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Plant–microbe symbioses span a continuum from pathogenic to mutualistic, with functional consequences for both organisms in the symbiosis. In order to increase sustainable food and fuel production in the future, it is imperative that we harness these symbioses. The tree genus Populus is an excellent model system for studies examining plant–microbe interactions due to the wealth of genomic information available and the molecular tools that have been developed to manipulate Populus–microbe symbioses. In this review, we highlight how Populus can serve as a model system to explore plant–microbe interactions. Specifically, we highlight research linking Populus–microbe interactions from the gene to the ecosystem level. We explore why Populus is an excellent model for perennial plant systems, the molecular underpinnings of Populus–microbe interactions, how host genetics influence microbial community composition, and how microbial communities vary at fine spatial scales and between Populus spp. Furthermore, we explore how changes in the microbiome may affect ecosystem-level functions in managed and natural ecosystems. Understanding and manipulating these interactions in Populus has the potential to improve plant health and affect ecosystem sustainability and processes because Populus trees function as foundational species in many natural ecosystems and are also deployed in managed ecosystems for various agroforestry applications. [Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Background Microbe-microbe interactions between members of the plant rhizosphere are important but remain poorly understood. A more comprehensive understanding of the molecular mechanisms used by microbes to cooperate, compete, and persist has been challenging because of the complexity of natural ecosystems and the limited control over environmental factors. One strategy to address this challenge relies on studying complexity in a progressive manner, by first building a detailed understanding of relatively simple subsets of the community and then achieving high predictive power through combining different building blocks (e.g., hosts, community members) for different environments. Herein, we coupled this reductionist approach with high-resolution mass spectrometry-based metaproteomics to study molecular mechanisms driving community assembly, adaptation, and functionality for a defined community of ten taxonomically diverse bacterial members of Populus deltoides rhizosphere co-cultured either in a complex or defined medium. Results Metaproteomics showed this defined community assembled into distinct microbiomes based on growth media that eventually exhibit composition and functional stability over time. The community grown in two different media showed variation in composition, yet both were dominated by only a few microbial strains. Proteome-wide interrogation provided detailed insights into the functional behavior of each dominant member as they adjust to changing community compositions and environments. The emergence and persistence of select microbes in these communities were driven by specialization in strategies including motility, antibiotic production, altered metabolism, and dormancy. Protein-level interrogation identified post-translational modifications that provided additional insights into regulatory mechanisms influencing microbial adaptation in the changing environments. Conclusions This study provides high-resolution proteome-level insights into our understanding of microbe-microbe interactions and highlights specialized biological processes carried out by specific members of assembled microbiomes to compete and persist in changing environmental conditions. Emergent properties observed in these lower complexity communities can then be re-evaluated as more complex systems are studied and, when a particular property becomes less relevant, higher-order interactions can be identified.

Small secreted proteins (SSPs) are less than 250 amino acids in length and are actively transported out of cells through conventional protein secretion pathways or unconventional protein secretion pathways. In plants, SSPs have been found to play important roles in various processes, including plant growth and development, plant response to abiotic and biotic stresses, and beneficial plant–microbe interactions. Over the past 10 years, substantial progress has been made in the identification and functional characterization of SSPs in several plant species relevant to agriculture, bioenergy, and horticulture. Yet, there are potentially a lot of SSPs that have not been discovered in plant genomes, which is largely due to limitations of existing computational algorithms. Recent advances in genomics, transcriptomics, and proteomics research, as well as the development of new computational algorithms based on machine learning, provide unprecedented capabilities for genome-wide discovery of novel SSPs in plants. In this review, we summarize known SSPs and their functions in various plant species. Then we provide an update on the computational and experimental approaches that can be used to discover new SSPs. Finally, we discuss strategies for elucidating the biological functions of SSPs in plants.

The integral role of microbial communities in plant growth and health is now widely recognized, and, increasingly, the constituents of the microbiome are being defined. While phylogenetic surveys have revealed the taxa present in a microbiome and show that this composition can depend on, and respond to, environmental perturbations, the challenge shifts to determining why particular microbes are selected and how they collectively function in concert with their host. In this study, we targeted the isolation of representative bacterial strains from environmental samples of Populus roots using a direct plating approach and compared them to amplicon-based sequencing analysis of root samples. The resulting culture collection contains 3,211 unique isolates representing 10 classes, 18 orders, 45 families, and 120 genera from 6 phyla, based on 16S rRNA gene sequence analysis. The collection accounts for ∼50% of the natural community of plant-associated bacteria as determined by phylogenetic analysis. Additionally, a representative set of 553 had their genomes sequenced to facilitate functional analyses. The top sequence variants in the amplicon data, identified as Pseudomonas, had multiple representatives within the culture collection. We then explore a simplified microbiome, comprised of 10 strains representing abundant taxa from environmental samples, and tested for their ability to reproducibly colonize Populus root tissue. The 10-member simplified community was able to reproducibly colonize on Populus roots after 21 days, with some taxa found in surface-sterilized aboveground tissue. This study presents a comprehensive collection of bacteria isolated from Populus for use in exploring microbial function and community inoculation experiments to understand basic concepts of plant and environmental selection. IMPORTANCE Microbial communities play an integral role in the health and survival of their plant hosts. Many studies have identified key members in these communities and led to the use of synthetic communities for elucidating their function; however, these studies are limited by the available cultured bacterial representatives. Here, we present a bacterial culture collection comprising 3,211 isolates that is representative of the root community of Populus. We then demonstrate the ability to examine underlying microbe-microbe interactions using a synthetic community approach. This culture collection will allow for the greater exploration of the microbial community function through targeted experimentation and manipulation.

