Align to Define: Ecologically Meaningful Populations from Genomes.

Microbes in the same community but with distinct niches can have unique long stretches of perfect sequence identity due to recent genetic exchange. Arevalo et al. (2019) use this as a starting point for defining ecologically-relevant populations within a community and to identify the genes that appear to be driving divergence between populations.

Starch-Coated Magnetic Iron Oxide Nanoparticles for Affinity Purification of Recombinant Proteins.

Starch-coated magnetic iron oxide nanoparticles have been synthesized by a simple, fast, and cost-effective co-precipitation method with cornstarch as a stabilizing agent. The structural and magnetic characteristics of the synthesized material have been studied by transmission electron microscopy, Mössbauer spectroscopy, and vibrating sample magnetometry. The nature of bonds between ferrihydrite nanoparticles and a starch shell has been examined by Fourier transform infrared spectroscopy. The data on the magnetic response of the prepared composite particles have been obtained by magnetic measurements. The determined magnetic characteristics make the synthesized material a good candidate for use in magnetic separation. Starch-coated magnetic iron oxide nanoparticles have been tested as an affinity sorbent for one-step purification of several recombinant proteins (cardiac troponin I, survivin, and melanoma inhibitory activity protein) bearing the maltose-binding protein as an auxiliary fragment. It has been shown that, due to the highly specific binding of this fragment to the starch shell, the target fusion protein is selectively immobilized on magnetic nanoparticles and eluted with the maltose solution. The excellent efficiency of column-free purification, high binding capacity of the sorbent (100-500 µg of a recombinant protein per milligram of starch-coated magnetic iron oxide nanoparticles), and reusability of the obtained material have been demonstrated.

Interactions of Chemically Synthesized Ferrihydrite Nanoparticles with Human Serum Transferrin: Insights from Fluorescence Spectroscopic Studies.

Human serum transferrin (HST) is a glycoprotein involved in iron transport that may be a candidate for functionalized nanoparticles to bind and target cancer cells. In this study, the effects of the simple and doped with cobalt (Co) and copper (Cu) ferrihydrite nanoparticles (Fh-NPs, Cu-Fh-NPs, and Co-Fh-NPs) were studied by spectroscopic and molecular approaches. Fluorescence spectroscopy revealed a static quenching mechanism for all three types of Fh-NPs. All Fh-NPs interacted with HST with low affinity, and the binding was driven by hydrogen bonding and van der Waals forces for simple Fh-NPs and by hydrophobic interactions for Cu-Fh-NPs and Co-Fh-NPs binding, respectively. Of all samples, simple Fh-NPs bound the most to the HST binding site. Fluorescence resonance energy transfer (FRET) allowed the efficient determination of the energy transfer between HST and NPs and the distance at which the transfer takes place and confirmed the mechanism of quenching. The denaturation of the HST is an endothermic process, both in the case of apo HST and HST in the presence of the three types of Fh-NPs. Molecular docking studies revealed that Fh binds with a low affinity to HST (Ka = 9.17 × 103 M-1) in accord with the fluorescence results, where the interaction between simple Fh-NPs and HST was described by a binding constant of 9.54 × 103 M-1.

Exploring the Conformation and Thermal Stability of Human Serum Albumin Corona of Ferrihydrite Nanoparticles.

In the last few years, a great amount of attention has been given to nanoparticles research due to their physicochemical properties that allow their use in analytical instruments or in promising imaging applications on biological systems. The use of ferrihydrite nanoparticles (Fh-NPs) in practical applications implies a particular control of their magnetic properties, stability, biocompatibility, interaction with the surface of the target, and low toxicity. In this study, the formation and organization of human serum albumin (HSA) molecules around the simple Fh-NPs and Fh-NPs doped with Co and Cu were examined by Scanning Electron Microscopy (SEM) and Atomic Force Microscopy (AFM) in terms of morphology and particle size. The topology of all Fh-NPs shows an organized area of HSA around each type of Fh-NP. Molecular docking studies were used in order to determine the probable location of the ferrihydrite in the HSA structure. The thermal stability of these nanohybrids was further investigated by fluorimetry, using 214-Trp residue from HSA as a spectral sensor. The denaturation temperature (Tm) was determined, and stabilization of the HSA structure in the presence of Fh-NPs was discussed. This study could be a starting point for the development of different applications targeting the structure and stability of Fh-NPs complexes with proteins.

