Shelf Life and Simulated Gastrointestinal Tract Survival of Selected Commercial Probiotics During a Simulated Round-Trip Journey to Mars.

To enhance the gastrointestinal health of astronauts, probiotic microorganisms are being considered for inclusion on long-duration human missions to the Moon and Mars. Here we tested three commercial probiotics- strain BB536, strain DDS-1, and spores of strain HU58-for their survival to some of the conditions expected to be encountered during a 3-year, round trip voyage to Mars. All probiotics were supplied as freeze-dried cells in capsules at a titer of >10 colony forming units per capsule.

Mechanotransduction in Prokaryotes: A Possible Mechanism of Spaceflight Adaptation.

Our understanding of the mechanisms of microgravity perception and response in prokaryotes (Bacteria and Archaea) lag behind those which have been elucidated in eukaryotic organisms. In this hypothesis paper, we: (i) review how eukaryotic cells sense and respond to microgravity using various pathways responsive to unloading of mechanical stress; (ii) we observe that prokaryotic cells possess many structures analogous to mechanosensitive structures in eukaryotes; (iii) we review current evidence indicating that prokaryotes also possess active mechanosensing and mechanotransduction mechanisms; and (iv) we propose a complete mechanotransduction model including mechanisms by which mechanical signals may be transduced to the gene expression apparatus through alterations in bacterial nucleoid architecture, DNA supercoiling, and epigenetic pathways.

Comparison of transcriptome profiles from two separate missions to the International Space Station.

The human spaceflight environment is notable for the unique factor of microgravity, which exerts numerous physiologic effects on macroscopic organisms, but how this environment may affect single-celled microbes is less clear. In an effort to understand how the microbial transcriptome responds to the unique environment of spaceflight, the model Gram-positive bacterium was flown on two separate missions to the International Space Station in experiments dubbed BRIC-21 and BRIC-23. Cells were grown to late-exponential/early stationary phase, frozen, then returned to Earth for RNA-seq analysis in parallel with matched ground control samples.

Transcriptomic responses of Serratia liquefaciens cells grown under simulated Martian conditions of low temperature, low pressure, and CO-enriched anoxic atmosphere.

Results from previous experiments indicated that the Gram-negative α-proteobacterium Serratia liquefaciens strain ATCC 27592 was capable of growth under low temperature (0 °C), low pressure (0.7 kPa), and anoxic, CO-dominated atmosphere-conditions intended to simulate the near-subsurface environment of Mars. To probe the response of its transcriptome to this extreme environment, S.

Alterations in the Spectrum of Spontaneous Rifampicin-Resistance Mutations in the Gene after Cultivation in the Human Spaceflight Environment.

The effect of exposure to the human spaceflight environment on growth, mutagenic frequency, and spectrum of mutations to rifampicin resistance (Rif) was investigated. cells were cultivated in Biological Research in Canister-Petri Dish Fixation Units (BRIC-PDFUs) on two separate missions to the International Space Station (ISS), dubbed BRIC-18 and BRIC-21, with matching asynchronous ground controls. No statistically significant difference in either growth or in the frequency of mutation to Rif was found in either experiment.

Cultivation in Space Flight Produces Minimal Alterations in the Susceptibility of Bacillus subtilis Cells to 72 Different Antibiotics and Growth-Inhibiting Compounds.

Past results have suggested that bacterial antibiotic susceptibility is altered during space flight. To test this notion, cells were cultivated in matched hardware, medium, and environmental conditions either in space flight microgravity on the International Space Station, termed flight (FL) samples, or at Earth-normal gravity, termed ground control (GC) samples. The susceptibility of FL and GC samples was compared to 72 antibiotics and growth-inhibitory compounds using the Omnilog phenotype microarray (PM) system.

Cultivation of Staphylococcus epidermidis in the Human Spaceflight Environment Leads to Alterations in the Frequency and Spectrum of Spontaneous Rifampicin-Resistance Mutations in the rpoB Gene.

Bacteria of the genus Staphylococcus are persistent inhabitants of human spaceflight habitats and represent potential opportunistic pathogens. The effect of the human spaceflight environment on the growth and the frequency of mutations to antibiotic resistance in the model organism Staphylococcus epidermidis strain ATCC12228 was investigated. Six cultures of the test organism were cultivated in biological research in canisters-Petri dish fixation units for 122 h on orbit in the International Space Station (ISS) as part of the SpaceX-3 resupply mission.

Evolution in the Bacillaceae.

The family Bacillaceae constitutes a phenotypically diverse and globally ubiquitous assemblage of bacteria. Investigation into how evolution has shaped, and continues to shape, this family has relied on several widely ranging approaches from classical taxonomy, ecological field studies, and evolution in soil microcosms to genomic-scale phylogenetics, laboratory, and directed evolution experiments. One unifying characteristic of the Bacillaceae, the endospore, poses unique challenges to answering questions regarding both the calculation of evolutionary rates and claims of extreme longevity in ancient environmental samples.

