[Show abstract][Hide abstract] ABSTRACT: Within boreal and temperate forest ecosystems the majority of trees and shrubs form beneficial relationships with mutualistic ectomycorrhizal fungi (ECM) that support plant health through increased access to nutrients as well as aiding in stress and pest tolerance. The intimate interaction between fungal hyphae and plant roots result in a new symbiotic 'organ' called the ECM root tip. Little is understood concerning the metabolic re-programming that favors the formation of this hybrid tissue in compatible interactions and what prevents the formation of ECM root tips in incompatible interactions. We show here that the metabolic changes during favorable colonization between the ECM fungus Laccaria bicolor and its compatible host, Populus trichocarpa, are characterized by shifts in aromatic acid, organic acid, and fatty acid metabolism. We demonstrate that this extensive metabolic re-programming is repressed in incompatible interactions and that more defensive compounds are produced or retained. We also demonstrate that L. bicolor can metabolize a number of secreted defensive compounds and that the degradation of some of these compounds produce immune response metabolites (e.g., salicylic acid from salicin). Therefore, our results suggest that the metabolic responsiveness of plant roots to L. bicolor is a determinant factor in fungal:host interactions.
[Show abstract][Hide abstract] ABSTRACT: Engineered gene circuits offer an opportunity to harness biological systems for biotechnological and biomedical applications. However, reliance on native host promoters for the construction of circuit elements, such as logic gates, can make the implementation of predictable, independently functioning circuits difficult. In contrast, T7 promoters offer a simple orthogonal expression system for use in a variety of cellular backgrounds and even in cell-free systems. Here we develop a T7 promoter system that can be regulated by two different transcriptional repressors for the construction of a logic gate that functions in cells and in cell-free systems. We first present LacI repressible T7lacO promoters that are regulated from a distal lac operator site for repression. We next explore the positioning of a tet operator site within the T7lacO framework to create T7 promoters that respond to tet and lac repressors and realize an IMPLIES gate. Finally, we demonstrate that these dual input sensitive promoters function in an E. coli cell-free protein expression system. Our results expand the utility of T7 promoters in cell based as well as cell-free synthetic biology applications.
PLoS ONE 10/2013; 8(10):e78442. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Realizing the potential of cell free systems will require development of ligand sensitive gene promoters that control gene expression in response to a ligand of interest. Here, we describe an approach to designing ligand sensitive transcriptional control in cell free systems that is based on the combination of a DNA aptamer that binds thrombin and the T7 bacteriophage promoter. Placement of the aptamer near the T7 promoter, and using a primarily single stranded template, results in up to a five-fold change in gene expression in a ligand concentration dependent manner. We further demonstrate that the sensitivity to thrombin concentration and the fold change in expression can be tuned by altering the position of the aptamer. The results described here pave the way for the use of DNA aptamers to achieve modular regulation of transcription in response to a wide variety of ligands in cell free systems.
[Show abstract][Hide abstract] ABSTRACT: A new synthesis approach is described that allows the direct incorporation of fluorescent labels into the volume or body of SiO2 nanoparticles. In this process, fluorescent Alexa Fluor dyes with different emission wavelengths were covalently incorporated into the SiO2 nanoparticles during their formation by the hydrolysis of tetraethoxysilane. The dye molecules were homogeneously distributed throughout the SiO2 nanoparticles. The quantum yields of the Alexa Fluor volume-labeled SiO2 nanoparticles were much higher than nanoparticles labeled using conventional organic dyes. The size of the resulting nanoparticles was controlled using microemulsion reaction media with sizes in the range of 20-100 nm and a polydispersity of <15%. In comparison with conventional surface tagged particles created by post-synthesis modification, this process maintains the physical and surface chemical properties that have the most pronounced effect on colloidal stability and interactions with their surroundings. These volume-labeled nanoparticles have proven to be extremely robust, showing excellent signal strength, negligible photobleaching, and minimal loss of functional organic components. The native or "free" surface of the volume-labeled particles can be altered to achieve a specific surface functionality without altering fluorescence. Their utility was demonstrated for visualizing the association of surface-modified fluorescent particles with cultured macrophages. Differences in particle agglomeration and cell association were clearly associated with differences in observed nanoparticle toxicity. The capacity to maintain particle fluorescence while making significant changes to surface chemistry makes these particles extremely versatile and useful for studies of particle agglomeration, uptake, and transport in environmental and biological systems.
