DNA-guided crystallization of colloidal nanoparticles.

Many nanometre-sized building blocks will readily assemble into macroscopic structures. If the process is accompanied by effective control over the interactions between the blocks and all entropic effects, then the resultant structures will be ordered with a precision hard to achieve with other fabrication methods. But it remains challenging to use self-assembly to design systems comprised of different types of building blocks-to realize novel magnetic, plasmonic and photonic metamaterials, for example. A conceptually simple idea for overcoming this problem is the use of 'encodable' interactions between building blocks; this can in principle be straightforwardly implemented using biomolecules. Strategies that use DNA programmability to control the placement of nanoparticles in one and two dimensions have indeed been demonstrated. However, our theoretical understanding of how to extend this approach to three dimensions is limited, and most experiments have yielded amorphous aggregates and only occasionally crystallites of close-packed micrometre-sized particles. Here, we report the formation of three-dimensional crystalline assemblies of gold nanoparticles mediated by interactions between complementary DNA molecules attached to the nanoparticles' surface. We find that the nanoparticle crystals form reversibly during heating and cooling cycles. Moreover, the body-centred-cubic lattice structure is temperature-tuneable and structurally open, with particles occupying only approximately 4% of the unit cell volume. We expect that our DNA-mediated crystallization approach, and the insight into DNA design requirements it has provided, will facilitate both the creation of new classes of ordered multicomponent metamaterials and the exploration of the phase behaviour of hybrid systems with addressable interactions.

Genome sequence of the plant growth promoting endophytic bacterium Enterobacter sp. 638.

Enterobacter sp. 638 is an endophytic plant growth promoting gamma-proteobacterium that was isolated from the stem of poplar (Populus trichocarpaxdeltoides cv. H11-11), a potentially important biofuel feed stock plant. The Enterobacter sp. 638 genome sequence reveals the presence of a 4,518,712 bp chromosome and a 157,749 bp plasmid (pENT638-1). Genome annotation and comparative genomics allowed the identification of an extended set of genes specific to the plant niche adaptation of this bacterium. This includes genes that code for putative proteins involved in survival in the rhizosphere (to cope with oxidative stress or uptake of nutrients released by plant roots), root adhesion (pili, adhesion, hemagglutinin, cellulose biosynthesis), colonization/establishment inside the plant (chemiotaxis, flagella, cellobiose phosphorylase), plant protection against fungal and bacterial infections (siderophore production and synthesis of the antimicrobial compounds 4-hydroxybenzoate and 2-phenylethanol), and improved poplar growth and development through the production of the phytohormones indole acetic acid, acetoin, and 2,3-butanediol. Metabolite analysis confirmed by quantitative RT-PCR showed that, the production of acetoin and 2,3-butanediol is induced by the presence of sucrose in the growth medium. Interestingly, both the genetic determinants required for sucrose metabolism and the synthesis of acetoin and 2,3-butanediol are clustered on a genomic island. These findings point to a close interaction between Enterobacter sp. 638 and its poplar host, where the availability of sucrose, a major plant sugar, affects the synthesis of plant growth promoting phytohormones by the endophytic bacterium. The availability of the genome sequence, combined with metabolome and transcriptome analysis, will provide a better understanding of the synergistic interactions between poplar and its growth promoting endophyte Enterobacter sp. 638. This information can be further exploited to improve establishment and sustainable production of poplar as an energy feedstock on marginal, non-agricultural soils using endophytic bacteria as growth promoting agents.

Complete genome sequence of Clostridium sp. strain BNL1100, a cellulolytic mesophile isolated from corn stover.

We present the full genome sequence of Clostridium sp. strain BNL1100, a Gram-positive, endospore-forming, lignocellulolytic bacterium isolated from a corn stover enrichment culture. The 4,613,747-bp genome of strain BNL1100 contains 4,025 putative protein-coding genes, of which 103 are glycoside hydrolases, the highest detected number in cluster III clostridia.

