Radiation-resistant Bacterium Research Articles

Evidence for the Cloning of Deinococcus radiodurans DNA Fragments That Render Escherichia coli Radiation Resistant

DNA from the radiation-resistant bacterium Deinococcus radiodurans was isolated and used to generate a cosmid library. This cosmid library was grown in Escherichia coli and radiation-resistant E. coli were isolated. Following exposure to 1000 Gy the radiation-resistant transformants exhibited a survival of approximately 10(-1) instead of the 10(-11) exhibited by the nontransformed E. coli. Smaller fragments of DNA were subcloned from the radiation-resistant E. coli; these fragments bestow similar levels of radiation resistance (ratio of slopes = 6.8) to native E. coli upon transfection.

Radiation resistence of microorganisms from radiation sterilization processing environments

The radiation resistance of microorganisms was examined on the samples of dust collected from the radiation sterilization processing environments including assembly, storage, and sterilization plant areas. The isolation of radiation resistant strains was performed by irradiation with screening doses ranging from 10 to 35 kGy and test pieces containing 10 6 to 10 8 CFU in dried serum-broth, representing 100 to 5000 colonies of primary cultures of microorganisms from 7 different sites. In an examination of 16900 colonies of aerobic microorganisms from 3 hygienically controlled production sites and 4 uncontrolled ones, 30 strains of bacteria were isolated. Of those 15 were classified as genus Bacillus, 9 as Micrococcus and 6 as Sarcina. All of the 15 strains of Gram positive sporeforming aerobic rods exhibited an exponential decrease in the surviving fraction as a function of dose, indicating that the inactivation of spores of aerobic rods is a consequence of a single energy deposition into the target. All strains were found to be moderately resistant to radiation with D-6 values (dose required to reduce survival to 6 log cycles) between 18 and 26 kGy. All of the isolated Gram positive cocci showed inactivation curves having a shoulder, indicating that different processes are involved in the inactivation of these cells, e.g. accumulation of sublethal lesions, or final repair capacity of potential lethal lesions. Moderate radiation resistance was observed in 13 strains with D-6 values between 16 to 30 kGy. Two slow-growing, red pigmented strains tentatively classified as genus Micrococcus isolated from uncontrolled sites (human dwellings) were exceptionally resistant with D-6 more than 45 kGy. For hygienically controlled sites, Gram positive spereforming rods composed two thirds of the resistant microflora, while Gram positive cocci comprised one third. For hygienically uncontrolled sites this ratio was reversed. An assumption is made that one isolated strain has grown up from one CFU in the original microflora. In the light of the observed proportion of radiation resistant bacteria in microflora from hygienically controlled radiation sterilization processing environments, and assuming an initial contamination of 50 CFU per product unit, there is no more than one chance of a contaminated item remaining within a population of 10 7 items subjected to sterilization by exposure to a radiation dose of 25 kGy.

Lack of ultraviolet mutagenesis in radiation-resistant bacteria

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.

Radiation-resistant micro-organisms isolated from textiles.

Towels from private homes and public offices and underwear contaminated by being used by employees at a public health laboratory were examined for occurrence of radiation-resistant bacteria and fungi. Three different methods were used for isolation of the most resistant organisms, one with multiplication of the microbial population prior to an irradiation used for selection, and two without this multiplication and with the organisms placed on membrane filters or in situ on the textiles, respectively. A total of 44 different strains were isolated. Differences in the three methods used for selection of the most radiation-resistant microorganisms were not reflected in the results. 16 pigment-producing Gram-positive cocci, tentatively classified as Micrococcus radiodurans, were the most radiation-resistant and were isolated in about half of the examinations. Other Gram-positive cocci, non-spore forming rods, some Nocardia and Candida parapsilosis strains and two Bacillus strains constituted the rest of the collection. Wtih few exceptions dose-response curves for the strains were upward convex. D-6 values were determined to be between 1.5 megarad for the most radiation sensitive, a Candida, and 5.7 megarad for the most resistant,, tentatively classified as M. radiodurans. The D-6 values for the Bacillus strains were in both cases 1.8 megarad, consistent with a D-value of 0.3 megarad. The same resistance is reported to be the maximum resistance for B pumilus, strain E601, commonly used as reference strain in the literature on radiation sterilization of medical devices and supplies.

