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UV-induced mutation in the very radiation-resistant bacterium Micrococcus radiodurans was investigated. Forward mutation of the wild type to resistance to trimethoprim and reversion of a temperature-sensitive mutant were studied. Both the wild type and the temperature-sensitive mutant were found to be non-mutable by UV-radiation, but were sensitive to mutation by N -methyl- N ′-nitro- N -nitrosoguanidine (NTG). The wild type was also resistant to mutation by 4-nitroquinoline- N -oxide (4NQO). Neither strain was sensitized to UV-induced mutation by exposure to caffeine after irradiation. It is concluded that, unlike other bacteria in which high resistance to UV-induced killing is associated with sensitivity to UV-induced mutation, repair of UV-induced damage in M. radiodurans is accurate.

When a culture of the temperature-sensitive DNA mutant Micrococcus radiodurans tsI is irradiated with a sublethal dose of ultraviolet or ionizing radiation and is plated immediately, all the bacteria give rise, after 36 h incubation, to colonies identical to those derived from unirradiated bacteria. However, when the irradiated population is held at its restrictive temperature (39 degrees C) (restrictive temperature holding) for 3 h before being plated, less than 0-1% of the surviving bacteria give rise to normal colonies, the rest producing, after incubation for 96 h, small malformed colonies. Qualitatively, the same effect is observed when u.v.-irradiated wild-type M. radiodurans is incubated at 39 degrees C in the presence of nalidixic acid before plating. Compared with the loss of viability, the loss of normal colony development as a function of the radiation dose is sensitive, having I/e values of 210 ergs/mm2 for u.v. radiation and of 4 to 5 krad for 60Co gamma-radiation. These are identical to the radiation dose-response values of a recombination-deficient mutant of M. radiodurans. At first the abnormal colonies consist entirely of giant bacteria but eventually a few bacteria with normal morphology appear and because of their much faster generation time a highly sectored colony results. These colonies can be "rescued" by plating the irradiated bacteria held at 39 degrees C on agar containing pantoyl lactone, their growth being identical to that of unirradiated bacteria. Abnormal colony development is not a general phenomenon in temperature-sensitive mutants of M. radiodurans but occurs in those mutants which are sensitized to radiation when held at 39 degrees C. It is concluded that these abnormal colonies are produced as a result of a defect in a recombination function and that this function is also involved in the regulation of normal cell division.

The survival of biological activity in irradiated transforming deoxyribonucleic acid (DNA) has been assayed in the wild type and a radiation-sensitive mutant of Micrococcus radiodurans . The frequency of transformation with unirradiated DNA was lower in the mutant to about the same extent as the mutant's increased sensitivity to radiation. However, in both the wild type and the mutant, the irradiated DNA that was incorporated into the bacterial genome was repaired to the same extent as determined by the loss of transforming activity with increasing radiation dose. This applied to DNA irradiated either with ionizing or ultraviolet (UV) radiation. The rate of inactivation of biological activity after UV radiation was the same in any of the DNA preparations tested. For ionizing radiation, the rate of inactivation varied up to 40-fold, depending on the DNA preparation used, but for any one preparation was the same whether assayed in the wild type or the radiation-sensitive mutant. When recipient bacteria were irradiated with ionizing or UV radiation immediately before transformation, the frequency of transformation with unirradiated DNA fell, rapidly and exponentially in the case of the sensitive mutant but in a more complicated fashion in the wild type. The repair of DNA irradiated with ionizing radiation was approximately the same whether assayed in unirradiated or irradiated hosts. Thus, irradiation of the host reduced the integration of DNA but not its repair.

Micrococcus radiodurans , a red-pigmented bacterium, is very resistant both to ionizing and to ultra-violet radiations. The dose-response curves in both cases are sigmoidal with high extra-polation numbers. Two non-pigmented mutants of M . radiodurans have been isolated. One of these shows the same type of survival curve as the wild type while the other has an exponential survival curve. Energy transfer reactions including the possible role of pigment have been excluded as a mechanism of resistance. Similarly, resistance is not due to unusual amounts or unique properties of the bacterial DNA . Survivors of both sensitive and resistant bacteria show a lag period following irradiation. In the case of sensitive bacteria no change occurs in the radiosensitivity of the cells during this period. On the other hand, M . radiodurans recovers during the lag period, at the end of which it has become as resistant as the unirradiated cells. All available evidence for the loss of viability in bacteria points to DNA as the target. Recovery of M . radiodurans from radiation damage, therefore, is concluded to be due to repair of damaged DNA by an active cellular, probably enzymic process which occurs during the lag period. The large shoulder of the survival curve and the high extrapolation number are a reflexion of the efficiency of the repair system. The significance of the sigmoidal and exponential survival curves of resistant bacteria is discussed and a theory put forward which postulates two types of damage to the genetic structures, each having a specific repair system. Some experimental evidence in support of this theory is produced.

Similarity of Repair of Ionizing and Ultra-Violet Radiation Damage in Micrococcus radiodurans

A mutant of micrococcus radiodurans which is deficient in recombination has been isolated after treatment of the wild type with N-methyl-N'-nitro-N-nitrosoguanidine. We have called this mutant Micrococcus radiodurans rec30. The efficiency of recombination in this mutant, as measured by transformation, is less than 0.01% that of the wild type. It is 15 times more sensitive to the lethal action of ultraviolet radiation, 120 times more sensitive to ionizing radiation, and 300 times more sensitive to mitomycin C (MMC) than the wild type. It is probably inactivated by a single MMC-induced deoxyribonucleic acid cross-link per genome. The excision of ultraviolet-induced pyrimidine dimers is normal. There is no radiation-induced degradation of deoxyribonucleic acid. All spontaneous revertants selected for resistance to low levels of MMC had wild-type resistance to radiation and MMC, and the same efficiency of recombination as the wild type, suggesting that the recombination deficiency of the strain is due to a single mutation. Deoxyribonucleic acid from this mutant can transform M. radiodurans UV17 presumed deficient in an exr type gene to wild type.