[Show abstract][Hide abstract] ABSTRACT: Enteroaggregative Escherichia coli (EAEC) causes diarrhoea. The antibiotic of choice for treating EAEC infections is ciprofloxacin. EAEC differs from other subgroups of pathogenic E. coli by having a surface protein, dispersin, which has previously been shown to play an important role in ciprofloxacin susceptibility for EAEC model strain 042. To investigate further the role of dispersin in ciprofloxacin susceptibility, minimum inhibitory concentrations (MICs) were determined for 25 clinical isolates, including 15 with dispersin and 10 without. Dispersin-positive strains had a lower MIC than dispersin-negative strains. The mechanism of action behind this observation may be caused by dispersin (i) increasing the bacteria-antibiotic interaction or (ii) facilitating ciprofloxacin access to the intracellular target, DNA gyrase/topoisomerase. To test the role of dispersin in ciprofloxacin sensitivity, EAEC 042 as well as its isogenic mutants, dispersin mutant (042aap) and a mutant in the transporter apparatus gene aatA, believed to be involved in dispersin transport to the bacterial surface (042aatA), were utilised. As predicted, 042 had a higher sensitivity to ciprofloxacin than 042aap, but it was also found that the MIC of 042aatA was similar to 042aap. To address the question of the role of dispersin in ciprofloxacin susceptibility, the concentration of ciprofloxacin bound in biofilms of 042 and 042aap was quantified by treating bacteria with radiolabelled 2-(14)C-ciprofloxacin. The results showed that dispersin did not increase the amount of bound ciprofloxacin as a function of biomass, indicating instead that dispersin facilitates ciprofloxacin access to the intracellular target leading to increased antibiotic susceptibility.
International journal of antimicrobial agents 08/2013; · 3.03 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Bio-inspired bottom-up assembly and layer-by-layer (LbL) construction of inorganic materials from lithographically defined organic templates enables the fabrication of nanostructured systems under mild temperature and pH conditions. Such processes open the door to low-impact manufacturing and facile recycling of hybrid materials for energy, biology and information technologies. Here, templated LbL assembly of silica was achieved using a combination of electron beam lithography, chemical lift-off and aqueous solution chemistry. Nanopatterns of lines, honeycomb-lattices, and dot arrays were defined in polymer resist using electron beam lithography. Following development, exposed areas of silicon were functionalized with a vapor deposited amine-silane monolayer. Silicic acid solutions of varying pH and salt content were reacted with the patterned organic amine-functional templates. Vapor treatment and solution reaction could be repeated, allowing LbL deposition. Conditions for the silicic acid deposition had a strong effect on thickness of each layer and the morphology of the amorphous silica formed. 'Defects' in the arrays of silica nanostructures were minor and do not affect the overall organization of the layers. The bio-inspired method described here facilitates the bottom-up assembly of inorganic nanostructures defined in three-dimensions and provides a path, via LbL processing, for the construction of layered hybrid materials under mild conditions.
[Show abstract][Hide abstract] ABSTRACT: Metal and metal oxide nanoparticles are among the most commonly used nanomaterials and their potential for adversely affecting environmental systems raises concern. Complex microbial consortia underlie environmental processes, and the potential toxicity of nanoparticles to microbial systems, and the consequent impacts on trophic balances, is particularly worrisome. The diverse array of metal and metal oxides, the different sizes and shapes that can be prepared and the variety of possible surface coatings complicate assessments of toxicity. Further muddling biocidal interpretations are the diversity of microbes and their intrinsic tolerances to stresses. Here, we review a range of studies focused on nanoparticle-microbial interactions in an effort to correlate the physical-chemical properties of engineered metal and metal oxide nanoparticles to their biological response. General conclusions regarding the parent material of the nanoparticle and the nanoparticle's size and shape on potential toxicity can be made. However, the surface coating of the material, which can be altered significantly by environmental conditions, can ameliorate or promote microbial toxicity. Understanding nanoparticle transformations and how the nanoparticle surface can be designed to control toxicity represents a key area for further study. Additionally, the vast array of microbial species and the structuring of these species within communities complicate extrapolations of nanoparticle toxicity in real world settings. Ultimately, to interpret the effect and eventual fate of engineered materials in the environment, an understanding of the relationship between nanoparticle properties and responses at the molecular, cellular and community levels will be essential.
