Synergistic response of extremophiles in cyanobacterial crusts against in-situ exposure to multiple stratospheric stresses

doi:10.21203/rs.3.rs-3868504/v1

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Synergistic response of extremophiles in cyanobacterial crusts against in-situ exposure to multiple stratospheric stresses

https://doi.org/10.21203/rs.3.rs-3868504/v1

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Background

The stratosphere, with its harsh conditions similar to the Martian surface, provides a unique and relatively accessible environment for studying the adaptation of extremophiles in anticipation of extraterrestrial colonization applications. However, we are just beginning to understand the synergistic response of microbial communities under this Mars-like near space. Using cyanobacterial crusts from deserts, as a multitrophic model system, we loaded it on a balloon-borne astrobiology platform for direct exposure to multiple stratospheric stresses such as high radiation and extreme temperature fluctuations. We then performed multi-omics analyses to delineate the underlying composition alterations and metabolic response at the community level.

Results

After short-term in-situ exposure, a significant shift in the community composition of active members was observed that the relative abundance of photoautotrophs declined while chemotrophic abundance increased. We tracked the changes in metabolic processes against the stratospheric ambiance and found that life history strategies associated with resource acquisition, growth potential, and stress tolerance were regulated distinctively in different microbial categories. For instance, α-proteobacteria and chloroflexi tended to enhance the strategies related to the ability of stress tolerance, while actinobacteria assigned more resources to reinforce their growth potential. Cyanobacteria contributed to the promotion of different strategies, indicating their significant adaptability differentiation. Moreover, we demonstrated the unique thriving of Scytonema, a diazotrophic genus of cyanobacteria, attributed to its synthesis of anti-ultraviolet scytonemin, diverse material, and energy acquisition. These synergistic responses further induced interspecies mutualistic nutrient interactions, thus promoting the retention of organic carbon and nitrogen within the community, finally maintaining the stability of biocrusts in extreme environments of the stratosphere.

Conclusions

Our study underscores the adaptive resilience of cyanobacterial crusts under stratospheric conditions, with a notable shift in microbial composition and metabolic strategies. The robustness of Scytonema, particularly its unique survival capabilities, highlights its potential for extraterrestrial colonization. These findings expand our understanding of microbial proliferation in extreme environments, providing valuable insights for future astrobiological endeavors.

Balloon-borne platform

Biological soil crusts

Cyanobacteria

Life history strategies

Metabolic activity

Metagenome-assembled genomes

Metatranscriptome

Near space

The stratosphere, located 20 ~ 50 km above sea level, is characterized by intense ultraviolet radiation (UV, ~ 100 W/m²), energetic ions, low temperatures (~ -36°C), low pressure (~ 1 kPa), and aridity (relative humidity < 1%), often referred to as a Mars-like environment ^{[1, 2]}. Its unique features make it an ideal site for extraterrestrial environment research ^{[1, 2]}. Although various bacterial and archaeal strains, particularly spore-forming ones ^{[3, 4]}, have been recently collected and isolated from the stratospheric environment ^{[5, 6]}, understanding the response and adaptation of Earth’s extremophiles to near space in-situ exposure remains crucial for astrobiology.

The stratospheric multiple stresses, especially UV radiation than drought and low temperature and pressure, pose significant challenges to microbial survival ^{[1, 7, 8]}. The majority of strains, such as those belonging to the genera Bacillus and Halobacterium, show decreased survival rates, while some can persist through unknown mechanisms ^[2]. In addition, non-spore-forming bacteria and archaea employ distinct strategies like higher genomic GC content, aggregate formation, and stress repair protein expression to withstand adversity ^[6]. However, current research in the International Space Station primarily focuses on microbiology related to human health, such as gut microbiota dynamics and pathogenic bacteria resistance, and merely examines the influence of microgravity ^[9–12] (but see ref. ^[13]). It is of pivotal importance to delineate the synergistic response of natural multitrophic communities directly to complex stresses of the stratosphere, as an inescapable step toward the potential application for extraterrestrial colonization.