Bacterial membranes are complex mixtures of lipids and proteins, the combination of which confers biophysical properties that allows cells to respond to environmental conditions. Carotenoids are sterol analogs that are important for regulating membrane dynamics. The membrane of Pantoea sp. YR343 is characterized by the presence of the carotenoid zeaxanthin, and a carotenoid-deficient mutant, ΔcrtB, displays defects in root colonization, reduced secretion of indole-3-acetic acid, and defects in biofilm formation. Here we demonstrate that the loss of carotenoids results in changes to the membrane lipid composition in Pantoea sp. YR343, including increased amounts of unsaturated fatty acids in the ΔcrtB mutant membranes. These mutant cells displayed less fluid membranes in comparison to wild type cells as measured by fluorescence anisotropy of whole cells. Studies with artificial systems, however, have shown that carotenoids impart membrane rigidifying properties. Thus, we examined membrane fluidity using spheroplasts and vesicles composed of lipids extracted from either wild type or mutant cells. Interestingly, with the removal of the cell wall and membrane proteins, ΔcrtB vesicles were more fluid than vesicles made from lipids extracted from wild type cells. In addition, carotenoids appeared to stabilize membrane fluidity during rapidly changing temperatures. Taken together, these results suggest that Pantoea sp. YR343 compensates for the loss of carotenoids by changing lipid composition, which together with membrane proteins, results in reduced membrane fluidity. These changes may influence the abundance or function of membrane proteins that are responsible for the physiological changes observed in the ΔcrtB mutant cells.

Graphical abstract, Microbial communities colonize plant tissues and contribute to host function. How these communities form and how individual members contribute to shaping the microbial community are not well understood. Synthetic microbial communities, where defined individual isolates are combined, can serve as valuable model systems for uncovering the organizational principles of communities. Using genome-defined organisms, systematic analysis by computationally-based network reconstruction can lead to mechanistic insights and the metabolic interactions between species. In this study, 10 bacterial strains isolated from the Populus deltoides rhizosphere were combined and passaged in two different media environments to form stable microbial communities. The membership and relative abundances of the strains stabilized after around 5 growth cycles and resulted in just a few dominant strains that depended on the medium. To unravel the underlying metabolic interactions, flux balance analysis was used to model microbial growth and identify potential metabolic exchanges involved in shaping the microbial communities. These analyses were complemented by growth curves of the individual isolates, pairwise interaction screens, and metaproteomics of the community. A fast growth rate is identified as one factor that can provide an advantage for maintaining presence in the community. Final community selection can also depend on selective antagonistic relationships and metabolic exchanges. Revealing the mechanisms of interaction among plant-associated microorganisms provides insights into strategies for engineering microbial communities that can potentially increase plant growth and disease resistance. Further, deciphering the membership and metabolic potentials of a bacterial community will enable the design of synthetic communities with desired biological functions.

Membrane organization plays an important role in signaling, transport, and defense. In eukaryotes, the stability, organization, and function of membrane proteins are influenced by certain lipids and sterols, such as cholesterol. Bacteria lack cholesterol, but carotenoids and hopanoids are predicted to play a similar role in modulating membrane properties. We have previously shown that the loss of carotenoids in the plant-associated bacteria Pantoea sp. YR343 results in changes to membrane biophysical properties and leads to physiological changes, including increased sensitivity to reactive oxygen species, reduced indole-3-acetic acid secretion, reduced biofilm and pellicle formation, and reduced plant colonization. Here, using whole cell and membrane proteomics, we show that the deletion of carotenoid production in Pantoea sp. YR343 results in altered membrane protein distribution and abundance. Moreover, we observe significant differences in the protein composition of detergent-resistant membrane fractions from wildtype and mutant cells, consistent with the prediction that carotenoids play a role in organizing membrane microdomains. These data provide new insights into the function of carotenoids in bacterial membrane organization and identify cellular functions that are affected by the loss of carotenoids.

We identified two poplar (Populus sp.)-associated microbes, the fungus, Mortierella elongata strain AG77, and the bacterium, Burkholderia strain BT03, that mutually promote each other's growth. Using culture assays in concert with a novel microfluidic device to generate time-lapse videos, we found growth specific media differing in pH and pre-conditioned by microbial growth led to increased fungal and bacterial growth rates. Coupling microfluidics and comparative metabolomics data results indicated that observed microbial growth stimulation involves metabolic exchange during two ordered events. The first is an emission of fungal metabolites, including organic acids used or modified by bacteria. A second signal of unknown nature is produced by bacteria which increases fungal growth rates. We find this symbiosis is initiated in part by metabolic exchange involving fungal organic acids.

Bacterial endophytes that colonize Populus trees contribute to nutrient acquisition, prime immunity responses, and directly or indirectly increase both above- and below-ground biomasses. Endophytes are embedded within plant material, so physical separation and isolation are difficult tasks. Application of culture-independent methods, such as metagenome or bacterial transcriptome sequencing, has been limited due to the predominance of DNA from the plant biomass. Here, we describe a modified differential and density gradient centrifugation-based protocol for the separation of endophytic bacteria from Populus roots. This protocol achieved substantial reduction in contaminating plant DNA, allowed enrichment of endophytic bacteria away from the plant material, and enabled single-cell genomics analysis. Four single-cell genomes were selected for whole-genome amplification based on their rarity in the microbiome (potentially uncultured taxa) as well as their inferred abilities to form associations with plants. Bioinformatics analyses, including assembly, contamination removal, and completeness estimation, were performed to obtain single-amplified genomes (SAGs) of organisms from the phyla Armatimonadetes , Verrucomicrobia , and Planctomycetes , which were unrepresented in our previous cultivation efforts. Comparative genomic analysis revealed unique characteristics of each SAG that could facilitate future cultivation efforts for these bacteria. IMPORTANCE Plant roots harbor a diverse collection of microbes that live within host tissues. To gain a comprehensive understanding of microbial adaptations to this endophytic lifestyle from strains that cannot be cultivated, it is necessary to separate bacterial cells from the predominance of plant tissue. This study provides a valuable approach for the separation and isolation of endophytic bacteria from plant root tissue. Isolated live bacteria provide material for microbiome sequencing, single-cell genomics, and analyses of genomes of uncultured bacteria to provide genomics information that will facilitate future cultivation attempts.

