healing and is centrally responsible for continuous fatty degeneration of the infarcted myocardium contributing to adverse remodeling of the heart. Specifically, we show that the fatty degeneration of the hemorrhagic MI zone stems from iron-induced macrophage activation, lipid peroxidation, foam cell formation, ceroid production, foam cell apoptosis and iron recycling. We also demonstrate that timely reduction of iron within the hemorrhagic MI zone reduces fatty infiltration and directs the heart towards favorable remodeling. Collectively, our findings elucidate why some, but not all, MIs are destined to CHF and help define a potential therapeutic strategy to mitigate post-MI CHF independent of MI size.

Microbe-Encapsulated Silica Gel Biosorbents for Selective Extraction of Scandium from Coal Byproducts.

Scandium (Sc) has great potential for use in aerospace and clean energy applications, but its supply is currently limited by a lack of commercially viable deposits and the environmental burden of its production. In this work, a biosorption-based flow-through process was developed for extraction of Sc from low-grade feedstocks. A microbe-encapsulated silica gel (MESG) biosorbent was synthesized through sol-gel encapsulation of Arthrobacter nicotianae, a bacterium that selectively adsorbs Sc. Microscopic imaging revealed a high cell loading and macroporous structure, which enabled rapid mass transport and adsorption/desorption of metal ions. The biosorbent displayed high Sc selectivity against lanthanides and major base metals, with the exception of Fe(III). Following pH adjustment to remove Fe(III) from an acid leachate prepared from lignite coal, a packed-bed column loaded with the MESG biosorbent exhibited near-complete Sc separation from lanthanides; the column eluate had a Sc enrichment factor of 10.9, with Sc constituting 96.4% of the total rare earth elements. The MESG biosorbent exhibited no significant degradation with regard to both adsorption capacity and physical structure after 10 adsorption/desorption cycles. Overall, our results suggest that the MESG biosorbent offers an effective and green alternative to conventional liquid-liquid extraction for Sc recovery.

A green sulfur bacterium from epsomitic Hot Lake, Washington, USA.

Hot Lake is a small heliothermal and hypersaline lake in far north-central Washington State (USA) and is limnologically unusual because MgSO4 rather than NaCl is the dominant salt. In late summer, the Hot Lake metalimnion becomes distinctly green from blooms of planktonic phototrophs. In a study undertaken over 60 years ago, these blooms were predicted to include green sulfur bacteria, but no cultures were obtained. We sampled Hot Lake and established enrichment cultures for phototrophic sulfur bacteria in MgSO4-rich sulfidic media. Most enrichments turned green or red within 2 weeks, and from green-colored enrichments, pure cultures of a lobed green sulfur bacterium (phylum Chlorobi) were isolated. Phylogenetic analyses showed the organism to be a species of the prosthecate green sulfur bacterium Prosthecochloris. Cultures of this Hot Lake phototroph were halophilic and tolerated high levels of sulfide and MgSO4. In addition, unlike all recognized species of Prosthecochloris, the Hot Lake isolates grew at temperatures up to 45 °C, indicating an adaptation to the warm summer temperatures of the lake. Photoautotrophy by Hot Lake green sulfur bacteria may contribute dissolved organic matter to anoxic zones of the lake, and their diazotrophic capacity may provide a key source of bioavailable nitrogen, as well.

Impacts of anthropogenic gadolinium on the activity of the ammonia oxidizing bacterium Nitrosomonas europaea.

Widespread use of gadolinium-based contrast agents in medical imaging has resulted in increased Gd inputs to municipal wastewater treatment plants. Others have reported that typical wastewater treatment does not attenuate Gd, resulting in discharges to natural waters. However, whether elevated Gd impacts the performance of biological treatment has not been investigated. We examined whether gadolinium chloride or Gd chelated with diethylenetriaminepentaacetic acid (DTPA) affected the activity of the model nitrifying bacterium Nitrosomonas europaea. At nominal GdCl3 additions ranging from 1 to 500 µM, no impact was observed compared to the control. Most (>98%) of the added Gd precipitated, and extracellular GdPO4 nanoparticles were observed. When chelated with DTPA, Gd remained soluble, but no statistically significant impact on ammonia oxidation was observed until the highest concentrations tested. At 300 and 500 µM Gd-DTPA, a temporary reduction of nitrite production relative to the control (effect size 1.3 mg l-1 and 1.5 mg l-1, respectively, at 24 h) was seen. By itself, DTPA was highly inhibitory. Modeling suggested that DTPA likely chelated other metals, but adjusting the concentrations of the most abundant metals in the medium, calcium and magnesium, indicated that lowering their free ion activities was probably not the cause of inhibition. Complexation of other essential metals was more likely. Our studies indicate that while the low bioavailability of Gd may limit its ecosystem impacts, the role of synthetic ligands used with Gd and other rare earth elements should be considered as the production, use and disposal of these elements increases.

