Effect of culture conditions on the performance of lignocellulose-degrading synthetic microbial consortia.

In this study, we examined a synthetic microbial consortium, composed of two selected bacteria, i.e., Citrobacter freundii so4 and Sphingobacterium multivorum w15, next to the fungus Coniochaeta sp. 2T2.1, with respect to their fate and roles in the degradation of wheat straw (WS). A special focus was placed on the effects of pH (7.2, 6.2, or 5.2), temperature (25 versus 28 °C), and shaking speed (60 versus 180 rpm). Coniochaeta sp. 2T2.1 consistently had a key role in the degradation process, with the two bacteria having additional roles. Whereas temperature exerted only minor effects on the degradation, pH and shaking speed were key determinants of both organismal growth and WS degradation levels. In detail, the three-partner degrader consortium showed significantly higher WS degradation values at pH 6.2 and 5.2 than at pH 7.2. Moreover, the two bacteria revealed up to tenfold enhanced final cell densities (ranging from log8.0 to log9.0 colony forming unit (CFU)/mL) in the presence of Coniochaeta sp. 2T2.1 than when growing alone or in a bacterial bi-culture, regardless of pH range or shaking speed. Conversely, at 180 rpm, fungal growth was clearly suppressed by the presence of the bacteria at pH 5.2 and pH 6.2, but not at pH 7.2. In contrast, at 60 rpm, the presence of the bacteria fostered fungal growth. In these latter cultures, oxygen levels were significantly lowered as compared to the maximal levels found at 180 rpm (about 5.67 mg/L, ~ 62% of saturation). Conspicuous effects on biomass appearance pointed to a fungal biofilm-modulating role of the bacteria.Key points• Coniochaeta sp. 2T2.1 has a key role in wheat straw (WS) degradation.• Bacterial impact shifts when conditions change.• pH and shaking speed are key drivers of the growth dynamics and WS degradation.

Adaptative transcriptional response of Dietzia cinnamea P4 strain to sunlight simulator.

Responses to sunlight exposure of the oil-degrading Dietzia cinnamea P4 strain were evaluated by transcriptional levels of SOS genes, photoreactivation and genes involved in tolerance to high levels of reactive oxygen species. The P4 strain was exposed for 1 and 2 h and the magnitude of level changes in the mRNA was evaluated by qPCR. The results described the activation of the SOS system, with the decline of the repressor lexA gene levels and the concomitant increase of recA and uvrAD genes levels. The genes that participate in the photoreactivation process were also responsive to sunlight. The phrB gene encoding deoxyribodipyrimidine photo-lyase had its expression increased after 1-h exposure, while the phytAB genes showed a progressive increase over the studied period. The protective genes against reactive oxygen species, catalases, superoxides, peroxidases, and thioredoxins, had their expression rates detected under the conditions validated in this study. These results show a fast and coordinated response of genes from different DNA repair and tolerance mechanisms employed by strain P4, suggesting a complex concerted protective action against environmental stressors.

Oxidative damage induced by H2O2 reveals SOS adaptive transcriptional response of Dietzia cinnamea strain P4.

The oxidative stress response of the highly resistant actinomycete Dietzia cinnamea P4 after treatment with hydrogen peroxide (H2O2) was assessed in order to depict the possible mechanisms underlying its intrinsic high resistance to DNA damaging agents. We used transcriptional profiling to monitor the magnitude and kinetics of changes in the mRNA levels after exposure to different concentrations of H2O2 at 10 min and 1 h following the addition of the stressor. Catalase and superoxide dismutase genes were induced in different ways, according to the condition applied. Moreover, alkyl hydroperoxide reductase ahpCF, thiol peroxidase, thioredoxin and glutathione genes were upregulated in the presence of H2O2. Expression of peroxidase genes was not detected during the experiment. Overall results point to an actinomycete strain endowed with a set of enzymatic defenses against oxidative stress and with the main genes belonging to a functional SOS system (lexA, recA, uvrD), including suppression of lexA repressor, concomitantly to recA and uvrD gene upregulation upon H2O2 challenge.

The multi-omics promise in context: from sequence to microbial isolate.