Most stratospheric exposure experiments to date largely concentrated on heterotrophs, leaving a research gap in the study of photoautotrophs in such high-brightness environments ^{[14, 15]}. Nonetheless, cyanobacteria, particularly genera like Chroococcidiopsis and Nostoc, present an excellent opportunity for these investigations ^[13]. Some studies have found these cyanobacteria maintain activities only when mixed with simulated Martian soil, underscoring the key role of physical shielding for survival ^[16]. As a model system for community ecology ^[17], natural biocrusts in drylands formed by filamentous cyanobacteria and their exopolysaccharides with mineral particles provide a desirable material for studying multitrophic responses of microbiota against stratospheric stresses^[18]. As the primary colonizers in the early geological age of Earth, biocrusts played a crucial role in improving soil and atmospheric conditions ^[19]. Given the similarities between Mars and early Earth, cyanobacterial crusts are considered promising candidates for colonizing and modifying extraterrestrial environments ^{[16, 20, 21]}. However, most current research focuses on physiological changes such as photosynthetic rate, pigment contents, antioxidant enzymes, and extracellular polysaccharides^{[18, 20, 22]}. Alternatively, meta-omics approaches, including metatranscriaptomics and metagenome-assembled-genomes (MAGs), offer comprehensive insights into the functional potential of specific taxa and are increasingly applied in astrobiology research ^[23–25]. Our study delved into the shift in microbial metabolism to provide an in-depth understanding of how the biocrust community synergistically responds to stratospheric exposure.

To bridge the gap in tracking microbial responses under extreme conditions, we affixed cyanobacterial crusts to a helium-inflated high-altitude balloon and exposed to the stratosphere at an altitude of 32 km for approximately 200 min. Meanwhile, biocrusts exposed on the ground of the launch site served as the control group, and a simulated experiment was designed to investigate the relative impact of UV radiation versus temperature on community dynamics. We then employed a suite of multi-omics approaches to conduct a synchronous analysis across different groups. The metabolic mechanisms were scrutinized through (i) metabolic alterations characterized by active pathways of the entire community, (ii) metabolic potential of highly resistant taxa (MAGs), (iii) co-occurrence network among active genes and predominant microbial taxa, and (iv) shifts in life-history strategies.

We hypothesize that intense radiation and water scarcity could impair photosynthetic activity while potentiating the utilization of chemical energy ^{[26, 27]}, stress tolerance ^{[22, 28]}, antioxidant C1 metabolism ^[29], acetaldehyde shunt ^[30], and photorespiration ^[31]. Dominant genera like Microcoleus, which are not drought-tolerant, would show decreased activity or a mixotrophic state due to the utilization of extracellular organic matter ^{[32, 33]}, but highly UV-resistant genera like Scytonema, Nostoc, and Chroococcidiopsis may have a competitive advantage ^[34]. Hence, given the multiple stresses, the microbiota is anticipated to display unique resource allocation patterns within their life-history strategies ^{[39, 40]}. Furthermore, interactions, especially those involving quorum sensing and nutrient exchange, could contribute to increased community resistance ^[35–37]. Ultimately, we deduce that cyanobacteria facilitate heterotrophic growth in response to irradiation stress ^[38], thereby enhancing the activity of heterotrophs such as actinobacteria ^{[26, 39, 40]}. To our knowledge, this study is the first comprehensive exploration of metabolic responses of biological communities to Mars-like environments, presenting valuable insights into the colonization potential of extremophiles on extraterrestrial planets.

Biocrusts collection and loading

Cyanobacterial crusts were collected from the southeastern edge of the Tengger Desert in June 2019. To ensure sample homogeneity, we selected a designated plot away from shrubs (approximately 2 m × 3 m). Intact biocrust samples (approximately 2 mm thick) were delicately peeled off using a sterile spatula after pressing a 15 cm sterile petri dish upside down into the topsoil. Each petri dish of biocrusts was divided into five aliquots: one for flight exposure, one for ground control, and the others for simulated experiments (see below). The dry sample was promptly transported to the laboratory under controlled room temperature and placed in a sterile sample pool (3.6 cm in diameter, 2 mm thick) with a small 0.2 µm air filtering hole at the bottom. To ensure adequate exposure of the biocrusts to stresses, transparent fused quartz glass was affixed on top of each sample pool, allowing 93% transmission of UV.

Flight exposure and simulated experiments

Sample pools containing natural cyanobacterial crusts were transported to the field experimental base in Ulan Middle Banner, Inner Mongolia. A high-altitude balloon, equipped with a meteorological sensor, a positioning module, a temperature sensor, and an illumination sensor, carried the sample pools for the exposure of cyanobacterial crusts. The process of high-altitude balloon flight has been specifically described elsewhere ^[41]. Two treatments were conducted at the experimental site: flight exposed treatment (hereafter referred to as S) and ground control treatment (G), each with three replicates. The equivalent temperature and light intensity of the samples in both treatments were recorded. The balloon ascended at 6:00 am and attained an altitude of 32 km at 7:48 am, then the cyanobacterial crusts were exposed to the stratospheric environment. After 3 hours and 17 minutes of exposure, the lid was closed and the payload descended with a parachute. Meanwhile, the ground control group were also completed its treatment. Both flight and control samples were crushed, stored in ample dry ice, and transported to the laboratory at -80°C for subsequent DNA, RNA, and protein extraction.

