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Ultraviolet (UV) radiation did not induce rifampicin-resistant mutants in populations of the taxonomically-related radiation-resistant bacteria Deinococcus radiodurans, D. radiopugnans, D. radiophilus and D. proteolyticus, although such mutants arose spontaneously at a low frequency and at a high frequency after treatment of cultures with N-nitroso compounds. The radiation-resistant bacteria Arthrobacter radiotolerans and P-30-A were also UV-immutable whereas the more radiation-sensitive Pseudomonas radiora was UV-mutable. We conclude that the radiation-resistant bacteria repair UV-induced DNA damage accurately and lack an error-prone pathway for the repair of such damage.

SUMMARY: In an attempt to isolate a strain of Rhizobium trifolii which could be highly mutated by ultraviolet light (u.v.), a u.v.-sensitive mutant was isolated using a semi-selective procedure. The mutant was not only 85 times more sensitive than the wild-type to the lethal effects of u.v., but was mutated at u.v. doses which had little mutagenic effect on the wild-type. Its sensitivity to the mutagenic agents methyl methanesulphonate and gamma rays was unaltered, but its spontaneous mutation frequencies for two antibiotic resistances were increased. The mutation conferring u.v. sensitivity was mapped on the chromosome of Rhizobium leguminosarum 300 in a position between the markers ser-2 and ade-88. Unsuccessful attempts were made to transfer into the u.v.-sensitive mutant any one of a number of plasmids known to decrease the lethality of u.v. and enhance its mutagenicity.

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.

The lethal and mutagenic effects of methyl methanesulphonate (MMS), ethyl methanesulphonate (EMS), and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) can be dissociated in a mitomycin C (MTC)-sensitive mutant, strain 302, of Micrococcus radiodurans. As regards lethality 302 is extremely sensitive, compared with the wild type, to MTC and decarbamoyl MTC (DCMTC), slightly sensitive to EMS, MNNG, nitrous acid, 7-bromomethylbenz[alpha]anthracene (BrMBA), and N-acetoxy-N-2-acetylaminofluorene (AAAF), and resistant to MMS, hydroxylamine, and ICR 191G. As regards mutability it is, compared to the wild type, very sensitive to MMS, EMS, and MNNG, and slightly sensitive to hydroxylamine and nitrous acid but not to any other agent examined. Alkaline sucrose gradient studies indicate the 302 does not incise DNA containing BrMBA adducts, although it does incise DNA damaged by AAAF but probably not to the same extent as wild type. We put forward the hypothesis that the hypermutability of 302 is due to the non-removal of bases or nucleotides, modified in exocyclic positions, which have altered base-pairing capabilities, while lethality results from the non-removal of bases or nucleotides, also modified in exocyclic positions, which no longer form hydrogen-bonded base pairs.

Summary: Transformation frequencies greater than 1 % for some single markers were obtained in Micrococcus radiodurans when bacteria in the exponential phase of growth were resuspended in fresh growth medium containing 0.03 M-Ca2+ before being incubated with transforming DNA. Mg2+, Sr2+ or Zn2+ could not replace Ca2- in giving high frequencies of transformation. The time required for the maximum expression of transformed markers was 2 to 3 h for resistance to rifampicin and acriflavin and 6 to 8 h for resistance to erythromycin, kanamycin and streptomycin. The comparative frequency of transformants at maximum expression for each resistance marker was: kanamycin, 1; streptomycin, 1; acriflavin, 4; erythromycin, 25; rifampicin, 64. Cultures were competent during all stages of exponential growth, the frequency of transformants only falling during stationary phase. The minimum time for DNA to be taken up by M. radiodurans into a DNAase-resistant form was between 3 and 6 s. From 6 s to 10 min exposure to DNA, the number of transformants increased non-linearly with time as though the process was inducible. The transformation frequency was directly proportional to the DNA concentration up to 1 μg ml−1, although even at 88 μg ml−1 the bacteria were not saturated. Attempts to measure the fraction of cells which were competent, using the unlinked marker technique, gave values well in excess of one. These were interpreted in terms of multiple genome copies and approximate values of between 0.25 and 0.72 were derived for the competent fractions.

