Nobuo Munakata

Mapping of the genes controlling excision repair of Pyrimidine photoproducts in Bacillus subtilis

SummaryTwenty nine uvr mutations of Bacillus subtilis Marburg have been classified into two groups by mutual transformation crosses. One, uvrA, contained 20 mutations and involved uvrAl which had been isolated by Reiter and Strauss (1965) and linked to the hisA gene by Hoch and Anagnostopoulos (1970). Another group, uvrB, contained 9 mutations and the linkage relationship has been established as argA-polA-uvrB-leu-pheA. Both of them seemed to govern the incision reaction upon DNA containing pyrimidine dimers or spore photoproducts. It thus seems that in B. subtilis, as in Escherichia coli, the process of excision repair is controlled by the products of two or more genes not closely linked in the chromosome.

Genetic analysis of a mutant ofBacillus subtilis producing ultraviolet-sensitive spores

SummaryA mutant ofBacillus subtilis, uvssp-42-1, producing UV-sensitive spores was studied genetically. By treatment of the cells with DNA prepared from auvr strain two types,uvs-42 (Hcr−) andssp-1 (Hcr+), of transformants producing UV-resistant spores were obtained. Only strains having both types of mutations together produced UV-sensitive spores.

Adaptive response to simple alkylating agents in Bacillus subtilis cells

Abstract When the cells of Bacillus subtilis Marburg strains were grown in the presence of N -methyl- N ′-nitro- N -nitrosoguanidine (MNNG) at a non-toxic concentration, they acquired resistance with regard to both lethality (enhancement of survival) and mutagenicity (reduction of mutation frequency) to subsequent challenge with MNNG at higher concentrations. Thus, like Escherichia coli cells (Samson and Cairns, 1977), B. subtilis cells respond adaptively to MNNG. Protein synthesis was necessary for the adaptation, and an activity to destroy specifically O 6 -methylguanine residues in methylated DNA was present in the extracts of the adapted cells. Therefore, this response is attributable to an error-free DNA-repair system induced by the exposure to MNNG. The adaptive response was observed with the cells carrying uvrA, polA or recA mutation, indicating this to be independent of the excision repair or recA -dependent repair. For the acquisition of the resistance to MNNG, methylating or ethylating agents were effective, but neither N -butyl- N -nitrosorea (BNU) nor N -propyl- N ′-nitro- N -nitrosoguanidine (PNNG) was effective. The cells pre-treated with MNNG became resistant to the challenge with alkynitrosoureas or N -alkyl- N ′-nitro- N -nitrosoguanidine, but not to the challenge with methyl methanesulphonate (MMS) or dimethyl sulphate (DMS). Thus, the target of the induced DNA-repair activity is likely to be O -alkylated bases in DNA. Though the phenomenology of the adaptive response seems similar to that in E. coli , the following two lines of evidence indicate that the underlying mechanisms may not be identical: (1) in B. subtilis , the cells of polA strains are adapted both with regard to lethality and to mutagenicity; (2) in B. subtilis , MMS or DMS acts only as the inducer of the response.

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 by 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.

Mutation induction with UV- and X-radiations in spores and vegetative cells of Bacillus subtilis.

Spores and vegetative cells of Bacillus subtilis strains with various defects in DNA-repair capacities (hcr-, ssp-, hcr-ssp-) were irradiated with UV radiation or X-rays. Induced mutation frequency was determined from the observed frequency of prototrophic reversion of a suppressible auxotrophic mutation. At equal physical dose, after either UV- or X-irradiation, spores were more resistant to mutations as well as to killing than were vegetative cells. However, quantitative comparison revealed that, at equally lethal doses, spores and vegetative cells were almost equally mutable by X-rays whereas spores were considerably less mutable by UV than were vegetative cells. Thus, as judged from their mutagenic efficiency relative to the lethality, X-ray-induced damage in the spore DNA and the vegetative DNA were equally mutagenic, while UV-induced DNA photoproducts in the spore were less mutagenic than those in vegetative cells. Post-treatment of UV-irradiated cells with caffeine decreased the survival and the induced mutation frequency for either spores or vegetative cells for all the strains. In X-irradiated spores, however, a similar suppressing effect of caffeine was observed only for mutability of a strain lacking DNA polymerase I activity.