To assess the individual impact of stratospheric stresses on cyanobacterial crusts, we conducted three simulated experiments using the same batch of biocrusts: temperature, UV, and the combined effect of temperature with UV. Biocrusts were placed in the same flight sample pool. Temperature was adjusted every 5 minutes to simulate the flight process, and radiation intensity was simulated using 400 µW/cm² UV-B. Then, the samples were processed following the aforementioned method.

The frozen cyanobacterial crusts were subjected to grinding and sieving (180 µm). Approximately 0.5 g of each sample was weighted and total nucleic acids were extracted using the RNeasy PowerSoil Total RNA Kit (Qiagen, Germany). Total DNA was then separated using the DNA Elution, and RNA was further purified using the DNase Max Kit (Qiagen, Germany). The quality and purity of nucleic acids were evaluated using NanoDrop 2000c (Thermo Fisher Scientific, USA) and agarose gel electrophoresis. The method for protein isolation from the crusts primarily based on a previous study ^[42].

Multi-omics sequencing, assembly, and annotation

Metagenomic and metatranscriptomic libraries were sequenced using Illumina platforms. The sequencing data underwent quality evaluation, adapter trimming, and filtering of low-quality reads. High-quality contigs were assembled, and open reading frames (ORFs) from contigs (≥ 300 bp) were predicted. Non-redundant gene sets were constructed, and alignment of gene reads, as well as taxonomic and functional annotation were performed according to our established methods ^[27]. Proteins that met quality standards were analyzed by Q-Exactive Plus Orbitrap mass spectrometry (Thermo Fisher Scientific, USA), followed by taxonomic and functional annotations.

Metagenome binning and MAG metabolic reconstruction

Metagenomic raw reads were quality-checked, assembled, and binned using the metaWRAP v1.3.2 pipeline with default parameters ^[43]. Average nucleotide identity was calculated by the fastANI tool with a 95% threshold (https://github.com/ParBLiSS/FastANI). The completeness and contamination of MAGs were evaluated by CheckM v1.0.18 ^[44]. MAGs were classified using GTDB-TK v1.5.1 (referencing GTDB-R06-RS202) ^[45]. A phylogenetic tree was constructed using high-quality cyanobacterial MAGs (completeness > 80%, contamination < 5%) and reference genomes obtained from NCBI GeneBank. Amino acid sequences were aligned using MAFFT v7.471 with the ‘-auto’ option ^[46], and the alignments were further refined using TrimAl v1.2.59 ^[47]. An evolutionary tree was constructed using IQ-TREE v2.0.5 with the optimal model ^[48], and then visualized in Interactive Trees of Life (iTOL v6) ^[49]. We calculated the relative abundance of MAGs in metagenome and metatranscriptome using CoverM (https://github.com/wwood/coverm) and normalized it by transcripts per kilobase million (TPM). Functional annotation was performed using KofamKOALA online tool (https://www.genome.jp/tools/kofamkoala/). Gapseq v1.1 ^[50] was used for metabolic reconstruction, referring the MetaCyc ^[51] and TCDB ^[52] databases. The AntiSmash database was also incorporated for the annotation of secondary metabolites synthesis genomic clusters. The phylogenetic analysis of nifH and scyA involved accessing these genes from NCBI GeneBank and merging them with our data to build the evolutionary tree. After aligning the target genes, transcript read counts mapped onto the genes were calculated using BEDTools ^[53], and normalized based on TPM for relative abundance analysis.

Data analysis

Heatmaps were generated using TBtools ^[54], while volcano plots, bubble plots of KEGG enrichment analysis, ternary plots, and three-dimensional graphs were plotted using the packages ggplot, clusterProfiler, ggtern, and plot3D in R-4.2.1, respectively. The package Deseq2 was utilized to assess significant differences in relative abundance and expression levels of taxa and functions between the flight exposed group and the others. Spearman correlation was calculated using the package psych. Network diagrams were visualized using Gephi (https://github.com/gephi/gephi/releases/tag/v0.9.2), with the Fruchterman Reingold algorithm layout used for clustering and evaluating modularity. The community-level metabolic inferences were based on KEGG metabolic networks.

