[Show abstract][Hide abstract] ABSTRACT: The bacterial microbiota of plants is diverse, with 1,000s of operational taxonomic units (OTUs)
associated with any individual plant. In this work, we used phenotypic analysis, comparative
genomics, and metabolic models to investigate the differences between 19 sequenced Pseudomonas
fluorescens strains. These isolates represent a single OTU and were collected from the rhizosphere
and endosphere of Populus deltoides. While no traits were exclusive to either endosphere or
rhizosphere P. fluorescens isolates, multiple pathways relevant for plant-bacterial interactions are
enriched in endosphere isolate genomes. Further, growth phenotypes such as phosphate
solubilization, protease activity, denitrification and root growth promotion are biased towards
endosphere isolates. Endosphere isolates have significantly more metabolic pathways for plant
signaling compounds and an increased metabolic range that includes utilization of energy rich
nucleotides and sugars, consistent with endosphere colonization. Rhizosphere P. fluorescens have
fewer pathways representative of plant-bacterial interactions but show metabolic bias towards
chemical substrates often found in root exudates. This work reveals the diverse functions that may
contribute to colonization of the endosphere by bacteria and are enriched among closely related
Frontiers in Microbiology 09/2015; DOI:10.3389/fmicb.2015.01118 · 3.99 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Nanomaterial based drug delivery systems allow for the independent tuning of the surface chemical and physical properties that affect their biodistribution in vivo and the therapeutic payloads that they are intended to deliver. Additionally, the added therapeutic and diagnostic value of their inherent material properties often provides extra functionality. Iron based nanomaterials with their magnetic properties and easily tailorable surface chemistry are of particular interest as model systems. In this study the core radius of the iron oxide nanoparticles (NPs) was 14.08 ± 3.92 nm while the hydrodynamic radius of the NPs, as determined by Dynamic Light Scattering (DLS), was between 90 – 110 nm. In this study, different approaches were explored to create radiolabeled NPs that are stable in solution. The NPs were functionalized with polycarboxylate or polyamine surface functional groups. Polycarboxylate functionalized NPs had a zeta potential of -35 mV and polyamine functionalized NPs had a zeta potential of +40 mV. The polycarboxylate functionalized NPs were chosen for in vivo biodistribution studies and hence were radiolabeled with 14C, with a final activity of 0.097 nCi/mg of NPs. In chronic studies, the biodistribution profile is tracked using low level radiolabeled proxies of the nanoparticles of interest. Conventionally, these radiolabeled proxies are chemically similar but not chemically identical to the non-radiolabeled NPs of interest. This study is novel as different approaches were explored to create radiolabeled NPs that are stable, possess a hydrodynamic radius of <100 nm and most importantly they exhibit an identical surface chemical functionality as their non-radiolabeled counterparts. Identical chemical functionality of the radiolabeled probes to the non-radiolabeled probes was an important consideration to generate statistically similar biodistribution data sets using multiple imaging and detection techniques. The radiolabeling approach described here is applicable to the synthesis of a large class of nanomaterials with multiple core and surface functionalities. This work combined with the biodistribution data suggests that the radiolabeling schemes carried out in this study have broad implications for use in pharmacokinetic studies for a variety of nanomaterials.
[Show abstract][Hide abstract] ABSTRACT: Biochemical separations are the heart of diagnostic assays and purification methods for biologics. On-chip miniaturization and modularization of separation procedures will enable the development of customized, portable devices for personalized health-care diagnostics and point-of-use production of treatments. In this report, we describe the design and fabrication of miniature ion exchange, size exclusion and affinity chromatography modules for on-chip clean-up of recombinantly-produced proteins. Our results demonstrate that these common separations techniques can be implemented in microfluidic modules with performance comparable to conventional approaches. We introduce embedded 3-D microfluidic interconnects for integrating micro-scale separation modules that can be arranged and reconfigured to suit a variety of fluidic operations or biochemical processes. We demonstrate the utility of the modular approach with a platform for the enrichment of enhanced green fluorescent protein (eGFP) from Escherichia coli lysate through integrated affinity and size-exclusion chromatography modules.
Lab on a Chip 02/2015; 15(8). DOI:10.1039/C5LC00094G · 6.12 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Motivation: To assess the potential of different types of sequence data combined with de novo and hybrid assembly approaches to improve existing draft genome sequences.
Results: Illumina, 454 and PacBio sequencing technologies were used to generate de novo and hybrid genome assemblies for four different bacteria, which were assessed for quality using summary statistics (e.g. number of contigs, N50) and in silico evaluation tools. Differences in predictions of multiple copies of rDNA operons for each respective bacterium were evaluated by PCR and Sanger sequencing, and then the validated results were applied as an additional criterion to rank assemblies. In general, assemblies using longer PacBio reads were better able to resolve repetitive regions. In this study, the combination of Illumina and PacBio sequence data assembled through the ALLPATHS-LG algorithm gave the best summary statistics and most accurate rDNA operon number predictions. This study will aid others looking to improve existing draft genome assemblies.
Availability and implementation: All assembly tools except CLC Genomics Workbench are freely available under GNU General Public License.
Supplementary data are available at Bioinformatics online.
[Show abstract][Hide abstract] ABSTRACT: The attachment and arrangement of microbes onto a substrate is influenced by both the biochemical and physical surface properties. In this report, we develop lectin-functionalized substrates containing patterned, three-dimensional polymeric structures of varied shapes and densities and use these to investigate the effects of topology and spatial confinement on lectin-mediated microbe immobilization. Films of poly(glycidyl methacrylate)-block-4,4-dimethyl-2-vinylazlactone (PGMA-b-PVDMA) were patterned on silicon surfaces into line arrays or square grid patterns with 5 μm wide features and varied pitch. The patterned films had three-dimensional geometries with 900 nm film thickness. After surface functionalization with wheat germ agglutinin, the size of Pseudomonas fluorescens aggregates immobilized was dependent on the pattern dimensions. Films patterned as parallel lines or square grids with a pitch of 10 μm or less led to the immobilization of individual microbes with minimal formation of aggregates. Both geometries allowed for incremental increases in aggregate size distribution with each increase in pitch. These engineered surfaces combine spatial confinement with affinity-based capture to control the extent of microbe adhesion and aggregation, and can also be used as a platform to investigate intercellular interactions and biofilm formation in microbial populations of controlled sizes.
[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. DOI:10.1371/journal.pone.0078442 · 3.23 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; 42(5). DOI:10.1016/j.ijantimicag.2013.07.007 · 4.30 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.
Lab on a Chip 12/2012; 6(4). DOI:10.1063/1.4765337 · 6.12 Impact Factor
[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. DOI:10.1128/JB.01243-12 · 2.81 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.