Basidiomycetes to the rescue: Mycoremediation of metal-organics co-contaminated soils.

The increasing need for metals leads to contaminated post-mining landscapes. At the same time, the contamination with organic, recalcitrant contamination increases. This poses a problem of reuse of large areas, often co-contaminated with both, metals, and organic pollutants. For the remediation of areas contaminated with multiple contaminants and combining many stress factors, technical solutions including groundwater treatment, where necessary, have been devised. However, this is applied to highly contaminated, small sites. The reuse of larger, co-contaminated landscapes remains a major challenge. Mycoremediation with fungi offers a good option for such areas. Fungi cope particularly well with heterogeneous conditions due to their adaptability and their large hyphal network. This chapter summarizes the advantages of basidiomycetes with a focus on wood rot fungi in terms of their ability to tolerate metals, radionuclides, and organic contaminants such as polycyclic aromatic hydrocarbons. It also shows how these fungi can reduce toxicity of contaminants to other organisms including plants to allow for restored land-use. The processes based on diverse molecular mechanisms are introduced and their use for mycoremediation is discussed.

Biofilm formation in Gram-positives as an answer to combined salt and metal stress.

Biofilm formation can lead to tolerance against stressors like antibiotics, toxic metals, salts, and other environmental contaminants. Halo- and metal-tolerant bacilli and actinomycete strains isolated from a former uranium mining and milling site in Germany were shown to form biofilm in response to salt and metal treatment; specifically, Cs and Sr exposition led to biofilm formation. Since the strains were obtained from soil samples, a more structured environment was tested using expanded clay to provide porous structures resembling the natural environment. There, accumulation of Cs could be shown for Bacillus sp. SB53B, and high Sr accumulation ranging from 75% to 90% was seen with all isolates tested. We could, therefore, show that biofilms in a structured environment like soil will contribute to the water purification obtained by the passage of water through the critical zone of soil, providing an ecosystem benefit that can hardly be overestimated.

Extremophile Metal Resistance: Plasmid-Encoded Functions in Streptomyces mirabilis.

The extreme metal tolerance of up to 130 mM NiSO4 in Streptomyces mirabilis P16B-1 was investigated. Genome sequencing revealed the presence of a large linear plasmid, pI. To identify plasmid-encoded determinants of metal resistance, a newly established transformation system was used to characterize the predicted plasmid-encoded loci nreB, hoxN, and copYZ. Reintroduction into the plasmid-cured S. mirabilis ΔpI confirmed that the predicted metal transporter gene nreB constitutes a nickel resistance factor, which was further supported by its heterologous expression in Escherichia coli. In contrast, the predicted nickel exporter gene hoxN decreased nickel tolerance, while copper tolerance was enhanced. The predicted copper-dependent transcriptional regulator gene copY did not induce tolerance toward either metal. Since genes for transfer were identified on the plasmid, its conjugational transfer to the metal-sensitive Streptomyces lividans TK24 was checked. This resulted in acquired tolerance toward 30 mM nickel and additionally increased the tolerance toward copper and cobalt, while oxidative stress tolerance remained unchanged. Intracellular nickel concentrations decreased in the transconjugant strain. The high extracellular nickel concentrations allowed for biomineralization. Plasmid transfer could also be confirmed into the co-occurring actinomycete Kribbella spp. in soil microcosms. IMPORTANCE Living in extremely metal-rich environments requires specific adaptations, and often, specific metal tolerance genes are encoded on a transferable plasmid. Here, Streptomyces mirabilis P16B-1, isolated from a former mining area and able to grow with up to 130 mM NiSO4, was investigated. The bacterial chromosome, as well as a giant plasmid, was sequenced. The plasmid-borne gene nreB was confirmed to confer metal resistance. A newly established transformation system allowed us to construct a plasmid-cured S. mirabilis as well as an nreB-rescued strain in addition to confirming nreB encoding nickel resistance if heterologously expressed in E. coli. The potential of intra- and interspecific plasmid transfer, together with the presence of metal resistance factors on that plasmid, underlines the importance of plasmids for transfer of resistance factors within a bacterial soil community.