Biotransformation of ferulic acid to 4-vinylguaiacol by Enterobacter soli and E. aerogenes.

We investigated the conversion of ferulic acid to 4-vinylguaiacol (4-VG), vanillin, vanillyl alcohol, and vanillic acid by five Enterobacter strains. These high-value chemicals are usually synthesized by chemical methods but biological synthesis adds market value. Ferulic acid, a relatively inexpensive component of agricultural crops, is plentiful in corn hulls, cereal bran, and sugar-beet pulp. Two Enterobacter strains, E. soli, and E. aerogenes, accumulated 550-600 ppm amounts of 4-VG when grown in media containing 1,000 ppm ferulic acid; no accumulations were observed with the other strains. Decreasing the amount of ferulic acid present in the media increased the conversion efficiency. When ferulic acid was supplied in 500, 250, or 125 ppm amounts E. aerogenes converted ~72 % of the ferulic acid present to 4-VG while E. soli converted ~100 % of the ferulic acid to 4-VG when supplied with 250 or 125 ppm amounts of ferulic acid. Also, lowering the pH improved the conversion efficiency. At pH 5.0 E. aerogenes converted ~84 % and E. soli converted ~100 % of 1,000 ppm ferulic acid to 4-VG. Only small, 1-5 ppm, accumulations of vanillin, vanillyl alcohol, and vanillic acid were observed. E. soli has a putative phenolic acid decarboxylase (PAD) that is 168 amino acids long and is similar to PADs in other enterobacteriales; this protein is likely involved in the bioconversion of ferulic acid to 4-VG. E. soli or E. aerogenes might be useful as a means of biotransforming ferulic acid to 4-VG.

Complete genome of the plant growth-promoting rhizobacterium Pseudomonas putida BIRD-1.

We report the complete sequence of the 5.7-Mbp genome of Pseudomonas putida BIRD-1, a metabolically versatile plant growth-promoting rhizobacterium that is highly tolerant to desiccation and capable of solubilizing inorganic phosphate and iron and of synthesizing phytohormones that stimulate seed germination and plant growth.

Continuous phase transformation in nanocube assemblies.

The phase behavior of 3D assemblies of nanocubes in a ligand-rich solution upon solvent evaporation was experimentally investigated using small-angle x-ray scattering and electron microscopy. We observed a continuous transformation of assemblies between simple cubic and rhombohedral phases, where a variable angle of rhombohedral structure is determined by ligand thickness. We established a quantitative relationship between the particle shape evolution from cubes to quasispheres and the lattice distortion during the transformation, with a pathway exhibiting the highest known packing.

Biosupported bimetallic Pd-Au nanocatalysts for dechlorination of environmental contaminants.

Biologically produced monometallic palladium nanoparticles (bio-Pd) have been shown to catalyze the dehalogenation of environmental contaminants, but fail to efficiently catalyze the degradation of other important recalcitrant halogenated compounds. This study represents the first report of biologically produced bimetallic Pd/Au nanoparticle catalysts. The obtained catalysts were tested for the dechlorination of diclofenac and trichlorethylene. When aqueous bivalent Pd(II) and trivalent Au(III) ions were both added to concentrations of 50 mg L(-1) and reduced simultaneously by Shewanella oneidensis in the presence of H(2), the resulting cell-associated bimetallic nanoparticles (bio-Pd/Au) were able to dehalogenate 78% of the initially added diclofenac after 24 h; in comparison, no dehalogenation was observed using monometallic bio-Pd or bio-Au. Other catalyst-synthesis strategies did not show improved dehalogenation of TCE and diclofenac compared with bio-Pd. Synchrotron-based X-ray diffraction, (scanning) transmission electron microscopy and energy dispersive X-ray spectroscopy indicated that the simultaneous reduction of Pd and Au supported on cells of S. oneidensis resulted in the formation of a unique bimetallic crystalline structure. This study demonstrates that the catalytic activity and functionality of possibly environmentally more benign biosupported Pd-catalysts can be improved by coprecipitation with Au.

