Pre-symbiotic response of the compatible host spruce and low-compatibility host pine to the ectomycorrhizal fungus Tricholoma vaccinum.

Mutualistic ectomycorrhizal symbiosis requires the exchange of signals even before direct contact of the partners. Volatiles, and specifically volatile terpenoids, can be detected at a distance and may trigger downstream signaling and reprogramming of metabolic responses. The late-stage ectomycorrhizal fungus Tricholoma vaccinum shows high host specificity with its main host spruce, Picea abies, while rarely associations can be found with pine, Pinus sylvestris. Hence, a comparison of the host and the low-compatibility host's responses can untangle differences in early signaling during mycorrhiza formation. We investigated sesquiterpenes and identified different patterns of phytohormone responses with spruce and pine. To test the specific role of volatiles, trees were exposed to the complete volatilome of the fungus versus volatiles present when terpene synthases were inhibited by rosuvastatin. The pleiotropic response in spruce included three non-identified products, a pyridine derivative as well as two diterpenes. In pine, other terpenoids responded to the fungal signal. Using exposure to the fungal volatilome with or without terpene synthesis inhibited, we could find a molecular explanation for the longer time needed to establish the low-compatibility interaction.

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.

Ectomycorrhizal Community Shifts at a Former Uranium Mining Site.

Ectomycorrhizal communities at young oak, pine, and birch stands in a former uranium mining site showed a low diversity of morphotypes with a preference for contact and short-distance exploration strategies formed by the fungi Russulaceae, Inocybaceae, Cortinariaceae, Thelephoraceae, Rhizopogonaceae, Tricholomataceae, as well as abundant Meliniomyces bicolor. In order to have better control over abiotic conditions, we established pot experiments with re-potted trees taken from the sites of direct investigation. This more standardized cultivation resulted in a lower diversity and decreased prominence of M. bicolor. In addition, the exploration strategies shifted to include long-distance exploration types. To mimic secondary succession with a high prevalence of fungal propagules present in the soil, inoculation of re-potted trees observed under standardized conditions for two years was used. The super-inoculation increased the effect of lower abundance and diversity of morphotypes. The contact morphotypes correlated with high Al, Cu, Fe, Sr, and U soil contents, the dark-colored short-distance exploration type did not show a specific preference for soil characteristics, and the medium fringe type with rhizomorphs on oaks correlated with total nitrogen. Thus, we could demonstrate that field trees, in a species-dependent manner, selected for ectomycorrhizal fungi with exploration types are likely to improve the plant's tolerance to specific abiotic conditions.

Salt and Metal Tolerance Involves Formation of Guttation Droplets in Species of the Aspergillus versicolor Complex.

Three strains of the Aspergillus versicolor complex were isolated from a salty marsh at a former uranium mining site in Thuringia, Germany. The strains from a metal-rich environment were not only highly salt tolerant (up to 20% NaCl), but at the same time could sustain elevated Cs and Sr (both up to 100 mM) concentrations as well as other (heavy) metals present in the environment. During growth experiments when screening for differential cell morphology, the occurrence of guttation droplets was observed, specifically when elevated Sr concentrations of 25 mM were present in the media. To analyze the potential of metal tolerance being promoted by these excretions, proteomics and metabolomics of guttation droplets were performed. Indeed, proteins involved in up-regulated metabolic activities as well as in stress responses were identified. The metabolome verified the presence of amino sugars, glucose homeostasis-regulating substances, abscisic acid and bioactive alkaloids, flavones and quinones.

Blue Light-Dependent Pre-mRNA Splicing Controls Pigment Biosynthesis in the Mushroom Terana caerulea.

Light induces the production of ink-blue pentacyclic natural products, the corticin pigments, in the cobalt crust mushroom Terana caerulea. Here, we describe the genetic locus for corticin biosynthesis and provide evidence for a light-dependent dual transcriptional/cotranscriptional regulatory mechanism. Light selectively induces the expression of the corA gene encoding the gateway enzyme, the first described mushroom polyporic acid synthetase CorA, while other biosynthetic genes for modifying enzymes necessary to complete corticin assembly are induced only at lower levels. The strongest corA induction was observed following exposure to blue and UV light. A second layer of regulation is provided by the light-dependent splicing of the three introns in the pre-mRNA of corA. Our results provide insight into the fundamental organization of how mushrooms regulate natural product biosynthesis. IMPORTANCE The regulation of natural product biosyntheses in mushrooms in response to environmental cues is poorly understood. We addressed this knowledge gap and chose the cobalt crust mushroom Terana caerulea as our model. Our work discovered a dual-level regulatory mechanism that connects light as an abiotic stimulus with a physiological response, i.e., the production of dark-blue pigments. Exposure to blue light elicits strongly increased transcription of the gene encoding the gateway enzyme, the polyporic acid synthetase CorA, that catalyzes the formation of the pigment core structure. Additionally, light is a prerequisite for the full splicing of corA pre-mRNA and, thus, its proper maturation. Dual transcriptional/cotranscriptional light-dependent control of fungal natural product biosynthesis has previously been unknown. As it allows the tight control of a key metabolic step, it may be a much more prevalent mechanism among these organisms.

Ectomycorrhizal Influence on the Dynamics of Sesquiterpene Release by Tricholoma vaccinum.

Tricholoma vaccinum is an ectomycorrhizal basidiomycete with high host specificity. The slow-growing fungus is able to produce twenty sesquiterpenes, including α-barbatene, sativene, isocaryophyllene, α-cuprenene, ß-cedrene, ß-copaene, 4-epi-α-acoradiene, and chamigrene in axenic culture. For the three major compounds, Δ6-protoilludene, ß-barbatene, and an unidentified oxygenated sesquiterpene (m/z 218.18), changed production during co-cultivation with the ectomycorrhizal partner tree, Picea abies, could be shown with distinct dynamics. During the mycorrhizal growth of T. vaccinum-P. abies, Δ6-protoilludene and the oxygenated sesquiterpene appeared at similar times, which warranted further studies of potential biosynthesis genes. In silico analyses identified a putative protoilludene synthesis gene, pie1, as being up-regulated in the mycorrhizal stage, in addition to the previously identified, co-regulated geosmin synthase, ges1. We therefore hypothesize that the sesquiterpene synthase pie1 has an important role during mycorrhization, through Δ6-protoilludene and/or its accompanied oxygenated sesquiterpene production.

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.

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.

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.

Metal adaptation and transport in hyphae of the wood-rot fungus Schizophyllum commune.

Fungi living in heavy metals and radionuclides contaminated environments, namely the Chernobyl Exclusion Zone need to be able to cope with these pollutants. In this study, the wood-rot fungus Schizophyllum commune was investigated for its metal tolerance mechanisms, and for its ability to transport such metals through its hyphae. Effects of temperature and pH on tolerance of Cs, Sr, Cd, and Zn were tested. At concentrations allowing for half-maximal growth, adapted strains were raised. The strontium-adapted strain, S. commune 12-43 Sr, showed transport of specifically Sr over distances on a cm-scale using split plates. The adaptation did not yield changes in cell or colony morphology. Intracellular metal localization was not changed, and gene expression profiles under metal stress growing on soil versus artificial medium showed a higher impact of a structured surface for growth on soil than with different metal concentrations. In the transcriptome, transporter genes were mostly down-regulated, while up-regulation was seen for genes involved in the secretory pathway under metal stress. A comparison of wildtype and adapted strains could confirm lower cellular stress levels leading to lack of glutathione S-transferase up-regulation in the adapted strain. Thus, we could show metal transport as well as specific mechanisms in metal stress avoidance.