Mechanism for microbial population collapse in a fluctuating resource environment.

Managing trade-offs through gene regulation is believed to confer resilience to a microbial community in a fluctuating resource environment. To investigate this hypothesis, we imposed a fluctuating environment that required the sulfate-reducer Desulfovibrio vulgaris to undergo repeated ecologically relevant shifts between retaining metabolic independence (active capacity for sulfate respiration) and becoming metabolically specialized to a mutualistic association with the hydrogen-consuming Methanococcus maripaludis Strikingly, the microbial community became progressively less proficient at restoring the environmentally relevant physiological state after each perturbation and most cultures collapsed within 3-7 shifts. Counterintuitively, the collapse phenomenon was prevented by a single regulatory mutation. We have characterized the mechanism for collapse by conducting RNA-seq analysis, proteomics, microcalorimetry, and single-cell transcriptome analysis. We demonstrate that the collapse was caused by conditional gene regulation, which drove precipitous decline in intracellular abundance of essential transcripts and proteins, imposing greater energetic burden of regulation to restore function in a fluctuating environment.

Effects of iron oxide nanoparticles on the gene expression profiles of cerebral endotheliocytes and astrocytes.

Iron oxide nanoparticles (IONPs) are considered as the most biocompatible magnetic materials suitable for biomedical applications. Nevertheless, there are many evidences of their toxicity for living organisms and partially neurotoxicity. The central nervous system is protected from undesirable substances circulating in the bloodstream by the blood-brain barrier (BBB). And even if being small enough, some nanoparticles could be able to penetrate cell membranes in other cells but will often be delayed by the BBB cells. However, the neurotoxicity of iron oxide is described even in the cases when IONPs should not uptake to the nervous system by experimental design. The aim of this study was to investigate what molecular changes in the cells-components of BBB - endotheliocytes and underlying astrocytes - may be caused by IONPs in the blood vessels of the brain. For this, a two-layer in vitro BBB model was created, consisting of rat cerebral endothelial cells and astrocytes. It was revealed that 100 and 200 mg/L of the nanoparticles induce metabolism alteration in the cells under study. Using RNA-sequencing, the up-regulation of pro-inflammatory chemokines encoding genes and changes in the expression of genes associated with detoxification in the endotheliocytes were demonstrated under the influence of 100 mg/L IONPs.

Genome-scale modeling of light-driven reductant partitioning and carbon fluxes in diazotrophic unicellular cyanobacterium Cyanothece sp. ATCC 51142.

Genome-scale metabolic models have proven useful for answering fundamental questions about metabolic capabilities of a variety of microorganisms, as well as informing their metabolic engineering. However, only a few models are available for oxygenic photosynthetic microorganisms, particularly in cyanobacteria in which photosynthetic and respiratory electron transport chains (ETC) share components. We addressed the complexity of cyanobacterial ETC by developing a genome-scale model for the diazotrophic cyanobacterium, Cyanothece sp. ATCC 51142. The resulting metabolic reconstruction, iCce806, consists of 806 genes associated with 667 metabolic reactions and includes a detailed representation of the ETC and a biomass equation based on experimental measurements. Both computational and experimental approaches were used to investigate light-driven metabolism in Cyanothece sp. ATCC 51142, with a particular focus on reductant production and partitioning within the ETC. The simulation results suggest that growth and metabolic flux distributions are substantially impacted by the relative amounts of light going into the individual photosystems. When growth is limited by the flux through photosystem I, terminal respiratory oxidases are predicted to be an important mechanism for removing excess reductant. Similarly, under photosystem II flux limitation, excess electron carriers must be removed via cyclic electron transport. Furthermore, in silico calculations were in good quantitative agreement with the measured growth rates whereas predictions of reaction usage were qualitatively consistent with protein and mRNA expression data, which we used to further improve the resolution of intracellular flux values.

