Resistance of Bacillus endospores to extreme terrestrial and extraterrestrial environments.

Endospores of Bacillus spp., especially Bacillus subtilis, have served as experimental models for exploring the molecular mechanisms underlying the incredible longevity of spores and their resistance to environmental insults. In this review we summarize the molecular laboratory model of spore resistance mechanisms and attempt to use the model as a basis for exploration of the resistance of spores to environmental extremes both on Earth and during postulated interplanetary transfer through space as a result of natural impact processes.

Structure and expression pattern of a human MTG8/ETO family gene, MTGR1.

AML1-MTG8 fusion protein, which is produced from the rearranged gene formed between AML1 and MTG8 in myeloid leukemia with t(8;21) chromosomal translocation, plays an important role in the pathogenesis of leukemia. We previously showed that ectopically expressed AML1-MTG8 fusion protein is associated with an MTG8-like protein in the mouse myeloid precursor cell line L-G, and this association seemed to be required for AML1-MTG8 to stimulate proliferation. As a candidate cDNA for this MTG8-like protein, a 6.4 kb MTGR1 cDNA encoding human MTGR1b protein of 604 amino acids was isolated. Since this cDNA was shorter than the main mRNA (about 7.5 kb), the 5'-end of the MTGR1 cDNA was extended using Marathon Ready cDNA. When the newly obtained 5'-sequence was combined with the previous cDNA, the resultant MTGR1 cDNA (6995 bp), including exon 3 that the previous cDNA lacked, could encode MTGR1a protein of 575 amino acids. Transcripts of the MTGR1 gene were expressed ubiquitously in the human tissues and cell lines examined. PCR analyses of the cDNAs from human tissues showed the presence of various splicing variants with regard to the 5'-region including exons 1, 2 and 3. The MTGR1 gene consists of 14 exons and spans about 68 kb. The genomic structure of MTGR1 is highly similar to those of other MTG 8-family genes, MTG8 and MTG16. MTG16 was recently cloned from the translocation breakpoint of myeloid malignancies with t(16;21) chromosomal translocation.

Comparisons of spore dosimetry and spectral photometry of solar-UV radiation at four sites in Japan and Europe.

In order to develop monitoring and assessment systems of biologically effective doses of solar-UV radiation, concurrent measurements of spectral photometry and spore dosimetry were conducted in summer months at four sites in Japan and Europe. Effectiveness spectra were derived by multiplying spectral irradiance in 0.5 nm steps between 290 and 400 nm with the inactivation efficiency of the spores determined using monochromatic radiation of fine wavelength resolution. Shapes of the effectiveness spectra were very similar at the four sites exhibiting major peaks at 303.5, 305.0, 307.5 and 311.0 nm. The dose rates for spore inactivation from direct survival measurements and from calculations by the integration of the effectiveness spectra were compared for 174 data points. The ratios (observed/calculated) of the two values were concordant with a mean of 1.26 (+/- 0.24 standard deviation [SD]). The possible causes for the variations and slightly larger observed values are discussed.

Spore dosimetry of solar UV radiation: applications to monitoring of daily irradiance and personal exposure.

Environmental UV radiation can be quantified using spore dosimetry, which measures the inactivation of repair-deficient Bacillus subtilis spores dried on a membrane filter. The system exhibits highly selective sensitivity to UV radiation, not being affected by various environmental adversities, such as high and low temperature and humidity. Biologically-effective dose rate and cumulative dose of ambient radiation are measurable under various conditions at various places on the earth, including tropical, temperate, and polar sites. Applications to monitor the exposure at the surface of organisms including humans and plants have also been advanced.

Base substitution spectra of nalidixylate resistant mutations induced by monochromatic soft X and 60Co gamma-rays in Bacillus subtilis spores.

Bacillus subtilis spores were exposed to three types of photons, monochromatic soft X-rays with the energy corresponding to the absorption peak of phosphorus K-shell electron (2,153 eV) and with the slightly lower energy (2,147 eV), and 60Co gamma-rays. From the irradiated spores, 233 mutants exhibiting nalidixic acid resistance were isolated, and together with 94 spontaneous mutants, the sequence changes in the 5'-terminal region of the gyrA gene coding for DNA gyrase subunit A were determined. Among eighteen alleles of the gyrA mutations, eight were single-base substitutions, nine were tandem double-base substitutions, and one was a double substitution skipping a middle base pair. About 6% of the radiation-induced mutations were tandem double-base substitutions, whereas none was observed among the spontaneous ones. Among spontaneous mutations, A:T and G:C pairs were equally subjected to mutations, whereas the substitutions from G:C pairs and those to A:T pairs predominated among those induced with soft X-rays. The peak-energy X-rays were more effective in killing and causing mutations than the low-energy X-rays, however, there seemed no base-change events uniquely attributable to phosphorus K-shell absorption.