The numbers and diversity of microbes in ecosystems within and around us is unmatched, yet most of these microorganisms remain recalcitrant to in vitro cultivation. Various high-throughput molecular techniques, collectively termed multi-omics, provide insights into the genomic structure and metabolic potential as well as activity of complex microbial communities. Nonetheless, pure or defined cultures are needed to (1) decipher microbial physiology and thus test multi-omics-based ecological hypotheses, (2) curate and improve database annotations and (3) realize novel applications in biotechnology. Cultivation thus provides context. In turn, we here argue that multi-omics information awaits integration into the development of novel cultivation strategies. This can build the foundation for a new era of omics information-guided microbial cultivation technology and reduce the inherent trial-and-error search space. This review discusses how information that can be extracted from multi-omics data can be applied for the cultivation of hitherto uncultured microorganisms. Furthermore, we summarize groundbreaking studies that successfully translated information derived from multi-omics into specific media formulations, screening techniques and selective enrichments in order to obtain novel targeted microbial isolates. By integrating these examples, we conclude with a proposed workflow to facilitate future omics-aided cultivation strategies that are inspired by the microbial complexity of the environment.

Migration of Paraburkholderia terrae BS001 Along Old Fungal Hyphae in Soil at Various pH Levels.

The movement of bacterial cells along with fungal hyphae in soil (the mycosphere) has been reported in several previous studies. However, how local soil conditions affect bacterial migration direction in the mycosphere has not been extensively studied. Here, we investigated the influence of two soil parameters, pH and soil moisture content, on the migration, and survival, of Paraburkholderia terrae BS001 in the mycosphere of Lyophyllum sp. strain Karsten in microcosms containing a loamy sand soil. The data showed that bacterial movement along the hyphal networks took place in both the "forward" and the "backward" directions. Low soil pH strongly restricted bacterial survival, as well as dispersal in both directions, in the mycosphere. The backward movement was weakly correlated with the amount of fungal tissue formed in the old mycelial network. The initial soil moisture content, set at 12 versus 17% (corresponding to 42 and 60% of the soil water holding capacity), also significantly affected the bacterial dispersal along the fungal hyphae. Overall, the presence of fungal hyphae was found to increase the soil pH (under conditions of acidity), which possibly exerted protective effects on the bacterial cells. Finally, we provide a refined model that describes the bacterial migration patterns with fungal hyphae based on the new findings in this study.

The parA Region of Broad-Host-Range PromA Plasmids Is a Carrier of Mobile Genes.

The ecological competences in microbiomes are driven by the adaptive capabilities present within microbiome members. Horizontal gene transfer (HGT) promoted by plasmids provides a rapid adaptive strategy to microbiomes, an interesting feature considering the constantly changing conditions in most environments. This study examined the parA locus, found in the highly promiscuous PromA class of plasmids, as the insertion site for incoming genes. A novel PCR system was designed that enabled examining insertions into this locus. Microbiomes of mangrove sediments, salt marsh, mycosphere, and bulk soil revealed habitat-specific sets of insertions in this plasmid region. Furthermore, such habitats could be differentiated based on patterns of parA-inserted genes, and the genes carried by these plasmids. Thus, a suite of dioxygenase-related genes and transposase elements were found in oil-affected mangroves, whereas genes involved in nitrogen and carbon cycling were detected in salt marsh and soils. All genes detected could be associated with capabilities of members of the microbiome to adapt to and survive in each habitat. The methodology developed in this work was effective, sensitive, and practical, allowing detection of mobilized genes between microorganisms.

Halotolerant microbial consortia able to degrade highly recalcitrant plant biomass substrate.

The microbial degradation of plant-derived compounds under salinity stress remains largely underexplored. The pretreatment of lignocellulose material, which is often needed to improve the production of lignocellulose monomers, leads to high salt levels, generating a saline environment that raises technical considerations that influence subsequent downstream processes. Here, we constructed halotolerant lignocellulose degrading microbial consortia by enriching a salt marsh soil microbiome on a recalcitrant carbon and energy source, i.e., wheat straw. The consortia were obtained after six cycles of growth on fresh substrate (adaptation phase), which was followed by four cycles on pre-digested (highly-recalcitrant) substrate (stabilization phase). The data indicated that typical salt-tolerant bacteria made up a large part of the selected consortia. These were "trained" to progressively perform better on fresh substrate, but a shift was observed when highly recalcitrant substrate was used. The most dominant bacteria in the consortia were Joostella marina, Flavobacterium beibuense, Algoriphagus ratkowskyi, Pseudomonas putida, and Halomonas meridiana. Interestingly, fungi were sparsely present and negatively affected by the change in the substrate composition. Sarocladium strictum was the single fungal strain recovered at the end of the adaptation phase, whereas it was deselected by the presence of recalcitrant substrate. Consortia selected in the latter substrate presented higher cellulose and lignin degradation than consortia selected on fresh substrate, indicating a specialization in transforming the recalcitrant regions of the substrate. Moreover, our results indicate that bacteria have a prime role in the degradation of recalcitrant lignocellulose under saline conditions, as compared to fungi. The final consortia constitute an interesting source of lignocellulolytic haloenzymes that can be used to increase the efficiency of the degradation process, while decreasing the associated costs.