Complete Genome Sequence of Serratia liquefaciens Strain ATCC 27592.

We report the complete genome sequence of Serratia liquefaciens strain ATCC 27592, which was previously identified as capable of growth under low-pressure conditions. To the best of our knowledge, this is the first announcement of the complete genome sequence of an S. liquefaciens strain.

Evolution of Bacillus subtilis to enhanced growth at low pressure: up-regulated transcription of des-desKR, encoding the fatty acid desaturase system.

The atmospheric pressure on Mars ranges from 1-10 mbar, about 1% of Earth pressure (∼1013 mbar). Low pressure is a growth-inhibitory factor for terrestrial microorganisms on Mars, and a putative low-pressure barrier for growth of Earth bacteria of ∼25 mbar has been postulated. In a previous communication, we described the isolation of a strain of Bacillus subtilis that had evolved enhanced growth ability at the near-inhibitory low pressure of 50 mbar.

Exploring the low-pressure growth limit: evolution of Bacillus subtilis in the laboratory to enhanced growth at 5 kilopascals.

Growth of Bacillus subtilis cells, normally adapted at Earth-normal atmospheric pressure (∼101.3 kPa), was progressively inhibited by lowering of pressure in liquid LB medium until growth essentially ceased at 2.5 kPa.

Exposure of DNA and Bacillus subtilis spores to simulated martian environments: use of quantitative PCR (qPCR) to measure inactivation rates of DNA to function as a template molecule.

Several NASA and ESA missions are planned for the next decade to investigate the possibility of present or past life on Mars. Evidence of extraterrestrial life will likely rely on the detection of biomolecules, which highlights the importance of preventing forward contamination not only with viable microorganisms but also with biomolecules that could compromise the validity of life-detection experiments. The designation of DNA as a high-priority biosignature makes it necessary to evaluate its persistence in extraterrestrial environments and the effects of those conditions on its biological activity.

Bacterial spores in granite survive hypervelocity launch by spallation: implications for lithopanspermia.

Bacterial spores are considered good candidates for endolithic life-forms that could survive interplanetary transport by natural impact processes, i.e., lithopanspermia.

Persistence of biomarker ATP and ATP-generating capability in bacterial cells and spores contaminating spacecraft materials under earth conditions and in a simulated martian environment.

Most planetary protection research has concentrated on characterizing viable bioloads on spacecraft surfaces, developing techniques for bioload reduction prior to launch, and studying the effects of simulated martian environments on microbial survival. Little research has examined the persistence of biogenic signature molecules on spacecraft materials under simulated martian surface conditions. This study examined how endogenous adenosine-5'-triphosphate (ATP) would persist on aluminum coupons under simulated martian conditions of 7.

Bacillus endospores isolated from granite: close molecular relationships to globally distributed Bacillus spp. from endolithic and extreme environments.

As part of an ongoing effort to catalog spore-forming bacterial populations in environments conducive to interplanetary transfer by natural impacts or by human spaceflight activities, spores of Bacillus spp. were isolated and characterized from the interior of near-subsurface granite rock collected from the Santa Catalina Mountains, AZ. Granite was found to contain approximately 500 cultivable Bacillus spores and approximately 10(4) total cultivable bacteria per gram.

Bacillus subtilis spores on artificial meteorites survive hypervelocity atmospheric entry: implications for Lithopanspermia.

An important but untested aspect of the lithopanspermia hypothesis is that microbes situated on or within meteorites could survive hypervelocity entry from space through Earth's atmosphere. The use of high-altitude sounding rockets to test this notion was explored. Granite samples permeated with spores of Bacillus subtilis strain WN511 were attached to the exterior telemetry module of a sounding rocket and launched from White Sands Missile Range, New Mexico into space, reaching maximum atmospheric entry velocity of 1.

Essential cysteine residues in Bacillus subtilis spore photoproduct lyase identified by alanine scanning mutagenesis.

Endospore-forming bacteria (Bacillus and Clostridium spp.) are highly ultraviolet (UV) resistant and repair UV-induced DNA damage in part using the spore-specific DNA repair enzyme spore photoproduct (SP) lyase. SP lyase in all known sporeformers contains four conserved cysteine residues; three absolutely conserved residues are located at the "Radical SAM" consensus (C91xxxC95xxC98), which presumably participates in [4Fe-4S] cluster formation.

Bacterial endospores and their significance in stress resistance.