Sunlight mutagenesis: changes in mutational specificity during the irradiation of phage M13mp2.

We reported previously that the mutations in phage M13mp2, a single-stranded DNA phage, induced by sunlight exposure are predominated by G-to-C transversions. We have now made an unexpected observation that an exposure to sunlight for a short period of time results in induction mainly of C-to-T transitions while a longer exposure results in the induction of G-to-C transversions. This peculiar phenomenon suggests that DNA damage formed by initial sunlight exposure can be transformed during an elongated exposure. 7, 8-Dihydro-8-oxoguanine (8-oxoG) in DNA might be involved in the shift of the mutational specificity, as 8-oxoG was formed in the phage DNA upon the sunlight exposure. We also compared the mutagenic activity of UVB irradiation with that of sunlight exposure. The results demonstrate that the genotoxic properties of sunlight and UVB in phage M13mp2 mutagenesis are different. The shift in the mutational specificity associated with the dose of the sunlight may call for general cautions in the studies of agent-induced mutagenesis.

Comparative measurements of solar UV radiation with spore dosimetry at three European and two Japanese sites.

Spore dosimetry of solar UV radiation has been employed in several field intercomparison campaigns carried out in summer months in Japan and Europe. The dose-rate profiles, total daily doses and personal daily exposures have been determined and compared at five sites based on the spore inactivation dose (SID). The maximum dose rate (0.83 SID/min) and the maximum daily dose (197 SID) observed at subtropical Naha (26.2 degrees N) are about three times those observed at subarctic Abisko (68.4 degrees N). The amounts of personal exposure during three European campaigns are moderate and show a tendency of inverse relationships with the daily doses.

Genomic structure of the human RBP56/hTAFII68 and FUS/TLS genes.

We previously isolated RBP56 cDNA by PCR using mixed primers designed from the conserved sequences of the RNA binding domain of FUS/TLS and EWS proteins. RBP56 protein turned out to be hTAFII68 which was isolated as a TATA-binding protein associated factor (TAF) from a sub-population of TFIID complexes (Bertolotti A., Lutz, Y., Heard, D.J., Chambon, P., Tora, L., 1996. hTAFII68, a novel RNA/ssDNA-binding protein with homology to the proto-oncoproteins TLS/FUS and EWS is associated with both TFIID and RNA polymerase II. EMBO J. 15, 5022-5031). The RBP56/hTAFII68, FUS/TLS and EWS proteins comprise a sub-family of RNA binding proteins, which consist of an N-terminal Ser, Gly, Gln and Tyr-rich region, an RNA binding domain, a Cys2/Cys2 zinc finger motif and a C-terminal RGG-containing region. Rearrangement of the FUS/TLS gene and the EWS gene has been found in several types of malignant tumors, and the resultant fusion proteins play an important role in the pathogenesis of these tumors. In the present study, we determined the genomic structure of the RBP56/hTAFII68 gene. The RBP56/hTAFII68 gene spans about 37kb and consists of 16 exons from 33bp to 562bp. The longest exon, exon 15, encodes the C-terminal region containing 19 repeats of a degenerate DR(S)GG(G)YGG sequence. While the structure of the FUS/TLS gene has been reported previously, we determined the total DNA sequence of the FUS/TLS gene, consisting of 12kb. The RBP56/hTAFII68, FUS/TLS and EWS genes consist of similar numbers of exons. Comparison of the structures of these three genes showed that the organization of exons in the central part encoding a homologous RNA binding domain and a cysteine finger motif is highly conserved, and other exon boundaries are also located at similar sites, indicating that these three genes most likely originate from the same ancestor gene.

Monitoring of solar-UV exposure among schoolchildren in five Japanese cities using spore dosimeter and UV-coloring labels.