Disentangling mechanisms that mediate the balance between stochastic and deterministic processes in microbial succession.

Ecological succession and the balance between stochastic and deterministic processes are two major themes within microbial ecology, but these conceptual domains have mostly developed independent of each other. Here we provide a framework that integrates shifts in community assembly processes with microbial primary succession to better understand mechanisms governing the stochastic/deterministic balance. Synthesizing previous work, we devised a conceptual model that links ecosystem development to alternative hypotheses related to shifts in ecological assembly processes. Conceptual model hypotheses were tested by coupling spatiotemporal data on soil bacterial communities with environmental conditions in a salt marsh chronosequence spanning 105 years of succession. Analyses within successional stages showed community composition to be initially governed by stochasticity, but as succession proceeded, there was a progressive increase in deterministic selection correlated with increasing sodium concentration. Analyses of community turnover among successional stages--which provide a larger spatiotemporal scale relative to within stage analyses--revealed that changes in the concentration of soil organic matter were the main predictor of the type and relative influence of determinism. Taken together, these results suggest scale-dependency in the mechanisms underlying selection. To better understand mechanisms governing these patterns, we developed an ecological simulation model that revealed how changes in selective environments cause shifts in the stochastic/deterministic balance. Finally, we propose an extended--and experimentally testable--conceptual model integrating ecological assembly processes with primary and secondary succession. This framework provides a priori hypotheses for future experiments, thereby facilitating a systematic approach to understand assembly and succession in microbial communities across ecosystems.

Transcriptional Responses of the Bacterium Burkholderia terrae BS001 to the Fungal Host Lyophyllum sp. Strain Karsten under Soil-Mimicking Conditions.

In this study, the mycosphere isolate Burkholderia terrae BS001 was confronted with the soil fungus Lyophyllum sp. strain Karsten on soil extract agar plates in order to examine its transcriptional responses over time. At the initial stages of the experiment (T1-day 3; T2-day 5), contact between both partner organisms was absent, whereas in the final stage (T3-day 8), the two populations made intimate physical contact. Overall, a strong modulation of the strain BS001 gene expression patterns was found. First, the stationary-phase sigma factor RpoS, and numerous genes under its control, were strongly expressed as a response to the soil extract agar, and this extended over the whole temporal regime. In the system, B. terrae BS001 apparently perceived the presence of the fungal hyphae already at the early experimental stages (T1, T2), by strongly upregulating a suite of chemotaxis and flagellar motility genes. With respect to specific metabolism and energy generation, a picture of differential involvement in different metabolic routes was obtained. Initial (T1, T2) up- or downregulation of ethanolamine and mandelate uptake and utilization pathways was substituted by a strong investment, in the presence of the fungus, in the expression of putative metabolic gene clusters (T3). Specifically at T3, five clustered genes that are potentially involved in energy generation coupled to an oxidative stress response, and two genes encoding short-chain dehydrogenases/oxidoreductases (SDR), were highly upregulated. In contrast, the dnaE2 gene (related to general stress response; encoding error-prone DNA polymerase) was transcriptionally downregulated at this stage. This study revealed that B. terrae BS001, from a stress-induced state, resulting from the soil extract agar milieu, responds positively to fungal hyphae that encroach upon it, in a temporally dynamic manner. The response is characterized by phases in which the modulation of (1) chemotaxis, (2) metabolic activity, and (3) oxidative stress responses are key mechanisms.

The first acidobacterial laccase-like multicopper oxidase revealed by metagenomics shows high salt and thermo-tolerance.

