Taxonomic and functional analyses of intact microbial communities thriving in extreme, astrobiology-relevant, anoxic sites.

BACKGROUND: Extreme terrestrial, analogue environments are widely used models to study the limits of life and to infer habitability of extraterrestrial settings. In contrast to Earth's ecosystems, potential extraterrestrial biotopes are usually characterized by a lack of oxygen. METHODS: In the MASE project (Mars Analogues for Space Exploration), we selected representative anoxic analogue environments (permafrost, salt-mine, acidic lake and river, sulfur springs) for the comprehensive analysis of their microbial communities. We assessed the microbiome profile of intact cells by propidium monoazide-based amplicon and shotgun metagenome sequencing, supplemented with an extensive cultivation effort. RESULTS: The information retrieved from microbiome analyses on the intact microbial community thriving in the MASE sites, together with the isolation of 31 model microorganisms and successful binning of 15 high-quality genomes allowed us to observe principle pathways, which pinpoint specific microbial functions in the MASE sites compared to moderate environments. The microorganisms were characterized by an impressive machinery to withstand physical and chemical pressures. All levels of our analyses revealed the strong and omnipresent dependency of the microbial communities on complex organic matter. Moreover, we identified an extremotolerant cosmopolitan group of 34 poly-extremophiles thriving in all sites. CONCLUSIONS: Our results reveal the presence of a core microbiome and microbial taxonomic similarities between saline and acidic anoxic environments. Our work further emphasizes the importance of the environmental, terrestrial parameters for the functionality of a microbial community, but also reveals a high proportion of living microorganisms in extreme environments with a high adaptation potential within habitability borders. Video abstract.

The microbiome of alpine snow algae shows a specific inter-kingdom connectivity and algae-bacteria interactions with supportive capacities.

Mutualistic interactions within microbial assemblages provide a survival strategy under extreme conditions; however, little is known about the complexity of interaction networks in multipartite, free-living communities. In the present study, the interplay within algae-dominated microbial communities exposed to harsh environmental influences in the Austrian Alps was assessed in order to reveal the interconnectivity of eukaryotic and prokaryotic inhabitants. All analyzed snowfields harbored distinct microbial communities. Network analyses revealed that mutual exclusion prevailed among microalgae in the alpine environment, while bacteria were mainly positively embedded in the interaction networks. Especially members of Proteobacteria, with a high prevalence of Oxalobacteraceae, Pseudomonadaceae, and Sphingomonadaceae showed genus-specific co-occurrences with distinct microalgae. Co-cultivation experiments with algal and bacterial isolates confirmed beneficial interactions that were predicted based on the bioinformatic analyses; they resulted in up to 2.6-fold more biomass for the industrially relevant microalga Chlorella vulgaris, and up to 4.6-fold increase in biomass for the cryophilic Chloromonas typhlos. Our findings support the initial hypothesis that microbial communities exposed to adverse environmental conditions in alpine systems harbor inter-kingdom supportive capacities. The insights into mutualistic inter-kingdom interactions and the ecology of microalgae within complex microbial communities provide explanations for the prevalence and resilience of such assemblages in alpine environments.

Novel insights into plant-associated archaea and their functioning in arugula (Eruca sativa Mill.).

A plant's microbiota has various implications for the plant's health and performance; however, the roles of many microbial lineages, particularly Archaea, have not been explored in detail. In the present study, analysis of archaea-specific 16S rRNA gene fragments and shotgun-sequenced metagenomes was combined with visualization techniques to obtain the first insights into the archaeome of a common salad plant, arugula (Eruca sativa Mill.). The archaeal communities associated with the soil, rhizosphere and phyllosphere were distinct, but a high proportion of community members were shared among all analysed habitats. Soil habitats exhibited the highest diversity of Archaea, followed by the rhizosphere and the phyllosphere. The archaeal community was dominated by Thaumarchaeota and Euryarchaeota, with the most abundant taxa assigned to Candidatus Nitrosocosmicus, species of the 'Soil Crenarchaeotic Group' and, interestingly, Methanosarcina. Moreover, a large number of archaea-assigned sequences remained unassigned at lower taxonomic levels. Overall, analysis of shotgun-sequenced total-community DNA revealed a more diverse archaeome. Differences were evident at the class level and at higher taxonomic resolutions when compared to results from the 16S rRNA gene fragment amplicon library. Functional assessments primarily revealed archaeal genes related to response to stress (especially oxidative stress), CO2 fixation, and glycogen degradation. Microscopic visualizations of fluorescently labelled archaea in the phyllosphere revealed small scattered colonies, while archaea in the rhizosphere were found to be embedded within large bacterial biofilms. Altogether, Archaea were identified as a rather small but niche-specific component of the microbiomes of the widespread leafy green plant arugula.