Palladium nanoparticles produced by fermentatively cultivated bacteria as catalyst for diatrizoate removal with biogenic hydrogen.

A new biological inspired method to produce nanopalladium is the precipitation of Pd on a bacterium, i.e., bio-Pd. This bio-Pd can be applied as catalyst in dehalogenation reactions. However, large amounts of hydrogen are required as electron donor in these reactions resulting in considerable costs. This study demonstrates how bacteria, cultivated under fermentative conditions, can be used to reductively precipitate bio-Pd catalysts and generate the electron donor hydrogen. In this way, one could avoid the costs coupled to hydrogen supply. The catalytic activities of Pd(0) nanoparticles produced by different strains of bacteria (bio-Pd) cultivated under fermentative conditions were compared in terms of their ability to dehalogenate the recalcitrant aqueous pollutants diatrizoate and trichloroethylene. While all of the fermentative bio-Pd preparations followed first order kinetics in the dehalogenation of diatrizoate, the catalytic activity differed systematically according to hydrogen production and starting Pd(II) concentration in solution. Batch reactors with nanoparticles formed by Citrobacter braakii showed the highest diatrizoate dehalogenation activity with first order constants of 0.45 ± 0.02 h⁻¹ and 5.58 ± 0.6 h⁻¹ in batches with initial concentrations of 10 and 50 mg L⁻¹ Pd, respectively. Nanoparticles on C. braakii, used in a membrane bioreactor treating influent containing 20 mg L⁻¹ diatrizoate, were capable of dehalogenating 22 mg diatrizoate mg⁻¹ Pd over a period of 19 days before bio-Pd catalytic activity was exhausted. This study demonstrates the possibility to use the combination of Pd(II), a carbon source and bacteria under fermentative conditions for the abatement of environmental halogenated contaminants.

Rationally designed bacterial consortia to treat chronic immune-mediated colitis and restore intestinal homeostasis.

Environmental factors, mucosal permeability and defective immunoregulation drive overactive immunity to a subset of resident intestinal bacteria that mediate multiple inflammatory conditions. GUT-103 and GUT-108, live biotherapeutic products rationally designed to complement missing or underrepresented functions in the dysbiotic microbiome of IBD patients, address upstream targets, rather than targeting a single cytokine to block downstream inflammation responses. GUT-103, composed of 17 strains that synergistically provide protective and sustained engraftment in the IBD inflammatory environment, prevented and treated chronic immune-mediated colitis. Therapeutic application of GUT-108 reversed established colitis in a humanized chronic T cell-mediated mouse model. It decreased pathobionts while expanding resident protective bacteria; produced metabolites promoting mucosal healing and immunoregulatory responses; decreased inflammatory cytokines and Th-1 and Th-17 cells; and induced interleukin-10-producing colonic regulatory cells, and IL-10-independent homeostatic pathways. We propose GUT-108 for treating and preventing relapse for IBD and other inflammatory conditions characterized by unbalanced microbiota and mucosal permeability.

Sequencing of multiple clostridial genomes related to biomass conversion and biofuel production.

Modern methods to develop microbe-based biomass conversion processes require a system-level understanding of the microbes involved. Clostridium species have long been recognized as ideal candidates for processes involving biomass conversion and production of various biofuels and other industrial products. To expand the knowledge base for clostridial species relevant to current biofuel production efforts, we have sequenced the genomes of 20 species spanning multiple genera. The majority of species sequenced fall within the class III cellulosome-encoding Clostridium and the class V saccharolytic Thermoanaerobacteraceae. Species were chosen based on representation in the experimental literature as model organisms, ability to degrade cellulosic biomass either by free enzymes or by cellulosomes, ability to rapidly ferment hexose and pentose sugars to ethanol, and ability to ferment synthesis gas to ethanol. The sequenced strains significantly increase the number of noncommensal/nonpathogenic clostridial species and provide a key foundation for future studies of biomass conversion, cellulosome composition, and clostridial systems biology.

Stepwise surface encoding for high-throughput assembly of nanoclusters.

