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Common treatment for venous leg wounds includes topical wound dressings with compression. At each dressing change, wounds are debrided and washed; however, the effect of the washing procedure on the wound microbiome has not been studied. We hypothesized that wound washing may alter the wound microbiome. To characterize microbiome changes with respect to wound washing, swabs from 11 patients with chronic wounds were sampled before and after washing, and patient microbiomes were characterized using 16S rRNA sequencing and culturing. Microbiomes across patient samples prior to washing were typically polymicrobial but varied in the number and type of bacterial genera present. Proteus and Pseudomonas were the dominant genera in the study. We found that washing does not consistently change microbiome diversity but does cause consistent changes in microbiome composition. Specifically, washing caused a decrease in the relative abundance of the most highly represented genera in each patient cluster. The finding that venous leg ulcer wound washing, a standard of care therapy, can induce changes in the wound microbiome is novel and could be potentially informative for future guided therapy strategies.

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.

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.

Premise Plant endophytic bacterial strains can influence plant traits such as leaf area and root length. Yet, the influence of more complex bacterial communities in regulating overall plant phenotype is less explored. Here, in two complementary experiments, we tested whether we can predict plant phenotype response to changes in microbial community composition. Methods In the first study, we inoculated a single genotype of Populus deltoides with individual root endophytic bacteria and measured plant phenotype. Next, data from this single inoculation were used to predict phenotypic traits after mixed three-strain community inoculations, which we tested in the second experiment. Results By itself, each bacterial endophyte significantly but weakly altered plant phenotype relative to noninoculated plants. In a mixture, bacterial strain Burkholderia BT03, constituted at least 98% of community relative abundance. Yet, plant resource allocation and tissue nutrient concentrations were disproportionately influenced by Pseudomonas sp. GM17, GM30, and GM41. We found a 10% increase in leaf mass fraction and an 11% decrease in root mass fraction when replacing Pseudomonas GM17 with GM41 in communities containing both Pseudomonas GM30 and Burkholderia BT03. Conclusions Our results indicate that interactions among endophytic bacteria may drive plant phenotype over the contribution of each strain individually. Additionally, we have shown that low-abundance strains contribute to plant phenotype challenging the assumption that the dominant strains will drive plant function.

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.

Bacterially produced volatile organic compounds (VOCs) can modify growth patterns of eukaryotic hosts and competing/cohabiting microbes. These compounds have been implicated in skin disorders and attraction of biting pests. Current methods to detect and characterize VOCs from microbial cultures can be laborious and low-throughput, making it difficult to understand the behavior of microbial populations. In this work we present an efficient method employing gas chromatography/mass spectrometry with autosampling to characterize VOC profiles from solid-phase bacterial cultures. We compare this method to complementary plate-based assays and measure the effects of growth media and incubation temperature on the VOC profiles from a well-studied Pseudomonas aeruginosa PAO1 system. We observe that P. aeruginosa produces longer chain VOCs, such as 2-undecanone and 2-undecanol in higher amounts at 37 °C than 30 °C. We demonstrate the throughput of this method by studying VOC profiles from a representative collection of skin bacterial isolates under three parallel growth conditions. We observe differential production of various aldehydes and ketones depending on bacterial strain. This generalizable method will support screening of bacterial populations in a variety of research areas.

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.

In order to persist in nature, RNA viruses have evolved strategies to grow in diverse host environments. To better understand how such strategies might work, we used qRT-PCR to measure viral RNA species during cellular infections by a model RNA virus, vesicular stomatitis virus (VSV). Absolute levels of the VSV major transcript and genome were measured for infections in BHK and PC3 cells, across different multiplicities of infection (MOI 1, 10, 100), in the absence or presence of protein synthesis, as well as in cells in an interferon-activated anti-viral state. While viral genome replication was delayed in more resistant host cells, kinetic modeling of these data revealed a simple linear relationship between the mRNA production rate and genome levels under all tested conditions. These results indicate that while viral transcription and genome replication both depend on the availability of the viral RNA-dependent RNA polymerase and host cellular resources, transcription proceeds without apparent limits on these resources. Biotechnol. Bioeng. 2015;112: 1655–1662. © 2015 Wiley Periodicals, Inc.