Metagenomics is a powerful tool that allows identifying enzymes with novel properties from the unculturable component of microbiomes. However, thus far only a limited number of laccase or laccase -like enzymes identified through metagenomics has been subsequently biochemically characterized. This work describes the successful bio-mining of bacterial laccase-like enzymes in an acidic bog soil metagenome and the characterization of the first acidobacterial laccase-like multicopper oxidase (LMCO). LMCOs have hitherto been mostly studied in fungi and some have already found applications in diverse industries. However, improved LMCOs are in high demand. Using molecular screening of a small metagenomic library (13,500 clones), a gene encoding a three-domain LMCO (LacM) was detected, showing the highest similarity to putative copper oxidases of Candidatus Solibacter (Acidobacteria). The encoded protein was expressed in Escherichia coli, purified by affinity chromatography and biochemically characterized. LacM oxidized a variety of phenolic substrates, including two standard laccase substrates (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), k cat/k M  = 8.45 s-1 mM-1; 2,6-dimethoxyphenol (2,6-DMP), k cat/k M  = 6.42 s-1 mM-1), next to L-3,4-dihydroxyphenylalanine (L-DOPA), vanillic acid, syringaldazine, pyrogallol, and pyrocatechol. With respect to the latter two lignin building blocks, LacM showed the highest catalytic activity (k cat/k M  = 173.6 s-1 mM-1) for pyrogallol, with ca. 20% activity preserved even at pH 8.0. The enzyme was thermostable and heat-activated in the interval 40-60 °C, with an optimal activity on ABTS at 50 °C. It was rather stable at high salt concentration (e.g., 34% activity preserved at 500 mM NaCl) and in the presence of organic solvents. Remarkably, LacM decolored azo and triphenylmethane dyes, also in the absence of redox mediators.

Denitrifying bacterial communities display different temporal fluctuation patterns across Dutch agricultural soils.

Considering the great agronomic and environmental importance of denitrification, the aim of the present study was to study the temporal and spatial factors controlling the abundance and activity of denitrifying bacterial communities in a range of eight agricultural soils over 2 years. Abundance was quantified by qPCR of the nirS, nirK and nosZ genes, and the potential denitrification enzyme activity (DEA) was estimated. Our data showed a significant temporal variation considerably high for the abundance of nirK-harboring communities, followed by nosZ and nirS communities. Regarding soil parameters, the abundances of nosZ, nirS and nirK were mostly influenced by organic material, pH, and slightly by NO3-, respectively. Soil texture was the most important factor regulating DEA, which could not be explained by the abundance of denitrifiers. Analyses of general patterns across lands to understand the soil functioning is not an easy task because the multiple factors influencing processes such as denitrification can skew the data. Careful analysis of atypical sites are necessary to classify the soils according to trait similarity and in this way reach a better predictability of the denitrifiers dynamics.

A multi-substrate approach for functional metagenomics-based screening for (hemi)cellulases in two wheat straw-degrading microbial consortia unveils novel thermoalkaliphilic enzymes.

BACKGROUND: Functional metagenomics is a promising strategy for the exploration of the biocatalytic potential of microbiomes in order to uncover novel enzymes for industrial processes (e.g. biorefining or bleaching pulp). Most current methodologies used to screen for enzymes involved in plant biomass degradation are based on the use of single substrates. Moreover, highly diverse environments are used as metagenomic sources. However, such methods suffer from low hit rates of positive clones and hence the discovery of novel enzymatic activities from metagenomes has been hampered. RESULTS: Here, we constructed fosmid libraries from two wheat straw-degrading microbial consortia, denoted RWS (bred on untreated wheat straw) and TWS (bred on heat-treated wheat straw). Approximately 22,000 clones from each library were screened for (hemi)cellulose-degrading enzymes using a multi-chromogenic substrate approach. The screens yielded 71 positive clones for both libraries, giving hit rates of 1:440 and 1:1,047 for RWS and TWS, respectively. Seven clones (NT2-2, T5-5, NT18-17, T4-1, 10BT, NT18-21 and T17-2) were selected for sequence analyses. Their inserts revealed the presence of 18 genes encoding enzymes belonging to twelve different glycosyl hydrolase families (GH2, GH3, GH13, GH17, GH20, GH27, GH32, GH39, GH53, GH58, GH65 and GH109). These encompassed several carbohydrate-active gene clusters traceable mainly to Klebsiella related species. Detailed functional analyses showed that clone NT2-2 (containing a beta-galactosidase of ~116 kDa) had highest enzymatic activity at 55 °C and pH 9.0. Additionally, clone T5-5 (containing a beta-xylosidase of ~86 kDa) showed > 90% of enzymatic activity at 55 °C and pH 10.0. CONCLUSIONS: This study employed a high-throughput method for rapid screening of fosmid metagenomic libraries for (hemi)cellulose-degrading enzymes. The approach, consisting of screens on multi-substrates coupled to further analyses, revealed high hit rates, as compared with recent other studies. Two clones, 10BT and T4-1, required the presence of multiple substrates for detectable activity, indicating a new avenue in library activity screening. Finally, clones NT2-2, T5-5 and NT18-17 were found to encode putative novel thermo-alkaline enzymes, which could represent a starting point for further biotechnological applications.

