Whole-genome shotgun optical mapping of Deinococcus radiodurans.

A whole-genome restriction map of Deinococcus radiodurans, a radiation-resistant bacterium able to survive up to 15,000 grays of ionizing radiation, was constructed without using DNA libraries, the polymerase chain reaction, or electrophoresis. Very large, randomly sheared, genomic DNA fragments were used to construct maps from individual DNA molecules that were assembled into two circular overlapping maps (2.6 and 0.415 megabases), without gaps. A third smaller chromosome (176 kilobases) was identified and characterized. Aberrant nonlinear DNA structures that may define chromosome structure and organization, as well as intermediates in DNA repair, were directly visualized by optical mapping techniques after gamma irradiation.

Genome sequence of the radioresistant bacterium Deinococcus radiodurans R1.

The complete genome sequence of the radiation-resistant bacterium Deinococcus radiodurans R1 is composed of two chromosomes (2,648,638 and 412,348 base pairs), a megaplasmid (177,466 base pairs), and a small plasmid (45,704 base pairs), yielding a total genome of 3,284, 156 base pairs. Multiple components distributed on the chromosomes and megaplasmid that contribute to the ability of D. radiodurans to survive under conditions of starvation, oxidative stress, and high amounts of DNA damage were identified. Deinococcus radiodurans represents an organism in which all systems for DNA repair, DNA damage export, desiccation and starvation recovery, and genetic redundancy are present in one cell.

Genome of the extremely radiation-resistant bacterium Deinococcus radiodurans viewed from the perspective of comparative genomics.

The bacterium Deinococcus radiodurans shows remarkable resistance to a range of damage caused by ionizing radiation, desiccation, UV radiation, oxidizing agents, and electrophilic mutagens. D. radiodurans is best known for its extreme resistance to ionizing radiation; not only can it grow continuously in the presence of chronic radiation (6 kilorads/h), but also it can survive acute exposures to gamma radiation exceeding 1,500 kilorads without dying or undergoing induced mutation. These characteristics were the impetus for sequencing the genome of D. radiodurans and the ongoing development of its use for bioremediation of radioactive wastes. Although it is known that these multiple resistance phenotypes stem from efficient DNA repair processes, the mechanisms underlying these extraordinary repair capabilities remain poorly understood. In this work we present an extensive comparative sequence analysis of the Deinococcus genome. Deinococcus is the first representative with a completely sequenced genome from a distinct bacterial lineage of extremophiles, the Thermus-Deinococcus group. Phylogenetic tree analysis, combined with the identification of several synapomorphies between Thermus and Deinococcus, supports the hypothesis that it is an ancient group with no clear affinities to any of the other known bacterial lineages. Distinctive features of the Deinococcus genome as well as features shared with other free-living bacteria were revealed by comparison of its proteome to the collection of clusters of orthologous groups of proteins. Analysis of paralogs in Deinococcus has revealed several unique protein families. In addition, specific expansions of several other families including phosphatases, proteases, acyltransferases, and Nudix family pyrophosphohydrolases were detected. Genes that potentially affect DNA repair and recombination and stress responses were investigated in detail. Some proteins appear to have been horizontally transferred from eukaryotes and are not present in other bacteria. For example, three proteins homologous to plant desiccation resistance proteins were identified, and these are particularly interesting because of the correlation between desiccation and radiation resistance. Compared to other bacteria, the D. radiodurans genome is enriched in repetitive sequences, namely, IS-like transposons and small intergenic repeats. In combination, these observations suggest that several different biological mechanisms contribute to the multiple DNA repair-dependent phenotypes of this organism.

A DNA damage-responsive Drosophila melanogaster gene is also induced by heat shock.

A gene isolated by screening Drosophila melanogaster tissue culture cells for DNA damage regulation was also found to be regulated by heat shock. After UV irradiation or heat shock, induction is at the transcriptional level and results in the accumulation of a 1.0-kilobase polyadenylated transcript. The restriction map of the clone bears no resemblance to the known heat shock genes, which are shown to be uninduced by UV irradiation.

Engineering a recombinant Deinococcus radiodurans for organopollutant degradation in radioactive mixed waste environments.

