Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Bacillus subtilis spores, PROTECT experiment, Space-exposed and Mars-exposed vs. Earth-control

ABSTRACT: Because of their ubiquity and resistance to spacecraft decontamination, bacterial spores are considered likely potential forward contaminants on robotic missions to Mars. Thus it is important to understand their global responses to long-term exposure to space or Mars environments. As part of the PROTECT experiment, spores of B. subtilis 168 were exposed to real space conditions and to simulated martian conditions for 559 days in low Earth orbit mounted on the EXPOSE-E exposure platform outside the European Columbus module on the International Space Station. Upon return, spores were germinated, total RNA extracted and fluorescently labeled, and used to probe a custom Bacillus subtilis microarray to identify genes preferentially activated or repressed relative to ground control spores. Increased transcript levels were detected for a number of stress-related regulons responding to DNA damage (SOS response, SPβ prophage induction), protein damage (CtsR/Clp system), oxidative stress (PerR regulon) and cell envelope stress (SigV regulon). Spores exposed to space demonstrated a much broader and more severe stress response than spores exposed to simulated Mars conditions. The results are discussed in the context of planetary protection for a hypothetical journey of potential forward contaminant spores from Earth to Mars and their subsequent residence on Mars. Two-color microarrays were performed comparing germination of Space-exposed or Mars-exposed vs. ground-control (Earth) spores.

ORGANISM(S): Bacillus subtilis subsp. subtilis str. 168

SUBMITTER: Wayne Nicholson

PROVIDER: E-GEOD-37124 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Abstract Background The extraordinarily resistant bacterium Deinococcus radiodurans withstands harsh environmental conditions present in outer space. Deinococcus radiodurans was exposed for one year outside the International Space Station within Tanpopo orbital mission to investigate microbial survival and space travel. In addition, a ground-based simulation experiment with conditions, mirroring those from Low Earth orbit, was performed. Methods We monitored Deinococcus radiodurans cells during early stage of recovery after Low Earth orbit exposure using electron microscopy tools. Furthermore, proteomic, transcriptomic and metabolomic analyses were performed to identify molecular mechanisms responsible for the survival of Deinococcus radiodurans in Low Earth orbit. Results D. radiodurans cells exposed to low Earth orbit conditions do not exhibit any morphological damage. However, an accumulation of numerous outer-membrane associated vesicles was observed. On levels of proteins and transcripts, a multifaceted response was detected to alleviate cell stress. The UvrABC endonuclease excision repair mechanism was triggered to cope with DNA damage. Defense against reactive oxygen species is mirrored by the increased abundance of catalases and is accompanied by the increased abundance of putrescine which works as scavenging molecule. In addition, several proteins and mRNAs, responsible for regulatory and transporting functions showed increased abundances. The decrease in primary metabolites indicate alternations in the energy status, which is needed to repair damaged molecules. Conclusion Low Earth orbit induced molecular rearrangements trigger multiple components of metabolic stress response and regulatory networks in exposed microbial cells. Presented results show that the non-sporulating bacterium Deinococcus radiodurans survived long-term Low Earth orbit exposure if wavelength below 200 nm are not present, which mirrors the UV spectrum of Mars, where CO2 effectively provides a shield below 190 nm. These results should be considered in the context of planetary protection concerns and the development of new sterilization techniques for future space missions.

Project description:The members of the genus Clostridium, such as the other spore-forming anaerobic bacteria, have a complex and strictly regulated life-cycle but very little is known about genetic pathways involved at different stages. Clostridium sporogenes, Gram positive bacterium usually involved in food spoilage and frequently isolated from late blowed cheese is genetically indistinguishable from proteolytic Clostridium botulinum and is the non-neurotoxigenic counterpart often used as exemplar for the toxic subtypes. In this work we have performed a microscopy study combined with a custom array-based analysis of C. sporogenes cycle, from dormant spores to early stationary phase. We identified in spores a total of 211 transcripts validating the ipothesis that mRNAs are abundant and strikingly different from those present in growing cells. The spores transcripts included genes responsible of different life-sustaining functions, suggesting the theory of transcripts entrapment or of a basic poly-functional genes activation for future steps. In addition, after three hours from the beginning of the germination process, a 20% of the total up-regulated genes were temporally expressed in germinating spores. The vegetative condition appears to be the more active in terms of gene transcription and protein synthesis and also genes for germination and sporulation factors seem to be expressed at this point. These results suggest that spores are not silent entities and a wider knowledge about genetic pathways involved in Clostridium life cycle could be of help for a better understanding also of pathogenic clostridia types. RNA was isolated from spores, germinating spores (3h), outgrowth/transitional mid-log vegetative cells and early stationary cells of Clostridium sporogenes UC9000 previously isolated from milk. There were 3 biological replicates (independent cultures) for each condition. Probes corresponding to 3400 genomic CDSs of C. botulinum A ATCC 3502 were synthesized in three replicates randomly distributed on the chip.

Project description:Spermatophyte pollen tubes and root hairs have been used as single-cell-type model systems toward understanding the molecular processes underlying polar growth of plant cells. Horsetail (Equisetum arvense L.) is a perennial herb species in Equisetopsida, which creates separately growing spring and summer stems in its life cycle. The mature chlorophyllous spores produced from spring stems can germinate without dormancy. Here we report the cellular features and protein expression patterns in five stages of horsetail spore germination (mature spores, rehydrated spores, double-celled spores, germinated spores, and spores with protonemal cells). Using 2-DE combined with mass spectrometry, 80 proteins were found to be differentially expressed upon spore germination. Among them, proteins involved in photosynthesis, protein turnover, and energy supply were over-represented. Thirteen proteins appeared as isoforms on the gels, indicating the potential importance of post-translational modification. In addition, the dynamic changes of ascorbate peroxidase, peroxiredoxin, and dehydroascorbate reductase implied that reactive oxygen species homeostasis is critical in regulating cell division and tip-growth. The time course of germination and diverse expression patterns of proteins in photosynthesis, energy supply, lipid and amino acid metabolism indicated that heterotrophic and autotrophic metabolism were necessary in light-dependent germination of the spores. Twenty-six proteins were involved in protein synthesis, folding, and degradation, indicating that protein turnover is vital to spore germination and rhizoid tip-growth. Furthermore, the altered abundance of small G protein Ran1, 14-3-3 protein, actin, and Caffeoyl-CoA O-methyltransferase revealed that signaling transduction, vesicle trafficking, cytoskeleton dynamics, and cell wall modulation were critical to cell division and polar growth. These findings provide up-to-date evidences for understanding fern spore asymmetric division and rhizoid polar growth.

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Bacillus subtilis spores, PROTECT experiment, Space-exposed and Mars-exposed vs. Earth-control

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