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PubMed1991

Heterozygosity and instability of amplified chromosomal insertions in the radioresistant bacterium Deinococcus radiodurans.

Natural transformation, duplication insertion, and plasmid transformation in Deinococcus radiodurans, a bacterium that contains 4 to 10 chromosomes per cell, were studied. Duplication insertions were often heterozygous, with some chromosomes containing highly amplified insertions and others containing no insertions. Large amplified regions were apparently deleted by intrachromosomal recombination, generating as by-products extrachromosomal circles consisting of multiple tandem repeats of the amplified sequence. The circles were of heterogenous integer sizes, containing as many as 10 or more amplification units. Two strains that are defective in natural transformation and sensitive to DNA-damaging agents were further characterized. Both strains were defective in duplication insertion. While on strain was normal for plasmid transformation, the other was totally defective in this regard, suggesting that plasmid transfer in D. radiodurans may require recombinational functions.

Chromosomes, Bacterial↗
PubMed1991

Phosphatidylglyceroylalkylamine, a novel phosphoglycolipid precursor in Deinococcus radiodurans.

We report here the structure of a previously uncharacterized phospholipid in the radiation-resistant bacterium Deinococcus radiodurans. This phospholipid, designated lipid 4, was shown by chemical analysis, HF hydrolysis, and nuclear magnetic resonance spectroscopy to be phosphatidylglyceroylalkylamine. Lipid 4 thus contains the unusual lipid constituents glyceric acid and alkylamines, which have previously been identified in two complex phosphoglycolipids from this organism. By [32P]phosphate pulse-chase labeling techniques, lipid 4 was shown to be the precursor of the complex phosphoglycolipids alpha-galactosyl- and alpha-N-acetylglucosaminylphosphatidylglyceroylalkylamine. While phosphatidylglyceroylalkylamine is rapidly biosynthesized from Pi, its subsequent glycosylation occurs much more slowly. Therefore, we conclude that the final glycosylation step is the rate-limiting event in the biosynthesis of the complex phosphoglycolipids alpha-galactosyl- and alpha-N-acetylglucosaminyl-phosphatidylglyceroylalkylamine.

Acetates↗
PubMed1991

A highly conserved repeated chromosomal sequence in the radioresistant bacterium Deinococcus radiodurans SARK.

A DNA fragment containing a portion of a DNA damage-inducible gene from Deinococcus radiodurans SARK hybridized to numerous fragments of SARK genomic DNA because of a highly conserved repetitive chromosomal element. The element is of variable length, ranging from 150 to 192 bp, depending on the absence or presence of one or two 21-bp sequences located internally. A putative translational start site of the damage-inducible gene is within the reiterated element. The element contains dyad symmetries that suggest modes of transcriptional and/or translational control.

Amino Acid Sequence↗
PubMed1992

Fatty acids are precursors of alkylamines in Deinococcus radiodurans.

Deinococcus radiodurans contains novel phospholipids of which the structures of three have been previously described. These three lipids contain both fatty acids and alkylamines. Both the fatty acid and alkylamine constituents were found to be composed of a mixture of species, of which C15, C16, and C17 saturated and monounsaturated alkyl chains predominated. Alkylamines contained a relatively higher proportion of saturated species. Progression of bacterial growth through the mid-log to stationary phases was accompanied by an increase in the proportions of C15 and C17 alkyl chains in both fatty acid and alkylamine constituents. Radiolabeled palmitic acid was found to be rapidly incorporated into both fatty acid and alkylamine components of phosphatidylglyceroylalkylamine, which is the precursor of the more-complex phosphoglycolipids found in major amounts in D. radiodurans. After culturing D. radiodurans in the presence of a mixture of palmitic acids labeled with 14C and 3H in the 1 and 9,10 positions, respectively, the same 14C/3H ratio was recovered in both fatty acid and alkylamine constituents, strongly suggesting that alkylamines are derived from intact fatty acids rather than by a de novo pathway. The results identify a novel product of fatty acid metabolism which has not to date been observed in any other organism.

Alkylation↗
PubMed1993

Identification, sequencing, and targeted mutagenesis of a DNA polymerase gene required for the extreme radioresistance of Deinococcus radiodurans.

