Thierry K. S. Janssens

Molecular mechanisms of heavy metal tolerance and evolution in invertebrates.

Following the genomics revolution, our knowledge of the molecular mechanisms underlying defenses against stress has been greatly expanded. Under strong selective pressure many animals may evolve an enhanced stress tolerance. This can be achieved by altering the structure of proteins (through mutations in the coding regions of genes) or by altering the amount of protein (through changes in transcriptional regulation). The latter type of evolution can be achieved by substitutions in the promoter of the gene of interest (cis‐regulatory change) or by altering the structure or amount of transcriptional regulator proteins (trans‐regulatory change). The metallothionein system is one of the best studied stress response systems in the context of heavy metals. Metallothionein expression is assumed to be regulated by metal transcription factor 1 (MTF‐1); however, up to now the involvement of MTF‐1 has only been proven for some vertebrates and Drosophila. Data on invertebrates such as nematodes and earthworms suggest that other mechanisms of metallothionein induction may be present. A detailed study of Cd tolerance was done for a species of soil‐living springtail, Orchesella cincta. The metallothionein gene of this species is overexpressed in metal‐exposed field populations. Analysis of the metallothionein promoter has demonstrated extensive polymorphisms that have a functional significance, as shown in bioreporter assays. In a study comparing 20 different populations, the frequency of a high‐expresser promoter allele was positively correlated with the concentration of metals in soil, especially Cd. The springtail study shows that cis‐regulatory change of genes involved in the cellular stress response may contribute to evolution of metal tolerance.

Adaptive differences in gene expression associated with heavy metal tolerance in the soil arthropod Orchesella cincta

Field‐selected tolerance to heavy metals has been reported for Orchesella cincta (Arthropoda: Collembola) populations occurring at metal‐contaminated mining sites. This tolerance correlated with heritable increase in metal excretion efficiency, less pronounced cadmium (Cd)‐induced growth reduction and overexpression of the metallothionein gene. We applied transcriptomics to determine differential gene expression caused by this abiotic stress in reference and Cd‐tolerant populations. Many cDNAs responded to Cd exposure in the reference population. Significantly fewer clones were Cd responsive in tolerant animals. Analysis of variance revealed transcripts that interact between Cd exposure and population. Hierarchical cluster analysis of these clones identified two major groups. The first one contained cDNAs that were up‐regulated by Cd in the reference culture but non‐responsive or down‐regulated in tolerant animals. This cluster was also characterized by elevated constitutive expression in the tolerant population. Gene ontology analysis revealed that these cDNAs were involved in structural integrity of the cuticle, anti‐microbial defence, calcium channel‐blocking, sulphur assimilation and chromatin remodelling. The second group consisted of cDNAs down‐regulated in reference animals but not responding or slightly up‐regulated in tolerant animals. Their functions involved carbohydrate metabolic processes, Ca2+‐dependent stress signalling, redox state, proteolysis and digestion. The reference population showed a strong signature of stress‐induced genome‐wide perturbation of gene expression, whereas the tolerant animals maintained normal gene expression upon Cd exposure. We confirmed the micro‐evolutionary processes occurring in soil arthropod populations and suggest a major contribution of gene regulation to the evolution of a stress‐adapted phenotype.

Additive genetic variation of transcriptional regulation: metallothionein expression in the soil insect Orchesella cincta

Field-selected metal tolerance in Orchesella cincta is correlated with overexpression of the single copy cadmium (Cd) inducible metallothionein (mt). Previously, we have demonstrated large phenotypic variation in mt gene expression, and a higher frequency of high-expression phenotypes in a tolerant population. Here, we describe midparent–offspring regression analysis of mt gene expression in a laboratory culture originating from a noncontaminated natural population. Families were either not exposed (n=47) or exposed to 0.5 μmol Cd per gram dry food (n=46). Mean mt gene expressions normalized to 28S rRNA and β-actin RNA were generated using real-time RT-PCR applied to parents and offspring RNA and subjected to regression analysis. A significant heritability (h2) for mt gene expression was estimated between 0.36 (β-actin normalized) and 0.46 (28S normalized) in Cd exposed families. Nontreated families did not yield a significant h2 value. Restriction Fragment Length Polymorphism analysis of the metallothionein promoter sequence revealed eight promoter alleles that show structural variation. Three alleles show increased frequencies in families with high mt expression. Another gene, croquemort (isolated from a differential screening for 1 μmole Cd treatment) showed no h2 of gene expression in response to 0.5 μmol Cd. This gene codes for a receptor-protein involved in recognition of apoptotic cells and may participate in the general stress response. The present data suggest that evolution of metal tolerance in O. cincta can occur in the field by selection for high mt expression due to structural changes in mt cis-regulation.

