[Ecologic ultraviolet--a real mutagenic factor for vegetative cells of Bacillus subtilis]. / Ekologicheskii ul'trafiolet--real'nyi mutagennyi faktor dlia vegetativnykh kletok Bacillus subtilis.

The frequency of leu----Leu+ reversions represented mainly by suppressor mutations is increased in Bacillus subtilis uvr+ and uvr-1 cells after exposure to natural sunlight. Dependence of mutation yield on the time of exposure is linear (one hit kinetics) in case of the uvr-1 strain. In the uvr+ cells the yield of mutations is also linear, but only at short times of exposure, the curve bending and levelling off the plateau after 10-min cell illumination. It has been established in the experiments with optical filters that the mutagenic effect is related to wavelengths which correspond to the UVB zone of ecological UV. The mutagenesis caused by sunlight can be modified (weakened) by some post-irradiation treatments of bacteria, which also led to a decrease of mutations frequencies in B. subtilis uvr+ and uvr-1 cells after exposure to 254-nm UV. The data indicate that: 1) mutagenic influence of sunlight can be overcome only by the joint action of activities of the two cellular repair systems--photoreactivation and excision repair, 2) the real mutagenic effect of sunlight on such a non-photoreactivating organism as B. subtilis would not be enhanced with the increase of the UVB flow in sunlight spectrum.

[Manifestation of antimutagenic activity of "dark" repair under alternative levels of aeration of UV-irradiated bacteria]. / Proiavlenie antimutagennoi aktivnosti "temnovoi" reparatsii pri al'ternativnykh urovniakh aèratsii UF-obluchennykh bakterii,

The cease of aeration of UV-irradiated bacteria incubated in glucose-salt medium does not affect antimutagenic activity of excision repair in Escherichia coli cells but strongly inhibits that in Bacillus subtilis cells. It has been suggested that these differences are connected with various possibilities for energy (ATP) production in facultative anaerobe, which is E. coli, and obligate anaerobe, Bac. subtilis. The absence of noticeable influence of the aerobiosis----anaerobiosis shift on the kinetics of disappearance of potential mutations in E. coli cells is interpreted in terms of existence of a mechanism regulating the expenditure of cell energy reserve upon repair process. It is suggested that the low rate of disappearance of potential mutations observed in post-irradiation conditions favourable for protein synthesis is a consequence of limited supply of energy to repair process at some sites of cellular DNA, due to great expense of energy for protein synthesis.

[Determination of the initial rate of antimutagenic repair in UV-irradiated WP2 Escherichia coli cells]. / Opredelenie nachal'noi skorosti antimutagennoi reparatsii v UV-obluchennykh kletkakh Escherichia coli WP2.

The initial rates of antimutagenic dark repair were measured in Escherichia coli WP2 trpE65 cells irradiated by UV-light (11 J/m2) and then incubated in liquid media of various compositions. Samples were taken from suspension of incubated bacteria every 5 min following irradiation, mixed with acriflavine to block further repair and plated onto the selective medium containing acriflavine (1 micrograms/ml) to score the Trp+ mutations. The initial rate of antimutagenic repair was estimated from the kinetics of disappearance of mutations in several successive probes. It appeared to depend on the composition of a medium, to establish just after placing irradiated bacteria onto the medium and to decrease significantly in irradiated cells incubated under conditions favourable for growth. The decrease was not due to inhibition of postreplicative repair and was not caused by casaminoacids as such, but by combination of growth factors that provided the intensive protein synthesis. The decrease could be responsible for a strong mutational response of bacteria to irradiation because it secures the survival of premutagenic lesions in DNA till mutation fixation. It is suggested that metabolic regulation of the antimutagenic repair activity exists, based on an active switch of the energy flows required for several parallel metabolic pathways that proceed in irradiated cells.

[Excision repair does not protect Bacillus subtilis cells from inactivation by sunlight]. / Ekstsizionnaia reparatsiia ne zashchishchaet kletki Bacillus subtilis ot inaktivatsii solnechnym svetom.

