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 ∼500 cultivable Bacillus spores and ∼10(4) total cultivable bacteria per gram. Many of the Bacillus isolates produced a previously unreported diffusible blue fluorescent compound. Two strains of eight tested exhibited increased spore UV resistance relative to a standard Bacillus subtilis UV biodosimetry strain. Fifty-six isolates were identified by repetitive extragenic palindromic PCR (rep-PCR) and 16S rRNA gene analysis as most closely related to B. megaterium (15 isolates), B. simplex (23 isolates), B. drentensis (6 isolates), B. niacini (7 isolates), and, likely, a new species related to B. barbaricus (5 isolates). Granite isolates were very closely related to a limited number of Bacillus spp. previously found to inhabit (i) globally distributed endolithic sites such as biodeteriorated murals, stone tombs, underground caverns, and rock concretions and (ii) extreme environments such as Antarctic soils, deep sea floor sediments, and spacecraft assembly facilities. Thus, it appears that the occurrence of Bacillus spp. in endolithic or extreme environments is not accidental but that these environments create unique niches excluding most Bacillus spp. but to which a limited number of Bacillus spp. are specifically adapted

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

The effect of Bacillus subtilis exposure to the human spaceflight environment on growth, mutagenic frequency, and spectrum of mutations to rifampicin resistance (RifR) was investigated. B. subtilis 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 RifR was found in either experiment. However, nucleotide sequencing of the RifR regions of the rpoB gene from RifR mutants revealed dramatic differences in the spectrum of mutations between flight (FL) and ground control (GC) samples, including two newly discovered rpoB alleles in the FL samples (Q137R and L489S). The results strengthen the idea that exposure to the human spaceflight environment causes unique stresses on bacteria, leading to alterations in their mutagenic potential

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

The effect of Bacillus subtilis exposure to the human spaceflight environment on growth, mutagenic frequency, and spectrum of mutations to rifampicin resistance (RifR) was investigated. B. subtilis 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 RifR was found in either experiment. However, nucleotide sequencing of the RifR regions of the rpoB gene from RifR mutants revealed dramatic differences in the spectrum of mutations between flight (FL) and ground control (GC) samples, including two newly discovered rpoB alleles in the FL samples (Q137R and L489S). The results strengthen the idea that exposure to the human spaceflight environment causes unique stresses on bacteria, leading to alterations in their mutagenic potential

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 7.1 mbar, full-spectrum simulated martian radiation calibrated to 4 W m−2 of UV-C (200 to 280 nm), −10°C, and a Mars gas mix of CO2 (95.54%), N2 (2.7%), Ar (1.6%), O2 (0.13%), and H2O (0.03%). Cell or spore viabilities of Acinetobacter radioresistens, Bacillus pumilus, and B. subtilis were measured in minutes to hours, while high levels of endogenous ATP were recovered after exposures of up to 21 days. The dominant factor responsible for temporal reductions in viability and loss of ATP was the simulated Mars surface radiation; low pressure, low temperature, and the Mars gas composition exhibited only slight effects. The normal burst of endogenous ATP detected during spore germination in B. pumilus and B. subtilis was reduced by 1 or 2 orders of magnitude following, respectively, 8- or 30-min exposures to simulated martian conditions. The results support the conclusion that endogenous ATP will persist for time periods that are likely to extend beyond the nominal lengths of most surface missions on Mars, and planetary protection protocols prior to launch may require additional rigor to further reduce the presence and abundance of biosignature molecules on spacecraft surfaces

Slow Degradation Of Atp In Simulated Martian Environments Suggests Long Residence Times For The Biosignature Molecule On Spacecraft Surfaces On Mars

Prelaunch planetary protection protocols on spacecraft are designed to reduce the numbers and diversity of viable bioloads on surfaces in order to mitigate the forward contamination of planetary surfaces. In addition, there is a growing appreciation that prelaunch spacecraft cleaning protocols will be required to reduce the levels of biogenic signature molecules on spacecraft to levels that will not compromise life-detection experiments on landers. The biogenic molecule, adenosine triphosphate (ATP) was tested for long-term stability under simulated Mars surface conditions of high UV flux, low temperature, low pressure, Mars atmosphere, and clear-sky dust loading conditions. Data on UV-induced ATP degradation rates were then extrapolated to a diversity of global conditions using a radiative transfer model for UV on Mars. The UV-induced degradation of ATP tested at 4.1 W m-2 UVC (200-280 nm), -10 °C, 7.1 mb, 95% CO2 gas composition, and an atmospheric opacity of τ = 0.1 yielded a half-life for ATP of 1342 kJ m-2; or extrapolated to approximately 22 sols on equatorial Mars with an atmospheric opacity of τ = 0.5. Temperature was found to moderately affect ATP degradation rates under martian conditions; tests at -80 or 20 °C yielded ATP half-lives of 2594 or 1183 kJ m-2, respectively. The ATP degradation rates reported here are over 10 orders of magnitude slower than the UV-induced biocidal rates reported in the literature on the inactivation of strongly UV-resistant bacterial spores from Bacillus pumilus SAFR-032 [Schuerger, A.C., Richards, J.T., Newcombe, D.A., Venkateswaran, K.J., 2006. Icarus 181, 52-62]. Extrapolating results to global Mars conditions, residence times for a 99% reduction of ATP on spacecraft surfaces ranged from 158 sols on Sun-exposed surfaces to approximately 32,000 sols for the undersides of landers similar to Viking. However, spacecraft materials greatly affected the survival times of ATP under martian conditions. Stainless steel was found to enhance the UV degradation of ATP by over 2 orders of magnitude compared to ATP-doped iridited aluminum, graphite, and astroquartz coupons. Extrapolating these results to global conditions, ATP on stainless steel might be expected to persist between 2 and 320 sols for upper and lower surfaces of landers. Liquid chromatography-mass spectrometry data supported the conclusion that UV irradiation acted to remove the γ-phosphate group from ATP, and no evidence was observed for the UV-degradation of d-ribose or adenine moieties. Long residence times for ATP on spacecraft materials under martian conditions suggest that prelaunch cleaning protocols may need to be strengthened to mitigate against possible ATP contamination of life-detection experiments on Mars landers. © 2007 Elsevier Inc. All rights reserved