Recombinational micro-evolution of functionally different metallothionein promoter alleles from Orchesella cincta

BackgroundMetallothionein (mt) transcription is elevated in heavy metal tolerant field populations of Orchesella cincta (Collembola). This suggests that natural selection acts on transcriptional regulation of mt in springtails at sites where cadmium (Cd) levels in soil reach toxic values This study investigates the nature and the evolutionary origin of polymorphisms in the metallothionein promoter (pmt) and their functional significance for mt expression.ResultsWe sequenced approximately 1600 bp upstream the mt coding region by genome walking. Nine pmt alleles were discovered in NW-European populations. They differ in the number of some indels, consensus transcription factor binding sites and core promoter elements. Extensive recombination events between some of the alleles can be inferred from the alignment. A deviation from neutral expectations was detected in a cadmium tolerant population, pointing towards balancing selection on some promoter stretches. Luciferase constructs were made from the most abundant alleles, and responses to Cd, paraquat (oxidative stress inducer) and moulting hormone were studied in cell lines. By using paraquat we were able to dissect the effect of oxidative stress from the Cd specific effect, and extensive differences in mt induction levels between these two stressors were observed.ConclusionThe pmt alleles evolved by a number of recombination events, and exhibited differential inducibilities by Cd, paraquat and molting hormone. In a tolerant population from a metal contaminated site, promoter allele frequencies differed significantly from a reference site and nucleotide polymorphisms in some promoter stretches deviated from neutral expectations, revealing a signature of balancing selection. Our results suggest that the structural differences in the Orchesella cincta metallothionein promoter alleles contribute to the metallothionein -over-expresser phenotype in cadmium tolerant populations.

Soil Microbiome Is More Heterogeneous in Organic Than in Conventional Farming System

Organic farming system and sustainable management of soil pathogens aim at reducing the use of agricultural chemicals in order to improve ecosystem health. Despite the essential role of microbial communities in agro-ecosystems, we still have limited understanding of the complex response of microbial diversity and composition to organic and conventional farming systems and to alternative methods for controlling plant pathogens. In this study we assessed the microbial community structure, diversity and richness using 16S rRNA gene next generation sequences and report that conventional and organic farming systems had major influence on soil microbial diversity and community composition while the effects of the soil health treatments (sustainable alternatives for chemical control) in both farming systems were of smaller magnitude. Organically managed system increased taxonomic and phylogenetic richness, diversity and heterogeneity of the soil microbiota when compared with conventional farming system. The composition of microbial communities, but not the diversity nor heterogeneity, were altered by soil health treatments. Soil health treatments exhibited an overrepresentation of specific microbial taxa which are known to be involved in soil suppressiveness to pathogens (plant-parasitic nematodes and soil-borne fungi). Our results provide a comprehensive survey on the response of microbial communities to different agricultural systems and to soil treatments for controlling plant pathogens and give novel insights to improve the sustainability of agro-ecosystems by means of beneficial microorganisms.

Influence of adaptive evolution of cadmium tolerance on neutral and functional genetic variation in Orchesella cincta

Adaptation to environmental toxicants, such as metals, can affect population genetic diversity, both at neutral and selectable loci. At the transcriptional level, evolution of metal tolerance is possible due to the existence of polymorphisms in the cis-regulatory sequences of stress-responsive genes such as the metallothionein gene (mt). This study investigated the influence of cadmium adaptation on genetic diversity of soil-living Orchesella cincta (Collembola) populations in neutral (microsatellites and AFLP) and in functional (mt promoter) markers. Also, the influence of cis- and trans-acting factors on increased tolerance was addressed. No reduced genetic diversity was observed in two tolerant populations compared to five sensitive populations, either in neutral or in selectable markers. Extensive migration along with a large population size may explain the high genetic diversity measured. The metal-tolerant phenotype seems to be mostly influenced by genetic factors acting in cis on mt gene expression. The results suggest that certain promoter genotypes, which are found mainly or exclusively in Cd-tolerant populations, contribute to higher constitutive mt gene expression in individuals from these populations. However, more studies are needed to clearly unravel the influence of cis/trans-regulatory evolution in tolerant populations.

Narcotic mechanisms of acute toxicity of chlorinated anilines in Folsomia candida (Collembola) revealed by gene expression analysis

In order to clarify the mechanisms of reproductive toxicity in a QSAR approach, the transcriptional signatures upon the 2 day exposure to the 28 days EC₅₀ of a series of increasingly chlorinated aniline compounds and 1,2,3,4-tetrachlorobenzene were measured in Folsomia candida. In general, the transcriptional patterns associated with all compounds revealed toxicity at the cellular membranes and hence components of narcosis type I, but a principal component analysis revealed a deviating response by the pentachloroaniline and 2,3,5,6-tetrachloroaniline exposure. Moreover the expression of a subset of mainly biotransformation related genes showed a significant relationship with the logK(ow,) which stresses the presence of narcosis type I. This was confirmed by GO term enrichment at the level of cellular component. Genes involved in the three phases of xenobiotic biotransformation exhibited strict compound specific transcription patterns, which may reflect biotransformation processes in F. candida. Additional toxic mechanisms were especially observed for the 2,3,5,6-tetrachloroaniline, which possible works as an uncoupler or inhibitor of electron transport systems, which is revealed by the up-regulation of genes that encode different members of the electron transport chain. The aniline and 2,3,4-trichloroaniline exposure caused the induction of genes in the ROS defense system. Additional toxicity mechanisms were less clear, but they include the attack of microbial pathogens for the six other compounds and for 2,3,5,6-tetrachloroaniline an effect on mitochondrial protein folding.