A novel, nature-based alternative for photobioreactor decontaminations.

Large-scale microalgae cultivations are increasingly used for the production of animal feed, nutritional supplements and various high-value bioproducts. Due to the process size and other limitations, contaminations of microalgae fermentations with other photoautotrophic microorganism are frequently observed. In the present study, we explored the applicability of 5-isobutyl-2,3-dimethylpyrazine for the removal of contaminating microalgae from industrial photobioreactors. In order to select a representative microbial population for susceptibility experiments, reactor samples were obtained from a multi-stage cultivation process. Assignments of 18S rRNA gene fragment amplicons indicated that Haematococcus, Chlorella, and Scenedesmus were the three most frequently occurring microalgae genera in the selected reactors. Following the isolation of representative algae cultures, susceptibility tests were conducted with the antimicrobial pyrazine. It was demonstrated that all isolated contaminants are highly susceptible to the bioactive compound. The highest tolerance towards the alkylpyrazine was observed with Scenedesmus vacuolatus; solutions with 1.66% (v/v) of the active compound were required for its deactivation. Further tests with the vaporized pyrazine showed consistent reductions in the viability of treated microalgae. This pilot study provides evidence for the applicability of a novel, nature-based alternative for bioreactor decontaminations.

Enterobacteriaceae dominate the core microbiome and contribute to the resistome of arugula (Eruca sativa Mill.).

BACKGROUND: Arugula is a traditional medicinal plant and popular leafy green today. It is mainly consumed raw in the Western cuisine and known to contain various bioactive secondary metabolites. However, arugula has been also associated with high-profile outbreaks causing severe food-borne human diseases. A multiphasic approach integrating data from metagenomics, amplicon sequencing, and arugula-derived bacterial cultures was employed to understand the specificity of the indigenous microbiome and resistome of the edible plant parts. RESULTS: Our results indicate that arugula is colonized by a diverse, plant habitat-specific microbiota. The indigenous phyllosphere bacterial community was shown to be dominated by Enterobacteriaceae, which are well-equipped with various antibiotic resistances. Unexpectedly, the prevalence of specific resistance mechanisms targeting therapeutic antibiotics (fluoroquinolone, chloramphenicol, phenicol, macrolide, aminocoumarin) was only surpassed by efflux pump assignments. CONCLUSIONS: Enterobacteria, being core microbiome members of arugula, have a substantial implication in the overall resistome. Detailed insights into the natural occurrence of antibiotic resistances in arugula-associated microorganisms showed that the plant is a hotspot for distinctive defense mechanisms. The specific functioning of microorganisms in this unusual ecosystem provides a unique model to study antibiotic resistances in an ecological context.

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.

What Is the Role of Archaea in Plants? New Insights from the Vegetation of Alpine Bogs.

The Archaea represent a significant component of the plant microbiome, whereas their function is still unclear. Different plant species representing the natural vegetation of alpine bogs harbor a substantial archaeal community originating from five phyla, 60 genera, and 334 operational taxonomic units (OTUs). We identified a core archaeome for all bog plants and ecosystem-specific, so far unclassified Archaea In the metagenomic data set, Archaea were found to have the potential to interact with plants by (i) possible plant growth promotion through auxin biosynthesis, (ii) nutrient supply, and (iii) protection against abiotic (especially oxidative and osmotic) stress. The unexpectedly high degree of plant specificity supports plant-archaeon interactions. Moreover, functional signatures of Archaea reveal genetic capacity for the interplay with fungi and an important role in the carbon and nitrogen cycle: e.g., CO2 and N2 fixation. These facts reveal an important, yet unobserved role of the Archaea for plants as well as for the bog ecosystem.IMPORTANCEArchaea are still an underdetected and little-studied part of the plant microbiome. We provide first and novel insights into Archaea as a functional component of the plant microbiome obtained by metagenomic analyses. Archaea were found to have the potential to interact with plants by (i) plant growth promotion through auxin biosynthesis, (ii) nutrient supply, and (iii) protection against abiotic stress.

