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Wayne L Nicholson

Publications and source records attributed to Wayne L Nicholson.

17 recordsLinked to original sources

Survival and germinability of Bacillus subtilis spores exposed to simulated Mars solar radiation: implications for life detection and planetary protection.

Bacterial spores have been considered as microbial life that could survive interplanetary transport by natural impact processes or human spaceflight activity. Deposition of terrestrial microbes or their biosignature molecules onto the surface of Mars could negatively impact life detection experiments and planetary protection measures. Simulated Mars solar radiation, particularly the ultraviolet component, has been shown to reduce spore viability, but its effect on spore germination and resulting production of biosignature molecules has not been explored. We examined the survival and germinability of Bacillus subtilis spores exposed to simulated martian conditions that include solar radiation. Spores of B. subtilis that contain luciferase resulting from expression of an sspB-luxAB gene fusion were deposited on aluminum coupons to simulate deposition on spacecraft surfaces and exposed to simulated Mars atmosphere and solar radiation. The equivalent of 42 min of simulated Mars solar radiation exposure reduced spore viability by nearly 3 logs, while germination-induced bioluminescence, a measure of germination metabolism, was reduced by less than 1 log. The data indicate that spores can retain the potential to initiate germination-associated metabolic processes and produce biological signature molecules after being rendered nonviable by exposure to Mars solar radiation.

Aluminum↗

The population genetics of phenotypic deterioration in experimental populations of Bacillus subtilis.

Although many examples of trait loss exist in nature, the underlying population genetic mechanism responsible for the loss is usually unknown. Selective or neutral processes can result in the deterioration of a trait, and often one of these is inferred based on indirect evidence. Furthermore, selective pressures that are unique to particular environments and the effect these might have on the population genetic cause of trait loss are not well understood. Here we describe an experimental evolution system where two different environments were used for addressing the population genetic cause of trait loss throughout evolutionary time. We found that growth in minimal medium (i.e., prototrophy) was lost in all populations regardless of the experimental environment and that the pattern of trait loss in one environment was due to selection, whereas in the other environment the cause remains inconclusive.

Bacillus subtilis↗

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.

Alanine↗

The solar UV environment and bacterial spore UV resistance: considerations for Earth-to-Mars transport by natural processes and human spaceflight.

The environment in space and on planets such as Mars can be lethal to microorganisms because of the high vacuum and high solar radiation flux, in particular UV radiation, in such environments. Spores of various Bacillus species are among the organisms most resistant to the lethal effects of high vacuum and UV radiation, and as a consequence are of major concern for planetary contamination via unmanned spacecraft or even natural processes. This review focuses on the spores of various Bacillus species: (i) their mechanisms of UV resistance; (ii) their survival in unmanned spacecraft, space flight and simulated space flight and Martian conditions; (iii) the UV flux in space and on Mars; (iv) factors affecting spore survival in such high UV flux environments.

Bacillus↗

Bacillus subtilis spore survival and expression of germination-induced bioluminescence after prolonged incubation under simulated Mars atmospheric pressure and composition: implications for planetary protection and lithopanspermia.

Bacterial endospores in the genus Bacillus are considered good models for studying interplanetary transfer of microbes by natural or human processes. Although spore survival during transfer itself has been the subject of considerable study, the fate of spores in extraterrestrial environments has received less attention. In this report we subjected spores of a strain of Bacillus subtilis, containing luciferase resulting from expression of an sspB-luxAB gene fusion, to simulated martian atmospheric pressure (7-18 mbar) and composition (100% CO(2)) for up to 19 days in a Mars simulation chamber. We report here that survival was similar between spores exposed to Earth conditions and spores exposed up to 19 days to simulated martian conditions. However, germination-induced bioluminescence was lower in spores exposed to simulated martian atmosphere, which suggests sublethal impairment of some endogenous spore germination processes.

Atmospheric Pressure↗

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 1.2 km/s. Maximum recorded temperature during the flight was measured at 145 degrees C. The surfaces of the post-flight granite samples were swabbed and tested for recovery and survival of WN511 spores, using genetic markers and the unique DNA fingerprint of WN511 as recovery criteria. Spore survivors were isolated at high frequency, ranging from 1.2% to 4.4% compared with ground controls, from all surfaces except the forward-facing surface. Sporulation-defective mutants were noted among the spaceflight survivors at high frequency (4%). These experiments constitute the first report of spore survival to hypervelocity atmospheric transit, and indicate that sounding rocket flights can be used to model the high-speed atmospheric entry of bacteria-laden artificial meteorites.

Acceleration↗

Stochastic processes influence stationary-phase decisions in Bacillus subtilis.

