Prokaryotic, Microeukaryotic, and Fungal Composition in a Long-Term Polychlorinated Biphenyl-Contaminated Brownfield.

Polychlorinated biphenyls (PCBs) are recognized as persistent organic pollutants and accumulate in organisms, soils, waters, and sediments, causing major health and ecological perturbations. Literature reported PCB bio-transformation by fungi and bacteria in vitro, but data about the in situ impact of those compounds on microbial communities remained scarce while being useful to guide biotransformation assays. The present work investigated for the first time microbial diversity from the three-domains-of-life in a long-term contaminated brownfield (a former factory land). Soil samples were ranked according to their PCB concentrations, and a significant increase in abundance was shown according to increased concentrations. Microbial communities structure showed a segregation from the least to the most PCB-polluted samples. Among the identified microorganisms, Bacteria belonging to Gammaproteobacteria class, as well as Fungi affiliated to Saccharomycetes class or Pleurotaceae family, including some species known to transform some PCBs were abundantly retrieved in the highly polluted soil samples.

Datamining and functional environmental genomics reassess the phylogenetics and functional diversity of fungal monosaccharide transporters.

Sugar transporters are essential components of carbon metabolism and have been extensively studied to control sugar uptake by yeasts and filamentous fungi used in fermentation processes. Based on published information on characterized fungal sugar porters, we show that this protein family encompasses phylogenetically distinct clades. While several clades encompass transporters that seemingly specialized on specific "sugar-related" molecules (e.g., myo-inositol, charged sugar analogs), others include mostly either mono- or di/oligosaccharide low-specificity transporters. To address the issue of substrate specificity of sugar transporters, that protein primary sequences do not fully reveal, we screened "multi-species" soil eukaryotic cDNA libraries for mannose transporters, a sugar that had never been used to select transporters. We obtained 19 environmental transporters, mostly from Basidiomycota and Ascomycota. Among them, one belonged to the unusual "Fucose H+ Symporter" family, which is only known in Fungi for a rhamnose transporter in Aspergillus niger. Functional analysis of the 19 transporters by expression in yeast and for two of them in Xenopus laevis oocytes for electrophysiological measurements indicated that most of them showed a preference for D-mannose over other tested D-C6 (glucose, fructose, galactose) or D-C5 (xylose) sugars. For the several glucose and fructose-negative transporters, growth of the corresponding recombinant yeast strains was prevented on mannose in the presence of one of these sugars that may act by competition for the binding site. Our results highlight the potential of environmental genomics to figure out the functional diversity of key fungal protein families and that can be explored in a context of biotechnology. KEY POINTS: • Most fungal sugar transporters accept several sugars as substrates. • Transporters, belonging to 2 protein families, were isolated from soil cDNA libraries. • Environmental transporters featured novel substrate specificities.

Multi-metal tolerance of DHHC palmitoyl transferase-like protein isolated from metal contaminated soil.

The microbiota inhabiting in metal polluted environment develops strong defense mechanisms to combat pollution and sustain life. Investigating the functional genes of the eukaryotic microbiota inhabiting in these environments by using metatranscriptomics approach was the focus of this study. Size fractionated eukaryotic cDNA libraries (library A, < 0.5 kb, library B, 0.5-1.0 kb, and library C, > 1.0 kb) were constructed from RNA isolated from the metal contaminated soil. The library C was screened for Cadmium (Cd) tolerant genes by using Cd sensitive yeast mutant ycf1Δ by functional complementation assay, which yielded various clones capable of growing in Cd amended media. One of the Cd tolerant clones, PLCg39 was selected because of its ability to grow at high concentrations of Cd. Sequence analysis of PLCg39 showed homology with DHHC palmitoyl transferases, which are responsible for addition of palmitoyl groups to proteins and usually possess metal coordination domains. The cDNA PLCg39 was able to confer tolerance to Cd-sensitive (ycf1Δ), Copper-sensitive (cup1Δ) and Zn-sensitive (zrc1Δ) yeast mutants when grown at different concentrations of Cd (40-100 µM), Cu (150-1000 µM) and Zn (10-13 mM), respectively. The DHHC mutant akr1Δ transformed with PLCg39 rescued from the metal sensitivity indicating the role of DHHC palmitoyl transferase in metal tolerance. This study demonstrated that PLCg39 acts as a potential metal tolerant gene which could be used in bioremediation, biosensing and other biotechnological fields.

Metal induction of a Pisolithus albus metallothionein and its potential involvement in heavy metal tolerance during mycorrhizal symbiosis.