Isolation of radiation-resistant bacteria without exposure to irradiation

Resistance to desiccation was utilized in the selection of highly radiation-resistant asporogenous bacteria from non-irradiated sources. A bacterial suspension in phosphate buffer was dried in a thin film at 25 degrees C and 33% relative humidity. Storage under these conditions for 15 days or more reduced the number of radiation-sensitive bacteria. Further selection for radiation-resistant bacteria was obtained by irradiation of bacteria on velveteen in the replication process, thereby avoiding the toxic effect of irradiated media. The similarity of radiation resistance and identifying characteristics in irradiated and non-irradiated isolates should allay some concerns that highly radiation-resistant bacteria have been permanently altered by radiation selection.

The fine structure of Micrococcus radiophilus and Micrococcus radioproteolyticus.

The radiation resistant bacteria Micrococcus radiophilus and M. radioproteolyticus were studied by thin sectioning and freez-etching techniques and the two species were found to be similar in the fine structure. The only significant difference was in the appearance of the surfaces of the cell walls in freeze-etched preparations. Since the two species, together with M. radiodurans, possess a unique cell wall structure and a cell wall peptidoglycan, which is different from that of other micrococci and Gram-positive cocci, it is recommended that they be reclassified into a new genus.

Characterization of radiation-resistant vegetative bacteria in beef.

Ground beef contains numerous microorganisms of various types. The commonly recognized bacteria are associated with current problems of spoilage. Irradiation, however, contributes a new factor through selective destruction of the microflora. The residual microorganisms surviving a nonsterilizing dose are predominantly gram-negative coccobacilli. Various classifications have been given, e.g., Moraxella, Acinetobacter, Achromobacter, etc. For a more detailed study of these radiation-resistant bacteria occurring in ground beef, an enrichment procedure was used for isolation. By means of morphological and biochemical tests, most of the isolates were found to be Moraxella, based on current classifications. The range of growth temperatures was from 2 to 50 C. These bacteria were relatively heat sensitive, e.g., D10 of 5.4 min at 70 C or less. The radiation resistance ranged from D10 values of 273 to 2,039 krad. Thus, some were more resistant than any presently recognized spores. A reference culture of Moraxella osloensis was irradiated under conditions comparable to the enrichment procedure used with the ground beef. The only apparent changes were in morphology and penicillin sensitivity. However, after a few subcultures these bacteria reverted to the characteristics of the parent strain. Thus, it is apparent that these isolates are a part of the normal flora of ground beef and not aberrant forms arising from the irradiation procedure. The significance, if any, of these bacteria is not presently recognized.

Letter: The radiation-resistant red bacterium from rice.

Letter: The radiation-resistant red bacterium from rice.

Radiation-resistant Asporogenic Bacteria

Radiation-resistant Asporogenic Bacteria

Characterization of Radiation-Resistant Vegetative Bacteria in Beef1

Ground beef contains numerous microorganisms of various types. The commonly recognized bacteria are associated with current problems of spoilage. Irradiation, however, contributes a new factor through selective destruction of the microflora. The residual microorganisms surviving a nonsterilizing dose are predominantly gram-negative coccobacilli. Various classifications have been given, e.g., Moraxella, Acinetobacter, Achromobacter, etc. For a more detailed study of these radiation-resistant bacteria occurring in ground beef, an enrichment procedure was used for isolation. By means of morphological and biochemical tests, most of the isolates were found to be Moraxella, based on current classifications. The range of growth temperatures was from 2 to 50 C. These bacteria were relatively heat sensitive, e.g., D10 of 5.4 min at 70 C or less. The radiation resistance ranged from D10 values of 273 to 2,039 krad. Thus, some were more resistant than any presently recognized spores. A reference culture of Moraxella osloensis was irradiated under conditions comparable to the enrichment procedure used with the ground beef. The only apparent changes were in morphology and penicillin sensitivity. However, after a few subcultures these bacteria reverted to the characteristics of the parent strain. Thus, it is apparent that these isolates are a part of the normal flora of ground beef and not aberrant forms arising from the irradiation procedure. The significance, if any, of these bacteria is not presently recognized.

Accurate repair of ultraviolet-induced damage in Micrococcus radiodurans

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.

