Searching for new plastic-degrading enzymes from the plastisphere of alpine soils using a metagenomic mining approach.

Plastic materials, including microplastics, accumulate in all types of ecosystems, even in remote and cold environments such as the European Alps. This pollution poses a risk for the environment and humans and needs to be addressed. Using shotgun DNA metagenomics of soils collected in the eastern Swiss Alps at about 3,000 m a.s.l., we identified genes and their proteins that potentially can degrade plastics. We screened the metagenomes of the plastisphere and the bulk soil with a differential abundance analysis, conducted similarity-based screening with specific databases dedicated to putative plastic-degrading genes, and selected those genes with a high probability of signal peptides for extracellular export and a high confidence for functional domains. This procedure resulted in a final list of nine candidate genes. The lengths of the predicted proteins were between 425 and 845 amino acids, and the predicted genera producing these proteins belonged mainly to Caballeronia and Bradyrhizobium. We applied functional validation, using heterologous expression followed by enzymatic assays of the supernatant. Five of the nine proteins tested showed significantly increased activities when we used an esterase assay, and one of these five proteins from candidate genes, a hydrolase-type esterase, clearly had the highest activity, by more than double. We performed the fluorescence assays for plastic degradation of the plastic types BI-OPL and ecovio® only with proteins from the five candidate genes that were positively active in the esterase assay, but like the negative controls, these did not show any significantly increased activity. In contrast, the activity of the positive control, which contained a PLA-degrading gene insert known from the literature, was more than 20 times higher than that of the negative controls. These findings suggest that in silico screening followed by functional validation is suitable for finding new plastic-degrading enzymes. Although we only found one new esterase enzyme, our approach has the potential to be applied to any type of soil and to plastics in various ecosystems to search rapidly and efficiently for new plastic-degrading enzymes.

Long-term mercury contamination does not affect the microbial gene potential for C and N cycling in soils but enhances detoxification gene abundance.

Soil microorganisms are key transformers of mercury (Hg), a toxic and widespread pollutant. It remains uncertain, however, how long-term exposure to Hg affects crucial microbial functions, such as litter decomposition and nitrogen cycling. Here, we used a metagenomic approach to investigate the state of soil functions in an agricultural floodplain contaminated with Hg for more than 80 years. We sampled soils along a gradient of Hg contamination (high, moderate, low). Hg concentrations at the highly contaminated site (36 mg kg-1 dry soil on average) were approximately 10 times higher than at the moderately contaminated site (3 mg kg-1 dry soil) and more than 100 times higher than at the site with low contamination (0.25 mg kg-1 dry soil; corresponding to the natural background concentration in Switzerland). The analysis of the CAZy and NCyc databases showed that carbon and nitrogen cycling was not strongly affected with high Hg concentrations, although a significant change in the beta-diversity of the predicted genes was observed. The only functional classes from the CAZy database that were significantly positively overrepresented under higher Hg concentrations were genes involved in pectin degradation, and from the NCyc database dissimilatory nitrate reduction and N-fixation. When comparing between low and high Hg concentrations the genes of the EggNOG functional category of inorganic ion transport and metabolism, two genes encoding Hg transport proteins and one gene involved in heavy metal transport detoxification were among those that were highly significantly overrepresented. A look at genes specifically involved in detoxification of Hg species, such as the mer and hgc genes, showed a significant overrepresentation when Hg contamination was increased. Normalized counts of these genes revealed a dominant role for the phylum Proteobacteria. In particular, most counts for almost all mer genes were found in Betaproteobacteria. In contrast, hgc genes were most abundant in Desulfuromonadales. Overall, we conclude from this metagenomic analysis that long-term exposure to high Hg triggers shifts in the functional beta-diversity of the predicted microbial genes, but we do not see a dramatic change or breakdown in functional capabilities, but rather functional redundancy.