Niche availability and competitive loss by facilitation control proliferation of bacterial strains intended for soil microbiome interventions.

Microbiome engineering - the targeted manipulation of microbial communities - is considered a promising strategy to restore ecosystems, but experimental support and mechanistic understanding are required. Here, we show that bacterial inoculants for soil microbiome engineering may fail to establish because they inadvertently facilitate growth of native resident microbiomes. By generating soil microcosms in presence or absence of standardized soil resident communities, we show how different nutrient availabilities limit outgrowth of focal bacterial inoculants (three Pseudomonads), and how this might be improved by adding an artificial, inoculant-selective nutrient niche. Through random paired interaction assays in agarose micro-beads, we demonstrate that, in addition to direct competition, inoculants lose competitiveness by facilitating growth of resident soil bacteria. Metatranscriptomics experiments with toluene as selective nutrient niche for the inoculant Pseudomonas veronii indicate that this facilitation is due to loss and uptake of excreted metabolites by resident taxa. Generation of selective nutrient niches for inoculants may help to favor their proliferation for the duration of their intended action while limiting their competitive loss.

Spatial structure, chemotaxis and quorum sensing shape bacterial biomass accumulation in complex porous media.

Biological tissues, sediments, or engineered systems are spatially structured media with a tortuous and porous structure that host the flow of fluids. Such complex environments can influence the spatial and temporal colonization patterns of bacteria by controlling the transport of individual bacterial cells, the availability of resources, and the distribution of chemical signals for communication. Yet, due to the multi-scale structure of these complex systems, it is hard to assess how different biotic and abiotic properties work together to control the accumulation of bacterial biomass. Here, we explore how flow-mediated interactions allow the gut commensal Escherichia coli to colonize a porous structure that is composed of heterogenous dead-end pores (DEPs) and connecting percolating channels, i.e. transmitting pores (TPs), mimicking the structured surface of mammalian guts. We find that in presence of flow, gradients of the quorum sensing (QS) signaling molecule autoinducer-2 (AI-2) promote E. coli chemotactic accumulation in the DEPs. In this crowded environment, the combination of growth and cell-to-cell collision favors the development of suspended bacterial aggregates. This results in hot-spots of resource consumption, which, upon resource limitation, triggers the mechanical evasion of biomass from nutrients and oxygen depleted DEPs. Our findings demonstrate that microscale medium structure and complex flow coupled with bacterial quorum sensing and chemotaxis control the heterogenous accumulation of bacterial biomass in a spatially structured environment, such as villi and crypts in the gut or in tortuous pores within soil and filters.

Changes in structure and assembly of a species-rich soil natural community with contrasting nutrient availability upon establishment of a plant-beneficial Pseudomonas in the wheat rhizosphere.

BACKGROUND: Plant-beneficial bacterial inoculants are of great interest in agriculture as they have the potential to promote plant growth and health. However, the inoculation of the rhizosphere microbiome often results in a suboptimal or transient colonization, which is due to a variety of factors that influence the fate of the inoculant. To better understand the fate of plant-beneficial inoculants in complex rhizosphere microbiomes, composed by hundreds of genotypes and multifactorial selection mechanisms, controlled studies with high-complexity soil microbiomes are needed. RESULTS: We analysed early compositional changes in a taxa-rich natural soil bacterial community under both exponential nutrient-rich and stationary nutrient-limited growth conditions (i.e. growing and stable communities, respectively) following inoculation with the plant-beneficial bacterium Pseudomonas protegens in a bulk soil or a wheat rhizosphere environment. P. protegens successfully established under all conditions tested and was more abundant in the rhizosphere of the stable community. Nutrient availability was a major factor driving microbiome composition and structure as well as the underlying assembly processes. While access to nutrients resulted in communities assembled mainly by homogeneous selection, stochastic processes dominated under the nutrient-deprived conditions. We also observed an increased rhizosphere selection effect under nutrient-limited conditions, resulting in a higher number of amplicon sequence variants (ASVs) whose relative abundance was enriched. The inoculation with P. protegens produced discrete changes, some of which involved other Pseudomonas. Direct competition between Pseudomonas strains partially failed to replicate the observed differences in the microbiome and pointed to a more complex interaction network. CONCLUSIONS: The results of this study show that nutrient availability is a major driving force of microbiome composition, structure and diversity in both the bulk soil and the wheat rhizosphere and determines the assembly processes that govern early microbiome development. The successful establishment of the inoculant was facilitated by the wheat rhizosphere and produced discrete changes among other members of the microbiome. Direct competition between Pseudomonas strains only partially explained the microbiome changes, indicating that indirect interactions or spatial distribution in the rhizosphere or soil interface may be crucial for the survival of certain bacteria. Video Abstract.