[Show abstract][Hide abstract] ABSTRACT: Droplet based microfluidic systems provide an ideal platform for partitioning and manipulating aqueous samples for analysis. Identifying stable operating conditions under which droplets are generated is challenging yet crucial for real-world applications. A novel three-dimensional microfluidic platform that facilitates the consistent generation and gelation of alginate-calcium hydrogel microbeads for microbial encapsulation, over a broad range of backing pressures, in the absence of surfactants, is described. The unique three-dimensional design of the fluidic network utilizes a height difference at the junction between the aqueous sample injection and organic carrier channels to induce droplet formation via a surface tension enhanced self-shearing mechanism. Combined within a flow-focusing geometry, under constant pressure control, this arrangement facilitates predictable generation of droplets over a much broader range of operating conditions than conventional two-dimensional systems. The impact of operating pressures and geometry on droplet gelation, aqueous and organic material flow rates, microbead size and bead generation frequency are described. The system presented provides a robust platform for encapsulating single microbes in complex mixtures into individual hydrogel beads, and provides the foundation for the development of a complete system for sorting and analyzing microbes at the single cell level.
[Show abstract][Hide abstract] ABSTRACT: To aid in the investigation of the Populus deltoides microbiome, we generated draft genome sequences for 21 Pseudomonas strains and 19 other diverse bacteria isolated from Populus deltoides roots. Genome sequences for isolates similar to Acidovorax, Bradyrhizobium, Brevibacillus, Caulobacter, Chryseobacterium, Flavobacterium, Herbaspirillum, Novosphingobium, Pantoea, Phyllobacterium, Polaromonas, Rhizobium, Sphingobium, and Variovorax were generated.
Journal of bacteriology 11/2012; 194(21):5991-3. · 2.69 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background/Question/Methods
Populus trees represents a genetically diverse, ecologically widespread riparian genus, that have potential as cellulosic feedstocks for biofuels, and contain the first tree species to have a full genome sequence. These trees are also host to a wide variety of symbiotic microbial associations within their roots and rhizosphere, thus may serve as ideal models to study the breadth and mechanisms of interactions between plants and microorganisms. However, most of our knowledge of Populus microbial associations to date comes from greenhouse and plantation-based trees; there have been no efforts to comprehensively describe microbial communities of mature natural populations of Populus. We have compared root endophyte and rhizosphere samples collected from two dozen sites within two watershed populations of Populus deltoides in Tennessee and North Carolina over multiple seasons. 454 pyrosequencing has been applied to survey and quantify the microbial community associated with P. deltoides, using primers targeting the bacterial 16S rRNA gene and the fungal 28S rRNA gene. Genetic relatedness among the Populus trees was evaluated using 20 SSR markers chosen for distribution across all 19 linkage groups of the Populus genetic map. Soil physical, chemical and nutrient status, as well as tree growth and age characteristics were also evaluated.
Root endosphere and rhizosphere communities have been found to be composed of distinct assemblages of bacteria and fungi with largely non-overlapping OTU distributions. Within these distinct endophyte and rhizosphere habitats, community structure is also influenced by soil characteristics, watershed origin and plant genotype; while observed seasonal influences have been minimal. We have isolated cultures of over a thousand bacteria and fungi from these environments representing most of the dominant community members insitu. Many of these isolates show distinct growth-promoting phenotypes with Populus. These findings indicate that the characteristics of the Populus root/soil environment may represent a relatively strong selective force in shaping endophyte and rhizosphere microbial communities and their functions may have great importance upon the success of Populus sp. Forthcoming work in collaboration with JGI will explore more in depth the genetic basis of these associations within a common garden populations of P. trichocarpa containing over >1000 resequenced variants.