Thousands of waste sites around the world contain mixtures of toxic chlorinated solvents, hydrocarbon solvents, and radionuclides. Because of the inherent danger and expense of cleaning up such wastes by physicochemical methods, other methods are being pursued for cleanup of those sites. One alternative is to engineer radiation-resistant microbes that degrade or transform such wastes to less hazardous mixtures. We describe the construction and characterization of recombinant Deinococcus radiodurans, the most radiation-resistant organism known, expressing toluene dioxygenase (TDO). Cloning of the tod genes (which encode the multicomponent TDO) into the chromosome of this bacterium imparted to the strain the ability to oxidize toluene, chlorobenzene, 3,4-dichloro-1-butene, and indole. The recombinant strain was capable of growth and functional synthesis of TDO in the highly irradiating environment (60 Gy/h) of a 137Cs irradiator, where 5x10(8)cells/ml degraded 125 nmol/ml of chlorobenzene in 150 min. D. radiodurans strains were also tolerant to the solvent effects of toluene and trichloroethylene at levels exceeding those of many radioactive waste sites. These data support the prospective use of engineered D. radiodurans for bioremediation of mixed wastes containing both radionuclides and organic solvents.

Engineering Deinococcus radiodurans for metal remediation in radioactive mixed waste environments.

We have developed a radiation resistant bacterium for the treatment of mixed radioactive wastes containing ionic mercury. The high cost of remediating radioactive waste sites from nuclear weapons production has stimulated the development of bioremediation strategies using Deinococcus radiodurans, the most radiation resistant organism known. As a frequent constituent of these sites is the highly toxic ionic mercury (Hg) (II), we have generated several D. radiodurans strains expressing the cloned Hg (II) resistance gene (merA) from Escherichia coli strain BL308. We designed four different expression vectors for this purpose, and compared the relative advantages of each. The strains were shown to grow in the presence of both radiation and ionic mercury at concentrations well above those found in radioactive waste sites, and to effectively reduce Hg (II) to the less toxic volatile elemental mercury. We also demonstrated that different gene clusters could be used to engineer D. radiodurans for treatment of mixed radioactive wastes by developing a strain to detoxify both mercury and toluene. These expression systems could provide models to guide future D. radiodurans engineering efforts aimed at integrating several remediation functions into a single host.

Temperature-induced homeoviscous adaptation of Chinese hamster ovary cells.

Exponential and plateau phase Chinese hamster ovary cells were maintained for 3 days at 32, 37, 39 or 41 degrees C. The effect of growth temperature on the fluidity and composition of the cellular membranes, and on the ability of the cells to resist a subsequent heat treatment at 43 degrees C, was measured. Cells grown at temperatures above 37 degrees C displayed increased resistance or tolerance to a 43 degree C heat treatment, whereas cells grown at 32 degrees C were sensitized to heat. Extensive cell division was not required for expression of heat tolerance. Membrane fluidity, as determined by the degree of rotational mobility of the fluorescent probe diphenylhexatriene, decreased with increasing growth temperatures, but the relationship did not hold in exponential phase cells grown at 32 degrees C. The cholesterol : phospholipid molar ratio correlated with the fluorescence polarization values, suggesting that the cells are able to adjust membrane fluidity by varying the concentration of cholesterol. The results are compatible with the concept of homeoviscous adaptation: that organisms strive to maintain an optimal level of membrane fluidity and when grown at a different temperature will alter the lipid composition in order to maintain this level. Up until now, cholesterol has not been implicated in this process.

Extensive photodimerization of non-adjacent pyrimidines.

In a prior study we found that non-adjacent thymidyl residues in the single-stranded alternating copolymer poly[d(G-T)] are subject to photodimerization by germicidal lamp irradiation (lambda max 254 nm). The maximum yield of this photoproduct was 1% of the total thymine of poly[d(G-T)]. We now report that dimer formation in this polymer is increased to 10 to 40% thymine as dimer between non-adjacent pyrimidines, using near-ultraviolet irradiation (lambda max 310 nm) with or without acetone triplet-sensitization. As previously observed for 254 nm irradiation, dimer formation was nearly absent in double-stranded poly[d(G-T).d(C-A)]. These observations extend prior findings by demonstrating high-yield dimerization between non-adjacent pyrimidines via direct irradiation at environmentally relevant wavelengths (greater than or equal to 280 nm), and are potentially relevant to the mechanism of the ultraviolet light-induced targeted -1 frameshift mutation.