Geosmin synthase ges1 knock-down by siRNA in the dikaryotic fungus Tricholoma vaccinum.

Genetic manipulation for generating knock-out experiments is essential in deciphering the precise function of a gene. However, dikaryotic fungi pose the inherent challenge of having two allelic versions of each gene, one in each nucleus. In addition, they often are slow-growing and do not withstand protoplasting, which is why Agrobacterium tumefaciens-mediated transformation has been adapted. To obtain knock-out strains, however, is not feasible with a mere deletion construct transformation and screening for deletions in both nuclear copies. Hence, a convenient method using chemically synthesized dicer substrate interfering RNA (DsiRNA) for posttranscriptional interference of targeted mRNA was developed, based on the fungal dicer/argonaute system inherent in fungi for sequence recognition and degradation. A proof-of-principle using this newly established method for knock-down of the volatile geosmin is presented in the dikaryotic fungus Tricholoma vaccinum that is forming ectomycorrhizal symbiosis with spruce trees. The gene ges1, a terpene synthase, was transcribed with a 50-fold reduction in transcript levels in the knockdown strain. The volatile geosmin was slightly reduced, but not absent in the fungus carrying the knockdown construct pointing at low specificity in other terpene synthases known for that class of enzymes.

Biomineralization of Nickel Struvite Linked to Metal Resistance in Streptomyces mirabilis.

Biomineral formation is a common trait and prominent for soil Actinobacteria, including the genus Streptomyces. We investigated the formation of nickel-containing biominerals in the presence of a heavy-metal-resistant Streptomyces mirabilis P16B-1. Biomineralization was found to occur both in solid and liquid media. Minerals were identified with Raman spectroscopy and TEM-EDX to be either Mg-containing struvite produced in media containing no nickel, or Ni-struvite where Ni replaces the Mg when nickel was present in sufficient concentrations in the media. The precipitation of Ni-struvite reduced the concentration of nickel available in the medium. Therefore, Ni-struvite precipitation is an efficient mechanism for tolerance to nickel. We discuss the contribution of a plasmid-encoded nickel efflux transporter in aiding biomineralization. In the elevated local concentrations of Ni surrounding the cells carrying this plasmid, more biominerals occurred supporting this point of view. The biominerals formed have been quantified, showing that the conditions of growth do influence mineralization. This control is also visible in differences observed to biosynthetically synthesized Ni-struvites, including the use of sterile-filtered culture supernatant. The use of the wildtype S. mirabilis P16B-1 and its plasmid-free derivative, as well as a metal-sensitive recipient, S. lividans, and the same transformed with the plasmid, allowed us to access genetic factors involved in this partial control of biomineral formation.

Phytohormones and volatile organic compounds, like geosmin, in the ectomycorrhiza of Tricholoma vaccinum and Norway spruce (Picea abies).

The ectomycorrhizospheric habitat contains a diverse pool of organisms, including the host plant, mycorrhizal fungi, and other rhizospheric microorganisms. Different signaling molecules may influence the ectomycorrhizal symbiosis. Here, we investigated the potential of the basidiomycete Tricholoma vaccinum to produce communication molecules for the interaction with its coniferous host, Norway spruce (Picea abies). We focused on the production of volatile organic compounds and phytohormones in axenic T. vaccinum cultures, identified the potential biosynthesis genes, and investigated their expression by RNA-Seq analyses. T. vaccinum released volatiles not usually associated with fungi, like limonene and ß-barbatene, and geosmin. Using stable isotope labeling, the biosynthesis of geosmin was elucidated. The geosmin biosynthesis gene ges1 of T. vaccinum was identified, and up-regulation was scored during mycorrhiza, while a different regulation was seen with mycorrhizosphere bacteria. The fungus also released the volatile phytohormone ethylene and excreted salicylic and abscisic acid as well as jasmonates into the medium. The tree excreted the auxin, indole-3-acetic acid, and its biosynthesis intermediate, indole-3-acetamide, as well as salicylic acid with its root exudates. These compounds could be shown for the first time in exudates as well as in soil of a natural ectomycorrhizospheric habitat. The effects of phytohormones present in the mycorrhizosphere on hyphal branching of T. vaccinum were assessed. Salicylic and abscisic acid changed hyphal branching in a concentration-dependent manner. Since extensive branching is important for mycorrhiza establishment, a well-balanced level of mycorrhizospheric phytohormones is necessary. The regulation thus can be expected to contribute to an interkingdom language.