Soil-Derived Microbial Consortia Enriched with Different Plant Biomass Reveal Distinct Players Acting in Lignocellulose Degradation.

Here, we investigated how different plant biomass, and-for one substrate-pH, drive the composition of degrader microbial consortia. We bred such consortia from forest soil, incubated along nine aerobic sequential - batch enrichments with wheat straw (WS1, pH 7.2; WS2, pH 9.0), switchgrass (SG, pH 7.2), and corn stover (CS, pH 7.2) as carbon sources. Lignocellulosic compounds (lignin, cellulose and xylan) were best degraded in treatment SG, followed by CS, WS1 and WS2. In terms of composition, the consortia became relatively stable after transfers 4 to 6, as evidenced by PCR-DGGE profiles obtained from each consortium DNA. The final consortia differed by ~40 % (bacteria) and ~60 % (fungi) across treatments. A 'core' community represented by 5/16 (bacteria) and 3/14 (fungi) bands was discerned, next to a variable part. The composition of the final microbial consortia was strongly driven by the substrate, as taxonomically-diverse consortia appeared in the different substrate treatments, but not in the (WS) different pH one. Biodegradative strains affiliated to Sphingobacterium kitahiroshimense, Raoultella terrigena, Pseudomonas putida, Stenotrophomonas rhizophila (bacteria), Coniochaeta ligniaria and Acremonium sp. (fungi) were recovered in at least three treatments, whereas strains affiliated to Delftia tsuruhatensis, Paenibacillus xylanexedens, Sanguibacter inulus and Comamonas jiangduensis were treatment-specific.

Different inocula produce distinctive microbial consortia with similar lignocellulose degradation capacity.

Despite multiple research efforts, the current strategies for exploitation of lignocellulosic plant matter are still far from optimal, being hampered mostly by the difficulty of degrading the recalcitrant parts. An interesting approach is to use lignocellulose-degrading microbial communities by using different environmental sources of microbial inocula. However, it remains unclear whether the inoculum source matters for the degradation process. Here, we addressed this question by verifying the lignocellulose degradation potential of wheat (Triticum aestivum) straw by microbial consortia generated from three different microbial inoculum sources, i.e., forest soil, canal sediment and decaying wood. We selected these consortia through ten sequential-batch enrichments by dilution-to-stimulation using wheat straw as the sole carbon source. We monitored the changes in microbial composition and abundance, as well as their associated degradation capacity and enzymatic activities. Overall, the microbial consortia developed well on the substrate, with progressively-decreasing net average generation times. Each final consortium encompassed bacterial/fungal communities that were distinct in composition but functionally similar, as they all revealed high substrate degradation activities. However, we did find significant differences in the metabolic diversities per consortium: in wood-derived consortia cellobiohydrolases prevailed, in soil-derived ones ß-glucosidases, and in sediment-derived ones several activities. Isolates recovered from the consortia showed considerable metabolic diversities across the consortia. This confirmed that, although the overall lignocellulose degradation was similar, each consortium had a unique enzyme activity pattern. Clearly, inoculum source was the key determinant of the composition of the final microbial degrader consortia, yet with varying enzyme activities. Hence, in accord with Beyerinck's, "everything is everywhere, the environment selects" the source determines consortium composition.

Characterization of three plant biomass-degrading microbial consortia by metagenomics- and metasecretomics-based approaches.

