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We used microarrays to identify the causes of sporulation deficiencies in Bacillus subtilis after 6,000 generations of evolution. We found that sporulation loss did not result from large-scale deletions; therefore, it must have resulted from smaller indels and/or substitutions. Transcription patterns of one strain versus its ancestor showed that sporulation was not initiated and suggested that sporulation loss may be part of an overall decline in plasticity.

The role of DNA repair by nonhomologous end joining (NHEJ), homologous recombination, spore photoproduct lyase, and DNA polymerase I and genome protection via [alpha]/[beta]-type small, acid-soluble spore proteins (SASP) in Bacillus subtilis spore resistance to accelerated heavy ions (high-energy charged [HZE] particles) and X rays has been studied. Spores deficient in NHEJ and [alpha]/[beta]-type SASP were significantly more sensitive to HZE particle bombardment and X-ray irradiation than were the recA, polA, and splB mutant and wild-type spores, indicating that NHEJ provides an eflicient DNA double-strand break repair pathway during spore germination and that the loss of the [alpha]/[beta]-type SASP leads to a significant radiosensitivity to ionizing radiation, suggesting the essential function of these spore proteins as protectants of spore DNA against ionizing radiation.

RNA polymerase is a central macromolecular machine controlling the flow of information from genotype to phenotype, and insights into global transcriptional regulation can be gained by studying mutational perturbations in the enzyme. Mutations in the RNA polymerase [beta] subunit gene rpoB causing resistance to rifampin ([Rif.sup.r]) in Bacillus subtilis were previously shown to lead to alterations in the expression of a number of global phenotypes known to be under transcriptional control, such as growth, competence for transformation, sporulation, and germination (H. Maughan, B. Galeano, and W. L. Nicholson, J. Bacteriol. 186:2481-2486, 2004). To better understand the global etfects of rpoB mutations on metabolism, wild-type and 11 distinct congenic [Rif.sup.r] mutant strains of B. subtilis were tested for utilization of 95 substrates by use of Biolog GP2 MicroPlates. A number of alterations of substrate utilization patterns were observed in the [Rif.sup.r] mutants, including the utilization of novel substrates previously unknown in B. subtilis, such as gentiobiose, [beta]-methylD-glucoside, and D-psicose. The resuits indicate that combining global metabolic profiling with mutations in RNA polymerase provides a system-wide approach for uncovering previously unknown metabolic capabilities and further understanding global transcriptional control circuitry in B. subtilis.

Phenotypic loss is an important evolutionary force in nature but the mechanism (s) responsible for loss remains unclear. We used both simulation and multiple-regression approaches to analyze data on the loss of sporulation, a complex bacterial developmental process, during experimental evolution of Bacillus subtilis. Neutral processes of mutational degradation alone were sufficient to explain loss-of-sporulation ability in four of five populations, while evidence that selection facilitated mutational loss was found for only one population. These results are discussed in the context of the evolution of sporulation in particular and phenotypic loss in general.

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.icarus.2006.06.014 Byline: Andrew C. Schuerger (a), Wayne L. Nicholson (b) Keywords: Mars; Astrobiology Abstract: Robotic spacecraft are launched with finite levels of terrestrial microorganisms that are similar to the microbial communities within facilities in which spacecraft are assembled. In particular, spores of mesophilic aerobic Bacillus species are common spacecraft contaminants considered most likely to survive interplanetary transfer to Mars. During the cruise phase to Mars, and then again during surface operations, microbial bioloads are exposed to a diversity of biocidal factors that are likely to render the microbial species either dead or significantly inhibited from active metabolic activity and replication. We report here, for the first time, that interactive effects of low pressure, low temperature, and high CO.sub.2 atmospheres approaching conditions likely to be encountered on the martian surface strongly inhibit the growth and replication of seven common Bacillus spp. isolated from spacecraft. Tests were conducted within a small glass bell-jar system maintained in a low-temperature microbial incubator. Atmospheric pressures were controlled at 1013 (Earth-normal), 100, 50, 35, 25, or 15 mb, and temperatures were maintained at 30, 20, 15, 10, or 5a[degrees]C. Experiments were carried out for 48 h or 7 days under either Earth-normal O.sub.2/N.sub.2 or pure CO.sub.2 atmospheres. Results indicated that low pressure, low temperature, and high CO.sub.2 atmospheres, applied separately or in combination, were capable of inhibiting the growth and replication of B. pumilus SAFR-032, B. pumilus FO-36B, B. subtilis HA-101, B. subtilis 42HS-1, B. megaterium KL-197, B. licheniformis KL-196, and B. nealsonii FO-092 under simulated martian conditions. Endospores of all seven Bacillus spp. strains failed to germinate and grow at 25 mb at 30a[degrees]C. Although, vegetative cells of these strains exhibited a slightly greater ability to replicate at lower pressures than did endospores, vegetative cells of these species failed to grow at pressures below 25 mb. Interactive effects of these environmental parameters acted to generally increase the inhibitory nature of the low-pressure conditions on growth and replication of the seven Bacillus spp. tested. Author Affiliation: (a) Department of Plant Pathology, University of Florida, Bldg. M6-1025, Space Life Sciences Lab, Kennedy Space Center, FL 32899, USA (b) Department of Microbiology and Cell Science, University of Florida, Bldg. M6-1025, Space Life Sciences Lab, Kennedy Space Center, FL 32899, USA Article History: Received 15 December 2005; Revised 20 June 2006