The Pseudomonas genus contains a metabolically versatile group of organisms that are known to occupy numerous ecological niches, including the rhizosphere and endosphere of many plants. Their diversity influences the phylogenetic diversity and heterogeneity of these communities. On the basis of average amino acid identity, comparative genome analysis of >1,000 Pseudomonas genomes, including 21 Pseudomonas strains isolated from the roots of native Populus deltoides (eastern cottonwood) trees resulted in consistent and robust genomic clusters with phylogenetic homogeneity. All Pseudomonas aeruginosa genomes clustered together, and these were clearly distinct from other Pseudomonas species groups on the basis of pangenome and core genome analyses. In contrast, the genomes of Pseudomonas fluorescens were organized into 20 distinct genomic clusters, representing enormous diversity and heterogeneity. Most of our 21 Populus -associated isolates formed three distinct subgroups within the major P. fluorescens group, supported by pathway profile analysis, while two isolates were more closely related to Pseudomonas chlororaphis and Pseudomonas putida . Genes specific to Populus -associated subgroups were identified. Genes specific to subgroup 1 include several sensory systems that act in two-component signal transduction, a TonB-dependent receptor, and a phosphorelay sensor. Genes specific to subgroup 2 contain hypothetical genes, and genes specific to subgroup 3 were annotated with hydrolase activity. This study justifies the need to sequence multiple isolates, especially from P. fluorescens , which displays the most genetic variation, in order to study functional capabilities from a pangenomic perspective. This information will prove useful when choosing Pseudomonas strains for use to promote growth and increase disease resistance in plants.

The plant root microbiome is one of the most diverse and abundant biological communities known. Plant-associated bacteria can have a profound effect on plant growth and development, and especially on protection from disease and environmental stress. These organisms are also known to be a rich source of antibiotic and antifungal drugs. In order to better understand the ways bacterial communities influence plant health, we evaluated the diversity and uniqueness of the natural product gene clusters in bacteria isolated from poplar trees. The complex molecule clusters are abundant, and the majority are unique, suggesting a great potential to discover new molecules that could not only affect plant health but also could have applications as antibiotic agents., Natural products (NPs) isolated from bacteria have dramatically advanced human society, especially in medicine and agriculture. The rapidity and ease of genome sequencing have enabled bioinformatics-guided NP discovery and characterization. As a result, NP potential and diversity within a complex community, such as the microbiome of a plant, are rapidly expanding areas of scientific exploration. Here, we assess biosynthetic diversity in the Populus microbiome by analyzing both bacterial isolate genomes and metagenome samples. We utilize the fully sequenced genomes of isolates from the Populus root microbiome to characterize a subset of organisms for NP potential. The more than 3,400 individual gene clusters identified in 339 bacterial isolates, including 173 newly sequenced organisms, were diverse across NP types and distinct from known NP clusters. The ribosomally synthesized and posttranslationally modified peptides were both widespread and divergent from previously characterized molecules. Lactones and siderophores were prevalent in the genomes, suggesting a high level of communication and pressure to compete for resources. We then consider the overall bacterial diversity and NP variety of metagenome samples compared to the sequenced isolate collection and other plant microbiomes. The sequenced collection, curated to reflect the phylogenetic diversity of the Populus microbiome, also reflects the overall NP diversity trends seen in the metagenomic samples. In our study, only about 1% of all clusters from sequenced isolates were positively matched to a previously characterized gene cluster, suggesting a great opportunity for the discovery of novel NPs involved in communication and control in the Populus root microbiome. IMPORTANCE The plant root microbiome is one of the most diverse and abundant biological communities known. Plant-associated bacteria can have a profound effect on plant growth and development, and especially on protection from disease and environmental stress. These organisms are also known to be a rich source of antibiotic and antifungal drugs. In order to better understand the ways bacterial communities influence plant health, we evaluated the diversity and uniqueness of the natural product gene clusters in bacteria isolated from poplar trees. The complex molecule clusters are abundant, and the majority are unique, suggesting a great potential to discover new molecules that could not only affect plant health but also could have applications as antibiotic agents.

The use of naturally occurring antimicrobial peptides provides a promising route to selectively target pathogenic agents and to shape microbiome structure. Lantibiotics, such as duramycin, are one class of bacterially produced peptidic natural products that can selectively inhibit the growth of other bacteria. However, despite longstanding characterization efforts, the microbial selectivity and mode of action of duramycin are still obscure. We describe here a suite of biological, chemical and physical characterizations that shed new light on the selective and mechanistic aspects of duramycin activity. Bacterial screening assays have been performed using duramycin and Populus-derived bacterial isolates to determine species selectivity. Lipidomic profiles of selected resistant and sensitive strains show that the sensitivity of Gram-positive bacteria depends on the presence of phosphatidylethanolamine (PE) in the cell membrane. Further the surface and interface morphology were studied by high resolution Atomic Force Microscopy (AFM) and showed a progression of cellular changes in the cell envelope after treatment with duramycin for the susceptible bacterial strains. Together, these molecular and cellular level analyses provide insight into duramycin’s mode of action and a better understanding of its selectivity.

