Double mutations induced in Escherichia coli by ultraviolet light.

Double mutations to azide resistance and to bacteriophage T5 resistance of genes separated by more than 50 kilobases were induced in Escherichia coli WP2s in chemostat cultures by exposure to a single low dose of ultraviolet light. Frequencies of induced double mutations were three orders of magnitude greater than would be predicted by chance. Reversions from azide resistance and phage resistance occurred independently, showing that that the double mutation was not due to pleiotropic effects of a single gene mutation. These results support earlier findings which show that low doses of ultraviolet light induce multiple gene mutations in Bacillus subtilis over a similarly broad range.     

Photoreactivation of UV damage in Escherichia coli uvrA6: lethality is more effectively reversed than either premutagenic lesions or SOS induction

The effect of cyclobutyl pyrimidine dimers on cytotoxicity, induction of synthesis of the RecA and UmuC proteins, and mutagenesis was studied in Escherichia coli uvrA6 cells possessing excess amounts of photoreactivating enzyme. Exposure of 254 nm ultraviolet-irradiated (10 J/m2) cells to radiation from daylight fluorescent lamps reduced the amounts of thymine-containing dimers in a photoreactivating fluence-dependent manner, up to about 90% reduction at 5 min exposure. Of the lethal ultraviolet damage, 85% was photoreactivable (i.e. cyclobutyl pyrimidine dimers) and 15% was non-photoreactivable. An incident fluence of 1 J/m2 resulted in approximately a 5-fold increase in the synthesis of the RecA and UmuC proteins, as compared to the spontaneous level. If the UV-irradiated cell suspensions were illuminated with a fluorescent lamp at a dose which resulted in the full photoreactivation of viability, the yields of both proteins were reduced to 60% of the non-photoreactivated control cells. Furthermore, photoreactivation was shown to be more effective in the repair of lethal damage than in the repair of premutational damage. These experiments suggest that, among lethal damages, non-photoreactivable damage plays a more important role in both induction of the SOS functions and mutagenesis in uvrA6 cells than do cyclobutyl pyrimidine dimers.     

Photoreactivation of excystment delay in Colpoda, induced by far ultraviolet radiation

The surface of single cells of Dictyostelium discoideum during growth and aggregation has been observed by scanning electron microscopy (SEM). Our results show a series of morphological changes associated with the transition from growth to development which appear to be cellcontact dependent. In addition, from results using Concanavalin A as a biochemical probe, we conclude that the mannose-containing surface glycoproteins are accessible during growth, but become inaccessible during aggregation.     

Asymmetry of frameshift mutagenesis during leading and lagging-strand replication in Escherichia coli

Mutations in DNA, including frameshifts, may arise during DNA replication as a result of mistakes made by the DNA polymerase in copying the DNA template strands. In our efforts to better understand the factors that contribute to the accuracy of DNA replication, we have investigated whether frameshift mutations on the Escherichia coli chromosome occur differentially within the leading and lagging-strands of replication. The experimental system involves measurement of the reversion frequency for several defined lac frameshift alleles in pairs of strains in which the lac target is oriented in the two possible directions relative to the origin of chromosomal replication. Within these pairs any defined lac sequence will be subject to leading-strand replication in one orientation and to lagging-strand replication in the other. Fidelity differences between the two modes of replication can be observed as a differential lac reversion between the two strains. Our results, obtained with a series of lac alleles in a mismatch-repair-defective background, indicate that for at least some of the alleles there is indeed a difference in the fidelity of replication between the two modes of replication.     

Experimental evolution of ultraviolet radiation resistance in Escherichia coli

Ultraviolet (UV) light is a major cause of stress, mutation, and mortality in microorganisms, causing numerous forms of cellular damage. Nevertheless, there is tremendous variation within and among bacterial species in their sensitivity to UV light. We investigated direct and correlated responses to selection during exposure to UV. Replicate lines of Escherichia coli K12 were propagated for 600 generations, half with UV and half as a control without UV. All lines responded to selection, and we found strong positive and negative correlated responses to selection associated with increased UV resistance. Compared to Control populations, UV-selected populations increased in desiccation and starvation resistance approximately twofold but were 10 times more sensitive to hypersalinity. There was little evidence for a persistent large competitive fitness cost to UV resistance. These results suggest that natural variation in UV resistance may be maintained by trade-offs for resistance to other abiotic sources of mortality. We observed an average twofold increase in cell size by the UV-selected populations, consistent with a structural mode of adaptation to UV exposure having preadaptive and maladaptive consequences to other abiotic stresses.     

Ionizing and ultraviolet radiation-induced reversion of sequenced frameshift mutations in Escherichia coli: a new role for umuDC suggested by delayed photoreactivation

The ultraviolet (UV) and gamma radiation-induced reversion of the trpA21, trpA9813, and trpE9777 sequenced-frameshift mutations were studied in Escherichia coli K-12 with or without the plasmid pKM101. Radiation induced the reversion of all 3 frameshifts, and pKM101 enhanced this reversion 10-50-fold. Factors influencing the differential radiation revertability of frameshifts are discussed. The two most revertable frameshifts, trpE9777 and trpA9813, were used as probes to understand the role of the umuDC genes in radiation-induced frameshift reversion. Unlike the UV radiation-induced reversion of base-substitution mutations, the reversion of these frameshifts was not enhanced in a uvrA umuC strain by photoreactivation after a post-UV-irradiation incubation. The UmuDC proteins are suggested to have functions in the radiation induction of frameshifts that are more complex than are their functions in the induction of base substitutions.     

