Plasticity of a holobiont: desiccation induces fasting-like metabolism within the lichen microbiota.

The role of host-associated microbiota in enduring dehydration and drought is largely unknown. We have used lichens to study this increasingly important problem because they are the organisms that are optimally adapted to reoccurring hydration/dehydration cycles, and they host a defined and persistent bacterial community. The analysis of metatranscriptomic datasets from bacterial communities of the lung lichen (Lobaria pulmonaria (L.) Hoffm.), sampled under representative hydration stages, revealed significant structural shifts and functional specialization to host conditions. The hydrated samples showed upregulated transcription of transport systems, tRNA modification and various porins (Omp2b by Rhizobiales), whereas the desiccated samples showed different functions related to stress adaption prominently. Carbohydrate metabolism was activated under both conditions. Under dry conditions, upregulation of a specialized ketone metabolism indicated a switch to lipid-based nutrition. Several bacterial lineages were involved in a functional transition that was reminiscent of a 'fasting metaorganism'. Similar functional adaptions were assigned to taxonomically unrelated groups, indicating hydration-related specialization of the microbiota. We were able to show that host-associated bacterial communities are well adapted to dehydration by stress protection and changes of the metabolism. Moreover, our results indicate an intense interplay in holobiont functioning under drought stress.

Adaptions of Lichen Microbiota Functioning Under Persistent Exposure to Arsenic Contamination.

Host-associated microbiota play an important role in the health and persistence of more complex organisms. In this study, metagenomic analyses were used to reveal microbial community adaptations in three lichen samples as a response to different arsenic concentrations at the sampling sites. Elevated arsenic concentrations at a former mining site expanded the spectrum and number of relevant functions in the lichen-associated microorganisms. Apparent changes affected the abundance of numerous detoxification-related genes, they were substantially enhanced in arsenic-polluted samples. Complementary quantifications of the arsenite S-adenosylmethionine methyltransferase (arsM) gene showed that its abundance is not strictly responding to the environmental arsenic concentrations. The analyzed samples contained rather low numbers of the arsM gene with a maximum of 202 gene copies µl-1 in total community DNA extracts. In addition, bacterial isolates were screened for the presence of arsM. Positive isolates were exposed to different As(III) and As(V) concentrations and tolerated up to 30 mM inorganic arsenic in fluid media, while no substantial biotransformations were observed. Obtained data deepens our understanding related to adaptions of whole microbial communities to adverse environmental conditions. Moreover, this study provides the first evidence that the integrity of bacteria in the lichen holobiont is maintained by acquisition of specific resistances.

Deciphering functional diversification within the lichen microbiota by meta-omics.

BACKGROUND: Recent evidence of specific bacterial communities extended the traditional concept of fungal-algal lichen symbioses by a further organismal kingdom. Although functional roles were already assigned to dominant members of the highly diversified microbiota, a substantial fraction of the ubiquitous colonizers remained unexplored. We employed a multi-omics approach to further characterize functional guilds in an unconventional model system. RESULTS: The general community structure of the lichen-associated microbiota was shown to be highly similar irrespective of the employed omics approach. Five highly abundant bacterial orders-Sphingomonadales, Rhodospirillales, Myxococcales, Chthoniobacterales, and Sphingobacteriales-harbor functions that are of substantial importance for the holobiome. Identified functions range from the provision of vitamins and cofactors to the degradation of phenolic compounds like phenylpropanoid, xylenols, and cresols. CONCLUSIONS: Functions that facilitate the persistence of Lobaria pulmonaria under unfavorable conditions were present in previously overlooked fractions of the microbiota. So far, unrecognized groups like Chthoniobacterales (Verrucomicrobia) emerged as functional protectors in the lichen microbiome. By combining multi-omics and imaging techniques, we highlight previously overlooked participants in the complex microenvironment of the lichens.

Symbiotic Interplay of Fungi, Algae, and Bacteria within the Lung Lichen Lobaria pulmonaria L. Hoffm. as Assessed by State-of-the-Art Metaproteomics.

Lichens are recognized by macroscopic structures formed by a heterotrophic fungus, the mycobiont, which hosts internal autotrophic photosynthetic algal and/or cyanobacterial partners, referred to as the photobiont. We analyzed the structure and functionality of the entire lung lichen Lobaria pulmonaria L. Hoffm. collected from two different sites by state-of-the-art metaproteomics. In addition to the green algae and the ascomycetous fungus, a lichenicolous fungus as well as a complex prokaryotic community (different from the cyanobacteria) was found, the latter dominated by methanotrophic Rhizobiales. Various partner-specific proteins could be assigned to the different lichen symbionts, for example, fungal proteins involved in vesicle transport, algal proteins functioning in photosynthesis, cyanobacterial nitrogenase and GOGAT involved in nitrogen fixation, and bacterial enzymes responsible for methanol/C1-compound metabolism as well as CO-detoxification. Structural and functional information on proteins expressed by the lichen community complemented and extended our recent symbiosis model depicting the functional multiplayer network of single holobiont partners.1 Our new metaproteome analysis strongly supports the hypothesis (i) that interactions within the self-supporting association are multifaceted and (ii) that the strategy of functional diversification within the single lichen partners may support the longevity of L. pulmonaria under certain ecological conditions.