The NtrYX Two-Component System Regulates the Bacterial Cell Envelope.

Activity of the NtrYX two-component system has been associated with important processes in diverse bacteria, ranging from symbiosis to nitrogen and energy metabolism. In the facultative alphaproteobacterium Rhodobacter sphaeroides, loss of the two-component system NtrYX results in increased lipid production and sensitivity to some known cell envelope-active compounds. In this study, we show that NtrYX directly controls multiple properties of the cell envelope. We find that the response regulator NtrX binds upstream of cell envelope genes, including those involved in peptidoglycan biosynthesis and modification and in cell division. We show that loss of NtrYX impacts the cellular levels of peptidoglycan precursors and lipopolysaccharide and alters cell envelope structure, increasing cell length and the thickness of the periplasm. Cell envelope function is also disrupted in the absence of NtrYX, resulting in increased outer membrane permeability. Based on the properties of R. sphaeroides cells lacking NtrYX and the target genes under direct control of this two-component system, we propose that NtrYX plays a previously undescribed, and potentially conserved, role in the assembly, structure, and function of the cell envelope in a variety of bacteria.IMPORTANCE The bacterial cell envelope provides many important functions. It protects cells from harsh environments, serves as a selective permeability barrier, houses bioenergetic functions, defines sensitivity to antibacterial agents, and plays a crucial role in biofilm formation, symbiosis, and virulence. Despite the important roles of this cellular compartment, we lack a detailed understanding of the biosynthesis and remodeling of the cell envelope. Here, we report that the R. sphaeroides two-component signaling system NtrYX is a previously undescribed regulator of cell envelope processes, providing evidence that it is directly involved in controlling transcription of genes involved in cell envelope assembly, structure, and function in this and possibly other bacteria. Thus, our data report on a newly discovered process used by bacteria to assemble and remodel the cell envelope.

Infrared Optical Constants from Pressed Pellets of Powders: I. Improved n and k Values of (NH4)2SO4 from Single-Angle Reflectance.

In combination with other parameters, the real, n(v∼), and imaginary, k(v∼), components of the complex refractive index, n^ = n + ik, can be used to simulate the optical properties of a material in different forms, e.g., its infrared spectra. Ultimately, such n/k values can be used to generate a database of synthetic reflectance spectra for the different morphologies to which experimental data can be compared. But obtaining reliable values of the optical constants n/k for solid materials is challenging due to the lack of optical quality specimens, usually crystals, large enough to measure. An alternative to crystals is to press the powder into a uniform disk. We have produced pellets from ammonium sulfate, (NH4)2SO4, powder and derived the pellets' n and k values via single-angle reflectance using a specular reflectance device in combination with a Fourier transform infrared spectrometer. The single-angle technique measures amplitude of light reflected from the material as a function of wavelength over a wide spectral domain; the optical constants are determined from the reflectance data using the Kramers-Kronig relationship. We investigate several parameters associated with the pellets and pellet formation and their effects upon delivering the most reliable n/k values. Parameters studied include pellet diameter, mass, and density (void space), drying, grinding, sieving, and particle size in the pellet formation, as well as pressing pressure and duration. Of these parameters, using size-selected mixtures of dried, small (<50 µm) particles and pressing at ≥10 tons for at least 30 min were found key to forming highly reflective samples. Comparison of two sets of previous literature n(v∼) and k(v∼) values obtained from crystalline (NH4)2SO4 both as crystal reflectance as well as extinction spectra of aerosols measured in a flow tube shows reasonable agreement, but suggests the present values, as confirmed from two independent techniques, represent a substantial improvement for n/k values for (NH4)2SO4, also demonstrating promise to measure the optical constants of other materials.

Calcareous organic matter coatings sequester siderophores in alkaline soils.