Excision repair of ultraviolet radiation damage in toluene treated Escherichia coli

ATP independent excision repair of UV damage has been studied in $\text{E. coli}$ made permeable to nucleotides by treatment with toluene. In using this system, separation of the first step from the subsequent steps in the repair process is achieved. It was found that completion of repair is observed only in strains that have normal levels of DNA polymerase I.     

Cross-resistance relationships in Escherichia coli between ultraviolet radiation and nitrous acid

Zampieri, Antonio (Palo Alto Medical Research Foundation, Palo Alto, Calif.), and Joseph Greenberg. Cross-resistance relationships in Escherichia coli between ultraviolet radiation and nitrous acid. J. Bacteriol. 87:1094-1099. 1964.-A number of radiosensitive and radioresistant strains of Escherichia coli were tested for sensitivity to injury by nitrous acid. All the radioresistant strains, including 13 radioresistant mutants of strain S, B/r, Bpr5, and K-12, were found to be significantly more resistant to nitrous acid than were the radiosensitive strains S and B. The radioresistant mutants of strain S, Bpr5, and K-12 displayed similar responses to nitrous acid and were less resistant than was strain B/r. Strains B and S were indistinguishable on the basis of nitrous acid sensitivity. The survival curves of all strains examined were similar in shape to corresponding survival curves after ultraviolet radiation. The sensitivity to nitrous acid of the radiosensitive strains S and B, but not that of the radioresistant strains, was found to be greater on Tryptone medium than on Penassay medium, and greater on Penassay medium than on glucose-salts medium. Between 2 and 3% of the strain S survivors of nitrous acid treatment were radioresistant; 46 such radioresistant mutants were isolated and found to be identical in cross-resistance pattern with radioresistant types (R(3), R(4), or R(6)) previously described. The proportions in which these radioresistant types were found to occur were similar to those observed after selection by other radiomimetic agents.     

Randomness of base substitution mutations induced in the lacI gene of Escherichia coli by ionizing radiation

The lacI system of Escherichia coli provides a method for monitoring mutational events at a large number of sites. Using this system, we have previously determined the mutational spectra for gamma-ray and beta-particle emissions resulting from the decay of tritium. Analysis of these mutational spectra reveals that base substitution mutations induced by ionizing radiation are distributed nearly randomly throughout the lacI gene and include all detectable substitution events. The distribution of ionizing radiation-induced mutagenesis is similar to the low frequency of occurrence mutational events induced by other SOS-dependent mutagens. The lack of an apparent nonrandom or high frequency of occurrence component seen with other SOS-dependent mutagens can be best explained as the result of the random interaction of ionizing radiation with the DNA bases leading to production of a variety of base substitutions.     

Photoreactivation in phr mutants of Escherichia coli K-12.

We have investigated the genetics of photoreactivation in Escherichia coli K-12. We found that strains with point mutations or deletions in the phr gene showed a significant residual level of photoreactivation after exposure to large fluences of photoreactivating light. It had been previously proposed that a gene in the gal-att lambda interval is also involved in photoreactivation and that the residual photoreactivating activity might be due to this so-called phrA gene located at this interval. We found that deletions of the gal-att lambda region had no effect on either the rate or the final extent of photoreactivation observed in phr+ cells or phr mutants; however strains carrying the delta (gal-att lambda) deletions displayed increased sensitivity to near-UV radiation.     

Evidence for Two Mechanisms of Photoreactivation in Escherichia coli B

Escherichia coli B phr^-, which is not photoreactivable under certain conditions, has been shown to exhibit photoreactivation of killing in the logarithmic growth phase at 3341 A. Dependence of the reaction upon (a) wavelength, (b) dose, and (c) dose rate of the reactivating radiation, as well as upon (d) temperature during reactivation treatment, is very similar to that of photoprotection. We conclude that this photoreactivation is similar in mechanism to photoprotection, believed to be an indirect repair process, the initial step of which is non-enzymatic and leads to a growth-division delay. We therefore call the present phenomenon “indirect photoreactivation.” Similar studies suggest that indirect photoreactivation of killing occurs also in the parent strain, E. coli B (Harm). It has often been supposed that all photoreactivation results from a photoenzymatic reaction similar to that found to operate in vitro on transforming DNA. Our data provide the first evidence for two distinct types of photoreactivation of cell killing, one of which appears not to involve photoenzymes. These experiments also show that photoprotection results from intracellular events that can be induced by treatment after, as well as before, far ultraviolet irradiation.     

Sodium arsenite inhibits spontaneous and induced mutations in Escherichia coli

Sodium arsenite at a non-toxic concentration was found to inhibit strongly mutagenesis induced by ultraviolet light (UV), 4-nitroquinoline-1-oxide (4NQO), furylfuramide (AF-2) and methyl methane-sulfonate (MMS) as well as spontaneous mutation in the reversion assay of E. coli WP2uvrA/pKM101. The effect was not, however, seen in the case of the mutagenesis induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). In order to elucidate the mechanism of the mutation-inhibitory effect of sodium arsenite, its action on umuC gene expression and DNA-repair systems was investigated. It was found that sodium arsenite depressed beta-galactosidase induction, corresponding to the umuC gene expression. For UV-irradiated E. coli strains possessing different DNA-repair capacities, sodium arsenite decreased the UV survival rates of WP2, WP2uvrA[uvrA] and WP67[uvrA polA], increased those of SOS-uninducible strains having either the recA+ or uvrA+ such as CM571 [recA], CM561 [lexA(Ind-)] and CM611[uvrA lexA (Ind-)], and did not affect that of the uvrA recA double mutant, WP100. From these results, we assume that sodium arsenite may have at least two roles in its antimutagenesis: as an inhibitor of umuC gene expression, and as an enhancer of the error-free repairs depending on the uvrA and recA genes.