Resistance of blue-green algae to 60Co gamma radiation

Many characteristics of blue green algae suggest that they should be excellent organisms for the investigation of biological interaction with radiation, particularly ionizing radiation. Therefore, the relative resistance to gamma radiation from a 60Co source has been measured for 23 cyanophycean algae. Since the organisms represent a wide range of cell types it was necessary to use dry weight as the end point for viability determination. Using the radiation resistant bacterium Micrococcus radiodurans as a primary standard and the bacterium Sarcina lutea as a secondary standard, the algae have been compared with one another and with S. lutea using a dry weight end point; S. lutea has also been compared with M. radiodurans on a colony count basis. The algae show a wide range of radiation resistance and have been grouped into three categories: (1) low resistance, with an ld 90 of less than 400 krads, for example, Microcystis aeruginosa, Anacystis nidulans and some species of Schizothrix calcicola; (2) moderate resistance, (equal or better than M. radiodurans) with an ld 90 of more than 400 krads but less than 1200 krads, for example, Oscillatoria brevis, Anabaena variabilis and Scytonema hoffmannii; (3) high resistance, with an ld 90 of greater than 1200 krads, for example, Plectonema boryanum, Lyngbya estuarii, Nostoc muscorum and Microcoleus vaginatus.

The pitfalls of measuring DNA synthesis kinetics as exemplified in ultraviolet radiation studies

1. 1. Previous reports on the effect of ultraviolet irradiation on the incorporation of radioactive precursors of DNA by bacteria have presented the data as linear plots of counts/min vs. time. Using this type of plot one only obtains the apparent rate of DNA synthesis in bacterial cultures because the slope of these curves depends in part upon trivial parameters such as the initial viable cell number and the specific activity of the radioactive precursor. The true rate of DNA synthesis only depends upon the DNA content per cell and their generation time. Examples are given to demonstrate that linear plots can lead to erroneous conclusions concerning DNA syn thesis kinetics. 2. 2. We recommend a new protocol for studying the effect of radiation (and other agents) on macromolecular synthesis kinetics which circumvents the pitfalls inherent in previous methods: (1) use mutants requiring the labeled precursor ( e.g., thymine), prelabel the cells and keep the radioactivity present after irradiation (equivalent to the direct chemical determination of DNA); (2) plot the data as log counts/min vs. time (DNA synthesis is an exponential function in bacteria); and (3) determine the kinetics of cell growth at the same time aliquots are taken for the determination of incorporated radioactivity (one may then hope to deduce the biological relevance of the observed synthesis). The advantages of this protocol over previous methods are documented and discussed with special emphasis on the measurement of the extent of delay in DNA synthesis in radiation-resistant bacteria as a function of dose of ultraviolet radiation.

The isolation and some properties of radiation-sensitive mutants of Micrococcus radiodurans.

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.

Chemical study of the radiosensitization of Micrococcus sodonensis by iodine compounds.

IT has been shown that iodoacetamide1 (CH2ICONH2) and potassium iodide2 enhance the radiation inactivation of radiation resistant bacteria. Similar results have been obtained with iodoacetic acid3,4. Dean and Alexander2 found that sensitization with iodoacetamide was observed in. Micrococcus sodonensis only when the bacteria and sensitizer were irradiated together, indicating that sensitization was not caused by blocking of intracellular sulphydryl groups before irradiation or by the production of a stable toxic product. It was later demonstrated that some short lived transients of iodoacetamide were responsible for the sensitizing effect5. These transients would be formed by the reaction of iodoacetamide with the radiolytic products of water. The two principal species produced by irradiation of aqueous solutions are the solvated electron and the hydroxyl radical (OH). (For a comprehensive review of this subject see ref. 6.) The aim of the present investigation was to discover which of these species was involved in producing the toxic short lived transient and to determine the nature of this transient in iodoacetamide and potassium iodide. This could be done by studying the effect of scavengers for the solvated electron and the hydroxyl radical on the sensitizing ability of iodoacetamide and potassium iodide. Our results suggest that the iodine atom (I·), produced by the OH radical attack on the sensitizers, was responsible for the observed sensitization.

The fine structure of Micrococcus radiodurans.

The fine structure of the radiation-resistant bacterium, Micrococcus radiodurans, isolated by Anderson, was studied by electron microscopy of intact and disrupted cells using thin sectioning and negative staining techniques. The cytoplasm and nuclear structures are normal, but the cell wall and sheath are more complex than any so far described for a bacterium. The surface consists of four distinct layers, each having a characteristic fine structure, one of which has been tentatively identified as that responsible for maintaining the rigidity of the cells. Striations with a periodicity of 175 to 200 A are visible in thin sections of this layer, and a pseudo-hexagonal array of dark “holes” is seen in surface view of negatively-stained fragments. It is concluded that this layer is the main structural element of the cell wall of M. radiodurans. The other three layers of the surface have not been clearly located in thin sections; one of these layers has a well-defined pattern of hexagonally arranged units similar to that observed in Spirillum serpens by Murray but with different dimensions.