To monitor personal exposure to biologically effective solar-UV radiation, Bacillus subtilis spores on a membrane filter and UV-coloring labels were incorporated into a monitoring badge. The samples were covered with one of three types of filter sheet, dependent on the season, to reduce the amounts of exposure to measurable levels. Five fifth- or sixth-grade classes of primary schools, each consisting of 30-40 children, were chosen in northern (Sapporo), central (Tsukuba and Tokyo), and southern (Miyazaki and Naha) cities in Japan. In all four season, each child wore a badge on an upper arm for the entire waking hours, changing it daily, for a week. Upon collection of the badges, the survival of spores and the extent of coloration of the label were determined. The results were used to estimate the amount of daily exposure to biologically effective UV radiation, expressed as the value of spore inactivation dose. Unexpectedly, the average amounts of exposure were not directly correlated with the outdoor UV irradiance: in the two southern cities, despite high outdoor irradiance from spring to autumn, the average amounts of exposure were less than 3.1% of the average irradiance. Highly concentrated exposures occurred in two central cities on three days when extensive outdoor exercise took place. These results contradict the simple notion that children's exposure is in proportion to the outdoor UV irradiance, and support the view that the extent of solar-UV exposure is primarily determined by life-style rather than living location.

Induction of unique tandem-base change mutations in bacterial spores exposed to extreme dryness.

Despite the remarkable resistance to desiccation, Bacillus subtilis spores manifest indications of DNA damage when being kept in an extremely dry environment made by high vacuum. Spores of strain TKJ3422 (uvrA10 spl-1 recA4) with triple repair defects lost colony-forming capacity dependent on the duration and strength of the exposure. Mutations to rifampicin resistance were induced in the spores of the strain HA101 with wild-type repair capability and the strain TKJ6312 (uvrA10 spl-1) with double repair defects. The majority of nalidixic acid-resistant mutations induced by the exposure to high vacuum belonged to one particular allele gyrA12 carrying a tandem-base change, 5'-CA to 5'-TT, at codon 84 of the gyrA gene coding for DNA gyrase subunit A. This allele has never been found among more than 500 mutants obtained by various treatments other than vacuum exposure. These results indicate forced dehydration of DNA in the microenvironment of the spore core causes unique damage leading to lethal and mutagenic consequences.

Doses of solar ultraviolet radiation correlate with skin cancer rates in Japan.

We analyzed trends in the disease rate of skin cancers in the 1976-80 and 1986-90 intervals in the 27 university hospitals in Japan. We also measured doses of solar ultraviolet (UV) radiation at Sapporo, Kobe and Miyazaki to evaluate the relationship between the two in Japan. The rates of basal cell carcinoma (BCC) and actinic keratosis (AK) were higher in 1986-90 than in 1976-80, whereas the rate of squamous cell carcinoma (SCC) was lower in 1986-90 than in the earlier period. The rates of SCC, BCC and AK in the southern part of Japan were about five times higher than those in the north, and the average daily UV dose measured with a Robertson-Berger meter in 1995 was about 1.8 times higher in Miyazaki than in Kobe. That measured by MS-210D UV dosimeter in Sapporo was about 0.53 times lower than in Kobe. These results demonstrate that solar UV dose is higher in the southern part of Japan than that in the northern part, explaining the higher rate of non-melanoma skin cancer in southern part of Japan. A significant increase of AK and BCC may reflect the trend of UV increase in Japan.

p53 gene mutations in oral carcinomas from India.

In this study, we analyzed 53 oral squamous-cell carcinomas among Indians for the presence of alterations in the tumor-suppressor gene p53 by PCR-SSCP and sequencing methods. Our results showed that 21% (11/53) of oral carcinomas analyzed carried mutations within the exons 5-8 of the p53 gene. We have identified 11 single-base pair substitutions consisting of 10 mis-sense mutations and one at the splice acceptor site, and one deletion mutation involving 4 consecutive bases. The majority of the base substitutions were transitions (5 TA to CG and 5 GC to AT), while only one transversion (TA to GC) was observed. Probable hot-spots for the mutation induction were identified at codons 149 and 274, which have not been observed before in head-and-neck cancers. The mutational spectrum might have originated from base alkylations at guanine and thymine residues, caused by some alkylating agents. The present results are thus consistent with the involvement of tobacco-related nitrosoamines in the etiology of oral squamous-cell carcinoma.

Experimental correspondence between spore dosimetry and spectral photometry of solar ultraviolet radiation.

The biologically effective dose of solar UV radiation was estimated from the inactivation of UV-sensitive Bacillus subtilis spores. Two types of independent measurements were carried out concurrently at the Aerological Observatory in Tsukuba: one was the direct measurement of colony-forming survival that provided the inactivation dose per minute (ID/min) and the other was the measurement of the spectral irradiance by a Brewer spectrophotometer. To obtain the effective spectrum, the irradiance for each 1 nm wavelenght interval from 290 to 400 nm was multiplied with the efficiency for inactivation derived from the inactivation action spectrum of identically prepared spore samples. Integration of the effective spectrum provided the estimate for ID/min. The observed values of ID/min were closely concordant with the calculated values for the data obtained in four afternoons in 1993. The average ratio (+/- SD) between them was 1.24 (+/- 0.16) for 14 data points showing high inactivation rates (> 0.05 ID/min). Considering difficulties in the absolute dosimetry of UV radiation, the concordance was satisfactory and improved credibility of the two types of monitoring systems of biologically effective dose of solar UV radiation.