Bacterial endophytes: Molecular interactions with their hosts.

Plant growth promotion has been found associated with plants on the surface (epiphytic), inside (endophytic), or close to the plant roots (rhizospheric). Endophytic bacteria mainly have been researched for their beneficial activities in terms of nutrient availability, plant growth hormones, and control of soil-borne and systemic pathogens. Molecular communications leading to these interactions between plants and endophytic bacteria are now being unrevealed using multidisciplinary approaches with advanced techniques such as metagenomics, metaproteomics, metatranscriptomics, metaproteogenomic, microRNAs, microarray, chips as well as the comparison of complete genome sequences. More than 400 genes in both the genomes of host plant and bacterial endophyte are up- or downregulated for the establishment of endophytism and plant growth-promoting activity. The involvement of more than 20 genes for endophytism, about 50 genes for direct plant growth promotion, about 25 genes for biocontrol activity, and about 10 genes for mitigation of different stresses has been identified in various bacterial endophytes. This review summarizes the progress that has been made in recent years by these modern techniques and approaches.

Organic acids, siderophores, enzymes and mechanical pressure for black slate bioweathering with the basidiomycete Schizophyllum commune.

Although many fungi are known to be able to perform bioweathering of rocks and minerals, little information is available concerning the role of basidiomycetes in this process. The wood-rotting basidiomycete Schizophyllum commune was investigated for its ability to degrade black slate, a rock rich in organic carbon. Mechanical pressure of hyphae and extracellular polymeric substances was investigated for biophysical weathering. A mixed ß1-3/ß1-6 glucan, likely schizophyllan that is well known from S. commune, could be identified on black slate surfaces. Secretion of siderophores and organic acids as biochemical weathering agents was shown. Both may contribute to biochemical weathering in addition to enzymatic functions. Previously, the exoenzyme laccase was believed to attack organic the matter within the black slate, thereby releasing metals from the rock. Here, overexpression of laccase showed enhanced dissolution of quartz phases by etching and pitting. At the same time, the formation of a new secondary mineral phase, whewellite, could be demonstrated. Hence, a more comprehensive understanding of biophysical as well as biochemical weathering by S. commune could be reached and unexpected mechanisms like quartz dissolution linked to shale degradation.

The regulator of G-protein signalling Thn1 links pheromone response to volatile production in Schizophyllum commune.

The regulator of G-protein signalling, Thn1, is involved in sexual development through pheromone signalling in the mushroom forming basidiomycete Schizophyllum commune affecting hyphal morphology and mating interactions. Thn1 plays a key role in coordinating sesquiterpene production, pheromone response and sexual development. The gene thn1 is transcriptionally regulated in response to mating with a role in clamp cell development and hydrophobin gene transcription. Further, it negatively regulates cAMP signalling and secondary metabolism. Disruption of thn1 affects dikaryotization by reducing clamp fusion and development with predominant non-fused pseudoclamps. Enhanced protein kinase A (PKA) activities in Δthn1 strains indicate that Thn1 regulates pheromone signalling by de-activating G-protein α subunits, which control cAMP-dependent PKA. The repressed formation of aerial hyphae could be linked to a reduced metabolic activity and to a transcriptional down-regulation of hyd6 and sc3 hydrophobin genes. Thn1 was also shown to be necessary for the biosynthesis of sesquiterpenes and an altered spectrum of sesquiterpenes in Δthn1 is linked to transcriptional up-regulation of biosynthesis genes. Proteome analysis indicated changes in cytoskeletal structure affecting actin localization, linking the major regulator Thn1 to growth and development of S. commune. The results support a role for Thn1 in G-protein signalling connecting development and secondary metabolism.