Fitness-Conditional Genes for Soil Adaptation in the Bioaugmentation Agent Pseudomonas veronii 1YdBTEX2.

Strain inoculation (bioaugmentation) is a potentially useful technology to provide microbiomes with new functionalities. However, there is limited understanding of the genetic factors contributing to successful establishment of inoculants. This work aimed to characterize the genes implicated in proliferation of the monoaromatic compound-degrading Pseudomonas veronii 1YdBTEX2 in nonsterile polluted soils. We generated two independent mutant libraries by random minitransposon-delivered marker insertion followed by deep sequencing (Tn-seq) with a total of 5.0 × 105 unique insertions. Libraries were grown in multiple successive cycles for up to 50 generations either in batch liquid medium or in two types of soil microcosms with different resident microbial content (sand or silt) in the presence of toluene. Analysis of gene insertion abundances at different time points (passed generations of metapopulation growth), in comparison to proportions at start and to in silico generated randomized insertion distributions, allowed to define ~800 essential genes common to both libraries and ~2,700 genes with conditional fitness effects in either liquid or soil (195 of which resulted in fitness gain). Conditional fitness genes largely overlapped among all growth conditions but affected approximately twice as many functions in liquid than in soil. This indicates soil to be a more promiscuous environment for mutant growth, probably because of additional nutrient availability. Commonly depleted genes covered a wide range of biological functions and metabolic pathways, such as inorganic ion transport, fatty acid metabolism, amino acid biosynthesis, or nucleotide and cofactor metabolism. Only sparse gene sets were uncovered whose insertion caused fitness decrease exclusive for soils, which were different between silt and sand. Despite detectable higher resident bacteria and potential protist predatory counts in silt, we were, therefore, unable to detect any immediately obvious candidate genes affecting P. veronii biological competitiveness. In contrast to liquid growth conditions, mutants inactivating flagella biosynthesis and motility consistently gained strong fitness advantage in soils and displayed higher growth rates than wild type. In conclusion, although many gene functions were found to be important for growth in soils, most of these are not specific as they affect growth in liquid minimal medium more in general. This indicates that P. veronii does not need major metabolic reprogramming for proliferation in soil with accessible carbon and generally favorable growth conditions. IMPORTANCE Restoring damaged microbiomes is still a formidable challenge. Classical widely adopted approaches consist of augmenting communities with pure or mixed cultures in the hope that these display their intended selected properties under in situ conditions. Ecological theory, however, dictates that introduction of a nonresident microbe is unlikely to lead to its successful proliferation in a foreign system such as a soil microbiome. In an effort to study this systematically, we used random transposon insertion scanning to identify genes and possibly, metabolic subsystems, that are crucial for growth and survival of a bacterial inoculant (Pseudomonas veronii) for targeted degradation of monoaromatic compounds in contaminated nonsterile soils. Our results indicate that although many gene functions are important for proliferation in soil, they are general factors for growth and not exclusive for soil. In other words, P. veronii is a generalist that is not a priori hindered by the soil for its proliferation and would make a good bioaugmentation candidate.

A bistable prokaryotic differentiation system underlying development of conjugative transfer competence.