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.

Four genes concerned with the resistance of wild-type Micrococcus radiodurans to the lethal action of mitomycin-C (MTC), mtcA, mtcB, uvsA and uvsB, have been identified by isolating mutants sensitive to MTC. Two strains of M. radiodurans, 302 and 262 carrying mutations in mtcA and mtcB respectively, are between forty and sixty times as sensitive as the wild-type to MTC, only slightly more sensitive than the wild-type to ionizing (λ) radiation and have the same resistance as the wild-type to ultraviolet (u.v.) radiation. Strain 302 can be transformed at a high frequency to wild-type resistance to MTC with DNA from strain 262, and vice versa, indicating that mtcA and mtcB have different genetic locations. Two further strains of M. radiodurans, 303 and 263 having mutations in uvsA and uvsB respectively are only from four to eight times as sensitive as the wild-type to MTC, seven to thirteen times as sensitive to γ-radiation but between twenty to thirty-three times as sensitive to u.v. radiation. Strain 303 can be transformed with DNA from strain 263, or vice versa, to wild-type resistance to u.v. radiation, implying that uvsA and uvsB also have different genetic locations. M. radiodurans strain 301 which is mutant in both mtcA and uvsA, and strain 261 which is mutant in mtcB and uvsB are twenty to forty times as sensitive as the wild-type to both MTC and u.v. radiation and seven to ten times as sensitive to γ radiation. Neither mtcA and uvsA nor mtcB and uvsB are closely linked. None of the mutant strains is deficient in recombination, as measured by transformation. The repair of MTC-induced DNA damage in M. radiodurans must be different from that described for Escherichia coli.

SUMMARY: The efficiency of a variety of common mutagens in producing mutation in Rhizobium trifolii P3 was examined. Ethyl methanesulphonate, methyl methanesulphonate, decarbamoyl mitomycin C, nitrous acid and gamma radiation did not mutate R. trifolii P3. N-Methyl-N′-nitro-N-nitrosoguanidine (MNNG) and ultraviolet radiation were both mutagenic, the former being the more effective. Transposon mutagenesis with Tn5 yielded the same frequency and range of auxotrophs as did MNNG.

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.

Plasmids were found in strains representing all four species of the genus Deinococcus viz. D. radiodurans, D. radiopugnans, D. radiophilus and D. proteolyticus but were not found in the most intensively-investigated strain of the genus, D. radiodurans R1. Their sizes were calculated from electron micrographs. D. radiophilus yielded three size classes of plasmid while D. radiodurans Sark, D. proteolyticus and D. radiopugnans each yielded two. Attempts to cure D. radiophilus and D. radiodurans Sark of any of their plasmids, using a variety of methods, were unsuccessful. A 10.8 kbase pair (kb) plasmid from D. radiophilus, pUE1, was cloned into the PstI site of pAT153 and propagated in Escherichia coli HB101. The recombinant plasmid, pUE109 was subjected to single and double digestion with various restriction endonucleases and its restriction map constructed. The resistance of E. coli HB101 to ultraviolet radiation was not increased when pUE109 was introduced into it. Attempts to transform D. radiodurans with pUE109 failed to detect tetracycline-resistant transformants.

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.

A strain, 302, of Micrococcus radiodurans mutant in mtcA is very sensitive to the lethal effects of mitomycin C (MTC) and decarbamoyl MTC but is fully UV resistant. Alkaline sucrose gradient analysis of DNA from bacteria treated with 7-bromomethylbenz [ a ]-anthracene (BrMBA) indicates that 302 is unable to initiate excision repair of BrMBA-DNA adducts. 302 is hypermutable by methyl methanesulfonate (MMS), ethyl methanesulfonate (EMS) and N -methyl- N ′-nitro- N -nitro-soguanidine (MNNG). Both the wild type and 302 excise 7-methylguanine (7-MeG) and 3-methyladenine (3-MeA) from MNNG-methylated DNA but only the wild type excises O 6 -methylguanine ( O 6 -MeG). The mtcA gene product is therefore involved in the initial recognition and/or incision events in excision repair of MTC-mono- and di-adducts and also certain methyl and ethyl products in DNA.