SUMMARY: Two temperature-sensitive mutants of Micrococcus radiodurans defective in DNA synthesis, which were very resistant to the lethal effect of ultraviolet and ionizing radiation at permissive temperatures, became sensitive to radiation and also to the action of N-methyl-N'-nitro-N-nitrosoguanidine when held at the restrictive temperature of 39 °C. With M. radiodurans ts1 the sensitization began soon after transfer to 39 °C and reached a maximum 4 h later. During this period there was no loss of viability. After 4 h the shoulders of the ultraviolet and ionizing radiation survival curves had almost completely disappeared and the exponential part of the curves had doubled in slope. The size of the shoulder fell exponentially with the time the bacteria were held at 39 °C. Sensitization occurred in the presence of chloramphenicol. During the period the bacteria were held at 39 °C their ability to effect recombination as measured by transformation fell exponentially and was correlated with the rate of loss of the shoulder. This suggests that the repair which gives rise to the large shoulders of the radiation survival curves is of the post replication recombination type. The recovery of radiation resistance at 30 °C in bacteria which had been exposed to 39 °C for 75 min did not begin immediately. For 55 min there was no measurable increase in resistance but after 75 min substantial recovery had occurred and by 105 min was complete. Recovery of resistance did not occur in the presence of chloramphenicol even when the chloramphenicol was added 30 min after the bacteria had been at 30 °C. The sensitization to radiation was not a general property of temperature-sensitive (ts) mutants.

Various aspects of the repair of ultraviolet (UV) radiation-induced damage were compared in wild-type Micrococcus radiodurans and two UV-sensitive mutants. Unlike the wild type, the mutants are more sensitive to radiation at 265 nm than at 280 nm. The delay in deoxyribonucleic acid (DNA) synthesis following exposure to UV is about seven times as long in the mutants as in the wild type. All three strains excise UV-induced pyrimidine dimers from their DNA, although the rate at which cytosine-thymine dimers are excised is slower in the mutants. The three strains also mend the single-strand breaks that appear in the irradiated DNA as a result of dimer excision, although the process is less efficient in the mutants. It is suggested that the increased sensitivity of the mutants to UV radiation may be caused by a partial defect in the second step of dimer excision.

Micrococcus radiodurans is a red-pigmented, non-sporing bacterium which is extremely resistant to both ionizing1 and ultra-violet radiations2. The mechanisms of resistance are not known. In 1962 Kaplan and Zavarine3 examined the relationship between the base composition of DNA in eight species of bacteria and their resistance to X-radiation. On plotting the X-ray dose which reduced the surviving bacteria to 10 per cent on the exponential part of the survival curve (the D10) against the guanine cytosine (GC) content, a linear relationship was obtained in which resistance and GC content were inversely related, that is, resistance increased with lowering of the GC content. This was shown to apply to bacteria the GC content of which varied between 34 and 67 per cent of the total bases. On the other hand, preliminary experiments by the same authors3 with ultra-violet radiation indicated the reverse situation, namely, a direct relation between the GC content and resistance to ultra-violet radiation, that is, resistance increased with increasing GC content. Similar results have been reported by Haynes4. It follows that extreme resistance to both ionizing and ultra-violet radiations should be incompatible in the same organism. Since M. radiodurans is very resistant to both types of radiation it cannot fulfil both of the criteria which have been postulated for characterizing radiation resistance.

SUMMARY: Treatment of the radiation-resistant bacterium Micrococcus radiodurans with ultraviolet (u.v.) radiation and N-methyl-N'-nitro-N-nitrosoguanidine resulted in the isolation of two mutants highly sensitive to u.v. radiation. They were also sensitive to ionizing radiation and to the action of N-methyl-N'-nitro-N-nitrosoguanidime. The concentrations of sulphydryl groups in bacteria of the wild type and the mutants were not significantly different. Although the mutants were more sensitive to mitomycin C than the wild type the resistance of the latter was low. It is suggested that the DNA repair mechanism in the wild type operates very efficiently for the removal of single strand damage but not for that which involves cross-linking.

SUMMARY: Three temperature-sensitive mutants of Micrococcus radiodurans have been isolated which, unlike the wild-type, are unable to synthesize DNA at 39°. Synthesis of DNA stops immediately the bacteria are raised to the restrictive temperature. The mutants can be transformed normally for single genetic markers and can regain wild-type temperature resistance on incubation with DNA from wild-type at a frequency associated with single or very closely linked markers. Each mutant can also be transformed to wild-type temperature resistance with DNA from the other temperature-sensitive mutants. All three mutants are resistant to the lethal action of ionizing and ultraviolet radiations and at 39° are able to carry out all or most of the DNA repair functions associated with excision repair, but at a reduced rate compared with wild-type. The rate of excision at 39° of u.v.-induced thymine-containing pyrimidine dimers is four to five times slower in the mutants than in the wild-type.

IT is known that the frequency of lysogenic response in a culture of Salmonella typhimurium strain LT2, infected with the temperate bacterio-phage P22, can be influenced by the ratio of phage to bacterium (multiplicity of infection)1–3, and by modifying the metabolism of the infected cells soon after infection, for example, by the addition of chloramphenicol4. With a high multiplicity of infection a high frequency of lysogenic response is obtained. Using a low multiplicity of infection a low lysogenic response results. If these infected cells are exposed to chloramphenicol (25 µgm./ml.) 5 min. after infection and the chloramphenicol removed by dilution 15 min. later, then practically every cell infected becomes lysogenic.