Self-assembly offers a promising method to organize functional nanoscale objects into two-dimensional (2D) and 3D superstructures for exploiting their collective effects. On the other hand, many unique phenomena emerge after arranging a few nanoscale objects into clusters, the so-called artificial molecules. The strategy of using biomolecular linkers between nanoparticles has proven especially useful for construction of such nanoclusters. However, conventional solution-based reactions typically yield a broad population of multimers or isomers of clusters; furthermore, the efficiency of fabrication is often limited. Here, we describe a novel high-throughput method for designing and fabricating clusters using DNA-encoded nanoparticles assembled on a solid support in a stepwise manner. This method efficiently imparts particles with anisotropy during their assembly and disassembly at a surface, generating remarkably high yields of well-defined dimer clusters and Janus (two-faced) nanoparticles. The method is scalable and modular, assuring large quantities of clusters of designated sizes and compositions.

Concomitant microbial generation of palladium nanoparticles and hydrogen to immobilize chromate.

The catalytic properties of various metal nanoparticles have led to their use in environmental remediation. Our aim is to develop and apply an efficient bioremediation method based on in situ biosynthesis of bio-Pd nanoparticles and hydrogen. C. pasteurianum BC1 was used to reduce Pd(II) ions to form Pd nanoparticles (bio-Pd) that primarily precipitated on the cell wall and in the cytoplasm. C. pasteurianum BC1 cells, loaded with bio-Pd nanoparticle in the presence of glucose, were subsequently used to fermentatively produce hydrogen and to effectively catalyze the removal of soluble Cr(VI) via reductive transformation to insoluble Cr(III) species. Batch and aquifer microcosm experiments using C. pasteurianum BC1 cells loaded with bio-Pd showed efficient reductive Cr(VI) removal, while in control experiments with killed or viable but Pd-free bacterial cultures no reductive Cr(VI) removal was observed. Our results suggest a novel process where the in situ microbial production of hydrogen is directly coupled to the catalytic bio-Pd mediated reduction of chromate. This process offers significant advantages over the current groundwater treatment technologies that rely on introducing preformed catalytic nanoparticles into groundwater treatment zones and the costly addition of molecular hydrogen to above ground pump and treat systems.

COVID-19 and the Gut Microbiome: More than a Gut Feeling.

Due to its fundamental role in the induction, training, and function of the immune system, it is critical to include characterizations of the gut microbiome in clinical trials and studies that aim to broaden our understanding of coronavirus disease 2019 (COVID-19). Understanding the "gut-lung axes," where gut microbiome composition influences the lung's susceptibility to viral infections and viral infections of the lung alter gut microbiome composition toward proinflammatory functional dysbiosis, will be critical in addressing COVID-19, including disease progression, the importance of preexisting conditions, and the risk for developing complications. These insights may further help to develop better intervention strategies for COVID-19 and other diseases caused by respiratory viruses.

Genome survey and characterization of endophytic bacteria exhibiting a beneficial effect on growth and development of poplar trees.

The association of endophytic bacteria with their plant hosts has a beneficial effect for many different plant species. Our goal is to identify endophytic bacteria that improve the biomass production and the carbon sequestration potential of poplar trees (Populus spp.) when grown in marginal soil and to gain an insight in the mechanisms underlying plant growth promotion. Members of the Gammaproteobacteria dominated a collection of 78 bacterial endophytes isolated from poplar and willow trees. As representatives for the dominant genera of endophytic gammaproteobacteria, we selected Enterobacter sp. strain 638, Stenotrophomonas maltophilia R551-3, Pseudomonas putida W619, and Serratia proteamaculans 568 for genome sequencing and analysis of their plant growth-promoting effects, including root development. Derivatives of these endophytes, labeled with gfp, were also used to study the colonization of their poplar hosts. In greenhouse studies, poplar cuttings (Populus deltoides x Populus nigra DN-34) inoculated with Enterobacter sp. strain 638 repeatedly showed the highest increase in biomass production compared to cuttings of noninoculated control plants. Sequence data combined with the analysis of their metabolic properties resulted in the identification of many putative mechanisms, including carbon source utilization, that help these endophytes to thrive within a plant environment and to potentially affect the growth and development of their plant hosts. Understanding the interactions between endophytic bacteria and their host plants should ultimately result in the design of strategies for improved poplar biomass production on marginal soils as a feedstock for biofuels.