It has recently been proposed that phenotypic variation in clonal populations of bacterial species results from intracellular "noise," i.e., random fluctuations in levels of cellular molecules, which would be predicted to be insensitive to selective pressure. To test this notion, we propagated five populations of Bacillus subtilis for 5,000 generations with selection for one phenotype: the decision to sporulate. In support of the noise hypothesis, we report that none of the populations responded to selection by improving their efficiency of sporulation, indicating that intracellular noise is independent of heritable genotype.

Bacillus subtilis↗

Novel rpoB mutations conferring rifampin resistance on Bacillus subtilis: global effects on growth, competence, sporulation, and germination.

Previously, spontaneous rifampin resistance mutations were isolated in cluster I of the rpoB gene, resulting in amino acid replacements (Q469R, H482R, H482Y, or S487L) in the Bacillus subtilis RNA polymerase beta subunit (W. L. Nicholson and H. Maughan, J. Bacteriol. 184:4936-4940, 2002). In this study, each amino acid change in the beta subunit was observed to result in its own unique spectrum of effects on growth and various developmental events, including sporulation, germination, and competence for transformation. The results thus establish the important role played by the RNA polymerase beta subunit, not only in the catalytic aspect of transcription, but also in the regulation of major developmental events in B. subtilis.

Alleles↗

Using thermal inactivation kinetics to calculate the probability of extreme spore longevity: implications for paleomicrobiology and lithopanspermia.

Thermal inactivation kinetics with extrapolation were used to model the survival probabilities of spores of various Bacillus species over time periods of millions of years at the historical ambient temperatures (25-40 degrees C) encountered within the 250 million-year-old Salado formation, from which the putative ancient spore-forming bacterium Salibacillus marismortui strain 2-9-3 was recovered. The model indicated extremely low-to-moderate survival probabilities for spores of mesophiles. but surprisingly high survival probabilities for thermophilic spores. The significance of the results are discussed in terms of the survival probabilities of (i) terrestrial spores in ancient geologic samples and (ii) spores transported between planets within impact ejecta.

Bacillus↗

Spore UV and acceleration resistance of endolithic Bacillus pumilus and Bacillus subtilis isolates obtained from Sonoran desert basalt: implications for lithopanspermia.

Bacterial spores have been used as model systems for studying the theory of interplanetary transport of life by natural processes such as asteroidal or cometary impacts (i.e., lithopanspermia). Because current spallation theory predicts that near-surface rocks are ideal candidates for planetary ejection and surface basalts are widely distributed throughout the rocky planets, we isolated spore-forming bacteria from the interior of near-subsurface basalt rocks collected in the Sonoran desert near Tucson, Arizona. Spores were found to inhabit basalt at very low concentrations (</=28 colony-forming units/g) in these samples. Six isolates identified as being most closely related to Bacillus pumilus and one Bacillus subtilis isolate were recovered from near-subsurface basalt samples. Populations of purified spores prepared from the isolated strains were subjected to 254-nm UV and ballistics tests in order to assess their resistance to UV radiation and to extreme acceleration shock, two proposed lethal factors for spores during interplanetary transfer. Specific natural isolates of B. pumilus were found to be substantially more resistant to UV and extreme acceleration than were reference laboratory strains of B. subtilis, the benchmark organism, suggesting that spores of environmental B. pumilus isolates may be more likely to survive the rigors of interplanetary transfer.

Bacillus↗

Comparison of UV inactivation of spores of three encephalitozoon species with that of spores of two DNA repair-deficient Bacillus subtilis biodosimetry strains.

When exposed to 254-nm UV, spores of Encephalitozoon intestinalis, Encephalitozoon cuniculi, and Encephalitozoon hellem exhibited 3.2-log reductions in viability at UV fluences of 60, 140, and 190 J/m(2), respectively, and demonstrated UV inactivation kinetics similar to those observed for endospores of DNA repair-defective mutant Bacillus subtilis strains used as biodosimetry surrogates. The results indicate that spores of Encephalitozoon spp. are readily inactivated at low UV fluences and that spores of UV-sensitive B. subtilis strains can be useful surrogates in evaluating UV reactor performance.

Animals↗

UV resistance of Bacillus anthracis spores revisited: validation of Bacillus subtilis spores as UV surrogates for spores of B. anthracis Sterne.

Recent bioterrorism concerns have prompted renewed efforts towards understanding the biology of bacterial spore resistance to radiation with a special emphasis on the spores of Bacillus anthracis. A review of the literature revealed that B. anthracis Sterne spores may be three to four times more resistant to 254-nm-wavelength UV than are spores of commonly used indicator strains of Bacillus subtilis. To test this notion, B. anthracis Sterne spores were purified and their UV inactivation kinetics were determined in parallel with those of the spores of two indicator strains of B. subtilis, strains WN624 and ATCC 6633. When prepared and assayed under identical conditions, the spores of all three strains exhibited essentially identical UV inactivation kinetics. The data indicate that standard UV treatments that are effective against B. subtilis spores are likely also sufficient to inactivate B. anthracis spores and that the spores of standard B. subtilis strains could reliably be used as a biodosimetry model for the UV inactivation of B. anthracis spores.