In terms of resistance to extreme environmental stresses, the bacterial spore represents a pinnacle of evolution. Spores are highly resistant to a wide variety of physical stresses such as: wet and dry heat, UV and gamma radiation, oxidizing agents, chemicals, and extremes of both vacuum and ultrahigh hydrostatic pressure. Some of the molecular mechanisms underlying spore resistance properties have been elucidated in the laboratory, and involve both: (i) protection of vital spore macromolecules during dormancy, and (ii) repair of damaged macromolecules during germination.

Temporal secretion of a multicellulolytic system in Myxobacter sp. AL-1. Molecular cloning and heterologous expression of cel9 encoding a modular endocellulase clustered in an operon with cel48, an exocellobiohydrolase gene.

The Gram-negative soil micro-organism Myxobacter sp. AL-1 possesses at least five extracellular cellulases, the production of which is regulated by the growth cycle. We cloned the complete gene for one of these cellulases, termed cel9, which encoded a 67-kDa modular family 9 endoglycohydrolase, which was produced during the stationary phase of growth and was strongly enhanced by avicel.

The TRAP-like SplA protein is a trans-acting negative regulator of spore photoproduct lyase synthesis during Bacillus subtilis sporulation.

UV resistance of bacterial endospores derives from a unique DNA photochemistry in which the major UV photoproduct is the thymine dimer 5-thyminyl-5,6-dihydrothymine (spore photoproduct [SP]) instead of cyclobutane pyrimidine dimers. Repair of SP during spore germination is due in large part to the activity of the enzyme SP lyase encoded by splB, the second cistron of the splAB operon. Expression of the splAB operon in Bacillus subtilis is transcriptionally activated by the Esigma(G) form of RNA polymerase during morphological stage III in the developing forespore compartment, and SP lyase is packaged into the dormant spore.

Analysis of spore photoproduct lyase operon (splAB) function using targeted deletion-insertion mutations spanning the Bacillus subtilis operons ptsHI and splAB.

Germinating Bacillus subtilis spores repair UV-induced DNA damage in part using the enzyme spore photoproduct (SP) lyase. SP lyase is encoded by splB, the second cistron of the splAB operon. The splAB operon is transcribed during sporulation from the P1 promoter, which partially overlaps the transcriptional terminator of the upstream ptsHI operon, which in turn encodes the Hpr protein and Enzyme I components of the PEP:sugar phosphotransferase (PTS) system.

Molecular dissection of mutations in the Bacillus subtilis spore photoproduct lyase gene which affect repair of spore DNA damage caused by UV radiation.

In response to UV irradiation, Bacillus subtilis spore DNA accumulates the unique thymine dimer 5-thyminyl-5,6-dihydrothymine, or spore photoproduct (SP). SP is broken down into monomers during spore germination by the product of the spl gene which has been proposed to encode the enzyme SP lyase. The wild-type spl gene was cloned by complementation of a mutation designated spl-1; the putative spl gene product is a 40-kDa protein whose deduced amino acid sequence contains regions homologous to DNA photolyases.

Molecular cloning and characterization of the Bacillus subtilis spore photoproduct lyase (spl) gene, which is involved in repair of UV radiation-induced DNA damage during spore germination.

Upon UV irradiation, Bacillus subtilis spore DNA accumulates the novel thymine dimer 5-thyminyl-5,6-dihydrothymine. Spores can repair this "spore photoproduct" (SP) upon germination either by the uvr-mediated general excision repair pathway or by the SP-specific spl pathway, which involves in situ monomerization of SP to two thymines by an enzyme named SP lyase. Mutants lacking both repair pathways produce spores that are extremely sensitive to UV.

Effect of chromosome location of Bacillus subtilis forespore genes on their spo gene dependence and transcription by E sigma F: identification of features of good E sigma F-dependent promoters.

Translational lacZ fusions to forespore genes of Bacillus subtilis were not expressed in spoIIAC (sigma F) or spoIIIE mutants when the lacZ fusions were integrated at the loci of the same genes or at the SP beta locus. However, some of these genes, including gerA, gpr, spoIIIG (sigma G), and sspE, were expressed in spoIIIE mutants and spoIIIE spoIIIG double mutants (but not in spoIIAC mutants) when the lacZ fusions were integrated at the amyE locus. When tested, the beta-galactosidase made in these mutants was found only in the forespore, and the 5' ends of the mRNAs produced in these mutants were identical to those in a Spo+ background.

Effect of promoter mutations and upstream deletions on the expression of genes coding for small, acid-soluble spore proteins of Bacillus subtilis.

The sspB and sspE genes code for major small, acid-soluble proteins of Bacillus subtilis spores and are transcribed during sporulation by RNA polymerase containing sigma G. Analysis of the expression in vivo and the sigma G-dependent transcription in vitro of sspB and sspE genes carrying upstream deletions or point mutations in -10 and -35 promoter regions is consistent with sigma G being the only major transcriptional regulator of these genes. These data also provide information on the residues in -10 and -35 regions which are most important for sigma G recognition.