Scattered-light-sheet microscopy with sub-cellular resolving power.

Since its first demonstration over 100 years ago, scattering-based light-sheet microscopy has recently re-emerged as a key modality in label-free tissue imaging and cellular morphometry; however, scattering-based light-sheet imaging with subcellular resolution remains an unmet target. This is because related approaches inevitably superimpose speckle or granular intensity modulation on to the native subcellular features. Here, we addressed this challenge by deploying a time-averaged pseudo-thermalized light-sheet illumination. While this approach increased the lateral dimensions of the illumination sheet, we achieved subcellular resolving power after image deconvolution. We validated this approach by imaging cytosolic carbon depots in yeast and bacteria with increased specificity, no staining, and ultralow irradiance levels. Overall, we expect this scattering-based light-sheet microscopy approach will advance single, live cell imaging by conferring low-irradiance and label-free operation towards eradicating phototoxicity.

Complete genome sequences of six strains of the genus Methylobacterium.

The complete and assembled genome sequences were determined for six strains of the alphaproteobacterial genus Methylobacterium, chosen for their key adaptations to different plant-associated niches and environmental constraints.

Aerobic Methoxydotrophy: Growth on Methoxylated Aromatic Compounds by Methylobacteriaceae.

Pink-pigmented facultative methylotrophs have long been studied for their ability to grow on reduced single-carbon (C1) compounds. The C1 groups that support methylotrophic growth may come from a variety of sources. Here, we describe a group of Methylobacterium strains that can engage in methoxydotrophy: they can metabolize the methoxy groups from several aromatic compounds that are commonly the product of lignin depolymerization. Furthermore, these organisms can utilize the full aromatic ring as a growth substrate, a phenotype that has rarely been described in Methylobacterium. We demonstrated growth on p-hydroxybenzoate, protocatechuate, vanillate, and ferulate in laboratory culture conditions. We also used comparative genomics to explore the evolutionary history of this trait, finding that the capacity for aromatic catabolism is likely ancestral to two clades of Methylobacterium, but has also been acquired horizontally by closely related organisms. In addition, we surveyed the published metagenome data to find that the most abundant group of aromatic-degrading Methylobacterium in the environment is likely the group related to Methylobacterium nodulans, and they are especially common in soil and root environments. The demethoxylation of lignin-derived aromatic monomers in aerobic environments releases formaldehyde, a metabolite that is a potent cellular toxin but that is also a growth substrate for methylotrophs. We found that, whereas some known lignin-degrading organisms excrete formaldehyde as a byproduct during growth on vanillate, Methylobacterium do not. This observation is especially relevant to our understanding of the ecology and the bioengineering of lignin degradation.

Biogenic Ferrihydrite Nanoparticles Produced by Klebsiella oxytoca: Characterization, Physicochemical Properties and Bovine Serum Albumin Interactions.

The synthesis of nanoparticles inside microorganisms is an economical alternative to chemical and physical methods of nanoparticle synthesis. In this study, ferrihydrite nanoparticles synthesized by Klebsiella oxytoca bacterium in special conditions were characterized by scanning electron microscopy (SEM), energy-dispersive X-ray analysis (EDS), small-angle X-ray (SAXS), UV-Vis spectroscopy, fluorescence, fluorescence resonance energy transfer (FRET), and molecular docking. The morphology and the structure of the particles were characterized by means of SEM and SAXS. The elemental content was determined by means of the EDS method. The absorption properties of the ferrihydrite nanoparticles were investigated by UV-Vis spectroscopy. The binding mechanism of the biogenic ferrihydrite nanoparticles to Bovine Serum Albumin (BSA) protein, studied by fluorescence, showed a static and weak process, combined with FRET. Protein denaturation by temperature and urea in the presence of the ferrihydrite nanoparticles demonstrated their influence on the unfolding process. The AutoDock Vina and UCSF Chimera programs were used to predict the optimal binding site of the ferrihydrite to BSA and to find the location of the hydrophobic cavities in the sub-domain IIA of the BSA structure.