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.

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.

Complete genome sequences of the Serratia plymuthica strains 3Rp8 and 3Re4-18, two rhizosphere bacteria with antagonistic activity towards fungal phytopathogens and plant growth promoting abilities.

The Serratia plymuthica strains 3Rp8 and 3Re4-18 are motile, Gram-negative, non-sporulating bacteria. Strain 3Rp8 was isolated from the rhizosphere of Brassica napus L. and strain 3Re4-18 from the endorhiza of Solanum tuberosum L. Studies have shown in vitro activity against the soil-borne fungi Verticillium dahliae Kleb., Rhizoctonia solani Kühn, and Sclerotinia sclerotiorum. Here, we announce and describe the complete genome sequence of S. plymuthica 3Rp8 consisting of a single circular chromosome of 5.5 Mb that encodes 4954 protein-coding and 108 RNA-only encoding genes and of S. plymuthica 3Re4-18 consisting of a single circular chromosome of 5.4 Mb that encodes 4845 protein-coding and 109 RNA-only encoding genes. The whole genome sequences and annotations are available in NCBI under the locus numbers CP012096 and CP012097, respectively. The genome analyses revealed genes putatively responsible for the promising plant growth promoting and biocontrol properties including predicting factors such as secretion systems, iron scavenging siderophores, chitinases, secreted proteases, glucanases and non-ribosomal peptide synthetases, as well as unique genomic islands.

Biological Control of Lettuce Drop and Host Plant Colonization by Rhizospheric and Endophytic Streptomycetes.

Lettuce drop, caused by the soil borne pathogen Sclerotinia sclerotiorum, is one of the most common and serious diseases of lettuce worldwide. Increased concerns about the side effects of chemical pesticides have resulted in greater interest in developing biocontrol strategies against S. sclerotiorum. However, relatively little is known about the mechanisms of Streptomyces spp. as biological control agents against S. sclerotiorum on lettuce. Two Streptomyces isolates, S. exfoliatus FT05W and S. cyaneus ZEA17I, inhibit mycelial growth of Sclerotinia sclerotiorum by more than 75% in vitro. We evaluated their biocontrol activity against S. sclerotiorum in vivo, and compared them to Streptomyces lydicus WYEC 108, isolated from Actinovate®. When Streptomyces spp. (10(6) CFU/mL) were applied to S. sclerotiorum inoculated substrate in a growth chamber 1 week prior lettuce sowing, they significantly reduced the risk of lettuce drop disease, compared to the inoculated control. Interestingly, under field conditions, S. exfoliatus FT05W and S. cyaneus ZEA17I protected lettuce from drop by 40 and 10% respectively, whereas S. lydicus WYEC 108 did not show any protection. We further labeled S. exfoliatus FT05W and S. cyaneus ZEA17I with the enhanced GFP (EGFP) marker to investigate their rhizosphere competence and ability to colonize lettuce roots using confocal laser scanning microscopy (CLSM). The abundant colonization of young lettuce seedlings by both strains demonstrated Streptomyces' capability to interact with the host from early stages of seed germination and root development. Moreover, the two strains were detected also on 2-week-old roots, indicating their potential of long-term interactions with lettuce. Additionally, scanning electron microscopy (SEM) observations showed EGFP-S. exfoliatus FT05W endophytic colonization of lettuce root cortex tissues. Finally, we determined its viability and persistence in the rhizosphere and endorhiza up to 3 weeks by quantifying its concentration in these compartments. Based on these results we conclude that S. exfoliatus FT05W has high potential to be exploited in agriculture for managing soil borne diseases barely controlled by available plant protection products.

Comparisons of diazotrophic communities in native and agricultural desert ecosystems reveal plants as important drivers in diversity.