Although most studies of organic matter (OM) stabilization in soils have focused on adsorption to aluminosilicate and iron-oxide minerals due to their strong interactions with organic nucleophiles, stabilization within alkaline soils has been empirically correlated with exchangeable Ca. Yet the extent of competing processes within natural soils remains unclear because of inadequate characterization of soil mineralogy and OM distribution within the soil in relation to minerals, particularly in C poor alkaline soils. In this study, we employed bulk and surface-sensitive spectroscopic methods including X-ray diffraction, 57Fe-Mössbauer, and X-ray photoemission spectroscopy (XPS), and transmission electron microscopy (TEM) methods to investigate the minerology and soil organic C and N distribution on individual fine particles within an alkaline soil. Microscopy and XPS analyses demonstrated preferential sorption of Ca-containing OM onto surfaces of Fe-oxides and calcite. This result was unexpected given that the bulk combined amounts of quartz and Fe-containing feldspars of the soil constitute ~90% of total minerals and the surface atomic composition was largely Fe and Al (>10% combined) compared to Ca (4.2%). Soil sorption experiments were conducted with two siderophores, pyoverdine and enterobactin, to evaluate the adsorption of organic molecules with functional groups that strongly and preferentially bind Fe. A greater fraction of pyoverdine was adsorbed compared to enterobactin, which is smaller, less polar, and has a lower aqueous solubility. Using NanoSIMS to map the distribution of isotopically-labeled siderophores, we observed correlations with Ca and Fe, along with strong isotopic dilution with native C, indicating associations with OM coatings rather than with bare mineral surfaces. We propose a mechanism of adsorption by which organics aggregate within alkaline soils via cation bridging, favoring the stabilization of larger molecules with a greater number of nucleophilic functional groups.

Phenazine-1-Carboxylic Acid-Producing Bacteria Enhance the Reactivity of Iron Minerals in Dryland and Irrigated Wheat Rhizospheres.

Phenazine-1-carboxylic acid (PCA) is a broad-spectrum antibiotic produced by rhizobacteria in the dryland wheat fields of the Columbia Plateau. PCA and other phenazines reductively dissolve Fe and Mn oxyhydroxides in bacterial culture systems, but the impact of PCA upon Fe and Mn cycling in the rhizosphere is unknown. Here, concentrations of dithionite-extractable and poorly crystalline Fe were approximately 10% and 30-40% higher, respectively, in dryland and irrigated rhizospheres inoculated with the PCA-producing (PCA+) strain Pseudomonas synxantha 2-79 than in rhizospheres inoculated with a PCA-deficient mutant. However, rhizosphere concentrations of Fe(II) and Mn did not differ significantly, indicating that PCA-mediated redox transformations of Fe and Mn were transient or were masked by competing processes. Total Fe and Mn uptake into wheat biomass also did not differ significantly, but the PCA+ strain significantly altered Fe translocation into shoots. X-ray absorption near edge spectroscopy revealed an abundance of Fe-bearing oxyhydroxides and phyllosilicates in all rhizospheres. These results indicate that the PCA+ strain enhanced the reactivity and mobility of Fe derived from soil minerals without producing parallel changes in plant Fe uptake. This is the first report that directly links significant alterations of Fe-bearing minerals in the rhizosphere to a single bacterial trait.

Microbe Encapsulation for Selective Rare-Earth Recovery from Electronic Waste Leachates.

Rare earth elements (REEs) are indispensable components of many green technologies and of increasing demand globally. However, refining REEs from raw materials using current technologies is energy intensive and enviromentally damaging. Here, we describe the development of a novel biosorption-based flow-through process for selective REE recovery from electronic wastes. An Escherichia coli strain previously engineered to display lanthanide-binding tags on the cell surface was encapsulated within a permeable polyethylene glycol diacrylate (PEGDA) hydrogel at high cell density using an emulsion process. This microbe bead adsorbent contained a homogenous distribution of cells whose surface functional groups remained accessible and effective for selective REE adsorption. The microbe beads were packed into fixed-bed columns, and breakthrough experiments demonstrated effective Nd extraction at a flow velocity of up to 3 m/h at pH 4-6. The microbe bead columns were stable for reuse, retaining 85% of the adsorption capacity after nine consecutive adsorption/desorption cycles. A bench-scale breakthrough curve with a NdFeB magnet leachate revealed a two-bed volume increase in breakthrough points for REEs compared to non-REE impurities and 97% REE purity of the adsorbed fraction upon breakthrough. These results demonstrate that the microbe beads are capable of repeatedly separating REEs from non-REE metals in a column system, paving the way for a biomass-based REE recovery system.

Blastochloris tepida, sp. nov., a thermophilic species of the bacteriochlorophyll b-containing genus Blastochloris.

A new taxon is created for the thermophilic purple nonsulfur bacterium previously designated as Rhodopseudomonas strain GI. Strain GI was isolated from a New Mexico (USA) hot spring microbial mat and grows optimally above 40 °C and to a maximum of 47 °C. Strain GI is a bacteriochlorophyll b-containing species of purple nonsulfur bacteria and displays a budding morphology, typical of species of the genus Blastochloris. Although resembling the species Blc. viridis in many respects, the absorption spectrum, carotenoid content, and lipid fatty acid profile of strain GI is distinct from that of Blc. viridis strain DSM133T and other recognized Blastochloris species. Strain GI forms its own subclade within the Blastochloris clade of purple nonsulfur bacteria based on comparative 16S rRNA gene sequences, and its genome is significantly larger than that of strain DSM133T; average nucleotide identity between the genomes of Blc. viridis and strain GI was below 85%. Moreover, concatenated sequence analyses of PufLM and DnaK clearly showed strain GI to be distinct from both Blc. viridis and Blc. sulfoviridis. Because of its unique assortment of properties, it is proposed to classify strain GI as a new species of the genus Blastochloris, as Blc. tepida, sp.n., with strain GIT designated as the type strain (= ATCC TSD-138 = DSM 106918).