Shifts in the composition of active members

To shed light on the adaptation of biocrust microbiota in poly-extreme conditions, we first examined alterations in the community composition, particularly active members, upon stratospheric exposure. Our complementary multi-omics results, based on the molecular markers of metagenomics, metatranscriptomics, and metaproteomics, demonstrated a consistent pattern of abundant microbial taxa in cyanobacterial crusts, comprised of Cyanobacteria, Actinobacteria, α-Proteobacteria, Bacteroidetes, and Chloroflexi (Fig. S1 B, C, D), wherein Cyanobacteria accounted for 80 ~ 90% of the total active abundance. At the metatranscriptomic level, we observed opposite changes in the relative abundances between cyanobacteria and heterotrophs due to exposure. That is, the transcriptional activity of cyanobacteria declined while that of heterotrophs, like species within Propionibacteriales (Actinobacteria) and Rhodospirillales (α-Proteobacteria), increased compared to the ground control group (Fig. 2A). This tendency was in concert with the results of our metagenomic and metaproteomic datasets (Fig. S2). In previous studies, the reduction of relative abundance in cyanobacteria was often detected in biocrusts and attributed to the impacts of strong ambient stresses, including drought and elevated CO₂ concentration ^{[32, 55]}. However, it is noteworthy that, at the genus level, despite the decline occurring in the dominant Microcoleus spp. during the stratospheric exposure, the active abundance of Scytonema still significantly increased. It provided evidence for the inference that Scytonema is more resistant to radiation and extreme temperature ^[34], while Microcoleus tends to be greater resilient against wind erosion and disturbances ^{[56, 57]}. These cues about the community dynamics suggested that photoautotrophs sustain more damage than heterotrophs in the stratosphere, despite the unique exception that Scytonema diverges from the majority of cyanobacteria in its adaptation to hostile conditions.

Characteristics in metabolism patterns

To elucidate the active functional responses in the stratosphere, we compared functional gene expression between exposed and control groups. As expected, significant metabolic alterations were observed at the metagenome, metatranscriptome, and metaproteome levels, in addition to changes in community structure post-exposure (p._adj < 0.05, Fig. 2, Fig. S3 and S4). At the metatranscriptome level, a higher number and greater amplitude of upregulated KEGG Orthology groups (108 KOs, approximate 2 ~ 4 times) were noted compared to downregulated KOs (75 KOs, 0.5 ~ 1 times) (Fig. 2B, C). KEGG enrichment analysis primarily revealed significant changes in substance transport (Fig. 2D).

We found that upregulated functions primarily included resistance, substance transport (macromolecules and ions), intermediate product transformation (TCA cycle, gluconeogenesis, and fermentation), synthesis pathways [e.g., ribulose monophosphate pathway (RuMP), the glyoxylate cycle, biological nitrogen fixation (BNF), assimilatory sulfate/nitrate reduction], and xylose utilization; while downregulated functions were associated with light energy utilization, substance transport (iron/biotin), the serine pathway, and cell wall component degradation (Fig. 3A). Among upregulated metabolic processes (Fig. S5), cyanobacteria showed increased BNF, stress tolerance, and substance transport activity; actinobacteria dominated in xylose utilization, poly-beta-hydroxybutyrate (PHA)/long-chain acyl-CoA (FA) synthesis, the glyoxylate cycle, and assimilatory sulfate reduction; and α-proteobacteria were involved in PHA degradation. In downregulated metabolic processes, cyanobacteria were prominent in iron/biotin transport, the serine pathway, and peptidoglycan degradation, while chloroflexi played a significant role in hemicellulose degradation.

Given its capacity to uncover the metabolic potential of novel microorganisms, MAG-level analysis has found extensive application in diverse ecosystems, such as the deep sea and submarine shell ^{[23, 24]}. In our study, we assembled 163 MAGs, each with over 50% completeness and less than 10% contamination (Fig. S6). These MAGs were primarily composed of Cyanobacteria, Chloroflexi, α-Proteobacteria, Actinobacteria, and Bacteroidetes, to a large extent aligning with the community structure observed at the 16S rRNA gene level (Fig. S1 A). It indicated that our MAG data effectively encapsulate the functional potential of the primary taxa, thereby complementing our metatranscriptomic results. We then scrutinized the functional potential of highly abundant MAGs and correlated these findings with changes in active function at the community level. This approach helped us understand the contribution of specific taxa to functional expression (Fig. 3B). Our analysis revealed that among the upregulated KOs, α-proteobacteria exhibited a high potential for stress resistance, cyanobacteria showed capabilities for photoprotection and BNF, bacteroidetes and chloroflexi were involved in the RuMP pathway, and actinobacteria, α-proteobacteria, and chloroflexi demonstrated potential for the glyoxylate cycle. In terms of downregulated functions, cyanobacteria and chloroflexi stood out for their significant potential in cell wall degradation.