Diazotrophs provide the only biological source of fixed atmospheric nitrogen in the biosphere. Although they are the key player for plant-available nitrogen, less is known about their diversity and potential importance in arid ecosystems. We investigated the nitrogenase gene diversity in native and agricultural desert soil as well as within root-associated microbiota of medicinal plants grown in Egypt through the combination of nifH-specific qPCR, fingerprints, amplicon pyrosequencing and fluorescence in situ hybridization-confocal laser scanning microscopy. Although the diazotrophic microbiota were characterized by generally high abundances and diversity, statistically significant differences were found between both soils, the different microhabitats, and between the investigated plants (Matricaria chamomilla L., Calendula officinalis L. and Solanum distichum Schumach. and Thonn.). We observed a considerable community shift from desert to agriculturally used soil that demonstrated a higher abundance and diversity in the agro-ecosystem. The endorhiza was characterized by lower abundances and only a subset of species when compared to the rhizosphere. While the microbiomes of the Asteraceae were similar and dominated by potential root-nodulating rhizobia acquired primarily from soil, the perennial S. distichum generally formed associations with free-living nitrogen fixers. These results underline the importance of diazotrophs in desert ecosystems and additionally identify plants as important drivers in functional gene pool diversity.

Biotic stress shifted structure and abundance of Enterobacteriaceae in the lettuce microbiome.

Lettuce cultivars are not only amongst the most popular vegetables eaten raw, they are also involved in severe pathogen outbreaks world-wide. While outbreaks caused by Enterobacteriaceae species are well-studied, less is known about their occurrence in natural environments as well as the impact of biotic stress. Here, we studied the ecology of the human health-relevant bacterial family Enterobacteriaceae and assessed the impact of biotic disturbances by a soil-borne phytopathogenic fungus and Gastropoda on their structure and abundance in mesocosm and pot experiments. Using a polyphasic approach including network analyses of 16S rRNA gene amplicon libraries, quantitative PCR and complementary fluorescence in situ hybridization (FISH) microscopy we found substantial yet divergent Enterobacteriaceae communities. A similar spectrum of 14 genera was identified from rhizo- and phyllospheres but the abundance of Enterobacteriaceae was on average 3fold higher in phyllosphere samples. Both stress factors shifted the bacterial community of the leaf habitat, characterized by increases of species abundance and diversity. For the rhizosphere, we observed significant structural shifts of Enterobacteriaceae communities but also a high degree of resilience. These results could be confirmed by FISH microscopy but it was difficult to visualize phyllosphere communities. Additional inoculation experiments with Escherichia coli as model revealed their presence below the wax layer as well as in the endosphere of leaves. The observed presence influenced by stress factors and the endophytic life style of Enterobacteriaceae on lettuce can be an important aspect in relation to human health.

Rhizobiales as functional and endosymbiontic members in the lichen symbiosis of Lobaria pulmonaria L.

Rhizobiales (Alphaproteobacteria) are well-known beneficial partners in plant-microbe interactions. Less is known about the occurrence and function of Rhizobiales in the lichen symbiosis, although it has previously been shown that Alphaproteobacteria are the dominating group in growing lichen thalli. We have analyzed the taxonomic structure and assigned functions to Rhizobiales within a metagenomic dataset of the lung lichen Lobaria pulmonaria L. One third (32.2%) of the overall bacteria belong to the Rhizobiales, in particular to the families Methylobacteriaceae, Bradyrhizobiaceae, and Rhizobiaceae. About 20% of our metagenomic assignments could not be placed in any of the Rhizobiales lineages, which indicates a yet undescribed bacterial diversity. SEED-based functional analysis focused on Rhizobiales and revealed functions supporting the symbiosis, including auxin and vitamin production, nitrogen fixation and stress protection. We also have used a specifically developed probe to localize Rhizobiales by confocal laser scanning microscopy after fluorescence in situ hybridization (FISH-CLSM). Bacteria preferentially colonized fungal surfaces, but there is clear evidence that members of the Rhizobiales are able to intrude at varying depths into the interhyphal gelatinous matrix of the upper lichen cortical layer and that at least occasionally some bacteria also are capable to colonize the interior of the fungal hyphae. Interestingly, the gradual development of an endosymbiotic bacterial life was found for lichen- as well as for fungal- and plant-associated bacteria. The new tools to study Rhizobiales, FISH microscopy and comparative metagenomics, suggest a similar beneficial role for lichens than for plants and will help to better understand the Rhizobiales-host interaction and their biotechnological potential.