Differential sharing and distinct co-occurrence networks among spatially close bacterial microbiota of bark, mosses and lichens‬‬.

Knowledge of bacterial community host-specificity has increased greatly in recent years. However, the intermicrobiome relationships of unrelated but spatially close organisms remain little understood. Trunks of trees covered by epiphytes represent complex habitats with a mosaic of ecological niches. In this context, we investigated the structure, diversity and interactions of microbiota associated with lichens, mosses and the bare tree bark. Comparative analysis revealed significant differences in the habitat-associated community structures. Corresponding co-occurrence analysis indicated that the lichen microbial network is less complex and less densely interconnected than the moss- and bark-associated networks. Several potential generalists and specialists were identified for the selected habitats. Generalists belonged mainly to Proteobacteria, with Sphingomonas as the most abundant genus. The generalists comprise microorganisms with generally beneficial features, such as nitrogen fixation or other supporting functions, according to a metagenomic analysis. We argue that beneficial strains shared among hosts contribute to ecological stability of the host biocoenoses.

Understanding Microbial Multi-Species Symbioses.

Lichens are commonly recognized as a symbiotic association of a fungus and a chlorophyll containing partner, either green algae or cyanobacteria, or both. The fungus provides a suitable habitat for the partner, which provides photosynthetically fixed carbon as energy source for the system. The evolutionary result of the self-sustaining partnership is a unique joint structure, the lichen thallus, which is indispensable for fungal sexual reproduction. The classical view of a dual symbiosis has been challenged by recent microbiome research, which revealed host-specific bacterial microbiomes. The recent results about bacterial associations with lichens symbioses corroborate their notion as a multi-species symbiosis. Multi-omics approaches have provided evidence for functional contribution by the bacterial microbiome to the entire lichen meta-organism while various abiotic and biotic factors can additionally influence the bacterial community structure. Results of current research also suggest that neighboring ecological niches influence the composition of the lichen bacterial microbiome. Specificity and functions are here reviewed based on these recent findings, converging to a holistic view of bacterial roles in lichens. Finally we propose that the lichen thallus has also evolved to function as a smart harvester of bacterial symbionts. We suggest that lichens represent an ideal model to study multi-species symbiosis, using the recently available omics tools and other cutting edge methods.

Analyzing the antagonistic potential of the lichen microbiome against pathogens by bridging metagenomic with culture studies.

Naturally occurring antagonists toward pathogens play an important role to avoid pathogen outbreaks in ecosystems, and they can be applied as biocontrol agents for crops. Lichens present long-living symbiotic systems continuously exposed to pathogens. To analyze the antagonistic potential in lichens, we studied the bacterial community active against model bacteria and fungi by an integrative approach combining isolate screening, omics techniques, and high resolution mass spectrometry. The highly diverse microbiome of the lung lichen [Lobaria pulmonaria (L.) Hoffm.] included an abundant antagonistic community dominated by Stenotrophomonas, Pseudomonas, and Burkholderia. While antagonists represent 24.5% of the isolates, they were identified with only 7% in the metagenome; which means that they were overrepresented in the culturable fraction. Isolates of the dominant antagonistic genus Stenotrophomonas produced spermidine as main bioactive component. Moreover, spermidine-related genes, especially for the transport, were identified in the metagenome. The majority of hits identified belonged to Alphaproteobacteria, while Stenotrophomonas-specific spermidine synthases were not present in the dataset. Evidence for plant growth promoting effects was found for lichen-associated strains of Stenotrophomonas. Linking of metagenomic and culture data was possible but showed partly contradictory results, which required a comparative assessment. However, we have shown that lichens are important reservoirs for antagonistic bacteria, which open broad possibilities for biotechnological applications.

A novel assay for the detection of bioactive volatiles evaluated by screening of lichen-associated bacteria.

Volatile organic compounds (VOCs) produced by microorganisms are known both for their effect on pathogens and their role as mediators in various interactions and communications. Previous studies have demonstrated the importance of VOCs for ecosystem functioning as well as their biotechnological potential, but screening for bioactive volatiles remained difficult. We have developed an efficient testing assay that is based on two multi-well plates, separated by a sealing silicone membrane, two tightening clamps, and variable growth media, or indicators. The experiment design as presented here is a novel and robust technique to identify positive as well as negative VOC effects on the growth of a target organism and to test for specific substances e.g., hydrogen cyanide which can be detected with a suitable indicator. While the first pre-screening assay is primarily based on indicator color change and visible growth diameter reduction, we also introduce an advanced and quantitatively precise experiment design. This adaptation involves qPCR-based quantification of viable target cells after concluding the treatment with VOCs. Therefore, we chose preselected active isolates and compared the partial 16S rRNA gene copy number of headspace-exposed E. coli with non-treated controls. Separately obtained headspace SPME and GC/MS-based profiles of selected bacterial isolates revealed the presence of specific and unique signatures which suggests divergent modes of action. The assay was evaluated by screening 100 isolates of lung lichen-associated bacteria. Approximately one quarter of the isolates showed VOC-based antibacterial and/or antifungal activity; mainly Pseudomonas and Stenotrophomonas species were identified as producers of bioactive volatiles.