Adverse growth conditions can lead to decreased plant growth, productivity, and survival, resulting in poor yields or failure of crops and biofeedstocks. In some cases, the microbial community associated with plants has been shown to alleviate plant stress and increase plant growth under suboptimal growing conditions. A systematic understanding of how the microbial community changes under these conditions is required to understand the contribution of the microbiome to water utilization, nutrient uptake, and ultimately yield. Using a microbiome inoculation strategy, we studied how the belowground microbiome of Populus deltoides changes in response to diverse environmental conditions, including water limitation, light limitation (shading), and metal toxicity. While plant responses to treatments in terms of growth, photosynthesis, gene expression and metabolite profiles were varied, we identified a core set of bacterial genera that change in abundance in response to host stress. The results of this study indicate substantial structure in the plant microbiome community and identify potential drivers of the phytobiome response to stress. IMPORTANCE The identification of a common “stress microbiome” indicates tightly controlled relationships between the plant host and bacterial associates and a conserved structure in bacterial communities associated with poplar trees under different growth conditions. The ability of the microbiome to buffer the plant from extreme environmental conditions coupled with the conserved stress microbiome observed in this study suggests an opportunity for future efforts aimed at predictably modulating the microbiome to optimize plant growth.

Root hairs increase root surface area for better water uptake and nutrient absorption by the plant. Because they are small in size and often obscured by their natural environment, root hair morphology and function are difficult to study and often excluded from plant research. In recent years, microfluidic platforms have offered a way to visualize root systems at high resolution without disturbing the roots during transfer to an imaging system. The microfluidic platform presented here builds on previous plant-on-a-chip research by incorporating a two-layer device to confine the Arabidopsis thaliana main root to the same optical plane as the root hairs. This design enables the quantification of root hairs on a cellular and organelle level and also prevents z-axis drifting during the addition of experimental treatments. We describe how to store the devices in a contained and hydrated environment, without the need for fluidic pumps, while maintaining a gnotobiotic environment for the seedling. After the optical imaging experiment, the device may be disassembled and used as a substrate for atomic force or scanning electron microscopy while keeping fine root structures intact.

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. Contact: brownsd@ornl.gov Supplementary information: Supplementary data are available at Bioinformatics online.

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 (042 aap ) and a mutant in the transporter apparatus gene aatA , believed to be involved in dispersin transport to the bacterial surface (042 aatA ), were utilised. As predicted, 042 had a higher sensitivity to ciprofloxacin than 042 aap , but it was also found that the MIC of 042 aatA was similar to 042 aap . To address the question of the role of dispersin in ciprofloxacin susceptibility, the concentration of ciprofloxacin bound in biofilms of 042 and 042 aap 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.

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.

Candida albicans is among the most common human fungal pathogens, causing a broad range of infections, including life-threatening systemic infections. The cell wall of C. albicans is the interface between the fungus and the innate immune system. The cell wall is composed of an outer layer enriched in mannosylated glycoproteins (mannan) and an inner layer enriched in β-(1,3)-glucan and chitin. Detection of C. albicans by Dectin-1, a C-type signaling lectin specific for β-(1,3)-glucan, is important for the innate immune system to recognize systemic fungal infections. Increased exposure of β-(1,3)-glucan to the immune system occurs when the mannan layer is altered or removed in a process called unmasking. Nanoscale changes to the cell wall during unmasking were explored in live cells with atomic force microscopy (AFM). Two mutants, the cho1 Δ/Δ and kre5 Δ/Δ mutants, were selected as representatives that exhibit modest and strong unmasking, respectively. Comparisons of the cho1 Δ/Δ and kre5 Δ/Δ mutants to the wild type reveal morphological changes in their cell walls that correlate with decreases in cell wall elasticity. In addition, AFM tips functionalized with Dectin-1 revealed that the forces of binding of Dectin-1 to all of the strains were similar, but the frequency of binding was highest for the kre5 Δ/Δ mutant, decreased for the cho1 Δ/Δ mutant, and rare for the wild type. These data show that nanoscale changes in surface topology are correlated with increased Dectin-1 adhesion and decreased cell wall elasticity. AFM, using tips functionalized with immunologically relevant molecules, can map epitopes of the cell wall and increase our understanding of pathogen recognition by the immune system.

The biological function of the plant-microbiome system is the result of contributions from the host plant and microbiome members. The Populus root microbiome is a diverse community that has high abundance of β- and γ-Proteobacteria, both classes which include multiple plant-growth promoting representatives. To understand the contribution of individual microbiome members in a community, we studied the function of a simplified community consisting of Pseudomonas and Burkholderia bacterial strains isolated from Populus hosts and inoculated on axenic Populus cutting in controlled laboratory conditions. Both strains increased lateral root formation and root hair production in Arabidopsis plate assays and are predicted to encode for different functions related to growth and plant growth promotion in Populus hosts. Inoculation individually, with either bacterial isolate, increased root growth relative to uninoculated controls, and while root area was increased in mixed inoculation, the interaction term was insignificant indicating additive effects of root phenotype. Complementary data including photosynthetic efficiency, whole-transcriptome gene expression and GC-MS metabolite expression data in individual and mixed inoculated treatments indicate that the effects of these bacterial strains are unique and additive. These results suggest that the function of a microbiome community may be predicted from the additive functions of the individual members.

The complex interactions between plants and their microbiome can have a profound effect on the health and productivity of the plant host. A better understanding of the microbial mechanisms that promote plant health and stress tolerance will enable strategies for improving the productivity of economically-important plants. Pantoea sp. YR343 is a motile, rod-shaped bacterium isolated from the roots of Populus deltoides that possesses the ability to solubilize phosphate and produce the phytohormone indole-3-acetic acid. Pantoea sp. YR343 readily colonizes plant roots and does not appear to be pathogenic when applied to the leaves or roots of selected plant hosts. To better understand the molecular mechanisms involved in plant association and rhizosphere survival by Pantoea sp. YR343, we constructed a mutant in which the crtB gene encoding phytoene synthase was deleted. Phytoene synthase is responsible for converting geranylgeranyl pyrophosphate to phytoene, an important precursor to the production of carotenoids. As predicted, the ΔcrtB mutant is defective in carotenoid production, and shows increased sensitivity to oxidative stress. Moreover, we find that the ΔcrtB mutant is impaired in biofilm formation and production of indole-3-acetic acid. Finally we demonstrate that the ΔcrtB mutant shows reduced colonization of plant roots. Taken together, these data suggest that carotenoids are important for plant association and/or rhizosphere survival in Pantoea sp. YR343.

