Patricia Fajardo-Cavazos

Bacillus Endospores Isolated from Granite: Close Molecular Relationships to Globally Distributed Bacillus spp. from Endolithic and Extreme Environments

ABSTRACT 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 ∼104 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.

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. A fourth conserved cysteine, the function of which is unknown, is located at C141 in SP lyase from all Bacillus spp. sequenced to date. To probe the function of the fourth cysteine, each conserved cysteine in the B. subtilis SP lyase was systematically altered to alanine by site-directed mutagenesis. UV-visible spectroscopy of wild-type and mutant SP lyases indicated that C141 does not participate in [4Fe-4S] formation and redox chemistry; however, in vivo SP lyase activity was abolished in all mutants, indicating an essential role for C141 in SP lyase activity.

Exploring the Low-Pressure Growth Limit: Evolution of Bacillus subtilis in the Laboratory to Enhanced Growth at 5 Kilopascals

ABSTRACT 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. Growth inhibition was immediately reversible upon return to 101.3 kPa, albeit at a slower rate. A population of B. subtilis cells was cultivated at the near-inhibitory pressure of 5 kPa for 1,000 generations, where a stepwise increase in growth was observed, as measured by the turbidity of 24-h cultures. An isolate from the 1,000-generation population was obtained that showed an increase in fitness at 5 kPa when compared to the ancestral strain or a strain obtained from a parallel population that evolved for 1,000 generations at 101.3 kPa. The results from this preliminary study have implications for understanding the ability of terrestrial microbes to grow in low-pressure environments such as Mars.

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. Organisms within rock can only embark on an interplanetary journey if they survive ejection from the surface of the donor planet and the associated extremes of compressional shock, heating, and acceleration. Previous simulation experiments have measured each of these three stresses more or less in isolation of one another, and results to date indicate that spores of the model organism Bacillus subtilis can survive each stress applied singly. Few simulations, however, have combined all three stresses simultaneously. Because considerable experimental and theoretical evidence supports a spallation mechanism for launch, we devised an experimental simulation of launch by spallation using the Ames Vertical Gun Range (AVGR). B. subtilis spores were applied to the surface of a granite target that was impacted from above by an aluminum projectile fired at 5.4 km/s. Granite spall fragments were captured in a foam recovery fixture and then recovered and assayed for shock damage by transmission electron microscopy and for spore survival by viability assays. Peak shock pressure at the impact site was calculated to be 57.1 GPa, though recovered spall fragments were only very lightly shocked at pressures of 5-7 GPa. Spore survival was calculated to be on the order of 10(-5), which is in agreement with results of previous static compressional shock experiments. These results demonstrate that endolithic spores can survive launch by spallation from a hypervelocity impact, which lends further evidence in favor of lithopanspermia theory.

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. In addition to temporal and compartmental control of splAB expression, a second regulatory circuit which modulates the level of expression of splB-lacZ fusions without altering their developmental timing or compartmentalization is reported here. This second regulatory circuit involves the negative action of the splA gene product, a 79-amino-acid protein with approximately 50% similarity and 17% identity to TRAP, the tryptophan RNA-binding attenuation protein from B. subtilis and Bacillus pumilus.

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. We exposed DNA deposited on spacecraft-qualified aluminum coupons to a simulated martian environment for periods ranging from 1 minute to 1 hour and measured its ability to function as a template for replication in a quantitative polymerase chain reaction (qPCR) assay. We found that inactivation of naked DNA or DNA extracted from exposed spores of Bacillus subtilis followed a multiphasic UV-dose response and that a fraction of DNA molecules retained functionality after 60 minutes of exposure to simulated full-spectrum solar radiation in martian atmospheric conditions. The results indicate that forward-contaminant DNA could persist for considerable periods of time at the martian surface.

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. To explore mechanisms that enabled growth of the low-pressure-adapted strain, numerous genes differentially transcribed between the ancestor strain WN624 and low-pressure-evolved strain WN1106 at 50 mbar were identified by microarray analysis. Among these was a cluster of three candidate genes (des, desK, and desR), whose mRNA levels in WN1106 were higher than the ancestor on the microarrays. Up-regulation of these genes was confirmed by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis. The des, desK, and desR genes encode the Des membrane fatty acid (FA) desaturase, the DesK sensor kinase, and the DesR response regulator, respectively, which function to maintain membrane fluidity in acute response to temperature downshift. Pressure downshift caused an up-regulation of des mRNA levels only in WN1106, but expression of a des-lacZ transcriptional fusion was unaffected, which suggests that des regulation was different in response to temperature versus pressure downshift. Competition experiments showed that inactivation of the des gene caused a slight, but statistically significant, loss of fitness of strain WN1106 at 50 mbar. Further, analysis of membrane FA composition of cells grown at 1013 versus 50 mbar revealed a decrease in the ratio of unsaturated to saturated FAs but an increase in the ratio of anteiso- to iso-FAs. The present study represents a first step toward identification of molecular mechanisms by which B. subtilis could sense and respond to the novel environmental stress of low pressure.

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

ABSTRACT 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.

Complete Genome Sequence of Serratia liquefaciens Strain ATCC 27592

ABSTRACT 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.

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. Asynchronous ground controls (GCs) consisted of identical sets of cultures cultivated for 122 h in the ISS Environmental Simulator at Kennedy Space Center. S. epidermidis exhibited significantly lower viable counts but significantly higher frequencies of mutation to rifampicin (Rif) resistance in space vs. GC cultures. The spectrum of mutations in the rpoB gene leading to RifR was altered in S. epidermidis isolates cultivated in the ISS compared to GCs. The results suggest that the human spaceflight environment induces unique physiologic stresses on growing bacterial cells leading to changes in mutagenic potential.