Bacterial networks and co-occurrence relationships in the lettuce root microbiota.

Lettuce is one of the most common raw foods worldwide, but occasionally also involved in pathogen outbreaks. To understand the correlative structure of the bacterial community as a network, we studied root microbiota of eight ancient and modern Lactuca sativa cultivars and the wild ancestor Lactuca serriola by pyrosequencing of 16S rRNA gene amplicon libraries. The lettuce microbiota was dominated by Proteobacteria and Bacteriodetes, as well as abundant Chloroflexi and Actinobacteria. Cultivar specificity comprised 12.5% of the species. Diversity indices were not different between lettuce cultivar groups but higher than in L. serriola, suggesting that domestication lead to bacterial diversification in lettuce root system. Spearman correlations between operational taxonomic units (OTUs) showed that co-occurrence prevailed over co-exclusion, and complementary fluorescence in situ hybridization-confocal laser scanning microscopy (FISH-CLSM) analyses revealed that this pattern results from both potential interactions and habitat sharing. Predominant taxa, such as Pseudomonas, Flavobacterium and Sphingomonadaceae rather suggested interactions, even though these are not necessarily part of significant modules in the co-occurrence networks. Without any need for complex interactions, single organisms are able to invade into this microbial network and to colonize lettuce plants, a fact that can influence the susceptibility to pathogens. The approach to combine co-occurrence analysis and FISH-CLSM allows reliably reconstructing and interpreting microbial interaction networks.

Vegetable microbiomes: is there a connection among opportunistic infections, human health and our 'gut feeling'?

The highly diverse microbiomes of vegetables are reservoirs for opportunistic and emerging pathogens. In recent years, an increased consumption, larger scale production and more efficient distribution of vegetables together with an increased number of immunocompromised individuals resulted in an enhanced number of documented outbreaks of human infections associated with the consumption of vegetables. Here we discuss the occurrence of potential pathogens in vegetable microbiomes, the impact of farming and processing practices, and plant and human health issues. Based on these results, we discuss the question if vegetables can serve as a source of infection for immunocompromised individuals as well as possible solutions to avoid outbreaks. Moreover, the potentially positive aspects of the vegetables microbiome for the gut microbiota and human health are presented.

The impact of the pathogen Rhizoctonia solani and its beneficial counterpart Bacillus amyloliquefaciens on the indigenous lettuce microbiome.

Lettuce belongs to the most commonly raw eaten food worldwide and its microbiome plays an important role for both human and plant health. Yet, little is known about the impact of potentially occurring pathogens and beneficial inoculants of the indigenous microorganisms associated with lettuce. To address this question we studied the impact of the phytopathogenic fungus Rhizoctonia solani and the biological control agent Bacillus amyloliquefaciens FZB42 on the indigenous rhizosphere and phyllosphere community of greenhouse-grown lettuce at two plant stages. The rhizosphere and phyllosphere gammaproteobacterial microbiomes of lettuce plants showed clear differences in their overall and core microbiome composition as well as in corresponding diversity indices. The rhizosphere was dominated by Xanthomonadaceae (48%) and Pseudomonadaceae (37%) with Rhodanobacter, Pseudoxanthomonas, Dokdonella, Luteimonas, Steroidobacter, Thermomonas as core inhabitants, while the dominating taxa associated to phyllosphere were Pseudomonadaceae (54%), Moraxellaceae (16%) and Enterobacteriaceae (25%) with Alkanindiges, Pantoea and a group of Enterobacteriaceae unclassified at genus level. The preferential occurrence of enterics in the phyllosphere was the most significant difference between both habitats. Additional enhancement of enterics on the phyllosphere was observed in bottom rot diseased lettuce plants, while Acinetobacter and Alkanindiges were identified as indicators of healthy plants. Interestingly, the microbial diversity was enhanced by treatment with both the pathogen, and the co-inoculated biological control agent. The highest impact and bacterial diversity was found by Rhizoctonia inoculation, but FZB42 lowered the impact of Rhizoctonia on the microbiome. This study shows that the indigenous microbiome shifts as a consequence to pathogen attack but FZB42 can compensate these effects, which supports their role as biocontrol agent and suggests a novel mode of action.