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.

Synthetic biology offers great promise to a variety of applications through the forward engineering of biological function. Most efforts in this field have focused on employing living cells, yet cell-free approaches offer simpler and more flexible contexts. Here, we evaluate cell-free regulatory systems based on T7 promoter-driven expression by characterizing variants of TetR and LacI repressible T7 promoters in a cell-free context and examining sequence elements that determine expression efficiency. Using the resulting constructs, we then explore different approaches for composing regulatory systems, leading to the implementation of inducible negative feedback in Escherichia coli extracts and in the minimal PURE system, which consists of purified proteins necessary for transcription and translation. Despite the fact that negative feedback motifs are common and essential to many natural and engineered systems, this simple building block has not previously been implemented in a cell-free context. As a final step, we then demonstrate that the feedback systems developed using our cell-free approach can be implemented in live E. coli as well, illustrating the potential for using cell-free expression to fast track the development of live cell systems in synthetic biology. Our quantitative cell-free component characterizations and demonstration of negative feedback embody important steps on the path to harnessing biological function in a bottom-up fashion.

The Azospirillum brasilense chemotaxis-like Che1 signal transduction pathway was recently shown to modulate changes in adhesive cell surface properties that, in turn, affect cell-to-cell aggregation and flocculation behaviors rather than flagellar-mediated chemotaxis. Attachment to surfaces and root colonization may be functions related to flocculation. Here, the conditions under which A. brasilense wild-type Sp7 and che1 mutant strains attach to abiotic and biotic surfaces were examined using in vitro attachment and biofilm assays combined with atomic force microscopy and confocal microscopy. The nitrogen source available for growth is found to be a major modulator of surface attachment by A. brasilense and could be promoted in vitro by lectins, suggesting that it depends on interaction with surface-exposed residues within the extracellular matrix of cells. However, Che1-dependent signaling is shown to contribute indirectly to surface attachment, indicating that distinct mechanisms are likely underlying flocculation and attachment to surfaces in A. brasilense.

Enteroaggregative Escherichia coli (EAEC) are bacterial pathogens that cause watery diarrhoea, which is often persistent and can be inflammatory. The antibiotic ciprofloxacin is used to treat EAEC infections, but a full understanding of the antimicrobial effects of ciprofloxacin is needed for more efficient treatment of bacterial infections. In this study, it was found that sub-minimum inhibitory concentrations (sub-MICs) of ciprofloxacin had an inhibitory effect on EAEC adhesion to glass and mammalian HEp-2 cells. It was also observed that bacterial surface properties play an important role in bacterial sensitivity to ciprofloxacin. In an EAEC mutant strain where the hydrophobic positively charged surface protein dispersin was absent, sensitivity to ciprofloxacin was reduced compared with the wild-type strain. Identified here are several antimicrobial effects of ciprofloxacin at sub-MIC concentrations indicating that bacterial surface hydrophobicity affects the response to ciprofloxacin. Investigating the effects of sub-MIC doses of antibiotics on targeted bacteria could help to further our understanding of bacterial pathogenicity and elucidate future antibiotic treatment modalities.

Nanocrystallites have garnered substantial interest due to their various applications, including catalysis and medical research. Consequently important aspects of synthesis related to control of shape and size through economical and non-hazardous means are desirable. Highly efficient bioreduction-based fabrication approaches that utilize microbes and/or plant extracts are poised to meet these needs. Here we show that the γ -proteobacterium Shewanella oneidensis can reduce tetrachloroaurate (III) ions to produce discrete extracellular spherical gold nanocrystallites. The particles were homogeneously shaped with multiple size distributions and produced under ambient conditions at high yield, 88% theoretical maximum. Further characterization revealed that the particles consist of spheres in the size range of ∼2–50 nm, with an average size of 12 ± 5 nm. The nanoparticles were hydrophilic and resisted aggregation even after several months. Based on our experiments, the particles are likely fabricated by the aid of reducing agents present in the bacterial cell membrane and are capped by a detachable protein/peptide coat. Ultraviolet–visible and Fourier transform infrared spectroscopy, X-ray diffraction, energy dispersive X-ray spectra and transmission electron microscopy measurements confirmed the formation, surface characteristics and crystalline nature of the nanoparticles. The antibacterial activity of these gold nanoparticles was assessed using Gram-negative ( Escherichia coli and S. oneidensis ) and Gram-positive ( Bacillus subtilis ) bacterial species. Toxicity assessments showed that the particles were neither toxic nor inhibitory to any of these bacteria.

Interest in engineered nanostructures has risen in recent years due to their use in energy conservation strategies and biomedicine. To ensure prudent development and use of nanomaterials, the fate and effects of such engineered structures on the environment should be understood. Interactions of nanomaterials with environmental microorganisms are inevitable, but the general consequences of such interactions remain unclear, due to a lack of standard methods for assessing such interactions. Therefore, we have initiated a multianalytical approach to understand the interactions of synthesized nanoparticles with bacterial systems. These efforts are focused initially on cerium oxide nanoparticles and model bacteria in order to evaluate characterization procedures and the possible fate of such materials in the environment. The growth and viability of the Gram-negative species Escherichia coli and Shewanella oneidensis , a metal-reducing bacterium, and the Gram-positive species Bacillus subtilis were examined relative to cerium oxide particle size, growth media, pH, and dosage. A hydrothermal synthesis approach was used to prepare cerium oxide nanoparticles of defined sizes in order to eliminate complications originating from the use of organic solvents and surfactants. Bactericidal effects were determined from MIC and CFU measurements, disk diffusion tests, and live/dead assays. For E. coli and B. subtilis , clear strain- and size-dependent inhibition was observed, whereas S. oneidensis appeared to be unaffected by the particles. Transmission electron microscopy along with microarray-based transcriptional profiling was used to understand the response mechanism of the bacteria. Use of multiple analytical approaches adds confidence to toxicity assessments, while the use of different bacterial systems highlights the potential wide-ranging effects of nanomaterial interactions in the environment.