Diverse capacities for the adaptive response to DNA alkylation in Bacillus species and strains.

Our previous studies of Bacillus subtilis showed that the genes responsible for the adaptive response to DNA alkylation were organized as a divergent regulon, in contrast to scattered operons in Escherichia coli ada regulon. To study the generality and diversity of gene organization, several species and strains of Bacillus were examined for the responsiveness to DNA alkylation. B. cereus cells exhibited the highest resistance to MNNG treatment. When the cells were grown in the presence of MNNG, 3-methyladenine DNA glycosylase and two species of DNA methyltransferase were induced as in B. subtilis 168 cells. B. licheniformis 749 and B. amyloliquefaciens H cells exhibited a partial response that manifested itself as the induction of one species of DNA methyltransferase. On the other hand, B. thuringiensis var. Tohokuensis, B. megaterium KMT, and B. subtilis W23 cells were totally deficient in this response, and were hypersensitive to alkylating agents. To determine the cause of this deficiency in strain W23, we examined the genomic structure of the corresponding region where three genes (alkA, adaA, and adaB) were located in 168. No homologues for the three genes were detected in W23 DNA by Southern hybridization. Two genes (glmS and ndhF) flanking the adaptive response regulon in 168 were also present in W23. A sequence of about 2750 bp that carried the entire regulon in 168 was replaced with a sequence of about 250 bp that was unique to W23. At the ends of the conserved segments, palindromic sequences corresponding to the transcriptional termination sites of the adaB and glmS genes were observed. The regulon in 168 could be artificially replaced by the W23 sequence, and be regained through DNA-mediated transformation.

Polyethylene glycol-modified avidin: a novel agent for the selective extraction of biotinylated immune-complex in an aqueous two-phase system.

Chicken avidin was chemically modified with 2,4-bis[O-methoxypoly(ethylene glycol)]-6-chloro-s-triazine (activated PEG2) to form PEG-avidin. The PEG-avidin, in which 78% of the amino groups were modified, retained 49% of the active biotin-binding sites. The modified avidin was partitioned preferentially into the PEG-phase in an aqueous two-phase system (PEG/dextran). Using PEG-avidin, the immune-complex formed between biotinylated anti-mouse IgG and its antigen IgG (mouse) molecules, was successfully transferred into the PEG-phase in an aqueous two-phase system. This finding leads to the effective isolation of a specific antigen among various kinds of antigens by partitioning with a two-phase system using PEG-avidin.

Molecular characterization of thirteen gyrA mutations conferring nalidixic acid resistance in Bacillus subtilis.

We isolated 607 independent nalidixic acid-resistant mutants from Bacillus subtilis. A 163 bp DNA segment from a 5' portion of the gyrA gene was amplified from the DNA of each mutant strain. After heat denaturation, the product was subjected to gel electrophoresis to detect conformational polymorphism of single-strand DNA (PCR-SSCP analysis). Mobility patterns of the two DNA strands from all the mutant strains examined differed from those of the parental wild-type strains. The patterns were classified into 13 types, and the DNA sequence of each type was determined. A unique sequence alteration was found in mutants belonging to each of the 13 types, defining 13 gyrA alleles. Eight were single base pair substitutions, four were substitutions of two consecutive base pairs and one was a substitution of three consecutive base pairs. Only three amino acid residues (Ser-84, Ala-85, and Glu-88) were altered in the deduced amino acid sequences of the mutated genes. We conclude that molecular typing based on the PCR-SSCP method is a powerful technique for the exhaustive identification of allelic variants among mutants selected for a phenotypic trait.

Biologically effective dose of solar ultraviolet radiation estimated by spore dosimetry in Tokyo since 1980.

The biologically effective dose of solar UV radiation has been measured in Tokyo since 1980 using Bacillus subtilis spores. To determine the cumulative dose in a half day, several samples of UV-sensitive spores were exposed in successive intervals from the solar-noon time. Because fluence-survival curves were exponential, the number of lethal hits received by the spores was calculated for each interval and termed inactivation dose (ID). The total number of hits obtained in a half day (half-day ID) was correlated with the amount of global insolation by a power-function regression. The regression analyses were performed for the data collected on 35 days from 1980 to 1986 and for the data collected on 53 days from 1989 to 1991. The latter data set yielded significantly larger estimates of half-day ID relative to the insolation than the former. These analyses suggested that the biologically effective dose relative to the insolation increased about 30% at some time in the later part of 1980s at this location. Changes of solar activity, air pollution and stratospheric ozone layer were considered as potentially responsible for this increase, but identification of the causative factors requires further efforts.