Comprehensive Phylogenomics of Methylobacterium Reveals Four Evolutionary Distinct Groups and Underappreciated Phyllosphere Diversity.

Methylobacterium is a group of methylotrophic microbes associated with soil, fresh water, and particularly the phyllosphere, the aerial part of plants that has been well studied in terms of physiology but whose evolutionary history and taxonomy are unclear. Recent work has suggested that Methylobacterium is much more diverse than thought previously, questioning its status as an ecologically and phylogenetically coherent taxonomic genus. However, taxonomic and evolutionary studies of Methylobacterium have mostly been restricted to model species, often isolated from habitats other than the phyllosphere and have yet to utilize comprehensive phylogenomic methods to examine gene trees, gene content, or synteny. By analyzing 189 Methylobacterium genomes from a wide range of habitats, including the phyllosphere, we inferred a robust phylogenetic tree while explicitly accounting for the impact of horizontal gene transfer (HGT). We showed that Methylobacterium contains four evolutionarily distinct groups of bacteria (namely A, B, C, D), characterized by different genome size, GC content, gene content, and genome architecture, revealing the dynamic nature of Methylobacterium genomes. In addition to recovering 59 described species, we identified 45 candidate species, mostly phyllosphere-associated, stressing the significance of plants as a reservoir of Methylobacterium diversity. We inferred an ancient transition from a free-living lifestyle to association with plant roots in Methylobacteriaceae ancestor, followed by phyllosphere association of three of the major groups (A, B, D), whose early branching in Methylobacterium history has been heavily obscured by HGT. Together, our work lays the foundations for a thorough redefinition of Methylobacterium taxonomy, beginning with the abandonment of Methylorubrum.

Cross-Feeding of a Toxic Metabolite in a Synthetic Lignocellulose-Degrading Microbial Community.

The recalcitrance of complex organic polymers such as lignocellulose is one of the major obstacles to sustainable energy production from plant biomass, and the generation of toxic intermediates can negatively impact the efficiency of microbial lignocellulose degradation. Here, we describe the development of a model microbial consortium for studying lignocellulose degradation, with the specific goal of mitigating the production of the toxin formaldehyde during the breakdown of methoxylated aromatic compounds. Included are Pseudomonas putida, a lignin degrader; Cellulomonas fimi, a cellulose degrader; and sometimes Yarrowia lipolytica, an oleaginous yeast. Unique to our system is the inclusion of Methylorubrum extorquens, a methylotroph capable of using formaldehyde for growth. We developed a defined minimal "Model Lignocellulose" growth medium for reproducible coculture experiments. We demonstrated that the formaldehyde produced by P. putida growing on vanillic acid can exceed the minimum inhibitory concentration for C. fimi, and, furthermore, that the presence of M. extorquens lowers those concentrations. We also uncovered unexpected ecological dynamics, including resource competition, and interspecies differences in growth requirements and toxin sensitivities. Finally, we introduced the possibility for a mutualistic interaction between C. fimi and M. extorquens through metabolite exchange. This study lays the foundation to enable future work incorporating metabolomic analysis and modeling, genetic engineering, and laboratory evolution, on a model system that is appropriate both for fundamental eco-evolutionary studies and for the optimization of efficiency and yield in microbially-mediated biomass transformation.

Silica-Coated Iron Oxide Nanoparticles for DNA Isolation for Molecular Genetic Studies in Hematology.