Peatlands harbour more than one-third of terrestrial carbon leading to the argument that the bryophytes, as major components of peatland ecosystems, store more organic carbon in soils than any other collective plant taxa. Plants of the genus Sphagnum are important components of peatland ecosystems and are potentially vulnerable to changing climatic conditions. However, the response of Sphagnum to rising temperatures, elevated CO2 and shifts in local hydrology have yet to be fully characterized. In this review, we examine Sphagnum biology and ecology and explore the role of this group of keystone species and its associated microbiome in carbon and nitrogen cycling using literature review and model simulations. Several issues are highlighted including the consequences of a variable environment on plant-microbiome interactions, uncertainty associated with CO2 diffusion resistances and the relationship between fixed N and that partitioned to the photosynthetic apparatus. We note that the Sphagnum fallax genome is currently being sequenced and outline potential applications of population-level genomics and corresponding plant photosynthesis and microbial metabolic modelling techniques. We highlight Sphagnum as a model organism to explore ecosystem response to a changing climate and to define the role that Sphagnum can play at the intersection of physiology, genetics and functional genomics.

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., 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.

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 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.

Populations of RNA viruses can spontaneously produce variants that differ in genome size, sequence, and biological activity. Defective variants that lack essential genes can nevertheless reproduce by coinfecting cells with viable virus, a process that interferes with virus growth. How such defective interfering particles (DIPs) change in abundance and biological activity within a virus population is not known. Here, a prototype RNA virus, vesicular stomatitis virus (VSV), was cultured for three passages on BHK host cells, and passages were subjected to Illumina sequencing. Reads from the initial population, when aligned to the full-length viral sequence (11,161 nucleotides [nt]), distributed uniformly across the genome. However, during passages two plateaus in read counts appeared toward the 5′ end of the negative-sense viral genome. Analysis by normalization and a simple sliding-window approach revealed plateau boundaries that suggested the emergence and enrichment of at least two truncated species having medium (∼5,900 nt) and short (∼4,000 nt) genomes. Relative measures of full-length and truncated species based on read counts were validated by quantitative reverse transcription-PCR (qRT-PCR). Limit-of-detection analysis suggests that deep sequencing can be more sensitive than complementary measures for detecting and quantifying defective particles in a population. Further, particle counts from transmission electron microscopy, coupled with infectivity assays, linked the rise in smaller genomes with an increase in truncated particles and interference activity. In summary, variation in deep sequencing coverage simultaneously shows the size, location, and relative level of truncated-genome variants, revealing a level of population heterogeneity that is masked by other measures of viral genomes and particles. IMPORTANCE We show how deep sequencing can be used to characterize the emergence, diversity, and relative abundance of truncated virus variants in virus populations. Adaptation of this approach to natural isolates may elucidate factors that influence the stability and persistence of virus populations in nature.

Plant traits, such as root and leaf area, influence how plants interact with their environment and the diverse microbiota living within plants can influence plant morphology and physiology. Here, we explored how three bacterial strains isolated from thePopulusroot microbiome, influenced plant phenotype. We chose three bacterial strains that differed in predicted metabolic capabilities, plant hormone production and metabolism, and secondary metabolite synthesis. We inoculated each bacterial strain on a single genotype ofPopulus trichocarpaand measured the response of plant growth related traits (root:shoot, biomass production, root and leaf growth rates) and physiological traits (chlorophyll content, net photosynthesis, net photosynthesis at saturating light–Asat, and saturating CO2–Amax). Overall, we found that bacterial root endophyte infection increased root growth rate up to 184% and leaf growth rate up to 137% relative to non-inoculated control plants, evidence that plants respond to bacteria by modifying morphology. However, endophyte inoculation had no influence on total plant biomass and photosynthetic traits (net photosynthesis, chlorophyll content). In sum, bacterial inoculation did not significantly increase plant carbon fixation and biomass, but their presence altered where and how carbon was being allocated in the plant host.

[This corrects the article DOI: 10.1371/journal.pone.0155080.].

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.