Several metabolic processes that were upregulated in our study, such as those associated with stress resistance ^[28], inorganic chemistry energy utilization ^[26], xylose utilization ^[58], fermentation ^[59], C1 metabolism, and long-chain carbon balance ^[60], have also been identified in various biological stress experiments. Concurrently, the downregulation of cell wall degradation aligns with the effects of drought and high-temperature stress ^[61], a response thought to provide resistance by preserving cellular integrity ^[62]. The observed downregulation of the serine pathway could be attributed to the high energy demands and nitrogen deficiency ^[63]. Overall, the stratospheric poly-extreme conditions induced the upregulation of metabolic processes related to stress resistance, substance transport, the RuMP pathway, and the glyoxylate cycle. In contrast, there was a decline in the serine pathway and degradation of cell wall components, indicating a significant nitrogen deficiency within the community. At the phylum level, major phyla such as Cyanobacteria, Actinobacteria, α-Proteobacteria, Bacteroidetes, and Chloroflexi exhibited pathways that facilitate biomass synthesis ^[40]. This suggests that the dominant taxa actively modulate metabolic activities to maintain community stability under stressful conditions. Notably, the significant upregulation of synthetic pathways enhanced the potential of actinobacteria as chemoautotrophic primary producers ^{[26, 64]}.

Co-occurrence network of community metabolism

The aforementioned results underscore the capacity of organisms to actively modulate metabolism in response to stressful environments. Yet, the potential interactions between species and metabolic processes, which could further promote community survival, remain unexplored. To address this, we performed a co-occurrence network analysis on highly abundant genera and altered functions, yielding six highly connected modules through clustering (Fig. 4).

Module 1, primarily composed of cyanobacteria, was characterized by the coupling of extracellular organic matter degradation, energy production, resistance, and detoxification mechanisms, synthesis of branched-chain amino acid (BAa) and FA, assimilation of nitrate to ammonium, and assimilatory sulfite reduction. Biomass formation was facilitated by BNF ^[65], FA biosynthesis, and assimilation of fermentation products ^[59]. A significant elevation in β-glucosidase activity observed in an exposure physiological experiment corroborates our findings^[18]. This suggests that the dominant cyanobacteria exhibit mixotrophic characteristics, consistent with the heterotrophic growth of cyanobacteria in the presence of abundant organic carbon ^[66].

Module 2, dominated by low-abundance taxa, was associated with hydrolysis of organic matter (extracellular peptidoglycan), cofactors (Fe/biotin), penetrant transport, and the serine pathway. The main function of the extracellular hydrolases L-Alanine aminopeptidase (AAP) and β-N-acetylglucosaminidase (NAG) is to obtain nitrogen sources, indicating a mixotrophic state coupled with the serine pathway in a nitrogen-deficient environment.

Module 3, primarily composed of ascomycota fungi, was combined with extracellular oxidases, ethanol fermentation, and xylose utilization. Fungi have an adaptive advantage over bacteria due to their higher drought tolerance ^[61]. They can utilize extracellular organic compounds through polyphenol oxidase and xylose metabolism ^{[67, 68]}. Then, fermented products could be recycled into biomass ^[59]. This indicated that the primary stressful factor for Ascomycota is the scarcity of available carbon, offset by its unique metabolic capabilities.

Module 4 was predominantly composed of α-proteobacteria and chloroflexi, which were involved in the synthesis of BAa and PHA, BNF, and assimilatory nitrate reduction. These processes were accompanied by the influx of energy and nutrients in various forms and the activation of detoxification, promoting biomass synthesis ^{[64, 65]}. This module was characterized by nonoxygenic photosynthetic microorganisms that exhibit strong resistance and autotrophic growth. Meanwhile, the presence of heterotrophic processes, such as the transport of extracellular substances and the synthesis of high molecular weight organic carbon, indicates a mixotrophic mode of growth.

Module 5, primarily consisting of actinobacteria, engaged in xylose utilization, PHA synthesis, the RuMP pathway, the glyoxylate cycle, the influx of energy and nutrients, and detoxification processes. The RuMP pathway and glyoxylate cycle are commonly speculated to enhance biosynthesis ^{[30, 63]}. These pathways facilitate a mixotrophic metabolism characterized by C1 metabolism, C2 assimilation, and macromolecular accumulation. This metabolic versatility with the role of actinobacteria as primary producers sustained their biomass production under varying conditions ^[40].

Module 6, dominated by bacteroidetes, was coupled with extracellular hydrolases, substance transport, detoxification, PHA degradation, and ammonia assimilation. It was previously observed that bacteroidetes can upregulated assimilatory metabolism under stressful conditions ^[69]. This module mainly exhibited heterotrophic characteristics with a ‘selfish’ substrate utilization trait, providing a competitive advantage in resource-limited conditions ^[70].