The mechanisms and impact of horizontal gene transfer processes to distribute gene functions with potential adaptive benefit among prokaryotes have been well documented. In contrast, little is known about the life-style of mobile elements mediating horizontal gene transfer, whereas this is the ultimate determinant for their transfer fitness. Here, we investigate the life-style of an integrative and conjugative element (ICE) within the genus Pseudomonas that is a model for a widespread family transmitting genes for xenobiotic compound metabolism and antibiotic resistances. Previous work showed bimodal ICE activation, but by using single cell time-lapse microscopy coupled to combinations of chromosomally integrated single copy ICE promoter-driven fluorescence reporters, RNA sequencing and mutant analysis, we now describe the complete regulon leading to the arisal of differentiated dedicated transfer competent cells. The regulon encompasses at least three regulatory nodes and five (possibly six) further conserved gene clusters on the ICE that all become expressed under stationary phase conditions. Time-lapse microscopy indicated expression of two regulatory nodes (i.e., bisR and alpA-bisDC) to precede that of the other clusters. Notably, expression of all clusters except of bisR was confined to the same cell subpopulation, and was dependent on the same key ICE regulatory factors. The ICE thus only transfers from a small fraction of cells in a population, with an estimated proportion of between 1.7-4%, which express various components of a dedicated transfer competence program imposed by the ICE, and form the centerpiece of ICE conjugation. The components mediating transfer competence are widely conserved, underscoring their selected fitness for efficient transfer of this class of mobile elements.

Reproducible Propagation of Species-Rich Soil Bacterial Communities Suggests Robust Underlying Deterministic Principles of Community Formation.

Microbiomes are typically characterized by high species diversity but it is poorly understood how such system-level complexity can be generated and propagated. Here, we used soil microcosms as a model to study development of bacterial communities as a function of their starting complexity and environmental boundary conditions. Despite inherent stochastic variation in manipulating species-rich communities, both laboratory-mixed medium complexity (21 soil bacterial isolates in equal proportions) and high-diversity natural top-soil communities followed highly reproducible succession paths, maintaining 16S rRNA gene amplicon signatures prominent for known soil communities in general. Development trajectories and compositional states were different for communities propagated in soil microcosms than in liquid suspension. Compositional states were maintained over multiple renewed growth cycles but could be diverged by short-term pollutant exposure. The different but robust trajectories demonstrated that deterministic taxa-inherent characteristics underlie reproducible development and self-organized complexity of soil microbiomes within their environmental boundary conditions. Our findings also have direct implications for potential strategies to achieve controlled restoration of desertified land. IMPORTANCE There is now a great awareness of the high diversity of most environmental ("free-living") and host-associated microbiomes, but exactly how diverse microbial communities form and maintain is still highly debated. A variety of theories have been put forward, but testing them has been problematic because most studies have been based on synthetic communities that fail to accurately mimic the natural composition (i.e., the species used are typically not found together in the same environment), the diversity (usually too low to be representative), or the environmental system itself (using designs with single carbon sources or solely mixed liquid cultures). In this study, we show how species-diverse soil bacterial communities can reproducibly be generated, propagated, and maintained, either from individual isolates (21 soil bacterial strains) or from natural microbial mixtures washed from top-soil. The high replicate consistency we achieve both in terms of species compositions and developmental trajectories demonstrates the strong inherent deterministic factors driving community formation from their species composition. Generating complex soil microbiomes may provide ways for restoration of damaged soils that are prevalent on our planet.

Genome-wide gene expression changes of Pseudomonas veronii 1YdBTEX2 during bioaugmentation in polluted soils.