Ultraviolet-induced dimerization of non-adjacent pyrimidines. A potential mechanism for the targeted -1 frameshift mutation.

The DNA photoproduct responsible for the ultraviolet (u.v.)-induced targeted -1 frameshift mutation is unknown. Based on mutagenesis studies by others, we surmised that this lesion might be found in high abundance in single-stranded DNA. u.v. irradiation of the single-stranded alternating copolymer poly[d(G-T)] yielded a photoproduct that was characterized in detail. It consists of a thymine-thymine cyclobutane dimer of predominantly cis-syn configuration occurring between non-adjacent thymidyl residues on the same strand. Its formation is strongly inhibited in double-stranded DNA. A similar u.v. photoproduct was obtained in higher yield from the polypyrimidine alternating copolymer poly[d(C-T)] under conditions in which it is single-stranded. It is proposed that replication across the unrepaired photoproduct: (formula; see text) is the cause of the targeted u.v.-induced -1 frameshift mutation.

Recombination between a resident plasmid and the chromosome following irradiation of the radioresistant bacterium Deinococcus radiodurans.

Interplasmidic and intrachromosomal recombination in Deinococcus radiodurans has been studied recently and has been found to occur at high frequency following exposure to ionizing radiation. In the current work, we document plasmid-chromosome recombination following exposure of D. radiodurans to 1.75 Mrad (17.5 kGy) 60Co, when the plasmid is present in the cell at the time of irradiation. Recombination is assayed using both physical and allelic polymorphisms of homologous genes in the plasmid and chromosome. Recombination was found to be largely, but not entirely, recA-dependent. Crossovers occur frequently, and a significant fraction of these are non-reciprocal.

Gene expression in Deinococcus radiodurans.

We previously reported that the Escherichia coli drug-resistance determinants aphA (kanamycin-resistance) and cat (chloramphenicol-resistance) could be introduced to Deinococcus radiodurans by transformation methods that produce duplication insertion. However, both determinants appeared to require dramatic chromosomal amplification for expression of resistance. Additional studies described here, confirming this requirement for extensive amplification, led us to the use of promoter-probe plasmids in which the E. coli promoter has been deleted, leaving only coding sequences for the marker gene. We find that the insertion of D. radiodurans sequences immediately upstream from the promoterless drug-resistance determinant produces drug-resistant transformants without significant chromosomal amplification. Furthermore, a series of stable E. coli-D. radiodurans shuttle plasmids was devised by inserting fragments of D. radiodurans plasmid pUE10 in an E. coli plasmid directly upstream from a promoterless cat gene. These constructions replicated in D. radiodurans by virtue of the pUE10 replicon and expressed the cat determinant because of D. radiodurans promoter sequences in the pUE10 fragment. Of three such constructions, none expressed the cat gene in E. coli. Similar results were obtained using a promoterless tet gene. Translational fusions were made between D. radiodurans genes and E. coli 5'-truncated lacZ. Three fusions that produced high levels of beta Gal in D. radiodurans were introduced into E. coli, but beta Gal was produced in only one. The results demonstrate that the E. coli genes cat, tet and lacZ can be efficiently expressed in D. radiodurans if a D. radiodurans promoter is provided, and that D. radiodurans promoters often do not function as promoters in E. coli.

Sequencing, targeted mutagenesis and expression of a recA gene required for the extreme radioresistance of Deinococcus radiodurans.

Deinococcus radiodurans and other members of the same genus share extreme resistance to ionizing radiation and many other agents that damage DNA. A DNA damage-sensitive and natural transformation-deficient strain generated by chemical mutagenesis (strain rec30) was found to be defective in a gene that has extended homology with recA of Escherichia coli. Upon transformation with a chromosomal DNA fragment that contained this deinococcal recA gene from wild-type (wt) D. radiodurans both DNA damage resistance and full transformation competence were restored in the rec30 mutant. Targeted insertional mutagenesis of the deinococcal recA gene was used to construct a mutant isogenic with the wt. The insertional mutant was phenotypically indistinguishable from strain rec30, indicating that the recA defect alone was responsible for observed phenotypic alterations. For example, in the case of ionizing radiation, the D37 of the wt was about 1.75 Mrad, while the D37 of rec30 and the insertional mutant were both 25 krad, a 70-fold decrease. Evidence is presented that expression of the deinococcal recA gene in E. coli is lethal, suggesting that the mode of interaction of the deinococcal RecA protein with nucleic acids or other cellular proteins differs at least in part from RecA of E. coli.