The making of a mushroom: Mitosis, nuclear migration and the actin network.

Basidiomycetes feature a prolonged dikaryotic life stage. A dispute over open versus closed mitosis could be solved using in vivo fluorescence videomicroscopy of histone 2B::EGFP and Lifeact labeled Schizophyllum commune. It revealed nuclei to condense to approximately one fifth in diameter during mitotic prophase. In addition, the specifics of clamp cell formation typical of many basidiomycetes included an actin network at the future site of nuclear division, which allowed for cessation of nuclear movement and re-localization of one nucleus towards the emerging clamp cell, while the other divided along the hyphal axis. Subsequent fusion of the clamp cell to form the clamp connection restored the close association of the two nuclei in a very fast process after clamp fusion. Septation was preceded by actin patches and vesicles involved in formation of the actin ring.

Smelling the difference: Transcriptome, proteome and volatilome changes after mating.

Mushrooms, such as Schizophyllum commune, have a specific odor. Whether this is linked to mating, prerequisite for mushroom formation, or also found in monokaryotic, unmated strains, was investigated with a comprehensive study on the transcriptome and proteome of this model organism. Mating interactions were investigated using a complete, cytosolic proteome map for unmated, monokaryotic, as well as for mated, dikaryotic mycelia. The regulations of the proteome were compared to transcriptional changes upon mating and to changes in smell by volatilome studies. We could show a good overlap between proteome and transcriptome data, but extensive posttranslational regulation was identified for more than 80% of transcripts. This suggests down-stream regulation upon interaction of mating partners and formation of the dikaryon that is competent to form fruiting bodies. The volatilome was shown to respond to mating by a broader spectrum of volatiles and increased emission of the mushroom smell molecules 3-octanone and 1-octen-3-ol, as well as ethanol and ß-bisabolol in the dikaryon. Putatively involved biosynthetic proteins like alcohol dehydrogenases, Ppo-like oxygenases, or sesquiterpene synthases showed correlating transcriptional regulation depending on either mono- or dikaryotic stages.

An Animal-Like Cryptochrome Controls the Chlamydomonas Sexual Cycle.

Cryptochromes are known as flavin-binding blue light receptors in bacteria, fungi, plants, and insects. The animal-like cryptochrome (aCRY) of the green alga Chlamydomonas reinhardtii has extended our view on cryptochromes, because it responds also to other wavelengths of the visible spectrum, including red light. Here, we have investigated if aCRY is involved in the regulation of the sexual life cycle of C. reinhardtii, which is controlled by blue and red light at the steps of gametogenesis along with its restoration and germination. We show that aCRY is differentially expressed not only during the life cycle but also within the cell as part of the soluble and/or membrane-associated protein fraction. Moreover, localization of aCRY within the algal cell body varies between vegetative cells and the different cell types of gametogenesis. aCRY is significantly (early day) or to a small extent (late night) enriched in the nucleus in vegetative cells. In pregametes, gametes and dark-inactivated gametes, aCRY is localized over the cell body. aCRY plays an important role in the sexual life cycle of C. reinhardtii: It controls the germination of the alga, under which the zygote undergoes meiosis, in a positive manner, similar to the regulation by the blue light receptors phototropin and plant cryptochrome (pCRY). However, aCRY acts in combination with pCRY as a negative regulator for mating ability as well as for mating maintenance, opposite to the function of phototropin in these processes.