BACKGROUND: Bioaugmentation aims to use the capacities of specific bacterial strains inoculated into sites to enhance pollutant biodegradation. Bioaugmentation results have been mixed, which has been attributed to poor inoculant growth and survival in the field, and, consequently, moderate catalytic performance. However, our understanding of biodegradation activity mostly comes from experiments conducted under laboratory conditions, and the processes occurring during adaptation and invasion of inoculants into complex environmental microbiomes remain poorly known. The main aim of this work was thus to study the specific and different cellular reactions of an inoculant for bioaugmentation during adaptation, growth and survival in natural clean and contaminated non-sterile soils, in order to better understand factors limiting bioaugmentation. RESULTS: As inoculant we focused on the monoaromatic compound-degrading bacterium Pseudomonas veronii 1YdBTEX2. The strain proliferated in all but one soil types in presence and in absence of exogenously added toluene. RNAseq and differential genome-wide gene expression analysis illustrated both a range of common soil responses such as increased nutrient scavenging and recycling, expression of defense mechanisms, as well as environment-specific reactions, notably osmoprotection and metal homeostasis. The core metabolism of P. veronii remained remarkably constant during exponential growth irrespective of the environment, with slight changes in cofactor regeneration pathways, possibly needed for balancing defense reactions. CONCLUSIONS: P. veronii displayed a versatile global program, enabling it to adapt to a variety of soil environments in the presence and even in absence of its target pollutant toluene. Our results thus challenge the widely perceived dogma of poor survival and growth of exogenous inoculants in complex microbial ecosystems such as soil and provide a further basis to developing successful bioaugmentation strategies.

Insights into Mobile Genetic Elements of the Biocide-Degrading Bacterium Pseudomonas nitroreducens HBP-1.

The sewage sludge isolate Pseudomonas nitroreducens HBP-1 was the first bacterium known to completely degrade the fungicide 2-hydroxybiphenyl. PacBio and Illumina whole-genome sequencing revealed three circular DNA replicons: a chromosome and two plasmids. Plasmids were shown to code for putative adaptive functions such as heavy metal resistance, but with unclarified ability for self-transfer. About one-tenth of strain HBP-1's chromosomal genes are likely of recent horizontal influx, being part of genomic islands, prophages and integrative and conjugative elements (ICEs). P. nitroreducens carries two large ICEs with different functional specialization, but with homologous core structures to the well-known ICEclc of Pseudomonas knackmussii B13. The variable regions of ICEPni1 (96 kb) code for, among others, heavy metal resistances and formaldehyde detoxification, whereas those of ICEPni2 (171 kb) encodes complete meta-cleavage pathways for catabolism of 2-hydroxybiphenyl and salicylate, a protocatechuate pathway and peripheral enzymes for 4-hydroxybenzoate, ferulate, vanillin and vanillate transformation. Both ICEs transferred at frequencies of 10-6-10-8 per P. nitroreducens HBP-1 donor into Pseudomonas putida, where they integrated site specifically into tRNAGly-gene targets, as expected. Our study highlights the underlying determinants and mechanisms driving dissemination of adaptive properties allowing bacterial strains to cope with polluted environments.

Author Correction: Mechanistic insights into bacterial metabolic reprogramming from omics-integrated genome-scale models.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Mechanistic insights into bacterial metabolic reprogramming from omics-integrated genome-scale models.

Understanding the adaptive responses of individual bacterial strains is crucial for microbiome engineering approaches that introduce new functionalities into complex microbiomes, such as xenobiotic compound metabolism for soil bioremediation. Adaptation requires metabolic reprogramming of the cell, which can be captured by multi-omics, but this data remains formidably challenging to interpret and predict. Here we present a new approach that combines genome-scale metabolic modeling with transcriptomics and exometabolomics, both of which are common tools for studying dynamic population behavior. As a realistic demonstration, we developed a genome-scale model of Pseudomonas veronii 1YdBTEX2, a candidate bioaugmentation agent for accelerated metabolism of mono-aromatic compounds in soil microbiomes, while simultaneously collecting experimental data of P. veronii metabolism during growth phase transitions. Predictions of the P. veronii growth rates and specific metabolic processes from the integrated model closely matched experimental observations. We conclude that integrative and network-based analysis can help build predictive models that accurately capture bacterial adaptation responses. Further development and testing of such models may considerably improve the successful establishment of bacterial inoculants in more complex systems.

Computational redesign of the Escherichia coli ribose-binding protein ligand binding pocket for 1,3-cyclohexanediol and cyclohexanol.