Lead(II) resistance in Cupriavidus metallidurans CH34: interplay between plasmid and chromosomally-located functions.

Proteome and transcriptome analysis, combined with mutagenesis, were used to better understand the response of Cupriavidus metallidurans CH34 against lead(II). Structural Pb(II)-resistance genes of the pMOL30-encoded pbrUTRABCD operon formed the major line of defense against Pb(II). However, several general stress response mechanisms under the control of alternative sigma factors such as sigma24/rpoK, sigma32/rpoH and sigma28/fliA were also induced. In addition, the expression of the pbrR(2) cadA pbrC(2) operon of the CMGI-1 region and the chromosomally encoded zntA were clearly induced in the presence of Pb(II), although their respective gene products were not detected via proteomics. After inactivation of the pbrA, pbrB or pbrD genes, the expression of the pbrR(2) cadA pbrC(2) operon went up considerably. This points towards synergistic interactions between pbrUTRABCD and pbrR(2) cadA pbrC(2) to maintain a low intracellular Pb(II) concentration, where pbrR(2) cadA pbrC(2) gene functions can complement and compensate for the mutations in the pbrA and pbrD genes. This role of zntA and cadA to complement for the loss of pbrA was further confirmed by mutation analysis. The pbrB:: colonsTn(Km2) mutation resulted in the most significant decrease of Pb(II) resistance, indicating that Pb(II) sequestration, avoiding re-entry of this toxic metal ion, forms a critical step in the pbr-encoded Pb(II) resistance mechanism.

ArsR arsenic-resistance regulatory protein from Cupriavidus metallidurans CH34.

The Cupriavidus metallidurans CH34 arsR gene, which is part of the arsRIC(2)BC(1)HP operon, and its putative arsenic-resistance regulatory protein were identified and characterized. The arsenic-induced transcriptome of C. metallidurans CH34 showed that the genes most upregulated in the presence of arsenate were all located within the ars operon, with none of the other numerous heavy metal resistance systems present in CH34 being induced. A transcriptional fusion between the luxCDABE operon and the arsR promoter/operator (P/O) region was used to confirm the in vivo induction of the ars operon by arsenite and arsenate. The arsR gene was cloned into expression vectors allowing for the overexpression of the ArsR protein as either his-tagged or untagged protein. The ability of the purified ArsR proteins to bind to the ars P/O region was analyzed in vitro by gel mobility shift assays. ArsR showed an affinity almost exclusively to its own ars P/O region. Dissociation of ArsR and its P/O region was metal dependent, and based on decreasing degrees of dissociation three groups of heavy metals could be distinguished: As(III), Bi(III), Co(II), Cu(II), Ni(II); Cd(II); Pb(II) and Zn(II), while no dissociation was observed in the presence of As(V).

Elevated atmospheric CO2 affects soil microbial diversity associated with trembling aspen.

The effects of elevated atmospheric CO(2) (560 p.p.m.) and subsequent plant responses on the soil microbial community composition associated with trembling aspen was assessed through the classification of 6996 complete ribosomal DNA sequences amplified from the Rhinelander WI free-air CO(2) and O(3) enrichment (FACE) experiments microbial community metagenome. This in-depth comparative analysis provides an unprecedented, detailed and deep branching profile of population changes incurred as a response to this environmental perturbation. Total bacterial and eukaryotic abundance does not change; however, an increase in heterotrophic decomposers and ectomycorrhizal fungi is observed. Nitrate reducers of the domain bacteria and archaea, of the phylum Crenarchaea, potentially implicated in ammonium oxidation, significantly decreased with elevated CO(2). These changes in soil biota are evidence for altered interactions between trembling aspen and the microorganisms in its surrounding soil, and support the theory that greater plant detritus production under elevated CO(2) significantly alters soil microbial community composition.