Exploring functional contexts of symbiotic sustain within lichen-associated bacteria by comparative omics.

Symbioses represent a frequent and successful lifestyle on earth and lichens are one of their classic examples. Recently, bacterial communities were identified as stable, specific and structurally integrated partners of the lichen symbiosis, but their role has remained largely elusive in comparison to the well-known functions of the fungal and algal partners. We have explored the metabolic potentials of the microbiome using the lung lichen Lobaria pulmonaria as the model. Metagenomic and proteomic data were comparatively assessed and visualized by Voronoi treemaps. The study was complemented with molecular, microscopic and physiological assays. We have found that more than 800 bacterial species have the ability to contribute multiple aspects to the symbiotic system, including essential functions such as (i) nutrient supply, especially nitrogen, phosphorous and sulfur, (ii) resistance against biotic stress factors (that is, pathogen defense), (iii) resistance against abiotic factors, (iv) support of photosynthesis by provision of vitamin B12, (v) fungal and algal growth support by provision of hormones, (vi) detoxification of metabolites, and (vii) degradation of older parts of the lichen thallus. Our findings showed the potential of lichen-associated bacteria to interact with the fungal as well as algal partner to support health, growth and fitness of their hosts. We developed a model of the symbiosis depicting the functional multi-player network of the participants, and argue that the strategy of functional diversification in lichens supports the longevity and persistence of lichens under extreme and changing ecological conditions.

Microbial cargo: do bacteria on symbiotic propagules reinforce the microbiome of lichens?

According to recent research, bacteria contribute as recurrent associates to the lichen symbiosis. Yet, the variation of the microbiomes within species and across geographically separated populations remained largely elusive. As a quite common dispersal mode, lichens evolved vertical transmission of both fungal and algal partners in specifically designed mitotic propagules. Bacteria, if co-transmitted with these symbiotic propagules, could contribute to a geographical structure of lichen-associated microbiomes. The lung lichen was sampled from three localities in eastern Austria to analyse their associated bacterial communities by bar-coded pyrosequencing, network analysis and fluorescence in situ hybridization. For the first time, bacteria were documented to colonize symbiotic propagules of lichens developed for short-distance transmission of the symbionts. The propagules share the overall bacterial community structure with the thalli at class level, except for filamentous Cyanobacteria (Nostocophycideae), and with Alphaproteobacteria as predominant group. All three sampling sites share a core fraction of the microbiome. Bacterial communities of lichen thalli from the same sampling site showed higher similarity than those of distant populations. This variation and the potential co-dispersal of a microbiome fraction with structures of the host organism contribute new aspects to the 'everything is everywhere' hypothesis.

The cancer stem cell subtype determines immune infiltration of glioblastoma.

Immune cell infiltration varies widely between different glioblastomas (GBMs). The underlying mechanism, however, remains unknown. Here we show that TGF-beta regulates proliferation, migration, and tumorigenicity of mesenchymal GBM cancer stem cells (CSCs) in vivo and in vitro. In contrast, proneural GBM CSCs resisted TGF-beta due to TGFR2 deficiency. In vivo, a substantially increased infiltration of immune cells was observed in mesenchymal GBMs, while immune infiltrates were rare in proneural GBMs. On a functional level, proneural CSC lines caused a significantly stronger TGF-beta-dependent suppression of NKG2D expression on CD8(+) T and NK cells in vitro providing a mechanistic explanation for the reduced immune infiltration of proneural GBMs. Thus, the molecular subtype of CSCs TGF-beta-dependently contributes to the degree of immune infiltration.

Identification of CD133(-)/telomerase(low) progenitor cells in glioblastoma-derived cancer stem cell lines.

Glioblastoma multiforme (GBM) is paradigmatic for the investigation of cancer stem cells (CSC) in solid tumors. The CSC hypothesis implies that tumors are maintained by a rare subpopulation of CSC that gives rise to rapidly proliferating progenitor cells. Although the presence of progenitor cells is crucial for the CSC hypothesis, progenitor cells derived from GBM CSC are yet uncharacterized. We analyzed human CD133(+) CSC lines that were directly derived from CD133(+) primary astrocytic GBM. In these CSC lines, CD133(+)/telomerase(high) CSC give rise to non-tumorigenic, CD133(-)/telomerase(low) progenitor cells. The proliferation of the progenitor cell population results in significant telomere shortening as compared to the CD133(+) compartment comprising CSC. The average difference in telomere length as determined by a modified multi-color flow fluorescent in situ hybridization was 320 bp corresponding to 4-8 cell divisions. Taken together, we demonstrate that CD133(+) primary astrocytic GBM comprise proliferating, CD133(-)/telomerase(low) progenitor cell population characterized by low telomerase activity and shortened telomeres as compared to CSC.