To compete in complex microbial communities, bacteria must sense environmental changes and adjust cellular functions for optimal growth. Chemotaxis-like signal transduction pathways are implicated in the regulation of multiple behaviors in response to changes in the environment, including motility patterns, exopolysaccharide production, and cell-to-cell interactions. In Azospirillum brasilense, cell surface properties, including exopolysaccharide production, are thought to play a direct role in promoting flocculation. Recently, the Che1 chemotaxis-like pathway from A. brasilense was shown to modulate flocculation, suggesting an associated modulation of cell surface properties. Using atomic force microscopy, distinct changes in the surface morphology of flocculating A. brasilense Che1 mutant strains were detected. Whereas the wild-type strain produces a smooth mucosal extracellular matrix after 24 h, the flocculating Che1 mutant strains produce distinctive extracellular fibril structures. Further analyses using flocculation inhibition, lectin-binding assays, and comparison of lipopolysaccharides profiles suggest that the extracellular matrix differs between the cheA1 and the cheY1 mutants, despite an apparent similarity in the macroscopic floc structures. Collectively, these data indicate that disruption of the Che1 pathway is correlated with distinctive changes in the extracellular matrix, which likely result from changes in surface polysaccharides structure and/or composition.

Chronic lung infections in cystic fibrosis patients are primarily caused by Pseudomonas aeruginosa. Though difficult to counteract effectively, colistin, an antimicrobial peptide, is proving useful. However, the exact mechanism of action of colistin is not fully understood. In this study, atomic force microscopy (AFM) was used to evaluate, in a liquid environment, the changes in P. aeruginosa morphology and nanomechanical properties due to exposure to colistin. The results of this work revealed that after 1 h of colistin exposure the ratio of individual bacteria to those found to be arrested in the process of division changed from 1.9 to 0.4 and the length of the cells decreased significantly. Morphologically, it was observed that the bacterial surface changed from a smooth to a wrinkled phenotype after 3 h exposure to colistin. Nanomechanically, in untreated bacteria, the cantilever indented the bacterial surface significantly more than it did after 1 h of colistin treatment (P-value = 0.015). Concurrently, after 2 h of exposure to colistin, a significant increase in the bacterial spring constant was also observed. These results indicate that the antimicrobial peptide colistin prevents bacterial proliferation by repressing cell division. We also found that treatment with colistin caused an increase in the rigidity of the bacterial cell wall while morphologically the cell surface changed from smooth to wrinkled, perhaps due to loss of lipopolysaccharides (LPS) or surface proteins.

We review previous work and present new data on the application of atomic force microscopy (AFM) to study biomineral formation in diatoms, unicellular algae that make cell walls of silica. Previous studies examined a small subset of mostly larger diatom species, identifying a prevalence of large particulate silica on the nanoscale. We survey different structures including valves, girdle bands, and elongated spines called setae, in a variety of species, and show a diversity of nano- and meso-scale silica morphologies, even on different portions of the same structure. A general trend of highly organized mesoscale silica structure on the proximal face of cell wall components was observed, with less organized structure occurring on the distal face. The highly organized structures have features suggestive of an underlying linear template, which defines the area of initial silica polymerization. Such features have not been imaged with such clarity previously, demonstrating the advantages of AFM to image small differences in surface morphology and providing new insights and confirming evidence for models of diatom silica structure formation. In addition to its imaging capability, more developed application of AFM to map locations of organic template components on the nanoscale will greatly aid in elucidating mechanisms of diatom biosilica synthesis.

The development and critical evaluation of new technologies for identifying genetic polymorphisms will rapidly accelerate the discovery and diagnosis of disease-related genes. We report a novel way for distinguishing a new class of human DNA polymorphisms, short insertion/deletion polymorphisms (indels). A sensor with cylindrical pores named channel glass in combination with tandem hybridization, which uses a 5'-fluorescent labeled stacking probe and microarray-based short allele-specific oligonucleotide (capture probe) was investigated. This methodology allows indels to be detected individually and rapidly with small quantities of target DNA. This establishes a reliable quantitative test. Approaches for simultaneously hybridizing different targets to arrayed probes, designed to detect various indels in parallel, were examined. Five markers were consistently detected in a single hybridization. Possible factors impeding the hybridization reaction process are discussed.

The bacterial microbiota of plants is diverse, with 1000s 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 toward 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 toward 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 isolates.

Bacteria colonize plant roots to form a symbiotic relationship with the plant and can play in important role in promoting plant growth. Raman spectroscopy is a useful technique to study these bacterial systems and the chemical signals they utilize to interact with the plant. We present a Raman study of Pantoea YR343 that was isolated from the rhizosphere of Populus deltoides (Eastern Cottonwood). Pantoea sp. YR343 produce yellowish carotenoid pigment that play a role in protection against UV radiation, in the anti-oxidative pathways and in membrane fluidity. Raman spectroscopy is used to non-invasively characterize the membrane bound carotenoids. The spectra collected from a mutant strain created by knocking out the crtB gene that encodes a phytoene synthase responsible for early stage of carotenoid biosynthesis, lack the carotenoid peaks. Surface Enhanced Raman Spectroscopy is being employed to detect the plant phytoharmone indoleacetic acid that is synthesized by the bacteria. This work describes our recent progress towards utilizing Raman spectroscopy as a label free, non-destructive method of studying plant-bacteria interactions in the rhizosphere.