Aim: To develop magnetic nanoparticles (MNPs) based on iron oxide for DNA isolation from blood cells for quantitative molecular genetic analyses of the V617F mutation in the Januskinase 2 (JAK2) gene. Materials and Methods: MNPs were synthesized by the coprecipitation method and coated with tetraethyl orthosilicate (TEOS). The size and shape of the complexes were estimated using transmission electron microscopy. Twenty blood samples from patients with myeloproliferative disorders were used for DNA isolation with the MNPs. DNA quality and compatibility for molecular genetic studies of the JAK2 V617F mutation were investigated by gel electrophoresis and real-time polymerase chain reaction (RT-PCR). Results: The average amount of DNA isolated from 150 µL of whole blood was 75.2 ng when MNPs were used and 72.5 ng when standard silica sorbent was used. There was no DNA damage observed after interaction with MNPs. RT-PCR demonstrated similar values for the JAK2 V617F mutant DNA ratios in the samples after DNA isolation with MNPs and by standard sorption on silica. Conclusions: MNPs with silicate capsules of sufficient thickness were obtained and the undesirable damaging effect of iron oxides on nucleic acids during isolation from cells were eliminated. Designed MNPs allow obtaining intact DNA for molecular genetic studies using the example of the JAK2 V617F for study.

Biogenic Ferrihydrite Nanoparticles: Synthesis, Properties In Vitro and In Vivo Testing and the Concentration Effect.

Biogenic ferrihydrite nanoparticles were synthesized as a result of the cultivation of Klebsiella oxytoca microorganisms. The distribution of nanoparticles in the body of laboratory animals and the physical properties of the nanoparticles were studied. The synthesized ferrihydrite nanoparticles are superparamagnetic at room temperature, and the characteristic blocking temperature is 23-25 K. The uncompensated moment of ferrihydrite particles was determined to be approximately 200 Bohr magnetons. In vitro testing of different concentrations of ferrihydrite nanoparticles for the functional activity of neutrophilic granulocytes by the chemiluminescence method showed an increase in the release of primary oxygen radicals by blood phagocytes when exposed to a minimum concentration and a decrease in secondary radicals when exposed to a maximum concentration. In vivo testing of ferrihydrite nanoparticles on Wister rats showed that a suspension of ferrihydrite nanoparticles has chronic toxicity, since it causes morphological changes in organs, mainly in the spleen, which are characterized by the accumulation of hemosiderin nanoparticles (stained blue according to Perls). Ferrihydrite can also directly or indirectly stimulate the proliferation and intracellular regeneration of hepatocytes. The partial detection of Perls-positive cells in the liver and kidneys can be explained by the rapid elimination from organs and the high dispersion of the nanomaterial. Thus, it is necessary to carry out studies of these processes at the systemic level, since the introduction of nanoparticles into the body is characterized by adaptive-proliferative processes, accompanied by the development of cell dystrophy and tension of the phagocytic system.

Ferrihydrite nanoparticles insights: Structural characterization, lactate dehydrogenase binding and virtual screening assay.

The binding between the enzyme lactate dehydrogenase (LDH) and ferrihydrite nanoparticles (Fh-NPs) was investigated by means of small-angle neutron scattering (SANS), Fourier-transform infrared (FTIR) spectroscopy, fluorescence and Förster resonance energy transfer (FRET) and molecular docking. Fh-NPs - LDH compounds of dimensions under 100 nm are formed. The conformational changes and the mechanism of interaction between LDH and Fh-NPs simple and doped with Cu and Co, and the effect of these NPs on the thermal denaturation of LDH were monitored. The quenching mechanism is static, the binding occurring with moderate affinity, being mainly driven by hydrogen bonding and van der Waals forces. FRET occurs at a minimal distance of 2.55 nm. Thermal denaturation of LDH in the presence of simple and doped Fh-NPs shows that the thermodynamic parameters of protein unfolding are significantly changed with temperature. The denaturation temperature of LDH shifts to higher values in the presence of all Fh-NPs, than in the case of simple LDH. The docking approach estimates the energy corresponding to the best fit of the ferrihydrite in the LDH binding site near Trp. These results have direct implications on the uses of the complex of LDH with Fh-NPs in various biochemical, biological, or clinical applications.

Ferrihydrite nanoparticles interaction with model lipid membranes.