Transcriptomic responses of Serratia liquefaciens cells grown under simulated Martian conditions of low temperature, low pressure, and CO2-enriched anoxic atmosphere

Abstract 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, CO2-dominated atmosphere–conditions intended to simulate the near-subsurface environment of Mars. To probe the response of its transcriptome to this extreme environment, S. liquefaciens ATCC 27592 was cultivated under 4 different environmental simulations: 0 °C, 0.7 kPa, CO2 atmosphere (Condition A); 0 °C, ~101.3 kPa, CO2 atmosphere (Condition B); 0 °C, ~101.3 kPa, ambient N2/O2 atmosphere (Condition C); and 30 °C, ~101.3 kPa, N2/O2 atmosphere (Condition D; ambient laboratory conditions). RNA-seq was performed on ribosomal RNA-depleted total RNA isolated from triplicate cultures grown under Conditions A-D and the datasets generated were subjected to transcriptome analyses. The data from Conditions A, B, or C were compared to laboratory Condition D. Significantly differentially expressed transcripts were identified belonging to a number of KEGG pathway categories. Up-regulated genes under all Conditions A, B, and C included those encoding transporters (ABC and PTS transporters); genes involved in translation (ribosomes and their biogenesis, biosynthesis of both tRNAs and aminoacyl-tRNAs); DNA repair and recombination; and non-coding RNAs. Genes down-regulated under all Conditions A, B, and C included: transporters (mostly ABC transporters); flagellar and motility proteins; genes involved in phenylalanine metabolism; transcription factors; and two-component systems. The results are discussed in the context of Mars astrobiology and planetary protection

Slow degradation of ATP in simulated martian environments suggests long residence times for the biosignature molecule on spacecraft surfaces. Icarus 194:86–100

Abstract Prelaunch planetary protection protocols on spacecraft are designed to reduce the numbers and diversity of viable bioloads on surfaces in order to mitigate the forward contamination of planetary surfaces. In addition, there is a growing appreciation that prelaunch spacecraft cleaning protocols will be required to reduce the levels of biogenic signature molecules on spacecraft to levels that will not compromise life-detection experiments on landers. The biogenic molecule, adenosine triphosphate (ATP) was tested for long-term stability under simulated Mars surface conditions of high UV flux, low temperature, low pressure, Mars atmosphere, and clear-sky dust loading conditions. Data on UV-induced ATP degradation rates were then extrapolated to a diversity of global conditions using a radiative transfer model for UV on Mars. The UV-induced degradation of ATP tested at 4.1 W m −2 UVC (200-280 nm), −10 • C, 7.1 mb, 95% CO 2 gas composition, and an atmospheric opacity of τ = 0.1 yielded a half-life for ATP of 1342 kJ m −2 ; or extrapolated to approximately 22 sols on equatorial Mars with an atmospheric opacity of τ = 0.5. Temperature was found to moderately affect ATP degradation rates under martian conditions; tests at −80 or 20 • C yielded ATP half-lives of 2594 or 1183 kJ m −2 , respectively. The ATP degradation rates reported here are over 10 orders of magnitude slower than the UV-induced biocidal rates reported in the literature on the inactivation of strongly UV-resistant bacterial spores from Bacillus pumilus SAFR-032 [Schuerger, A.C., Richards, J.T., Newcombe, D.A., Venkateswaran, K.J., 2006. Icarus 181, 52-62]. Extrapolating results to global Mars conditions, residence times for a 99% reduction of ATP on spacecraft surfaces ranged from 158 sols on Sun-exposed surfaces to approximately 32,000 sols for the undersides of landers similar to Viking. However, spacecraft materials greatly affected the survival times of ATP under martian conditions. Stainless steel was found to enhance the UV degradation of ATP by over 2 orders of magnitude compared to ATP-doped iridited aluminum, graphite, and astroquartz coupons. Extrapolating these results to global conditions, ATP on stainless steel might be expected to persist between 2 and 320 sols for upper and lower surfaces of landers. Liquid chromatography-mass spectrometry data supported the conclusion that UV irradiation acted to remove the γ -phosphate group from ATP, and no evidence was observed for the UV-degradation of D-ribose or adenine moieties. Long residence times for ATP on spacecraft materials under martian conditions suggest that prelaunch cleaning protocols may need to be strengthened to mitigate against possible ATP contamination of life-detection experiments on Mars landers