Bacterial periplasmic-binding proteins have been acclaimed as general biosensing platform, but their range of natural ligands is too limited for optimal development of chemical compound detection. Computational redesign of the ligand-binding pocket of periplasmic-binding proteins may yield variants with new properties, but, despite earlier claims, genuine changes of specificity to non-natural ligands have so far not been achieved. In order to better understand the reasons of such limited success, we revisited here the Escherichia coli RbsB ribose-binding protein, aiming to achieve perceptible transition from ribose to structurally related chemical ligands 1,3-cyclohexanediol and cyclohexanol. Combinations of mutations were computationally predicted for nine residues in the RbsB binding pocket, then synthesized and tested in an E. coli reporter chassis. Two million variants were screened in a microcolony-in-bead fluorescence-assisted sorting procedure, which yielded six mutants no longer responsive to ribose but with 1.2-1.5 times induction in presence of 1 mM 1,3-cyclohexanediol, one of which responded to cyclohexanol as well. Isothermal microcalorimetry confirmed 1,3-cyclohexanediol binding, although only two mutant proteins were sufficiently stable upon purification. Circular dichroism spectroscopy indicated discernable structural differences between these two mutant proteins and wild-type RbsB. This and further quantification of periplasmic-space abundance suggested most mutants to be prone to misfolding and/or with defects in translocation compared to wild-type. Our results thus affirm that computational design and library screening can yield RbsB mutants with recognition of non-natural but structurally similar ligands. The inherent arisal of protein instability or misfolding concomitant with designed altered ligand-binding pockets should be overcome by new experimental strategies or by improved future protein design algorithms.

Genomic changes underlying host specialization in the bee gut symbiont Lactobacillus Firm5.

Bacteria that engage in long-standing associations with particular hosts are expected to evolve host-specific adaptations that limit their capacity to thrive in other environments. Consistent with this, many gut symbionts seem to have a limited host range, based on community profiling and phylogenomics. However, few studies have experimentally investigated host specialization of gut symbionts and the underlying mechanisms have largely remained elusive. Here, we studied host specialization of a dominant gut symbiont of social bees, Lactobacillus Firm5. We show that Firm5 strains isolated from honey bees and bumble bees separate into deep-branching host-specific phylogenetic lineages. Despite their divergent evolution, colonization experiments show that bumble bee strains are capable of colonizing the honey bee gut. However, they were less successful than honey bee strains, and competition with honey bee strains completely abolished their colonization. In contrast, honey bee strains of divergent phylogenetic lineages were able to coexist within individual bees. This suggests that both host selection and interbacterial competition play important roles in host specialization. Using comparative genomics of 27 Firm5 isolates, we found that the genomes of honey bee strains harbour more carbohydrate-related functions than bumble bee strains, possibly providing a competitive advantage in the honey bee gut. Remarkably, most of the genes encoding carbohydrate-related functions were not conserved among the honey bee strains, which suggests that honey bees can support a metabolically more diverse community of Firm5 strains than bumble bees. These findings advance our understanding of the genomic changes underlying host specialization.

Physiological and transcriptome changes induced by Pseudomonas putida acquisition of an integrative and conjugative element.

Integrative and conjugative elements (ICEs) comprise ubiquitous large mobile regions in prokaryotic chromosomes that transmit vertically to daughter cells and transfer horizontally to distantly related lineages. Their evolutionary success originates in maximized combined ICE-host fitness trade-offs, but how the ICE impacts on the host metabolism and physiology is poorly understood. Here we investigate global changes in the host genetic network and physiology of Pseudomonas putida with or without an integrated ICEclc, a model ICE widely distributed in proteobacterial genomes. Genome-wide gene expression differences were analyzed by RNA-seq using exponentially growing or stationary phase-restimulated cultures on 3-chlorobenzoate, an aromatic compound metabolizable thanks to specific ICEclc-located genes. We found that the presence of ICEclc imposes a variety of changes in global pathways such as cell cycle and amino acid metabolism, which were more numerous in stationary-restimulated than exponential phase cells. Unexpectedly, ICEclc stimulates cellular motility and leads to more rapid growth on 3-chlorobenzoate than cells carrying only the integrated clc genes. ICEclc also concomitantly activates the P. putida Pspu28-prophage, but this in itself did not provoke measurable fitness effects. ICEclc thus interferes in a number of cellular pathways, inducing both direct benefits as well as indirect costs in P. putida.