18 S rRNA degradation is not accompanied by altered rRNA transport at early times following irradiation of HeLa cells.

The effect of ionizing radiation (137Cs) on processing and transport of ribosomal RNA (rRNA) was studied by pulse-labeling HeLa S3 cells with [3H]uridine immediately prior to irradiation. This approach permits kinetic analysis of processing of 45 S rRNA (radiolabeled predominantly prior to irradiation) into its 28 S and 18 S rRNA daughter species following irradiation. By this technique, we have recently demonstrated an increase in the normal 28 S:18 S rRNA stoichiometric ratio of 1:1 to as high as 1.6:1 during the interval 5 to 20 h following irradiation of HeLa cells at greater than or equal to 7.5 Gy. Alterations in 28 S:18 S ratio were evaluated in greater detail at early times following irradiation, up to 2 h. The 28 S:18 S ratio was found to be maximal at 1 h after radiation, at about 2:1, following 5 or 10 Gy. Using a method for rapid separation of nucleus from cytoplasm, transport of rRNA from nucleus to cytoplasm was also evaluated during this period. Despite an increase in the rate of 45 S rRNA processing, as well as an increased 28 S:18 S ratio, no alterations in transport from nucleus to cytoplasm were detected. This lack of transport alteration suggests that accumulation of excess 28 S rRNA is restricted to the nucleus, where it may represent an early step in the process of radiation-induced cell killing.

Increased resistance to ionizing and ultraviolet radiation in Escherichia coli JM83 is associated with a chromosomal rearrangement.

Cells cope with radiation damage through several mechanisms: (1) increased DNA repair activity, (2) scavenging and inactivation of radiation-induced radical molecules, and (3) entry into a G0-like quiescent state. We have investigated a chromosomal rearrangement to elucidate further the molecular and genetic mechanisms underlying these phenomena. A mutant of Escherichia coli JM83 (phi 80dlacZ delta M15) was isolated that demonstrated significantly increased resistance to both ionizing and ultraviolet radiation. Surviving fractions of mutant and wild-type cells were measured following exposure to standardized doses of radiation. Increased radioresistance was directly related to a chromosomal alteration near the bacteriophage phi 80 attachment site (attB), as initially detected by the LacZ- phenotype of the isolate. Southern hybridization of chromosomal DNA from the mutant and wild-type E. coli JM83 strains indicated that a deletion had occurred. We propose that the deletion near the attB locus produces the radioresistant phenotype of the E. coli JM83 LacZ- mutant, perhaps through the alteration or inactivation of a gene or its controlling element(s).

18 S ribosomal RNA is degraded during ribosome maturation in irradiated HeLa cells.

The effects of ionizing radiation (137Cs) on processing of ribosomal RNA (rRNA) were studied by pulse-labeling HeLa S3 cells with [3H]uridine immediately prior to irradiation. The 45 S rRNA precursor, and its two major daughter species, 28 and 18 S rRNA, were separated by gel electrophoresis and the extent of radiolabel incorporation into each was determined at various times after irradiation. This approach permitted kinetic analysis of processing of the 45 S rRNA which had been predominantly synthesized (radiolabeled) prior to irradiation. Since they both derive from the same 45 S pre-rRNA transcript, 28 and 18 S rRNA are produced with a stoichiometry of 1:1, as observed in control cells in the present studies. However, within 1 h following 10 Gy an altered stoichiometry of 28 S:18 S rRNA was apparent, reaching 1.6:1 by 5-7 h following irradiation. This alteration was also observed following the higher dose of 20 Gy, but not following exposures of 5 Gy or less. The 18 S portion of the 45 S pre-rRNA is transcribed prior to the 28 S portion. Consequently, an increase in the 28 S/18 S ratio can only be due to degradation of the 18 S species during or after processing. This alteration may represent a response to radiation-induced growth arrest, by reducing the number of newly synthesized ribosomes that would otherwise be required for cell propagation.