[Show abstract][Hide abstract] ABSTRACT: Shewanella oneidensis is a metal reducing bacterium, which is of interest for bioremediation and clean energy applications. S. oneidensis biofilms play a critical role in several situations such as in microbial energy harvesting devices. Here, we use a microfluidic device to quantify the effects of hydrodynamics on the biofilm morphology of S. oneidensis. For different rates of fluid flow through a complex microfluidic device, we studied the spatiotemporal dynamics of biofilms, and we quantified several morphological features such as spatial distribution, cluster formation and surface coverage. We found that hydrodynamics resulted in significant differences in biofilm dynamics. The baffles in the device created regions of low and high flow in the same device. At higher flow rates, a nonuniform biofilm develops, due to unequal advection in different regions of the microchannel. However, at lower flow rates, a more uniform biofilm evolved. This depicts competition between adhesion events, growth and fluid advection. Atomic force microscopy (AFM) revealed that higher production of extra-cellular polymeric substances (EPS) occurred at higher flow velocities.
[Show abstract][Hide abstract] ABSTRACT: Colonization of plants by nonpathogenic Pseudomonas fluorescens strains can confer enhanced defense capacity against a broad spectrum of pathogens. Few studies, however, have linked defense pathway regulation to primary metabolism and physiology. In this study, physiological data, metabolites, and transcript profiles are integrated to elucidate how molecular networks initiated at the root-microbe interface influence shoot metabolism and whole-plant performance. Experiments with Arabidopsis thaliana were performed using the newly identified P. fluorescens GM30 or P. fluorescens Pf-5 strains. Co-expression networks indicated that Pf-5 and GM30 induced a subnetwork specific to roots enriched for genes participating in RNA regulation, protein degradation, and hormonal metabolism. In contrast, only GM30 induced a subnetwork enriched for calcium signaling, sugar and nutrient signaling, and auxin metabolism, suggesting strain dependence in network architecture. In addition, one subnetwork present in shoots was enriched for genes in secondary metabolism, photosynthetic light reactions, and hormone metabolism. Metabolite analysis indicated that this network initiated changes in carbohydrate and amino acid metabolism. Consistent with this, we observed strain-specific responses in tryptophan and phenylalanine abundance. Both strains reduced host plant carbon gain and fitness, yet provided a clear fitness benefit when plants were challenged with the pathogen P. syringae DC3000.
[Show abstract][Hide abstract] ABSTRACT: Due to their unique antimicrobial properties silver nanocrystallites have garnered substantial attention and are used extensively for biomedical applications as an additive to wound dressings, surgical instruments and bone substitute materials. They are also released into unintended locations such as the environment or biosphere. Therefore it is imperative to understand the potential interactions, fate and transport of nanoparticles with environmental biotic systems. Numerous factors including the composition, size, shape, surface charge, and capping molecule of nanoparticles are known to influence cell cytotoxicity. Our results demonstrate that the physical/chemical properties of the silver nanoparticles including surface charge, differential binding and aggregation potential, which are influenced by the surface coatings, are a major determining factor in eliciting cytotoxicity and in dictating potential cellular interactions. In the present investigation, silver nanocrystallites with nearly uniform size and shape distribution but with different surface coatings, imparting overall high negativity to high positivity, were synthesized. These nanoparticles included poly(diallyldimethylammonium) chloride-Ag, biogenic-Ag, colloidal-Ag (uncoated), and oleate-Ag with zeta potentials +45 ± 5, -12 ± 2, -42 ± 5, and -45 ± 5 mV, respectively; the particles were purified and thoroughly characterized so as to avoid false cytotoxicity interpretations. A systematic investigation on the cytotoxic effects, cellular response, and membrane damage caused by these four different silver nanoparticles was carried out using multiple toxicity measurements on mouse macrophage (RAW-264.7) and lung epithelial (C-10) cell lines. Our results clearly indicate that the cytotoxicity was dependent on various factors such as surface charge and coating materials used in the synthesis, particle aggregation, and the cell-type for the different silver nanoparticles that were investigated. Poly(diallyldimethylammonium)-coated Ag nanoparticles were found to be the most toxic, followed by biogenic-Ag and oleate-Ag nanoparticles, whereas uncoated or colloidal silver nanoparticles were found to be the least toxic to both macrophage and lung epithelial cells. Also, based on our cytotoxicity interpretations, lung epithelial cells were found to be more resistant to the silver nanoparticles than the macrophage cells, regardless of the surface coating.