Monthly Archives: April 2017

Article: Molecular Ecology Ressources

Posted on by

A new promising phylogenetic marker to study the diversity of fungal communities: the GLYCOSIDE HYDROLASE 63 gene L Pérez‐Izquierdo, E Morin, JP Maurice, F Martin, A Rincón, M Buée. Molecular Ecology Resources

Abstract

In molecular ecology, the development of efficient molecular markers for fungi remains an important research domain. Nuclear ribosomal internal transcribed spacer (ITS) region was proposed as universal DNA barcode marker for Fungi, but this marker was criticized for idel-induced alignment problems and its potential lack of phylogenetic resolution. Our main aim was to develop a new phylogenetic gene and a putative functional marker, from single-copy gene, to describe fungal diversity. Thus, we developed a series of primers to amplify a polymorphic region of the Glycoside Hydrolase GH63 gene, encoding exo-acting α-glucosidases, in Basidiomycetes. These primers were validated on 125 different fungal genomic DNAs and GH63 amplification yield was compared with that of already published functional markers targeting genes coding for laccases, N-acetylhexosaminidases, cellobiohydrolases and class II Peroxidases. Specific amplicons were recovered for 95% of the fungal species tested and GH63 amplification success was strikingly higher than rates obtained with other functional genes. We downloaded the GH63 sequences from 483 fungal genomes publicly available at the JGI MycoCosm database. GH63 was present in 461 fungal genomes belonging to all phyla, except Microsporidia and Neocallimastigomycotadivisions. Moreover, the phylogenetic trees built with both GH63 and Rpb1 protein sequences revealed that GH63 is also a promising phylogenetic marker. Finally, a very high proportion of GH63 proteins was predicted to be secreted. This molecular tool could be a new phylogenetic marker of fungal species as well as potential indicator of functional diversity of Basidiomycotes fungal communities in term of secretory capacities.

Article: Environmental pollution

Posted on by

Low effect of phenanthrene bioaccessibility on its biodegradation in diffusely contaminated soil M Crampon, A Cébron, F Portet-Koltalo, S Uroz, F Le Derf, J Bodilis Environmental Pollution

Abstract

This study focused on the role of bioaccessibility in the phenanthrene (PHE) biodegradation in diffusely contaminated soil, by combining chemical and microbiological approaches. First, we determined PHE dissipation rates and PHE sorption/desorption isotherms for two soils (PPY and Pv) presenting similar chronic PAH contamination, but different physico-chemical properties. Our results revealed that the PHE dissipation rate was significantly higher in the Pv soil compared to the PPY soil, while PHE sorption/desorption isotherms were similar. Interestingly, increases of PHE desorption and potentially of PHE bioaccessibility were observed for both soils when adding rhamnolipids (biosurfactants produced by Pseudomonas aeruginosa). Second, using 13C-PHE incubated in the same soils, we analyzed the PHE degrading bacterial communities. The combination of stable isotope probing (DNA-SIP) and 16S rRNA gene pyrosequencing revealed that Betaproteobacteria were the main PHE degraders in the Pv soil, while a higher bacterial diversity (Alpha-, Beta-, Gammaproteobacteria and Actinobacteria) was involved in PHE degradation in the PPY soil. The amendment of biosurfactants commonly used in biostimulation methods (i.e. rhamnolipids) to the two soils clearly modified the PHE sorption/desorption isotherms, but had no significant impact on PHE degradation rates and PHE-degraders identity. These results demonstrated that increasing the bioaccessibility of PHE has a low impact on its degradation and on the functional populations involved in this degradation.

Article: PloS one

Posted on by

Atypical protein disulfide isomerases (PDI): Comparison of the molecular and catalytic properties of poplar PDI-A and PDI-M with PDI-L1A B Selles, F Zannini, J Couturier, JP Jacquot, N Rouhier PloS one 12 (3), e0174753

Abstract

Protein disulfide isomerases are overwhelmingly multi-modular redox catalysts able to perform the formation, reduction or isomerisation of disulfide bonds. We present here the biochemical characterization of three different poplar PDI isoforms. PDI-A is characterized by a single catalytic Trx module, the so-called a domain, whereas PDI-L1a and PDI-M display an a-b-b’-a’ and a°-a-b organisation respectively. Their activities have been tested in vitro using purified recombinant proteins and a series of model substrates as insulin, NADPH thioredoxin reductase, NADP malate dehydrogenase (NADP-MDH), peroxiredoxins or RNase A. We demonstrated that PDI-A exhibited none of the usually reported activities, although the cysteines of the WCKHC active site signature are able to form a disulfide with a redox midpoint potential of -170 mV at pH 7.0. The fact that it is able to bind a [Fe2S2] cluster upon Escherichia coli expression and anaerobic purification might indicate that it does not have a function in dithiol-disulfide exchange reactions. The two other proteins were able to catalyze oxidation or reduction reactions, PDI-L1a being more efficient in most cases, except that it was unable to activate the non-physiological substrate NADP-MDH, in contrast to PDI-M. To further evaluate the contribution of the catalytic domains of PDI-M, the dicysteinic motifs have been independently mutated in each a domain. The results indicated that the two a domains seem interconnected and that the a° module preferentially catalyzed oxidation reactions whereas the a module catalyzed reduction reactions, in line with the respective redox potentials of -170 mV and -190 mV at pH 7.0. Overall, these in vitro results illustrate that the number and position of aand b domains influence the redox properties and substrate recognition (both electron donors and acceptors) of PDI which contributes to understand why this protein family expanded along evolution.