The selection of microbes by enrichment on plant biomass has been proposed as an efficient way to develop new strategies for lignocellulose saccharification. Here, we report an in-depth analysis of soil-derived microbial consortia that were trained to degrade once-used wheat straw (WS1-M), switchgrass (SG-M) and corn stover (CS-M) under aerobic and mesophilic conditions. Molecular fingerprintings, bacterial 16S ribosomal RNA (rRNA) gene amplicon sequencing and metagenomic analyses showed that the three microbial consortia were taxonomically distinct. Based on the taxonomic affiliation of protein-encoding sequences, members of the Bacteroidetes (e.g. Chryseobacterium, Weeksella, Flavobacterium and Sphingobacterium) were preferentially selected on WS1-M, whereas SG-M and CS-M favoured members of the Proteobacteria (e.g. Caulobacter, Brevundimonas, Stenotrophomonas and Xanthomonas). The highest degradation rates of lignin (~59 %) were observed with SG-M, whereas CS-M showed a high consumption of cellulose and hemicellulose. Analyses of the carbohydrate-active enzymes in the three microbial consortia showed the dominance of glycosyl hydrolases (e.g. of families GH3, GH43, GH13, GH10, GH29, GH28, GH16, GH4 and GH92). In addition, proteins of families AA6, AA10 and AA2 were detected. Analysis of secreted protein fractions (metasecretome) for each selected microbial consortium mainly showed the presence of enzymes able to degrade arabinan, arabinoxylan, xylan, ß-glucan, galactomannan and rhamnogalacturonan. Notably, these metasecretomes contain enzymes that enable us to produce oligosaccharides directly from wheat straw, sugarcane bagasse and willow. Thus, the underlying microbial consortia constitute valuable resources for the production of enzyme cocktails for the efficient saccharification of plant biomass.

Response of the bacterial community in oil-contaminated marine water to the addition of chemical and biological dispersants.

The use of dispersants in different stages of the oil production chain and for the remediation of water and soil is a well established practice. However, the choice for a chemical or biological dispersant is still a controversial subject. Chemical surfactants that persist long in the environment may pose problems of toxicity themselves; therefore, biosurfactants are considered to constitute an environmentally friendly and effective alternative. Nevertheless, the putative effects of such agents on the microbiomes of oil-contaminated and uncontaminated marine environments have not been sufficiently evaluated. Here, we studied the effects of the surfactant Ultrasperse II® and the surfactin (biosurfactant) produced by Bacillus sp. H2O-1 on the bacterial communities of marine water. Specifically, we used quantitative PCR and genetic fingerprint analyses to study the abundance and structure of the bacterial communities in marine water collected from two regions with contrasting climatic conditions. The addition of either chemical surfactant or biosurfactant influenced the structure and abundance of total and oil-degrading bacterial communities of oil-contaminated and uncontaminated marine waters. Remarkably, the bacterial communities responded similarly to the addition of oil and/or either the surfactant or the biosurfactant in both set of microcosms. After 30 days of incubation, the addition of surfactin enhanced the oil-degrading bacteria more than the chemical surfactant. However, no increase of hydrocarbon biodegradation values was observed, irrespective of the dispersant used. These data contribute to an increased understanding of the impact of novel dispersants on marine bacteriomes before commercial release into the environment.

Differential stress resistance and metabolic traits underlie coexistence in a sympatrically evolved bacterial population.

Following intermittent batch growth in Luria-Bertani (LB) broth for about 1000 generations, differentially evolved forms were found in a population of Escherichia coli cells. Studies on this population revealed the emergence of key polymorphisms, as evidenced by analysis of both whole genome sequences and transcription analysis. Here, we investigated the phenotypic nature of several key forms and found a remarkable (interactive) coexistence of forms which highlights the presence of different ecological roles pointing at a dichotomy in: (i) tolerance to environmental stresses and (ii) the capacity to utilize particular carbon sources such as galactose. Both forms differed from their common ancestor by different criteria. This apparent coexistence of two diverged forms points at the occurrence of niche partitioning as a consequence of dichotomous adaptive evolution. Remarkably, the two forms were shown to continue to coexist - in varying ratio's - in an experiment that cycled them through periods of nutrient feast (plentiful growth substrates) and famine (growth-restrictive - stress conditions). The results further indicated that the equilibrium of the coexistence was destroyed when one of the parameters was high tuned, jeopardizing the stability of the coexisting pair.

Resource pulses can alleviate the biodiversity-invasion relationship in soil microbial communities.