In Deinococcus radiodurans, the genes uvsC, uvsD, uvsE, and mtcA are all involved in the single-strand incision of UV-irradiated DNA, and mutations in at least two of them were required to produce an incisionless strain. One mutation must be in mtcA and one in uvsC, uvsD, or uvsE. Strains carrying single mutations in any one of the genes can incise DNA to the same extent as the wild-type strain. Neither the presence of EDTA nor the absence of protein synthesis affected the incision step. Strains deficient in DNA incision have greatly reduced DNA degradation after UV irradiation, and upon addition of chloramphenicol to the postirradiation medium, they do not undergo excessive DNA degradation as is seen in the wild-type strain and strains singly mutant in uvsC, uvsD, or uvsE. The strain singly mutant in mtcA also lacked chloramphenicol-enhanced DNA degradation and loss of viability but behaved similarly to the wild-type strain with respect to resumption of DNA synthesis and DNA degradation in the absence of chloramphenicol. It is proposed that two constitutive, cation-independent UV endonucleases are present in D. radiodurans: UV endonuclease alpha (the product of the mtcA gene), which incises in response to pyrimidine dimers, mitomycin C cross-links, bromomethylbenzanthracene adducts, and other alkylation damage, and UV endonuclease beta (the product of the uvsC, uvsD, and uvsE genes), which incises only in response to pyrimidine dimers. Both endonucleases have associated exonuclease activity. The exonucleolytic activity associated with UV endonuclease alpha requires a UV-induced protein to terminate (or control) its activity, whereas the exonucleolytic activity associated with UV endonuclease beta is slower acting and does not require the inducible terminator.

The resistance of Micrococcus radiodurans to the lethal and mutagenic action 3f ultraviolet (UV) light, ionising (γ) radiation, mitomycin C (MTC), nitrous acid (NA), hydroxylamine (HA), N -methyl- N ′-nitro- N -nitrosoguanidine (NG), ethylmethanesulphonate (EMS) and β-propiolactone (βPL) has been compared with that of Escherichia coli B/r. M. radiodurans was much more resistant than E. coli B/r to the lethal effects of UV light (by a factor of 33), γ-radiation (55), NG (15) and NA (62), showed intermediate resistance to MTC (4) and HA(7), but was sensitive to EMS (1) and βPL (2). M. radiodurans was very resistant to mutagens producing damage which can be repaired by a recombination system, indicating that it possesses an extremely efficient recombination repair mechanism. Both species were equally sensitive to mutation to trimethoprim resistance by NG, but M. radiodurans was more resistant the E. coli B/r to the other multagens tested, being non-mutable by UV light, γ-radiation, MTC and HA, and only slightly sensitive to mutation by NA, EMS, and βPL. The resistance of M. radiodurans to mutation by UV-light, γ-radiation and MTC is consistent with an hypothesis that recombination repair in M. radiodurans is accurate since these mutagens may depend on an “error-prone” recombination system for their mutagenic effect in E. coli B/r. However, because M. radiodurans is also resistant to mutagens such as HA and EMS, which are mutagenic in E. coli in the absence of an “error-prone” system, we propose that all the mutagens tested may have a common mode of action in E. coli B/r, but that this mutagenic pathway is missing in M. radiodurans .

Micrococcus radiodurans, a gram-positive, nonsporing, red-pigmented bacterium, is the type species of a small group of bacteria, the members of which are characterized by extreme resistance to both the lethal and mutagenic effects of ionizing and ultraviolet (UV) radiation. They show no loss of viability up to doses of 500 krad or 500 Jm-2 of ionizing or UV radiation, respectively, and this has made them particularly useful for studying aspects of DNA damage and repair in populations in which every member is a survivor. Those scientists working with Escherichia coli will realize what a luxury this is. Nevertheless, these bacteria have been regarded largely as a scientific curiosity (a recent proposal has been made to change their generic name to Deinococcus, meaning “strange berry”)! and the research effort put into them has been paltry compared with that devoted to E. coli. Thus, although the first paper on them was published twenty-seven years ago, this is the first review of the literature on their remarkable property of radiation resistance.

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.

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.