Exponentially growing Bacillus subtilis cells are highly sensitive to inactivating action of sunlight, the strain deficient in excision repair being every more sensitive than the uvr1 mutant. The inactivating effect is connected with the action of irradiation located in the left part of the spectrum (the whole UV region and some zones of the visible one). Increased sensitivity to sunlight disappeared when cells were exposed to sunlight in a liquid medium with casaminoacids (2 g/l). The inactivating effect was probably of photodynamic nature and was caused either by DNA lesions that were not removed by excision repair or by the damage which arose not in DNA.

[Increase in the fraction of non-repaired mutations in Escherichia coli cells, incubated in the absence of glucose after UV-irradiation]. / Uvelichenie fraktsii nerepariruemykh mutatsii v kletkakh Escherichia coli, proinkubirovannykh v otsutstvie gliukozy posle UF-oblucheniia.

In E. coli WP2 trpE65 cells irradiated with UV-dose of 11 J/m2, the additional small portion of induced Trp+ mutations became resistant to photoreactivation or "dark" (excision) repair after a short-termed (10-30 min) postirradiation incubation of bacteria in a minimal medium deprived of glucose and tryptophan. Since protein synthesis could not proceed in those cells because of the lack of energy and tryptophan, the data indicate that an unknown mechanism exists which imparts some mutations with the resistance to antimutagenic repair in the absence of the inducible mutagenic system. In the light of this result, one could suggest that the normal process of mutation fixation (that is the loss of sensitivity of mutations to photoreactivation or to excision repair in cells incubated in growth medium after irradiation) should not necessarily be a direct consequence of manifestation of the activity of an inducible mutagenic system.

Study of MFD-type repair in locus determining resistance of Escherichia coli to streptomycin.

The yield of induced mutations to streptomycin resistance (Str) in E. coli, UV-irradiated and temporarily incubated in liquid medium not permitting protein synthesis, depends upon the conditions of preirradiation growth and preirradiation treatment of the bacteria, i.e. on their physiological state at the moment of irradiation. This fact is not readily reconciled with a model postulating mutation production in the structural genes of E. coli during excision repair. A preferred explanation is offered, based on the assumption that the efficiency of mutagenesis at the rpsL (strA) locus is determined by interference of antimutagenic (generalized excision repair and MFD) and promutagenic (mutation fixation of excision repair) events. The participation of macromolecular syntheses in Str mutation fixation is suggested.

[The need for protein synthesis in UV-irradiated Escherichia coli cells for fixation of induced Str mutations]. / Neobkhodimost' sinteza belka v UF-obluchennykh kletkakh Escherichia coli dlia fiksatsii indutsirovannykh mutatsii Str.

The kinetics of accumulation of fixed Str mutations was determined during incubation in nutritional medium of Escherichia coli WP2 irradiated with 6.8 J/m2 either at log growth phase or after completion of DNA replication. Those Str mutations which lost ability for photoreactivation (fixation I) or susceptibility to antimutagenic activity of mfd-type (fixation II) were considered as fixed mutations. It was shown that both fixations occurred synchronously, starting in about 10 min after irradiation and being over in 40-50 min. In cells irradiated after completion of replication, fixation depended on protein synthesis de novo: chloramphenicol added to irradiated culture blocked fixation. An attempt to study the effect of chloramphenicol on fixation in a culture irradiated at the log phase failed, because of high lethal action of the antibiotic on such cells. Fixation could proceed in the presence of acriflavine. Possible mechanisms for fixation of Str mutations are discussed in connection with the fact of its dependence on protein synthesis.

UV-induced mutation fixation in Bacillus subtilis.