DNA deoxyribophosphodiesterase and an activity that cleaves DNA containing thymine glycol adducts in Deinococcus radiodurans.

Deinococcus radiodurans is the most radioresistant bacterium discovered to date. Recently it has been demonstrated that this organism contains the DNA repair enzyme uracil-DNA glycosylase and an apurinic/apyrimidinic (AP) endonuclease that may function as part of a DNA base excision repair pathway. We demonstrate here that a DNA deoxyribophosphodiesterase activity that acts on incised AP sites in DNA to remove deoxyribose-phosphate groups is found in lysates prepared from D. radiodurans cells. The partially purified activity was found to be smaller in size than the E. coli dRpase activity, with an estimated molecular weight of 25-30 kDa. In addition, an activity that recognizes and cleaves DNA containing thymine glycols was also detected, with a molecular weight of approximately 30 kDa. This enzyme may be analogous to the thymine glycol glycosylase/AP lyase endonuclease III of E. coli.

Evidence against enhancement of the radioresistance of Escherichia coli by cloned Deinococcus radiodurans DNA.

Deinococcus radiodurans genomic DNA, introduced to Escherichia coli in cloning vectors, has been reported to produce radioresistant E. coli that can be selected by gamma irradiation. In this report prior results are reassessed experimentally, and additional studies are presented. Results to date suggest that the acquired radioresistance of E. coli selected by gamma irradiation does not stem from expression of stable plasmid-encoded D. radiodurans sequences, and that acquired radioresistance is not readily transmitted to naive (unirradiated) E. coli by transformation of plasmid recovered from the radioresistant isolates. Several interpretations are discussed.

Formation of single and double strand breaks in DNA ultraviolet irradiated at high intensity.

The induction of single-strand breaks (SSB) by two quantum processes in DNA is well established. We now report that biphotonic processes result in double-strand breaks (DSB) as well. pUC19 and bacteriophage M13 RF DNA were irradiated using an excimer laser (248 nm) at intensities of 10(7), 10(9), 10(10) and 10(11) W/m2 and doses up to 30 kJ/m2. The proportion of DNA as supercoil, open circular, linear and short fragments was determined by gel electrophoresis. Linear molecules were noted at fluences where supercoiled DNA was still present. The random occurrence of independent SSB in proximity to each other on opposite strands (producing linear DNA) implies introduction of numerous SSB per molecule in the sample. If so, supercoiled DNA that has sustained no SSB should not be observed. A model accounting for the amounts of supercoiled, open circular, linear and shorter fragments of DNA due to SSB, DSB and Scissions (opposition of two independently occurring SSB producing an apparent DSB) was developed, our experimental data and those of others were fit to the model, and quantum yields determined for SSB and DSB formation at each intensity. Results showed that high intensity laser radiation caused an increase in the quantum yields for both SSB and DSB formation. The mechanism of DSB formation is unknown, and may be due to simultaneous cleavage of both strands in one biphotonic event or the biased introduction of an SSB opposite a preexisting SSB, requiring two biphotonic events.

Analysis by pulsed-field gel electrophoresis of DNA double-strand breakage and repair in Deinococcus radiodurans and a radiosensitive mutant.

Double-strand break (dsb) induction and rejoining after ionizing radiation was analysed in Deinococcus radiodurans and a radiosensitive mutant by pulsed-field gel electrophoresis. Following 2 kGy, migration of genomic DNA (not restriction cleaved) from the plug into the gel was extensive, but was not observed after 90 min postirradiation recovery. By this time D. radiodurans chromosomes were intact, as demonstrated by restoration of the Not I restriction cleavage pattern of 11 bands, which we found to be the characteristic pattern in unirradiated cells. Following the higher exposure of 4 kGy, dsb rejoining took approximately 180 min, twice as long as required following the 2 kGy exposure. Restoration of dsb in the radiosensitive mutant strain 112, which appears to be defective in recombination, was markedly retarded at both 2 and 4 kGy. The Not I restriction fragments of wild-type D. radiodurans and the radiosensitive mutant were identical, totaling 3.58 Mbp, equivalent to 2.36 x 10(9) daltons per chromosome.