Microbial community dynamics in uranium contaminated subsurface sediments under biostimulated conditions with high nitrate and nickel pressure.

BACKGROUND, AIM, AND SCOPE: The subsurface at the Oak Ridge Field Research Center represents an extreme and diverse geochemical environment that places different stresses on the endogenous microbial communities, including low pH, elevated nitrate concentrations, and the occurrence of heavy metals and radionuclides, including hexavalent uranium [U(VI)]. The in situ immobilization of U(VI) in the aquifer can be achieved through microbial reduction to relatively insoluble U(IV). However, a high redox potential due to the presence of nitrate and the toxicity of heavy metals will impede this process. Our aim is to test biostimulation of the endogenous microbial communities to improve nitrate reduction and subsequent U(VI) reduction under conditions of elevated heavy metals. MATERIALS AND METHODS: Column experiments were used to test the possibility of using biostimulation via the addition of ethanol as a carbon source to improve nitrate reduction in the presence of elevated aqueous nickel. We subsequently analyzed the composition of the microbial communities that became established and their potential for U(VI) reduction and its in situ immobilization. RESULTS: Phylogenetic analysis revealed that the microbial population changed from heavy metal sensitive members of the actinobacteria, alpha- and gamma-proteobacteria to a community dominated by heavy metal resistant (nickel, cadmium, zinc, and cobalt resistant), nitrate reducing beta- and gamma-proteobacteria, and sulfate reducing Clostridiaceae. Coincidentally, synchrotron X-ray absorption spectroscopy analyses indicated that the resulting redox conditions favored U(VI) reduction transformation to insoluble U(IV) species associated with soil minerals and biomass. DISCUSSION: This study shows that the necessary genetic information to adapt to the implemented nickel stress resides in the endogenous microbial population present at the Oak Ridge FRC site, which changed from a community generally found under oligotrophic conditions to a community able to withstand the stress imposed by heavy metals, while efficiently reducing nitrate as electron donor. Once nitrate was reduced efficient reduction and in situ immobilization of uranium was observed. CONCLUSIONS: This study provides evidence that stimulating the metabolism of the endogenous bacterial population at the Oak Ridge FRC site by adding ethanol, a suitable carbon source, results in efficient nitrate reduction under conditions of elevated nickel, and a decrease of the redox potential such that sulfate and iron reducing bacteria are able to thrive and create conditions favorable for the reduction and in situ immobilization of uranium. Since we have found that the remediation potential resides within the endogenous microbial community, we believe it will be feasible to conduct field tests. RECOMMENDATIONS AND PERSPECTIVES: Biostimulation of endogenous bacteria provides an efficient tool for the successful in situ remediation of mixed-waste sites, particularly those co-contaminated with heavy metals, nitrate and radionuclides, as found in the United States and other countries as environmental legacies of the nuclear age.

Engineered endophytic bacteria improve phytoremediation of water-soluble, volatile, organic pollutants.

Phytoremediation of highly water soluble and volatile organic xenobiotics is often inefficient because plants do not completely degrade these compounds through their rhizospheres. This results in phytotoxicity and/or volatilization of chemicals through the leaves, which can cause additional environmental problems. We demonstrate that endophytic bacteria equipped with the appropriate degradation pathway improve the in planta degradation of toluene. We introduced the pTOM toluene-degradation plasmid of Burkholderia cepacia G4 into B. cepacia L.S.2.4, a natural endophyte of yellow lupine. After surface-sterilized lupine seeds were successfully inoculated with the recombinant strain, the engineered endophytic bacteria strongly degraded toluene, resulting in a marked decrease in its phytotoxicity, and a 50-70% reduction of its evapotranspiration through the leaves. This strategy promises to improve the efficiency of phytoremediating volatile organic contaminants.