The co-occurrence network of community metabolism delineates the distinct metabolic advantages of mutual and complementary strategies within the biocrust community. Notably, α-proteobacteria, with their nitrogen-fixing capabilities, were found to be associated with chloroflexi, while the serine pathway was implicated in the degradation of nitrogen-rich organic substances. Cyanobacteria, in particular, were linked with a variety of extracellular enzymes and other members, potentially sharing cyanobacterial metabolic products ^{[38, 69, 70]}. This metabolic coupling among microbial taxa and their respective functions contributed to the pronounced retention of carbon and nitrogen within the community, underscoring the synergistic response to stabilize biocrusts in the stratosphere ^{[71, 72]}.

Life-history strategies

In addition to the regulation of interspecies relationships, the allocation of limited resources is pivotal for microbial survival in stressful environments. To this end, we categorized the altered functions within life-history strategies between the flight-exposed and ground control groups, where the upregulation means a higher level of expression in the exposure group, while downregulation indicates a higher level in the ground control group. A-strategy represents the functions involved in resource acquisition, P-strategy represents substrate synthesis and energy metabolism, and S-strategy represents stress resistance mechanisms. The distribution of three strategies across different taxa was visualized using ternary plots (Fig. 5). Comparative analysis between flight-exposed and ground control groups revealed a shift towards S-strategy (augmented stress tolerance) and P-strategy (enhanced biosynthesis and energy metabolism) following exposure, while A-strategy showed a reduction in resource utilization and an increase in substrate transport (Fig. 5A). The observed shift was in accordance with the metagenomic data (Fig. S7) and aligned with findings from other drought stress experiments ^[73], reflecting the principles of nutrient exchange ^{[36, 74, 75]} and mutualistic enhancement ^{[37, 74]} in microbial communities. Notably, this study was partly inconsistent with previous related stress research in that we did not detect obvious responses in biofilm formation ^[76] and motility ^[77] within the water-deficient stratospheric environment. It is noteworthy that filament sliding of bundle-forming cyanobacteria like Microcoleus through the pores of the upper 1 ~ 2 mm layer of biocrusts guarantee their stress tolerance against ambient fluctuations ^[78]. It to some extent explained the abovementioned decline of Microcoleus spp. in the relative abundance of the active fraction.

In the analysis of taxonomic contributions to functional changes (Fig. 5B), cyanobacteria were predominantly associated with enhanced substrate transport and synthesis, osmoregulation, and antioxidant activity. Actinobacteria were implicated in substrate synthesis, while α-proteobacteria were linked to DNA repair and cold resistance mechanisms. The ternary diagram revealed a marked shift in the life strategies of these principal taxa. The enhancement of P-strategy in actinobacteria indicated the proliferation of microbes belonging to this phylum, consistent with previous findings ^[26]. α-Proteobacteria and chloroflexi showed an increase in S-strategy, with no significant alterations in P-strategy, implying a constrained growth potential. Cyanobacteria displayed diversifying strategies in different genera. For example, the UV-A sensitive Microcoleus, typically inhabiting the top layer of biocrusts (approximately 0.2 mm), faces intense UV radiation, water scarcity, and limited shelter, suggesting that its growth may be curtailed when S-strategy is prioritized ^[79], despite a high activity of nutrient exchange across the cell membrane ^{[80, 81]}. In contrast, Scytonema, with its resistance to UV-A and high temperatures ^[82], is likely to thrive with the increasement of A-strategy and P-strategy.

Characteristic metabolism potential of cyanobacteria Scytonema

Given the array of life-history strategies within cyanobacteria and the fluctuating abundance of dominant genera, we sought to uncover the mechanisms that underpin the remarkable adaptability and performance of Scytonema in the stratospheric environment. We obtained 57 high-quality MAGs (completeness > 80% and contamination < 5%), including 13 cyanobacterial ones. An evolutionary tree, derived from these MAGs alongside reference genomes, positioned bin.90 in close phylogenetic proximity to Scytonema sp. NIES-4073 (Fig. 6A). Furthermore, heatmaps representing the relative abundance clustering of MAG potential (Fig. S8 A) and activity (Fig. S8 B) both indicated a robust correlation between bin.90 and upregulated MAGs. This evidence supports the classification of bin.90 as Scytonema, which exhibited an increased abundance after exposure. We then compared the metabolic potential across different taxa and highlighted the unique metabolic characteristics of Scytonema.