We present a unique approach combining biological manipulation with advanced imaging tools to examine silica cell wall synthesis in the diatom Thalassiosira pseudonana. The innate capabilities of diatoms to form complex 3D silica structures on the nano- to micro-scale exceed current synthetic approaches because they use a fundamentally different formation process. Understanding the molecular details of the process requires identifying structural intermediates and correlating their formation with genes and proteins involved. This will aid in development of approaches to controllably alter structure, facilitating the use of diatoms as a direct source of nanostructured materials. In T. pseudonana, distinct silica morphologies were observed during formation of different cell wall substructures, and three different scales of structural organization were identified. At all levels, structure formation correlated with optimal design properties for the final product. These results provide a benchmark of measurements and new insights into biosilicification processes, potentially also benefiting biomimetic approaches.

The biological impact of 24-h (“chronic”) chromium(VI) [Cr(VI) or chromate] exposure on Shewanella oneidensis MR-1 was assessed by analyzing cellular morphology as well as genome-wide differential gene and protein expression profiles. Cells challenged aerobically with an initial chromate concentration of 0.3 mM in complex growth medium were compared to untreated control cells grown in the absence of chromate. At the 24-h time point at which cells were harvested for transcriptome and proteome analyses, no residual Cr(VI) was detected in the culture supernatant, thus suggesting the complete uptake and/or reduction of this metal by cells. In contrast to the untreated control cells, Cr(VI)-exposed cells formed apparently aseptate, nonmotile filaments that tended to aggregate. Transcriptome profiling and mass spectrometry-based proteomic characterization revealed that the principal molecular response to 24-h Cr(VI) exposure was the induction of prophage-related genes and their encoded products as well as a number of functionally undefined hypothetical genes that were located within the integrated phage regions of the MR-1 genome. In addition, genes with annotated functions in DNA metabolism, cell division, biosynthesis and degradation of the murein (peptidoglycan) sacculus, membrane response, and general environmental stress protection were upregulated, while genes encoding chemotaxis, motility, and transport/binding proteins were largely repressed under conditions of 24-h chromate treatment.

The preparation of RNA samples has become the rate-limiting step when performing genome-scale analyses by DNA microarrays. Methods to improve throughput of RNA isolation from tissues are needed. The effects of bead size and composition for disrupting mouse tissues have been evaluated in small centrifuge tubes and optimized for RNA production. The resulting process is inexpensive, resistant to cross-contamination, and amenable to robotic processing. After optimization, very-high-quality RNA can be produced. Comparisons between RNAs isolated by beadmilling (followed by solid-phase purification) and those by conventional isolation processes show that RNA produced by beadmilling is suitable for microarray analyses. Parallel implementation of beadmilling will enable a high-throughput tissue-to-RNA processing system for large-scale microarray analyses.

DNA microarrays have become an established tool for gene expression profiling. Construction of these microarrays using immobilized cDNAs is a common experimental strategy. However, this is extremely laborious, requiring the preparation of hundreds or thousands of cDNA probes. To minimize this initial bottleneck, we developed a comprehensive high-throughput robotic system to prepare DNA probes suitable for microarray analysis with minimal user intervention. We describe an automated system using the Multi-PROBE® Nucleic Acid Purification Workstation to provide the liquid handling and other functions needed to optimize this process. We were able to carry out fully automated plasmid cDNA isolation, PCR assay setup, and PCR purification and also to direct the characterization and tracking of DNA probes during processing. Protocols began with the initial preparation of a plasmid DNA archive of bacterial stocks in parallel 96-well plates (192 samples/run) and continued through to the dilution and reformatting of chip-ready DNA probes in 384-well format. These and other probe production procedures and additional instrument systems were used to process fully a set of mouse cDNA clones that were then validated by differential gene expression analysis.

Goal 1 of Department of Energy's Genomes to Life (GTL) program seeks to identify and characterize the complete set of protein complexes within a cell. Goal 1 forms the foundation necessary to accomplish the other objectives of the GTL program, which focus on gene regulatory networks and molecular level characterization of interactions in microbial communities. Together this information would allow cells and their components to be understood in sufficient detail to predict, test and understand the responses of a biological system to its environment. The Center for Molecular and Cellular Systems has been established to identify and characterize protein complexes using high through-put analytical technologies.A dynamic research program is being developed that supports the goals of the Center by focusing on the development new capabilities for sample preparation and complex separations, molecular level identification of the protein complexes by mass spectrometry, characterization of the complexes in living cells by imaging techniques, and bioinformatics and computational tools for the collection and interpretation of data and formation of databases and tools to allow the data to be shared by the biological community.

Within boreal and temperate forest ecosystems, the majority of trees and shrubs form beneficial relationships with mutualistic ectomycorrhizal (ECM) fungi 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 results in a new symbiotic “organ” called the ECM root tip. Little is understood concerning the metabolic reprogramming 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 reprogramming 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 produces 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 fungus–host interactions.

Enzyme immobilization onto silicon substrates has been investigated by five different coupling procedures. The methods included covalent coupling either through a metal link reagent or silane reagents containing pendant amino or epoxide linkers, an entrapment technique using a thin layer of gelatin, or an adsorption technique using poly- l -lysine. These immobilization procedures were evaluated using glucose oxidase and a simple spectrophotometric method employing Fenton’s reagent. Retention of enzyme activity and surface loading were assessed. The immobilization techniques were also evaluated by electron microscopy to characterize the evenness of the surface coatings. All of the covalent coupling procedures led to surface loadings, approaching 1 pmol mm −2 ; however, the surfaces appeared irregular on a microscopic scale. The poly- l -lysine adsorption technique provided the smoothest surface. With the exception of the entrapment technique, all immobilization procedures provided immobilized enzyme that retained >75% activity after several weeks of storage.