The influence was studied of a temporary specific inhibition of post-radiation macromolecular synthesis and of preliminary UV irradiation on the kinetics of accumulation of fixed mutations, that is mutations insensitive to MFD, in UV-irradiated B. subtilis cells. From experimental results it is deduced that the entry of a pre-mutagenic lesion into a round of replication, initiated before irradiation, is not a fixing event in UV mutagenesis. For performance of fixation, the proceeding of replication, initiated after irradiation, and protein synthesis are necessary. In irradiated cells incubated in medium with lowered concentration of nitrogen sources, the antimutagenic activity of a uvr+-dependent repair system competes with the process of fixation for pre-mutagenic lesions and reduces the efficiency of mutagenesis. The most efficient fixation and mutagenesis occur at high concentrations of nitrogen sources in post-radiation medium, when the manifestation of antimutagenic activity appears to be blocked. The possible nature of a process leading to mutation fixation is discussed.

DNA repair in Proteus mirabilis. VI. Plasmid (R46-) mediated recovery and UV mutagenesis.

The expression of plasmid R46-mediated recovery and mutagenic function (s) was studied in P. mirabilis, which is normally either weakly or non-mutable after UV exposure. The plasmid was found to confer on P. mirabilis enhanced UV resistance as well as UV-induced mutability for various types of forward mutations and reversion of the thr273 mutation. The plasmid enhanced survival of UV-irradiated phages in P. mirabilis both in unirradiated host cells and with increased efficiency after UV-exposure of host cells, as is characteristic of UV-inducible phage reactivation. Spontaneous mutability of P. mirabilis harboring R46 was about 2 to 7 times higher than that of cells without plasmid, depending on the marker, repair type, and plating density of the cells used. All of these R46-mediated rescue and mutagenic functions require the rec672 + gene function. It is assumed that the plasmid R46 adds functions to P. mirabilis comparable to those deficient in umuC and uvm mutants of E. coli (Kato and Shinoura, 1977; Steinborn, 1978) and that P. mirabilis possesses functions homologous to those controlled in E. coli by the recA + and lexA + genes. The significance of plasmid-mediated rescue and mutagenic functions for bacteria which lack the misrepair branch of mutagenesis, is discussed.

Study of MFD in Bacillus subtilis.

The frequency of UV-induced extragenic suppressor reversions to leucine independence in B. subtilis carrying a leu8 mutation decreased when irradiated cells were temporarily incubated in medium deprived of nitrogen sources. This mutation frequency decline (MFD) was inhibited by acriflavine and was poorly expressed in a uvr1 mutant. Consequently, MFD may be considered as the manifestation of an anti-mutagenic activity of excision repair. MFD was decelerated and even vanished in cells subjected to prolonged starvation of nitrogen sources before irradiation. MFD was accelerated in bacteria that were first irradiated and incubated in nutritional medium for at least 30 min. The stimulation of MFD by UV exposure was observed only in the uvr+ strain and depended on protein synthesis after irradiation. It is assumed that different rates of MFD in cells of various pre-radiation histories reflect different levels of the excision-repair activity inherent in these cells.

[UV-mutagenesis in Bacillus subtilis. VII. Mutability of recA, recB and recF strains]. / Izuchenie UF-mutageneza u Bacillus subtilis. Soobshchenie VIII. Mutabil'nost' shtammov recA, recB, recF.

UV-light induces reversions to threonine-independence in BD170 Rec+ cells, and does not induce them in isogenic strain BD241 recF. Reversions to methionine-independence are induced in cells GSY1028 recB, and are not in isogenic strain GSY1025 recA. It is possible to construct, with the use of transformation, a viable strain carrying mutations recF and su, the latter imparts threinine-independence to cells. Hence, the absence of UV-induced reversions in recF (and probably in recA) cells can not be explained by the lethal effect of joining, in one genome, two mutations each of which decreases a viability of bacteria. Formation of UV-induced forward mutations which provide additional growth requirements to bacteria is not disturbed in strains recA and recF. Experimental data allow to conclude that UV-induced mutagenesis is not a function of any of two known mechanisms of recombination which function in Bacillus subtilis cells.