Considering the unique physiological traits of Scytonema, particularly its capabilities in BNF and the production of UV-resistant scytonemin, we investigated the correlation between these two traits and the observed upregulation in its active abundance. We first constructed phylogenetic trees for the key feature nifH and scyA genes, respectively, and quantified the relative abundance of their expression activity. The result did not indicate a significant change in gene activity (Fig. S9), which may reflect the robust drought resistance of Scytonema sp. NIES-4073 during nitrogen deprivation ^[83]. In contrast, the scyA phylogenetic tree (Fig. 6B) and heatmap of active expression (Fig. 6C) suggesting an association with increased scytonemin synthesis in Scytonema. A significant downregulation trend at both metagenomic and metatranscriptomic levels was observed for the scyA gene corresponding to bin.88 (Fig. 6C), which is phylogenetically close to Tricocoleus (Fig. 6A). To elucidate the strong resistance mechanisms of Scytonema (i.e., bin.90), we compared its metabolic potential with Tricocoleus (bin.88), encompassing stress resistance, energy acquisition, nutrient and cofactor uptake, and environmental responsiveness.

Firstly, Scytonema exhibits a robust ability to withstand a variety of stresses (Fig. 7A). (i) Oxidation resistance: Despite the low oxygen level (~ 0.17%) ^[5], intense UV radiation (100 W/m²) in the stratosphere presents a significant challenge ^[1]. Nevertheless, Scytonema (bin.90) is equipped with UV-A/B-resistant mycosporine-like amino acids (MAAs) to mitigate UV radiation damage. It also features a comprehensive antioxidant enzyme system for the conversion of reactive oxygen species (ROS) to H₂O, the glutathione disulfide/glutathione (GSSH/GSH) cycle, nonenzymatic antioxidants such as astaxanthin, vitamin E, and β-carotene, and NAD(P)H quinone dehydrogenase 1 (NQO1) for damage repair ^[84]. (ii) Osmotic resistance: bin.90 can synthesize succinoglycans, which have water-holding capacity ^[85], transport β-lysine, hydrophobic docosahexaenoic acid (DHA)/eicosapentaenoic acid (EPA), and regulators, such as putrescine, spermidine, peptides, and proline. (iii) Detoxification: the influx of aspartate for the synthesis of phytochelatins synthesis and the efflux of salts and metal ions. (iv) Other resistances: Synthesis of methionine to enhance tolerance to oxidative and heat stresses ^[86] and secretion of extracellular polysaccharides in response to various stresses ^[85].

Secondly, Scytonema can acquire energy from diverse sources. (i) Light energy: bin.90 can synthesize photosynthetic pigments such as phycocyanin and chlorophyll a, which are capable of absorbing a broad spectrum of light, under aerobic conditions ^[87]. (ii) Organic chemical energy: Utilizing cytochrome bd as a terminal oxidase and using succinate, pyruvate, and NADH for energy production in anaerobic environments ^[88]. In addition, it can ferment lactate and utilize small extracellular molecules such as serine and acetate for energy generation ^[31]. (iii) Inorganic chemical energy: Multiple energy-producing transport systems and absorbing H₂ ^[89].

Third, Scytonema can acquire nutrients and essential cofactors through various mechanisms. (i) bin.90 secrets extracellular lysozymes to degrade organic matter and hydrolyze long-chain acyl thioesters for carbon sources. (ii) It can oxidize sulfoquinovose to obtain carbon and sulfur sources ^[90]. (iii) It gains nitrogen sources through BNF and intracellular transport of various amino acids and polyamines such as spermine/putrescine. (iv) bin.90 can secret alkaline phosphatase to convert extracellular organic phosphorus into inorganic phosphorus for utilization. (v) It employs multiple iron transporters ^[91]. (vi) bin.90 can sense environmental changes, primarily by synthesizing and secreting homoserine lactones, which act as quorum-sensing molecules or intercellular communication transmitters ^[35].

Furthermore, as previously noted, the dominant Microcoleus showed an inverse activity trend compared to Scytonema (Fig. 2A), which can be interpreted from a metabolic standpoint. Given the closest evolutionary relationship between bin.87/89/96 and reference Microcoleus gemones (Fig. 6A, indicating similar metabolic potential), we contrasted bin.90 with bin.87/89/96 to demonstrate the strong resistance of Scytonema (Fig. 7B). It was primarily evident in the synthesis and secretion of scytonemin and MAAs for resistance to UV-A and UV-B, the ascorbic acid cycle, the biosynthesis of cofactors such as vitamin B1 (thiamine pyrophosphate) and dopamine, the translocation of neutral amino acids, and the efflux of osmotically active solutes. These advantageous functional potentials were predominantly linked to quorum sensing, UV resistance, and antioxidant capabilities.