Metallothioneins (MTs) are small, cysteine-rich peptides involved in intracellular sequestration of heavy metals in eukaryotes. We examined the role in metal homeostasis and detoxification of an MT from the ectomycorrhizal fungus Pisolithus albus (PaMT1). PaMT1 encodes a 35 amino acid-long polypeptide, with 7 cysteine residues; most of them part of a C-x-C motif found in other known basidiomycete MTs. The expression levels of PaMT1 increased as a function of increased external Cu and Cd concentrations and were higher with Cu than with Cd. Heterologous complementation assays in metal-sensitive yeast mutants indicated that PaMT1 encodes a polypeptide capable of conferring higher tolerance to both Cu and Cd. Eucalyptus tereticornis plantlets colonized with P. albus grown in the presence of Cu and Cd showed better growth compared with those with non-mycorrhizal plants. Higher PaMT1 expression levels were recorded in mycorrhizal plants grown in the presence of Cu and Cd compared with those in control mycorrhizal plants not exposed to heavy metals. These data provide the first evidence to our knowledge that fungal MTs could protect ectomycorrhizal fungi from heavy metal stress and in turn help the plants to establish in metal-contaminated sites.

Construction of sized eukaryotic cDNA libraries using low input of total environmental metatranscriptomic RNA.

BACKGROUND: Construction of high quality cDNA libraries from the usually low amounts of eukaryotic mRNA extracted from environmental samples is essential in functional metatranscriptomics for the selection of functional, full-length genes encoding proteins of interest. Many of the inserts in libraries constructed by standard methods are represented by truncated cDNAs due to premature stoppage of reverse transcriptase activity and preferential cloning of short cDNAs. RESULTS: We report here a simple and cost effective technique for preparation of sized eukaryotic cDNA libraries from as low as three microgram of total soil RNA dominated by ribosomal and bacterial RNA. cDNAs synthesized by a template switching approach were size-fractionated by two dimensional agarose gel electrophoresis prior to PCR amplification and cloning. Effective size selection was demonstrated by PCR amplification of conserved gene families specific of each size class. Libraries of more than one million independent inserts whose sizes ranged between one and four kb were thus produced. Up to 80% of the insert sequences were homologous to eukaryotic gene sequences present in public databases. CONCLUSIONS: A simple and cost effective technique has been developed to construct sized eukaryotic cDNA libraries from environmental samples. This technique will facilitate expression cloning of environmental eukaryotic genes and contribute to a better understanding of basic biological and/or ecological processes carried out by eukaryotic microbial communities.

Under explored roles of microbial ligninolytic enzymes in aerobic polychlorinated biphenyl transformation.

Polychlorinated biphenyls (PCBs) are persistent organic pollutants in the environment that are responsible for many adverse health effects. Bioremediation appears to be a healthy and cost-effective alternative for remediating PCB-contaminated environments. While some microbial species have been observed to be capable of transforming PCBs, only two different microbial pathways (rdh and bph pathways) have been described to be involved in PCB transformations. Ligninolytic enzymes have been observed or are under suspicion in some microbial PCB transformations. However, the role of these promising PCB-transforming enzymes, which are produced by fungi and some aerobic bacteria, is still unclear. The present review describes their role by identifying microbial PCB-transforming species and their reported ligninolytic enzymes whether proven or suspected to be involved in PCB transformations. There are several lines of evidence that ligninolytic enzymes are responsible for PCB transformations such as (1) the ability of purified laccases from Myceliophthora thermophila, Pycnoporus cinnabarinus, Trametes versicolor, Cladosporium sp, and Coprinus cumatus to transform hydroxy-PCBs; (2) the increased production of laccases and peroxidases by many fungi in the presence of PCBs; and (3) the enhanced PCB transformation by Pseudomonas stutzeri and Sinorhizobium meliloti NM after the addition of ligninolytic enzyme enhancers. However, if the involvement of ligninolytic enzymes in PCB transformation is clearly demonstrated in some fungal species, it does not seem to be implicated in all microbial species suggesting other still unknown metabolic pathways involved in PCB transformation and different from the bph and rdh pathways. Therefore, PCB transformation may involve several metabolic pathways, some involving ligninolytic enzymes, bph or rdh genes, and some still unknown, depending on the microbial species. In addition, current knowledge does not fully clarify the role of ligninolytic enzymes in PCB oxidation and dechlorination. Therefore, further studies focusing on purified ligninolytic enzymes are needed to clearly elucidate their role in PCB transformation.

Soil metatranscriptomics for mining eukaryotic heavy metal resistance genes.