In recent years was observed an increased interest towards the use of metal nanoparticles for various biomedical applications, such as therapeutics, delivery systems or imaging. As biological membranes are the first structures with which the nanoparticles interact, it is necessary to understand better the mechanisms governing these interactions. In the present paper we aim to characterize the effect of three different ferrihydrite nanoparticles (simple or doped with cooper or cobalt) on the fluidity of model lipid membranes. First we evaluated the physicochemical properties of the nanoparticles: size and composition. Secondly, their effect on lipid membranes was also evaluated using Laurdan, TMA-DPH and DPH fluorescence. Our results can help better understand the mechanisms involved in nanoparticles and membrane interactions.

The electron transfer system of syntrophically grown Desulfovibrio vulgaris.

Interspecies hydrogen transfer between organisms producing and consuming hydrogen promotes the decomposition of organic matter in most anoxic environments. Although syntrophic coupling between hydrogen producers and consumers is a major feature of the carbon cycle, mechanisms for energy recovery at the extremely low free energies of reactions typical of these anaerobic communities have not been established. In this study, comparative transcriptional analysis of a model sulfate-reducing microbe, Desulfovibrio vulgaris Hildenborough, suggested the use of alternative electron transfer systems dependent on growth modality. During syntrophic growth on lactate with a hydrogenotrophic methanogen, numerous genes involved in electron transfer and energy generation were upregulated in D. vulgaris compared with their expression in sulfate-limited monocultures. In particular, genes coding for the putative membrane-bound Coo hydrogenase, two periplasmic hydrogenases (Hyd and Hyn), and the well-characterized high-molecular-weight cytochrome (Hmc) were among the most highly expressed and upregulated genes. Additionally, a predicted operon containing genes involved in lactate transport and oxidation exhibited upregulation, further suggesting an alternative pathway for electrons derived from lactate oxidation during syntrophic growth. Mutations in a subset of genes coding for Coo, Hmc, Hyd, and Hyn impaired or severely limited syntrophic growth but had little effect on growth via sulfate respiration. These results demonstrate that syntrophic growth and sulfate respiration use largely independent energy generation pathways and imply that to understand microbial processes that sustain nutrient cycling, lifestyles not captured in pure culture must be considered.

Contribution of mobile genetic elements to Desulfovibrio vulgaris genome plasticity.

The genome of Desulfovibrio vulgaris strain DePue, a sulfate-reducing Deltaproteobacterium isolated from heavy metal-impacted lake sediment, was completely sequenced and compared with the type strain D. vulgaris Hildenborough. The two genomes share a high degree of relatedness and synteny, but harbour distinct prophage and signatures of past phage encounters. In addition to a highly variable phage contribution, the genome of strain DePue contains a cluster of open-reading frames not found in strain Hildenborough coding for the production and export of a capsule exopolysaccharide, possibly of relevance to heavy metal resistance. Comparative whole-genome microarray analysis on four additional D. vulgaris strains established greater interstrain variation within regions associated with phage insertion and exopolysaccharide biosynthesis.

Metabolic modeling of a mutualistic microbial community.

The rate of production of methane in many environments depends upon mutualistic interactions between sulfate-reducing bacteria and methanogens. To enhance our understanding of these relationships, we took advantage of the fully sequenced genomes of Desulfovibrio vulgaris and Methanococcus maripaludis to produce and analyze the first multispecies stoichiometric metabolic model. Model results were compared to data on growth of the co-culture on lactate in the absence of sulfate. The model accurately predicted several ecologically relevant characteristics, including the flux of metabolites and the ratio of D. vulgaris to M. maripaludis cells during growth. In addition, the model and our data suggested that it was possible to eliminate formate as an interspecies electron shuttle, but hydrogen transfer was essential for syntrophic growth. Our work demonstrated that reconstructed metabolic networks and stoichiometric models can serve not only to predict metabolic fluxes and growth phenotypes of single organisms, but also to capture growth parameters and community composition of simple bacterial communities.