An experimental evolution system having two different environments that were used for addressing the population genetic cause of trait loss all along evolutionary time is described. Results reveal that growth in prototrophy was lost in all populations regardless of the experimental environment, in one environment the pattern of trait loss was due to selection and in the other environment the cause remains inconclusive.

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.

RNA polymerase (RNAP) is a highly conserved macromolecular machine that contributes to the flow of genetic information from genotype to phenotype. In Bacillus subtilis, mutations in the rpoB gene encoding the b-subunit of RNAP have been shown to alter a number of global phenotypes, including growth, utilization of unusual nutrient sources, sporulation, germination, and production of secondary metabolites. In addition, the spectrum of mutations in rpoB leading to rifampin resistance (Rifr) can change dramatically depending upon the environment to which B. subtilis cells or spores are exposed. Rifr rpoB mutations have historically been associated with slower growth and reduced fitness; however, these assessments of fitness were conducted on limited collections of mutants in rich laboratory media that poorly reflect natural environments typically inhabited by B. subtilis. Using a novel deep-sequencing approach in addition to traditional measurements of growth rate, lag time, and pairwise competitions, we demonstrated that the competitive advantages of specific rpoB alleles differ depending on the growth environment in which they are determined. IMPORTANCE Microbial resistance to antibiotics is a growing threat to public health across the world. Historically, resistance to antibiotics has been associated with reduced fitness. A growing body of evidence indicates that resistance to rifampin, a frontline antibiotic used to treat mycobacterial and biofilm-associated infections, may increase fitness given an appropriate environment even in the absence of the selective antibiotic. Here, we experimentally confirm this phenomenon by directly comparing the fitness of multiple rifampin-resistant mutants of Bacillus subtilis in rich LB medium and an asparagine minimal medium. Our research demonstrates that the fitness cost of rifampin resistance can vary greatly depending upon the environment. This has important implications for understanding how microbes develop antimicrobial resistance in the absence of antibiotic selection. [ABSTRACT FROM AUTHOR]

Results from previous investigations into spontaneous rifampin resistance (Rifr) mutations in the Bacillus subtilis rpoB gene suggested that the spectrum of mutations depends on the growth environment. However, these studies were limited by low sample numbers, allowing for the potential distortion of the data by the presence of "jackpot" mutations that may have arisen early in the growth of a population. Here, we addressed this issue by performing fluctuation analyses to assess both the rate and spectrum of Rifr mutations in two distinct media: LB, a complete laboratory medium, and SMMAsn, a minimal medium utilizing L-asparagine as the sole carbon source. We cultivated 60 separate populations under each growth condition and determined the mutation rate to Rifr to be slightly but significantly higher in LB cultures. We then sequenced the relevant regions of rpoB to map the spectrum of Rifr mutations under each growth condition. We found a distinct spectrum of mutations in each medium; LB cultures were dominated by the H482Y mutation (27/53 or 51%), whereas SMMAsn cultures were dominated by the S487L mutation (24/51 or 47%). Furthermore, we found through competition experiments that the relative fitness of the S487L mutant was significantly higher in SMMAsn than in LB medium. We therefore conclude that both the spectrum of Rifr mutations in the B. subtilis rpoB gene and the fitness of resulting mutants are influenced by the growth environment. [ABSTRACT FROM AUTHOR]

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— Bifidobacterium longum strain BB536, Lactobacillus acidophilus strain DDS-1, and spores of Bacillus subtilis 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 >109 colony forming units per capsule. Parameters tested were survival to: (i) long-term storage at ambient conditions, (ii) simulated Galactic Cosmic Radiation and Solar Particle Event radiation provided by the NASA Space Radiation Laboratory, (iii) exposure to simulated gastric fluid, and (iv) exposure to simulated intestinal fluid. We found that radiation exposure produced minimal effects on the probiotic strains. However, we found that that the shelf-lives of the three strains, and their survival during passage through simulations of the upper GI tract, differed dramatically. We observed that only spores of B. subtilis were capable of surviving all conditions and maintaining a titer of >109 spores per capsule. The results indicate that probiotics consisting of bacterial spores could be a viable option for long-duration human space travel. [ABSTRACT FROM AUTHOR]

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.