The energetic basis for hydroxyapatite mineralization by amelogenin variants provides insights into the origin of amelogenesis imperfecta.

Small variations in the primary amino acid sequence of extracellular matrix proteins can have profound effects on the biomineralization of hard tissues. For example, a change in one amino acid within the amelogenin protein can lead to drastic changes in enamel phenotype, resulting in amelogenesis imperfecta, enamel that is defective and easily damaged. Despite the importance of these undesirable phenotypes, there is very little understanding of how single amino acid variation in amelogenins can lead to malformed enamel. Here, we aim to develop a thermodynamic understanding of how protein variants can affect steps of the biomineralization process. High-resolution, in situ atomic force microscopy (AFM) showed that altering one amino acid within the murine amelogenin sequence (natural variants T21 and P41T, and experimental variant P71T) resulted in an increase in the quantity of protein adsorbed onto hydroxyapatite (HAP) and the formation of multiple protein layers. Quantitative analysis of the equilibrium adsorbate amounts revealed that the protein variants had higher oligomer-oligomer binding energies. MMP20 enzyme degradation and HAP mineralization studies showed that the amino acid variants slowed the degradation of amelogenin by MMP20 and inhibited the growth and phase transformation of HAP. We propose that the protein variants cause malformed enamel because they bind excessively to HAP and disrupt the normal HAP growth and enzymatic degradation processes. The in situ methods applied to determine the energetics of molecular level processes are powerful tools toward understanding the mechanisms of biomineralization.

Incomplete cell disruption of resistant microbes.

Biomolecules for OMIC analysis of microbial communities are commonly extracted by bead-beating or ultra-sonication, but both showed varying yields. In addition to that, different disruption pressures are necessary to lyse bacteria and fungi. However, the disruption efficiency and yields comparing bead-beating and ultra-sonication of different biological material have not yet been demonstrated. Here, we show that ultra-sonication in a bath transfers three times more energy than bead-beating over 10 min. TEM imaging revealed intact gram-positive bacterial and fungal cells whereas the gram-negative bacterial cells were destroyed beyond recognition after 10 min of ultra-sonication. DNA extraction using 10 min of bead-beating revealed higher yields for fungi but the extraction efficiency was at least three-fold lower considering its larger genome. By our critical viewpoint, we encourage the review of the commonly used extraction techniques as we provide evidence for a potential underrepresentation of resistant microbes, particularly fungi, in ecological studies.

Quantitative Mapping of Oxidative Stress Response to Lithium Cobalt Oxide Nanoparticles in Single Cells Using Multiplexed in Situ Gene Expression Analysis.

Engineered nanoparticles (NPs) can negatively impact biological systems through induced generation of reactive oxygen species (ROS). Overproduced ROS cause biochemical damage and hence need to be effectively buffered by a sophisticated cellular oxidative stress response system. How this complex cellular system, which consists of multiple enzymes, responds to NP-induced ROS is largely unknown. Here, we apply a single cell analysis to quantitatively evaluate 10 key ROS responsive genes simultaneously to understand how the cell prioritizes tasks and reallocates resources in response to NP-induced oxidative stress. We focus on rainbow trout gill epithelial cells-a model cell type for environmental exposure-and their response to the massive generation of ROS induced by lithium cobalt oxide (LCO) NPs, which are extensively used as cathode materials in lithium ion batteries. Using multiplexed fluctuation localization imaging-based fluorescence in situ hybridization (fliFISH) in single cells, we found a shift in the expression of oxidative stress response genes with initial increase in genes targeting superoxide species, followed by increase in genes targeting peroxide and hydroxyl species. In contrast, Li+ and Co2+, at concentrations expected to be shed from the NPs, did not induce ROS generation but showed a potent inhibition of transcription for all 10 stress response genes. Taken together, our findings suggest a "two-hit" model for LCO NP toxicity, where the intact LCO NPs induce high levels of ROS that elicit sequential engagement of stress response genes, while the released metal ions suppress the expression of these genes. Consequently, these effects synergistically drive the exposed cells to become more vulnerable to ROS stress and damage.