Heavy metals are pollutants which affect all organisms. Since a small number of eukaryotes have been investigated with respect to metal resistance, we hypothesize that many genes that control this phenomenon remain to be identified. This was tested by screening soil eukaryotic metatranscriptomes which encompass RNA from organisms belonging to the main eukaryotic phyla. Soil-extracted polyadenylated mRNAs were converted into cDNAs and 35 of them were selected for their ability to rescue the metal (Cd or Zn) sensitive phenotype of yeast mutants. Few of the genes belonged to families known to confer metal resistance when overexpressed in yeast. Several of them were homologous to genes that had not been studied in the context of metal resistance. For instance, the BOLA ones, which conferred cross metal (Zn, Co, Cd, Mn) resistance may act by interfering with Fe homeostasis. Other genes, such as those encoding 110- to 130-amino-acid-long, cysteine-rich polypeptides, had no homologues in databases. This study confirms that functional metatranscriptomics represents a powerful approach to address basic biological processes in eukaryotes. The selected genes can be used to probe new pathways involved in metal homeostasis and to manipulate the resistance level of selected organisms.

Polychlorinated Biphenyl Transformation, Peroxidase and Oxidase Activities of Fungi and Bacteria Isolated from a Historically Contaminated Site.

Causing major health and ecological disturbances, polychlorinated biphenyls (PCBs) are persistent organic pollutants still recovered all over the world. Microbial PCB biotransformation is a promising technique for depollution, but the involved molecular mechanisms remain misunderstood. Ligninolytic enzymes are suspected to be involved in many PCB transformations, but their assessments remain scarce. To further inventory the capabilities of microbes to transform PCBs through their ligninolytic enzymes, we investigated the role of oxidase and peroxidase among a set of microorganisms isolated from a historically PCB-contaminated site. Among 29 isolated fungi and 17 bacteria, this work reports for the first time the PCB-transforming capabilities from fungi affiliated to Didymella, Dothiora, Ilyonectria, Naganishia, Rhodoturula, Solicoccozyma, Thelebolus and Truncatella genera and bacteria affiliated to Peribacillus frigotolerans, Peribacillus muralis, Bacillus mycoides, Bacillus cereus, Bacillus toyonensis, Pseudarthrobacter sp., Pseudomonas chlororaphis, Erwinia aphidicola and Chryseobacterium defluvii. In the same way, this is the first report of fungal isolates affiliated to the Dothiora maculans specie and Cladosporium genus that displayed oxidase (putatively laccase) and peroxidase activity, respectively, enhanced in the presence of PCBs (more than 4-fold and 20-fold, respectively, compared to controls). Based on these results, the observed activities are suspected to be involved in PCB transformation.

Optimized assay and storage conditions for enzyme activity profiling of ectomycorrhizae.

The aim of a joint effort by different research teams was to provide an improved procedure for enzyme activity profiling of field-sampled ectomycorrhizae, including recommendations on the best conditions and maximum duration for storage of ectomycorrhizal samples. A more simplified and efficient protocol compared to formerly published procedures was achieved by using manufactured 96-filter plates in combination with a vacuum manifold and by optimizing incubation times. Major improvements were achieved by performing the series of eight enzyme assays with a single series of root samples instead of two series, reducing the time needed for sample preparation, minimizing error-prone steps such as pipetting and morphotyping, and facilitating subsequent DNA analyses due to the reduced sequencing effort. The best preservation of samples proved to be storage in soil at 4-6 °C in the form of undisturbed soil cores containing roots. Enzyme activities were maintained for up to 4 weeks under these conditions. Short-term storage of washed roots and ectomycorrhizal tips overnight in water did not cause substantial changes in enzyme activity profiles. No optimal means for longer-term storage by freezing at -20 °C or storage in 100% ethanol were recommended.

Eukaryotic Cell Capture by Amplified Magnetic in situ Hybridization Using Yeast as a Model.

A non-destructive approach based on magnetic in situ hybridization (MISH) and hybridization chain reaction (HCR) for the specific capture of eukaryotic cells has been developed. As a prerequisite, a HCR-MISH procedure initially used for tracking bacterial cells was here adapted for the first time to target eukaryotic cells using a universal eukaryotic probe, Euk-516R. Following labeling with superparamagnetic nanoparticles, cells from the model eukaryotic microorganism Saccharomyces cerevisiae were hybridized and isolated on a micro-magnet array. In addition, the eukaryotic cells were successfully targeted in an artificial mixture comprising bacterial cells, thus providing evidence that HCR-MISH is a promising technology to use for specific microeukaryote capture in complex microbial communities allowing their further morphological characterization. This new study opens great opportunities in ecological sciences, thus allowing the detection of specific cells in more complex cellular mixtures in the near future.