In summary, the key advantages of Scytonema, particularly its increasing activity in the hostile stratospheric environment, are UV protection and thermal resistance. Moreover, Scytonema also exhibits resilience to multiple stresses, like absorbing a broad spectrum of light and generating diverse potential energies, ranging from strong oxidation to strong reduction. Given the nutrient limitations in the stratosphere ^[39], Scytonema secretes extracellular hydrolases to acquire organic carbon and nitrogen, absorbs various sources of carbon, nitrogen, phosphorus, sulfur, and iron, performs BNF, and synthesizes key cofactors, polyunsaturated fatty acids, and polysaccharides. Considering phosphorus as a limiting nutrient for primary productivity in terrestrial ecosystems ^[92], Scytonema likely has advantages in autotrophic metabolism. Photosynthesis evolved as a response to the selective pressure of UV radiation stress and a scarcity of electron donors in early Earth. The earliest photosynthetic organisms, known as procyanobacteria, are akin to modern filamentous cyanobacteria with heterocyst ^[93]. Therefore, the UV resistance, diverse energy acquisition mechanisms, and presence of heterocyst in Scytonema underscore its potential as a primary producer in extraterrestrial settings, highlighting its applications in the development of life support systems for extraterrestrial colonization.

In conclusion, this study utilized metagenomics (metagenome-assembled genome), metatranscriptomics, and metaproteomics techniques to reveal that cyanobacterial crusts exhibit enhanced stress resistance, modular interactions between taxonomic genera and metabolic functions, and diverse life-history strategy responses after short-term exposure to the stratosphere. The observed upregulation of functions related to resistance, material transport, and synthesis, as well as the conversion of intermediate metabolites, coupled with the downregulation related to carbon degradation from cell wall sources. This suggests that the biocrust community actively mitigates adversity by modulating intracellular metabolism and preserving cellular structural integrity. The modular correlation between multitrophic microbiota and functions indicates that mutualistic interactions facilitate community coexistence and promote heterotroph growth. The synergistic response at the community level ensures significant carbon-nitrogen retention within the biocrusts. Furthermore, the presence of diverse response patterns in life-history strategies among phyla indicates substantial adaptive differentiation, with Actinobacteria expected to exhibit growth, while α-Proteobacteria and Chloroflexi primarily demonstrate stress resistance. Comparative analysis of MAGs reveals that Scytonema (bin.90) could possess strong radiation resistance and multiple nutrients and energy acquisition pathways, which may contribute to its upregulation trend of active abundance. This study enhances our understanding of biological community responses to extreme environments, providing theoretical support for the application of cyanobacterial crusts on Mars. Future research can consider extending the exposure duration or incorporating day-night alternation to investigate variations in community response and thoroughly examine the superiority of axenic Scytonema strains in the stratospheric environment.

Acknowledgments

We thank Gaohong Wang from the Institute of Hydrobiology, Chinese Academy of Sciences for their assistance in the field experiment.

Funding

This project was supported by the National Natural Science Foundation of China (32370125 and 41877419); the Strategic Priority Research Program of the Chinese Academy of Science (XDA17010502); and the funding of the Key Laboratory of Algal Biology, Chinese Academy of Sciences.

Data availability

The 16S rRNA gene sequences, metagenome, metagenome-assembled genomes, and metatranscriptome datasets of in-situ exposure and ground control groups in this study are available in the NCBI database under project IDs PRJNA1051297, PRJNA1051671, and PRJNA1051300. The metatranscriptome dataset of the simulation group is available in the NCBI database under project ID PRJNA1052761. The mass spectrometry proteomics data has been deposited in iProX with the dataset ID IPX0007726000.

Author Contributions

C.-X.H. conceived and designed this research; Q.L. and H.-J.Y. performed the field study and measurements; X.Z. analyzed the raw data and wrote the original draft; C.-X.H. and H.L. revised the manuscript with contributions from all co-authors.

Competing Interests

The authors declare no competing interests.

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No competing interests reported.

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Synergistic response of extremophiles in cyanobacterial crusts against in-situ exposure to multiple stratospheric stresses

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Abstract

Background

Results

Conclusions

Figures

Background

Materials and methods

Biocrusts collection and loading

Flight exposure and simulated experiments

Multi-omics sequencing, assembly, and annotation

Metagenome binning and MAG metabolic reconstruction

Data analysis

Results and Discussion

Shifts in the composition of active members

Characteristics in metabolism patterns

Co-occurrence network of community metabolism

Life-history strategies

Characteristic metabolism potential of cyanobacteria Scytonema

Conclusions

Declarations

References

Additional Declarations

Supplementary Files

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