Complete alanine scanning of the Escherichia coli RbsB ribose binding protein reveals residues important for chemoreceptor signaling and periplasmic abundance.

The Escherichia coli RbsB ribose binding protein has been used as a scaffold for predicting new ligand binding functions through in silico modeling, yet with limited success and reproducibility. In order to possibly improve the success of predictive modeling on RbsB, we study here the influence of individual residues on RbsB-mediated signaling in a near complete library of alanine-substituted RbsB mutants. Among a total of 232 tested mutants, we found 10 which no longer activated GFPmut2 reporter expression in E. coli from a ribose-RbsB hybrid receptor signaling chain, and 13 with significantly lower GFPmut2 induction than wild-type. Quantitative mass spectrometry abundance measurements of 25 mutants and wild-type RbsB in periplasmic space showed four categories of effects. Some (such as D89A) seem correctly produced and translocated but fail to be induced with ribose. Others (such as N190A) show lower induction probably as a result of less efficient production, folding and translocation. The third (such as N41A or K29A) have defects in both induction and abundance. The fourth category consists of semi-constitutive mutants with increased periplasmic abundance but maintenance of ribose induction. Our data show how RbsB modeling should include ligand-binding as well as folding, translocation and receptor binding.

Draft Genome Sequence of Microbacterium foliorum Strain 122 Isolated from a Plant Growing in a Chronically Hydrocarbon-Contaminated Site.

Microbacterium foliorum strain 122 is a bacterial endophyte isolated from a Dactylis glomerata plant growing in a natural oil seep soil located in Oil Springs, Ontario, Canada. We present here a draft genome sequence of an endophytic strain that has promising potential in hydrocarbon degradation and plant growth promotion.

Draft Genome Sequence of Plantibacterflavus Strain 251 Isolated from a Plant Growing in a Chronically Hydrocarbon-Contaminated Site.

Plantibacter flavus isolate 251 is a bacterial endophyte isolated from an Achillea millefolium plant growing in a natural oil seep soil located in Oil Springs, Ontario, Canada. We present here a draft genome sequence of an infrequently reported genus Plantibacter, highlighting an endophytic lifestyle and biotechnological potential.

The Genome of the Toluene-Degrading Pseudomonas veronii Strain 1YdBTEX2 and Its Differential Gene Expression in Contaminated Sand.

The natural restoration of soils polluted by aromatic hydrocarbons such as benzene, toluene, ethylbenzene and m- and p-xylene (BTEX) may be accelerated by inoculation of specific biodegraders (bioaugmentation). Bioaugmentation mainly involves introducing bacteria that deploy their metabolic properties and adaptation potential to survive and propagate in the contaminated environment by degrading the pollutant. In order to better understand the adaptive response of cells during a transition to contaminated material, we analyzed here the genome and short-term (1 h) changes in genome-wide gene expression of the BTEX-degrading bacterium Pseudomonas veronii 1YdBTEX2 in non-sterile soil and liquid medium, both in presence or absence of toluene. We obtained a gapless genome sequence of P. veronii 1YdBTEX2 covering three individual replicons with a total size of 8 Mb, two of which are largely unrelated to current known bacterial replicons. One-hour exposure to toluene, both in soil and liquid, triggered massive transcription (up to 208-fold induction) of multiple gene clusters, such as toluene degradation pathway(s), chemotaxis and toluene efflux pumps. This clearly underlines their key role in the adaptive response to toluene. In comparison to liquid medium, cells in soil drastically changed expression of genes involved in membrane functioning (e.g., lipid composition, lipid metabolism, cell fatty acid synthesis), osmotic stress response (e.g., polyamine or trehalose synthesis, uptake of potassium) and putrescine metabolism, highlighting the immediate response mechanisms of P. veronii 1YdBTEX2 for successful establishment in polluted soil.

