Adaptive mechanisms of Bacillus to near space extreme environments

Earth's near space is an extreme atmosphere environment with high levels of radiation, low atmospheric pressure and dramatic temperature fluctuations. The region is above the flight altitude of aircraft but below the orbit of satellites, which has special and Mars-like conditions for investigating the survival and evolution of life. Technical limitations including flight devices, payloads and technologies/methodologies hinder microbiological research in near space. In this study, we investigated microbial survival and adaptive strategies in near space using a scientific balloon fight mission and multi-omics analyses. Methods for sample preparation, storage, protector and vessel were optimized to prepare the exposed microbial samples. After 3 h 17 min of exposure at a float altitude of ~32 km, only Bacillus strains were alive with survival efficiencies of 0-10^-6. Diverse mutants with significantly altered metabolites were generated, firstly proving that Earth's near space could be used as a new powerful microbial breeding platform. Multi-omics analyses of mutants revealed cascade changes at the genome, transcriptome and proteome levels. In response to environmental stresses, two mutants had similar proteome changes caused by different genomic mutations and mRNA expression levels. Metabolic network analysis combined with proteins' expression levels revealed that metabolic fluxes of EMP, PPP and purine synthesis-related pathways were significantly altered to increase/decrease inosine production. Further analysis showed that proteins related to translation, molecular chaperones, cell wall/membrane, sporulation, DNA replication/repair and anti-oxidation were significantly upregulated, enabling cells to efficiently repair DNA/protein damages and improve viability against environmental stress. Overall, these results revealed genetic and metabolic responses of Bacillus to the harsh conditions in near space, providing a research basis for bacterial adaptive mechanisms in extreme environments.