Exploring bacterial interspecific interactions for discovery of novel antimicrobial compounds

Recent studies indicated that the production of secondary metabolites by soil bacteria can be triggered by interspecific interactions. However, little is known to date about interspecific interactions between Gram‐positive and Gram‐negative bacteria. In this study, we aimed to understand how the interspecific interaction between the Gram‐positive Paenibacillus sp. AD87 and the Gram‐negative Burkholderia sp. AD24 affects the fitness, gene expression and the production of soluble and volatile secondary metabolites of both bacteria. To obtain better insight into this interaction, transcriptome and metabolome analyses were performed. Our results revealed that the interaction between the two bacteria affected their fitness, gene expression and the production of secondary metabolites. During interaction, the growth of Paenibacillus was not affected, whereas the growth of Burkholderia was inhibited at 48 and 72 h. Transcriptome analysis revealed that the interaction between Burkholderia and Paenibacillus caused significant transcriptional changes in both bacteria as compared to the monocultures. The metabolomic analysis revealed that the interaction increased the production of specific volatile and soluble antimicrobial compounds such as 2,5‐bis(1‐methylethyl)‐pyrazine and an unknown Pederin‐like compound. The pyrazine volatile compound produced by Paenibacillus was subjected to bioassays and showed strong inhibitory activity against Burkholderia and a range of plant and human pathogens. Moreover, strong additive antimicrobial effects were observed when soluble extracts from the interacting bacteria were combined with the pure 2,5‐bis(1‐methylethyl)‐pyrazine. The results obtained in this study highlight the importance to explore bacterial interspecific interactions to discover novel secondary metabolites and to perform simultaneously metabolomics of both, soluble and volatile compounds.

Fungus-associated bacteriome in charge of their host behavior

Bacterial-fungal interactions are widespread in nature and there is a growing number of studies reporting distinct fungus-associated bacteria. However, little is known so far about how shifts in the fungus-associated bacteriome will affect the fungal hosts lifestyle. In the present study, we describe for the first time the bacterial community associated with the saprotrophic fungus Mucor hiemalis, commonly found in soil and rhizosphere. Two broad-spectrum antibiotics that strongly altered the bacterial community associated with the fungus were applied. Our results revealed that the antibiotic treatment did not significantly reduce the amount of bacteria associated to the fungus but rather changed the community composition by shifting from initially dominating Alpha-Proteobacteria to dominance of Gamma-Proteobacteria. A novel approach was applied for the isolation of fungal-associated bacteria which also revealed differences between bacterial isolates obtained from the original and the antibiotic-treated M. hiemalis. The shift in the composition of the fungal-associated bacterial community led to significantly reduced fungal growth, changes in fungal morphology, behavior and secondary-metabolites production. Furthermore, our results showed that the antibiotic-treated isolate was more attractive and susceptible to mycophagous bacteria as compared to the original isolate. Overall, our study highlights the importance of the fungus-associated bacteriome for the hosts lifestyle and interactions and indicate that isolation with antibacterials is not sufficient to eradicate the associated bacteria.

Combined Transcriptomics Analysis for Classification of Adverse Effects As a Potential End Point in Effect Based Screening

Environmental risk assessment relies on the use of bioassays to assess the environmental impact of chemicals. Gene expression is gaining acceptance as a valuable mechanistic end point in bioassays and effect-based screening. Data analysis and its results, however, are complex and often not directly applicable in risk assessment. Classifier analysis is a promising method to turn complex gene expression analysis results into answers suitable for risk assessment. We have assembled a large gene expression data set assembled from multiple studies and experiments in the springtail Folsomia candida, with the aim of selecting a set of genes that can be trained to classify general toxic stress. By performing differential expression analysis prior to classifier training, we were able to select a set of 135 genes which was enriched in stress related processes. Classifier models from this set were used to classify two test sets comprised of chemical spiked, polluted, and clean soils and compared to another, more traditional classifier feature selection. The gene set presented here outperformed the more traditionally selected gene set. This gene set has the potential to be used as a biomarker to test for adverse effects caused by chemicals in springtails to provide end points in environmental risk assessment.