The overall folded (global) structure of mRNA may be critical to translation and turnover control mechanisms, but it has received little experimental attention. Presented here is a comparative analysis of the basic features of the global secondary structure of a synthetic mRNA and the same intracellular eukaryotic mRNA by dimethyl sulfate (DMS) structure probing. Synthetic MFA2 mRNA of Saccharomyces cerevisiae first was examined by using both enzymes and chemical reagents to determine single-stranded and hybridized regions; RNAs with and without a poly(A) tail were compared. A folding pattern was obtained with the aid of the mfold program package that identified the model that best satisfied the probing data. A long-range structural interaction involving the 5′ and 3′ untranslated regions and causing a juxtaposition of the ends of the RNA, was examined further by a useful technique involving oligo(dT)-cellulose chromatography and antisense oligonucleotides. DMS chemical probing of A and C nucleotides of intracellular MFA2 mRNA was then done. The modification data support a very similar intracellular structure. When low reactivity of A and C residues is found in the synthetic RNA, ≈70% of the same sites are relatively more resistant to DMS modification in vivo . A slightly higher sensitivity to DMS is found in vivo for some of the A and C nucleotides predicted to be hybridized from the synthetic structural model. With this small mRNA, the translation process and mRNA-binding proteins do not block DMS modifications, and all A and C nucleotides are modified the same or more strongly than with the synthetic RNA.

Polymerase chain reaction (PCR) assays were designed to amplify 56- and 99-base regions of the pmoA gene from Methylosinus trichosporium OB3b and Methylomicrobium albus BG8, two species of methanotrophic bacteria that are of interest for monitoring bioremediation activity. The PCR product sizes are in a mass range that is accessible to analysis by MALDI-TOF mass spectrometry. A rapid purification procedure using commercially available reversed-phase cartridges was applied prior to MALDI-TOF analysis. A small aliquot (1.5%, 1.5 microL) from a single 100-microL PCR reaction was sufficient for reliable detection. No cross-amplification products were observed when primers designed for one bacterial species were used with genomic DNA of the other species. The methodology described here has potential to allow less expensive and faster characterization of the ability of microbial populations to destroy pollutants in groundwater and soil at contaminated industrial sites.

Direct imaging with the atomic force microscope has been used to identify specific nucleotide sequences in plasmid DNA molecules. This was accomplished using EcoRI (Gln-111), a mutant of the restriction enzyme that has a 1000-fold greater binding affinity than the wild-type enzyme but with cleavage rate constants reduced by a factor of 10(4). ScaI-linearized plasmids with single (pBS+) and double (pGEM-luc and pSV-beta-galactosidase) EcoRI recognition sites were imaged, and the bound enzyme was localized to a 50- to 100-nt resolution. The high affinity for the EcoRI binding site exhibited by this mutant endonuclease, coupled with an observed low level of nonspecific binding, should prove valuable for physically mapping large DNA clones by direct atomic force microscope imaging.

Accurate monitoring and identification of Legionella species, the causative agents of Legionnaires' and other diseases, in environmental water sources is an important public health issue. Traditional culture methods often lack the sensitivity and specificity that can be attained using the polymerase chain reaction (PCR) to amplify targeted regions of the bacterial genome. Matrix-assisted laser desorption/ionization combined with time-of-flight (MALDI-TOF) mass spectrometry is shown to be useful for detection of 108- and 168-base PCR products specific to Legionella. A rapid purification aimed at removal of salts and unreacted primers is demonstrated. The addition of a synthetic DNA 20-mer to the MALDI sample facilitates aiming the laser at a favorable spot on the sample probe from which the PCR products can be detected.

The structure and function of microbial communities is deeply influenced by the physical and chemical architecture of the local microenvironment and the abundance of its community members. The complexity of this natural parameter space has made characterization of the key drivers of community development difficult. In order to facilitate these characterizations, we have developed a microwell platform designed to screen microbial growth and interactions across a wide variety of physical and initial conditions. Assembly of microbial communities into microwells was achieved using a novel biofabrication method that exploits well feature sizes for control of innoculum levels. Wells with incrementally smaller size features created populations with increasingly larger variations in inoculum levels. This allowed for reproducible growth measurement in large (20 μm diameter) wells, and screening for favorable growth conditions in small (5, 10 μm diameter) wells. We demonstrate the utility of this approach for screening and discovery using 5 μm wells to assemble P. aeruginosa colonies across a broad distribution of innoculum levels, and identify those conditions that promote the highest probability of survivial and growth under spatial confinement. Multi-member community assembly was also characterized to demonstrate the broad potential of this platform for studying the role of member abundance on microbial competition, mutualism and community succession.

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.

Rhizobium sp. strain PDO1-076 is a plant-associated bacterium isolated from Populus deltoides , and its draft genome sequence is reported.

The majority of bacteria in nature live in biofilms, where they are encased by extracellular polymeric substances (EPS) and adhere to various surfaces and interfaces. Investigating the process of biofilm formation is critical for advancing our understanding of microbes in their most common mode of living. Despite progress in characterizing the effect of various environmental factors on biofilm formation, work remains to be done in the realm of exploring the inter-relationship between hydrodynamics, microbial adhesion and biofilm growth. We investigate the impact of secondary flow structures, which are created due to semi-confined features in a microfluidic device, on biofilm formation of Shewanella oneidensis MR-1. Secondary flows are important in many natural and artificial systems, but few studies have investigated their role in biofilm formation. To direct secondary flows in the creeping flow regime, where the Reynolds number is low, we flow microbe-laden culture through microscale confinement features. We demonstrate that these confinement features can result in pronounced changes in biofilm dynamics as a function of the fluid flow rate.

A microcantilever-based biosensor is described. The enzyme glucose oxidase was immobilized on a micromachined silicon cantilever containing a gold coating, such as those used for atomic force microscopy. Specific, quantifiable deflection of the derivatized cantilevers was observed in the presence of the appropriate analyte. An analysis of the reaction energetics and the expected thermal response of the cantilever indicates that cantilever deflection is not simply a result of reaction-generated heat. This deflection appears to result from surface induced stresses. The combination of a highly specific enzyme and the microcantilever platform provides a unique approach for quantifying enzyme substrates without the complication of sample labeling.