Bacillus anthracis↗

Inactivation of vegetative cells, but not spores, of Bacillus anthracis, B. cereus, and B. subtilis on stainless steel surfaces coated with an antimicrobial silver- and zinc-containing zeolite formulation.

Stainless steel surfaces coated with paints containing a silver- and zinc-containing zeolite (AgION antimicrobial) were assayed in comparison to uncoated stainless steel for antimicrobial activity against vegetative cells and spores of three Bacillus species, namely, B. anthracis Sterne, B. cereus T, and B. subtilis 168. Under the test conditions (25 degrees C and 80% relative humidity), the zeolite coating produced approximately 3 log(10) inactivation of vegetative cells within a 5- to 24-h period, but viability of spores of the three species was not significantly affected.

Anti-Bacterial Agents↗

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. Our group has recently become interested in testing if the laboratory model of spore UV resistance is relevant to spore persistence in the environment. We have constructed a number of Bacillus subtilis strains which are defective in various DNA repair systems and spore structural components. Using spores of these strains, we have been exploring: (i) the types of damage induced in DNA by the UV-B and UV-A components of sunlight; (ii) the relative contribution of the major spore DNA repair systems to spore solar radiation resistance; and (iii) the role of spore structural components such as the spore coats and dipicolinic acid (DPA) in attenuation of the lethal and mutagenic effects of solar UV. The current data are reviewed with the ultimate goal of obtaining a complete model describing spore persistence and longevity in the terrestrial solar UV radiation environment.

Bacillus subtilis↗

UV photochemistry of DNA in vitro and in Bacillus subtilis spores at earth-ambient and low atmospheric pressure: implications for spore survival on other planets or moons in the solar system.

Two major parameters influencing the survival of Bacillus subtilis spores in space and on bodies within the Solar System are UV radiation and vacuum, both of which induce inactivating damage to DNA. To date, however, spore survival and DNA photochemistry have been explored only at the extremes of Earth-normal atmospheric pressure (101.3 kPa) and at simulated space vacuum (10(-3)-10(-6) Pa). In this study, wild-type spores, mutant spores lacking alpha/beta-type small, acid-soluble spore proteins (SASP), naked DNA, and complexes between SASP SspC and DNA were exposed simultaneously to UV (254 nm) at intermediate pressure (1-2 Pa), and the UV photoproducts cis,syn-thymine-thymine cyclobutane dimer (c,sTT), trans,syn-thymine-thymine cyclobutane dimer (t,sTT), and "spore photoproduct" (SP) were quantified. At 101.3 kPa, UV-treated wild-type spores accumulated only SP, but spores treated with UV radiation at 1-2 Pa exhibited a spectrum of DNA damage similar to that of spores treated at 10(-6) Pa, with accumulation of SP, c,sTT, and t,sTT. The presence or absence of alpha/beta-type SASP in spores was partly responsible for the shift observed between levels of SP and c,sTT, but not t,sTT. The changes observed in spore DNA photochemistry at 1-2 Pa in vivo were not reproduced by irradiation of naked DNA or SspC:DNA complexes in vitro, suggesting that factors other than SASP are involved in spore DNA photochemistry at low pressure.

Atmospheric Pressure↗

The spectrum of spontaneous rifampin resistance mutations in the rpoB gene of Bacillus subtilis 168 spores differs from that of vegetative cells and resembles that of Mycobacterium tuberculosis.

Mutations causing rifampin resistance in vegetative cells of Bacillus subtilis 168 have thus far been mapped to a rather restricted set of alterations at either Q469 or H482 within cluster I of the rpoB gene encoding the beta subunit of RNA polymerase. In this study, we demonstrated that spores of B. subtilis 168 exhibit a spectrum of spontaneous rifampin resistance mutations distinct from that of vegetative cells. In addition to the rpoB mutations Q469K, Q469R, and H482Y previously characterized in vegetative cells, we isolated a new mutation of rpoB, H482R, from vegetative cells. Additional new rifampin resistance mutations arising from spores were detected at A478N and most frequently at S487L. The S487L change is the predominant change found in rpoB mutations sequenced from rifampin-resistant clinical isolates of Mycobacterium tuberculosis. The observations are discussed in terms of the underlying differences of the DNA environment within dormant cells and vegetatively growing cells.

Amino Acid Sequence↗