Immunohistochemical examination of tumor-suppressor gene p53 product and pyrimidine dimer in solar keratosis.

In order to find biomarkers to measure the effects of UV irradiation, we examined the accumulation of p53 protein and pyrimidine dimers in 18 solar keratosis specimens. Frozen or AMeX-fixed solar keratosis specimens were immunohistochemically stained by anti-p53 mouse monoclonal antibody, pAb1801 and polyclonal anti-(pyrimidine dimer) antibody. Nuclear accumulation of p53 protein was found in 5/18 (28%) solar keratosis lesions. The percentage of cases showing nuclear p53 protein varied according to the histological type; in the bowenoid type it was 4/7 (57%); in the atrophic type it was 1/7 (14%). Nuclear pyrimidine dimers were not stained in solar keratosis, although the skin of UV-irradiated nude mice was positive. Accumulation of p53 protein is a good marker for early precancerous change caused by UV exposure.

Inactivation action spectra of Bacillus subtilis spores with monochromatic soft X rays (0.1-0.6 nm) of synchrotron radiation.

Five types of Bacillus subtilis spores differing in DNA repair and recombinational capacities were exposed in vacuum to monochromatic soft X rays from synchrotron radiation. The inactivation rate constants were obtained from exposure-survival curves upon irradiations at 12 wavelengths in the range of 0.1000 nm (12.40 keV) to 0.6000 nm (2.066 keV). Spores of two repair-deficient strains, UVS (uvrA ssp) and UVP (uvrA ssp polA), exhibited almost equal sensitivities to those of wild-type UVR+, while those of two recombination-deficient strains, RCE (recE) and RCF (recF), exhibited higher sensitivities in the whole wavelength range. This suggested that the repair of DNA damage produced by soft X rays was dependent on the recombinational capabilities. Inactivation action spectra based on photon fluence showed that the effectiveness of the radiation increased as the wavelengths became longer. Abrupt changes in the effectiveness occurred around the wavelengths corresponding to the absorption edges of K-shell electrons of phosphorus and calcium. In both cases, the sensitivity was the highest at the wavelengths of the resonance absorption peak, the next highest at those of the higher energy, and the lowest at the lower energy. Mass energy absorption coefficients of spores were obtained from the transmission of a flake made of spores. They were used to derive inactivation action spectra based on absorbed doses. In these spectra, basal levels of the sensitivity seemed constant, and enhancements of the sensitivity were observed consistent with the absorption by calcium and phosphorus. Thus calcium and phosphorus atoms were the predominant targets for the absorption events leading to the inactivation of spores in the wavelength range examined.

Molecular analysis of Bacillus subtilis ada mutants deficient in the adaptive response to simple alkylating agents.

Previously, we isolated and characterized six Bacillus subtilis ada mutants that were hypersensitive to methylnitroso compounds and deficient in the adaptive response to alkylation. Cloning of the DNA complementing the defects revealed the presence of an ada operon consisting of two tandem and partially overlapping genes, adaA and adaB. The two genes encoded proteins with methylphosphotriester-DNA methyltransferase and O6-methylguanine-DNA methyltransferase activities, respectively. To locate the six mutations, the ada operon was divided into five overlapping regions of about 350 bp. The fragments of each region were amplified by polymerase chain reaction and analyzed by gel electrophoresis to detect single-strand conformation polymorphism. Nucleotide sequences of the fragments exhibiting mobility shifts were determined. Three of the mutants carried sequence alterations in the adaA gene: the adaA1 and adaA2 mutants had a one-base deletion and insertion, respectively, and the adaA5 mutant had a substitution of two consecutive bases causing changes of two amino acid residues next to the presumptive alkyl-accepting Cys-85 residue. Three mutants carried sequence alterations in the adaB gene: the adaB3 mutant contained a rearrangement, the adaB6 mutant contained a base substitution causing a change of the presumptive alkyl-accepting Cys-141 to Tyr, and the adaB4 mutant contained a base substitution changing Leu-167 to Pro. The adaB mutants produced ada transcripts upon treatment with low doses of alkylating agents, whereas the adaA mutant did not. We conclude that the AdaA protein functions as the transcriptional activator of this operon, while the AdaB protein specializes in repair of alkylated residues in DNA.