The roles of species richness, resource use, and resource availability are central to many hypotheses explaining the diversity-invasion phenomenon but are generally not investigated together. Here, we created a large diversity gradient of soil microbial communities by either assembling communities of pure bacterial strains or removing the diversity of a natural soil. Using data on the resource-use capacities of the soil communities and an invader that were gathered from 71 carbon sources, we quantified the niches available to both constituents by using the metrics community niche and remaining niche available to the invader. A strong positive relationship between species richness and community niche across both experiments indicated the presence of resource complementarity. Moreover, community niche and the remaining niche available to the invader predicted invader abundance well. This suggested that increased competition in communities of higher diversity limits community invasibility and underscored the importance of resource availability as a key mechanism through which diversity hinders invasions. As a proof of principle, we subjected selected invaded communities to a resource pulse, which progressively uncoupled the link between soil microbial diversity and invasion and allowed the invader to rebound after nearly being eliminated in some communities. Our results thus show that (1) resource competition suppresses invasion, (2) biodiversity increases resource competition and decreases invasion through niche preemption, and (3) resource pulses that cannot be fully used, even by diverse communities, are favorable to invasion.

Characterization of a collection of plasmid-containing bacteria isolated from an on-farm biopurification system used for pesticide removal.

Biopurification systems (BPS) are complex soil-related and artificially-generated environments usually designed for the removal of toxic compounds from contaminated wastewaters. The present study has been conducted to isolate and characterize a collection of cultivable plasmid-carrying bacterial isolates recovered from a BPS established for the decontamination of wastewater generated in a farmyard. Out of 1400 isolates, a collection of 75 plasmid-containing bacteria was obtained, of which 35 representative isolates comprising in total at least 50 plasmids were chosen for further characterization. Bacterial hosts were taxonomically assigned by 16S ribosomal RNA gene sequencing and phenotypically characterized according to their ability to grow in presence of different antibiotics and heavy metals. The study demonstrated that a high proportion of the isolates was tolerant to antibiotics and/or heavy metals, highlighting the on-farm BPS enrichment in such genetic traits. Several plasmids conferring such resistances in the bacterial collection were detected to be either mobilizable or selftransmissible. Occurrence of broad host range plasmids of the incompatibility groups IncP, IncQ, IncN and IncW was examined with positive results only for the first group. Presence of the IS1071 insertion sequence, frequently associated with xenobiotics degradation genes, was detected in DNA obtained from 24 of these isolates, strongly suggesting the presence of yet-hidden catabolic activities in the collection of isolates. The results showed a remarkable diversity in the plasmid mobilome of cultivable bacteria in the BPS with the presence of abundant resistance markers of different types, thus providing a suitable environment to investigate the genetic structure of the mobile genetic pool in a model on-farm biofilter for wastewater decontamination in intensive agricultural production.

IncP-1 and PromA group plasmids are major providers of horizontal gene transfer capacities across bacteria in the mycosphere of different soil fungi.

Plasmids of the IncP-1ß group have been found to be important carriers of accessory genes that enhance the ecological fitness of bacteria, whereas plasmids of the PromA group are key agents of horizontal gene transfer in particular soil settings. However, there is still a paucity of knowledge with respect to the diversity, abundance, and involvement in horizontal gene transfer of plasmids of both groups in the mycosphere. Using triparental exogenous isolation based on the IncQ tracer plasmid pSUP104 as well as direct molecular detection, we analyzed the pool of mobilizer and self-transferable plasmids in mycosphere soil. Replicate mushroom types that were related to Russula, Inocybe, Ampulloclitocybe, and Galerina spp. were sampled from a forest soil area, and bulk soil was used as the control. The data showed that the levels of IncP-1ß plasmids are significantly raised across several of the mycospheres analyzed, whereas those of PromA group plasmids were similar across the mycospheres and corresponding bulk soil. Moreover, the frequencies of triparental exogenous isolation of mobilizer plasmids into a Pseudomonas fluorescens recipient strain were significantly elevated in communities from several mycospheres as compared with those from bulk soil. Molecular analysis of selected transconjugants, as well as from directly isolated strains, revealed the presence of plasmids of three size groups, i.e., (1) 40-45, (2) 50-60, and (3) ≥60 kb, across all isolations. Replicon typing using IncN, IncW and IncA/C proxies revealed no positive signals. In contrast, a suite of plasmids produced signals with IncP-1ß as well as PromA type replicon typing systems. Moreover, a selected subset of plasmids, obtained from the Inocybe and Galerina isolates, was transferred out further, revealing their capacities to transfer and mobilize across a broad host range.