Although clinostats have long been used in space microbiology studies as ground-based analogs of spaceflight, few studies to date have systematically compared -omics data from clinostats versus spaceflight. This study compared the transcriptomic response of the Gram-positive bacterium Bacillus subtilis flown in space with corresponding transcriptomes derived from 2-D clinostat (High Aspect Ratio Vessel: HARV) experiments performed under the same conditions of bacterial strain, growth medium, temperature, and incubation time. High-quality total RNA (RNA Integrity Number >9.6) was isolated from multiple biological replicates from each treatment, transcripts were quantified by RNA-seq, and raw data was processed through a previously described standardized bioinformatics pipeline. Transcriptome data sets from spaceflight-grown and corresponding clinostat-grown cells were compared by using three different methods: (i) principal component analysis, (ii) analysis of differentially expressed genes, and (iii) gene set enrichment analysis of KEGG pathways. All three analyses found a low degree of concordance between the spaceflight and corresponding clinostat transcriptome data sets, ranging from 0.9% to 5.3% concordance. These results are in agreement with prior studies that also revealed low concordances between spaceflight and clinostat transcriptomes of the Gram-negative bacteria Rhodospirillum rubrum and Pseudomonas aeruginosa. The results are discussed from the perspective of several potential confounding factors, and suggestions are offered with the aim of achieving increased concordance between clinostat and spaceflight data. [ABSTRACT FROM AUTHOR]

A population of Bacillus subtilis cells is cultivated at the near-inhibitory pressure of 5 kPa, where a stepwise increase in growth is observed. The results have helped in understanding the ability of terrestrial microbes to grow in low-pressure environments such as Mars.

Small, acid-soluble spore proteins (SASP)-defective mutants of Bacillus subtilis and spores deficient in dacB, a mutation leading to an increased core water content, are used for studying the relative contributions of SASP and increased core water content to spore resistance to germicidal and simulated environmental ultraviolet (UV) exposure. The results have shown that [alpha]/[beta]-type SASP and decreased spore core water content have played a major role in spore resistance to environmentally relevant UV wavelengths whereas SASP-[gamma] has not.

The amino acid sequences of two authentic AR/BDH-encoding genes from Saccharomyces cerevisiae and Bacillus cereus are used to identify highly similar protein encoded by the B. subtilis ydjL gene. A very small amount of 2,3-butanediol detected in very-late-stationary-phase 96-hour cultures of the ydjL::cat mutant revealed the existence of a second gene encoding a minor AR activity, which calls for a need to rename acetoin reductase/2,3-butanediol dehydrogenase (AR/BDH)-encoding gene ydjL to bdhA.

A study was conducted to determine the stability of endogenous ATP present within intact cells of common spacecraft contaminants located on the surface of spacecraft materials and exposed to simulated Martian conditions including full-spectrum solar radiation. The results showed that endogenous ATP would persist for time periods that are likely to extend beyond the nominal lengths of most surface missions on Mars.

This article illustrates the antimicrobial activity of the paints containing silver- and zinc-containing zeolite formulation against vegetative cells and spores of Bacillus species. Results show the paints cause about 3 log(sub)10 inactivation of vegetative cells within a 5- to 24-hour period at 25 C and 80% relative humidity. However, viability of spores is least affected.

DNA is considered a potential biomarker for life-detection experiments destined for Mars. Experiments were conducted to examine the photochemistry of bacterial DNA, either unprotected or within Bacillus subtilis spores, in response to exposure to simulated martian surface conditions consisting of the following: temperature (−10°C), pressure (0.7 kPa), atmospheric composition [CO2 (95.54%), N2 (2.7%), Ar (1.6%), O2 (0.13%), and H2O (0.03%)], and UV–visible–near IR solar radiation spectrum (200–1100 nm) calibrated to 4 W/m2 of UVC (200–280 nm). While the majority (99.9%) of viable spores deposited in multiple layers on spacecraft-qualified aluminum coupons were inactivated within 5 min, a detectable fraction survived for up to the equivalent of ∼115 martian sols. Spore photoproduct (SP) was the major lesion detected in spore DNA, with minor amounts of cyclobutane pyrimidine dimers (CPD), in the order TT CPD > TC CPD >> CT CPD. In addition, the (6-4)TC, but not the (6-4)TT, photoproduct was detected in spore DNA. When unprotected DNA was exposed to simulated martian conditions, all photoproducts were detected. Surprisingly, the (6-4)TC photoproduct was the major photoproduct, followed by SP ∼ TT CPD > TC CPD > (6-4)TT > CT CPD > CC CPD. Differences in the photochemistry of unprotected DNA and spore DNA in response to simulated martian surface conditions versus laboratory conditions are reviewed and discussed. The results have implications for the planning of future life-detection experiments that use DNA as the target, and for the long-term persistence on Mars of forward contaminants or their DNA. Key Words: Bacillus subtilis—DNA—Mars—Photochemistry—Spore—Ultraviolet. Astrobiology 18, 393–402. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]