Inactivation of transforming DNA by ultraviolet irradiation

Abstract Transforming activities of various markers of Bacillus subtilis were inactivated by UV irradiation to different degrees. The results were compared with the studies on the differential inactivation by heat denaturation and on the fractionation on a methylated albumin column, which suggested that these markers might reside on DNA molecules of different base compositions. The markers on GC-rich DNA molecules seemed to be more resistant to UV inactivation and vice versa. This conclusion was supported by comparing the photoreactivable sectors of two markers showing different UV sensitivities. Experimental results also indicated a correlation between the UV resistance and the frequency of transformation. It seems likely that the base composition in the marker region might be an important factor causing the difference in transformation frequency and that the UV irradiation might exaggerate the difference. In contrast, X-rays inactivated all the activities at almost similar rates. Minor differences might have been caused by certain indirect effects.

A mutant of Bacillus subtilis producing ultraviolet-sensitive spores

Abstract In general, bacterial spores are more resistant to ultraviolet (UV) irradiation than the vegetative cells of the same strain ( Zelle and Hollaender, 1955 ) ( Donnellan and Stafford, 1968 ). It is thought that the spores might be protected against UV effects by a special mechanism. This paper deals with isolation of a mutant of Bacillus subtilis producing UV-sensitive spores. This mutant was derived from a mutant producing UV-sensitive vegetative cells. In the new isolate, the spores were almost as sensitive to UV irradiation as the vegetative cells. Spore-specific protection or repair mechanism is discussed.

Two classes of Bacillus subtilis mutants deficient in the adaptive response to simple alkylating agents

SummarySix mutant strains of Bacillus subtilis hypersensitive to N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) were shown to be deficient in the adaptive response to MNNG and termed ada mutants (Morohoshi and Munakata 1985). All the mutations mapped between the attSPO2 and lin loci on the chromosome. The mutant and wild-type (ada+) cells contained similar constitutive levels of O6-methylguanine-DNA methyltransferase activity. Pretreatment with low concentrations of MNNG increased the activity about nine-fold in the ada+ cells, while it uniformly decreased the activity in the ada cells. The pretreatment of three mutants (ada-3, ada-4, and ada-6) as well as ada+, augumented the activity of methylpurine-DNA glycosylase and rendered the cells resistant to the lethal and mutagenic effects of N-propyl- or N-butyl-N′-nitro-N-nitrosoguanidine. With the rest of the mutant strains (ada-1, ada-2, and ada-5), neither of such responses was elicited by the pretreatment. Thus, the former ada strains seem to have a defect in the gene specifically involved in the induction of the methyltransferase, while the latter ada strains have a defect in the gene controlling the adaptive response as a whole.

Isolation of a Bacillus subtilis mutant defective in constitutive 06-alkylguanine-DNA alkyltransferase

A mutant of Bacillus subtilis defective in the constitutive activity of O6-alkylguanine-DNA alkyltransferase was isolated from a strain (ada-1) deficient in the adaptive response to DNA alkylation. Cells carrying the mutation dat-1 which was responsible for the defect in constitutive activity exhibited hypersensitivity for lethality and mutagenesis when challenged with methyl-nitroso compounds. The constitutive activity is independent of the adaptive response, and seems to function as a basal defense against environmental alkylating agents.

DNA glycosylase activities in the nematode, Caenorhabditis elegans

Abstract DNA glycosylases acting upon uracil- or 3-methyl-adenine-containing DNA have been detected in the sonic extracts of the nematode, Caenorhabditis elegans. 4 types of the asynchronously-growing worms, embryos obtained from gravid hermaphrodites, aseptically-hatched larvae, or dauer larvae. Uracil-DNA glcosylase activity was found in all 4 types of the extracts, and the activity was highest in the embryonic extract. In contrast, 3-methyladenine-DNA glycosylase activity was undetectable in the embryonic extract, while an equal level of activity was found in the other 3 types of the extracts. The results substantiate the ubiquity of base-excision repair in various organisms, and suggest that some of the repair functions may be developmentally regulated in multicellular animals.