Comparative genome analysis of Pseudomonas knackmussii†B13, the first bacterium known to degrade chloroaromatic compounds.

Pseudomonas knackmussii B13 was the first strain to be isolated in 1974 that could degrade chlorinated aromatic hydrocarbons. This discovery was the prologue for subsequent characterization of numerous bacterial metabolic pathways, for genetic and biochemical studies, and which spurred ideas for pollutant bioremediation. In this study, we determined the complete genome sequence of B13 using next generation sequencing technologies and optical mapping. Genome annotation indicated that B13 has a variety of metabolic pathways for degrading monoaromatic hydrocarbons including chlorobenzoate, aminophenol, anthranilate and hydroxyquinol, but not polyaromatic compounds. Comparative genome analysis revealed that B13 is closest to Pseudomonas denitrificans and Pseudomonas aeruginosa. The B13 genome contains at least eight genomic islands [prophages and integrative conjugative elements (ICEs)], which were absent in closely related pseudomonads. We confirm that two ICEs are identical copies of the 103 kb self-transmissible element ICEclc that carries the genes for chlorocatechol metabolism. Comparison of ICEclc showed that it is composed of a variable and a 'core' region, which is very conserved among proteobacterial genomes, suggesting a widely distributed family of so far uncharacterized ICE. Resequencing of two spontaneous B13 mutants revealed a number of single nucleotide substitutions, as well as excision of a large 220 kb region and a prophage that drastically change the host metabolic capacity and survivability.

Community-wide plasmid gene mobilization and selection.

Plasmids have long been recognized as an important driver of DNA exchange and genetic innovation in prokaryotes. The success of plasmids has been attributed to their independent replication from the host's chromosome and their frequent self-transfer. It is thought that plasmids accumulate, rearrange and distribute nonessential genes, which may provide an advantage for host proliferation under selective conditions. In order to test this hypothesis independently of biases from culture selection, we study the plasmid metagenome from microbial communities in two activated sludge systems, one of which receives mostly household and the other chemical industry wastewater. We find that plasmids from activated sludge microbial communities carry among the largest proportion of unknown gene pools so far detected in metagenomic DNA, confirming their presumed role of DNA innovators. At a system level both plasmid metagenomes were dominated by functions associated with replication and transposition, and contained a wide variety of antibiotic and heavy metal resistances. Plasmid families were very different in the two metagenomes and grouped in deep-branching new families compared with known plasmid replicons. A number of abundant plasmid replicons could be completely assembled directly from the metagenome, providing insight in plasmid composition without culturing bias. Functionally, the two metagenomes strongly differed in several ways, including a greater abundance of genes for carbohydrate metabolism in the industrial and of general defense factors in the household activated sludge plasmid metagenome. This suggests that plasmids not only contribute to the adaptation of single individual prokaryotic species, but of the prokaryotic community as a whole under local selective conditions.

Examining chemical compound biodegradation at low concentrations through bacterial cell proliferation.

We show proof of principle for assessing compound biodegradation at 1-2 mg C per L by measuring microbial community growth over time with direct cell counting by flow cytometry. The concept is based on the assumption that the microbial community will increase in cell number through incorporation of carbon from the added test compound into new cells in the absence of (as much as possible) other assimilable carbon. We show on pure cultures of the bacterium Pseudomonas azelaica that specific population growth can be measured with as low as 0.1 mg 2-hydroxybiphenyl per L, whereas in mixed community 1 mg 2-hydroxybiphenyl per L still supported growth. Growth was also detected with a set of fragrance compounds dosed at 1-2 mg C per L into diluted activated sludge and freshwater lake communities at starting densities of 10(4) cells per ml. Yield approximations from the observed community growth was to some extent in agreement with standard OECD biodegradation test results for all, except one of the examined compounds.