Deinococcus radiodurans and other species of the same genus share extreme resistance to ionizing radiation and many other agents that damage DNA. Two different DNA damage-sensitive strains generated by chemical mutagenesis were found to be defective in a gene that has extended DNA and protein sequence homology with polA of Escherichia coli. Both mutant strains lacked DNA polymerase, as measured in activity gels. Transformation of this gene from wild-type D. radiodurans restored to the mutants both polymerase activity and DNA damage resistance. A technique for targeted insertional mutagenesis in D. radiodurans is presented. This technique was employed to construct a pol mutant isogenic with the wild type (the first example of targeted mutagenesis in this eubacterial family). This insertional mutant lacked DNA polymerase activity and was even more sensitive to DNA damage than the mutants derived by chemical mutagenesis. In the case of ionizing radiation, the survival of the wild type after receiving 1 Mrad was 100% while survival of the insertional mutant extrapolated to 10(-24). These results demonstrate that the gene described here encodes a DNA polymerase and that defects in this pol gene cause a dramatic loss of resistance of D. radiodurans to DNA damage.

Amino Acid Sequence↗
PubMed1994

In vivo damage and recA-dependent repair of plasmid and chromosomal DNA in the radiation-resistant bacterium Deinococcus radiodurans.

Deinococcus radiodurans R1 and other members of this genus share extraordinary resistance to the lethal and mutagenic effects of ionizing radiation. We have recently identified a RecA homolog in strain R1 and have shown that mutation of the corresponding gene causes marked radiosensitivity. We show here that following high-level exposure to gamma irradiation (1.75 megarads, the dose required to yield 37% of CFU for plateau-phase wild-type R1), the wild-type strain repairs > 150 double-strand breaks per chromosome, whereas a recA-defective mutant (rec30) repairs very few or none. A heterologous Escherichia coli-D. radiodurans shuttle plasmid (pMD68) was constructed and found to be retained in surviving D. radiodurans R1 and rec30 following any radiation exposure up to the highest dose tested, 3 megarads. Plasmid repair was monitored in vivo following irradiation with 1.75 megarads in both R1/pMD68 and rec30/pMD68. Immediately after irradiation, plasmids from both strains contained numerous breaks and failed to transform E. coli. While irradiation with 1.75 megarads was lethal to rec30 cultures, a small amount of supercoiled plasmid was regenerated, but it lacked the ability to transform E. coli. In contrast, wild-type cultures showed a cell division arrest of about 10 h, followed by exponential growth. Supercoiled plasmid was regenerated at normal levels, and it readily transformed E. coli. These studies show that D. radiodurans retains a heterologous plasmid following irradiation and repairs it with the same high efficiency as its chromosomal DNA, while the repair defect in rec30 prevents repair of the plasmid. Taken together, the results of this study suggest that plasmid DNA damaged in vivo in D. radiodurans is repaired by recA-dependent mechanisms similar to those employed in the repair of chromosomal DNA.

Chromosomes, Bacterial↗
PubMed1994

Novel ionizing radiation-sensitive mutants of Deinococcus radiodurans.

Two new loci, irrB and irrI, have been identified in Deinococcus radiodurans. Inactivation of either locus results in a partial loss of resistance to ionizing radiation. The magnitude of this loss is locus specific and differentially affected by inactivation of the uvrA gene product. An irrB uvrA double mutant is more sensitive to ionizing radiation than is an irrB mutant. In contrast, the irrI uvrA double mutant and the irrI mutant are equally sensitive to ionizing radiation. The irrB and irrI mutations also reduce D. radiodurans resistance to UV radiation, this effect being most pronounced in uvrA+ backgrounds. Subclones of each gene have been isolated, and the loci have been mapped relative to each other. The irrB and irrI genes are separated by approximately 20 kb of intervening sequence that encodes the uvrA and pol genes.

Adenosine Triphosphatases↗
PubMed1994

Interplasmidic recombination following irradiation of the radioresistant bacterium Deinococcus radiodurans.

Deinococcus radiodurans R1 and other members of the eubacterial family Deinococcaceae are extremely resistant to ionizing radiation and many other agents that damage DNA. For example, after irradiation, D. radiodurans can repair > 100 DNA double-strand breaks per chromosome without lethality or mutagenesis, while most other organisms can survive no more than 2 or 3 double-strand breaks. The unusual resistance of D. radiodurans is recA dependent, but the repair pathway(s) is not understood. Recently, we described how a plasmid present in D. radiodurans (plasmid copy number, approximately 6 per cell; chromosome copy number, approximately 4 per cell) during high-dose irradiation undergoes extreme damage like the chromosome and is retained by the cell without selection and fully repaired with the same efficiency as the chromosome. In the current work, we have investigated the repair of two similar plasmids within the same cell. These two plasmids were designed to provide both restriction fragment polymorphisms and a drug selection indicator of recombination. This study presents a novel system of analysis of in vivo damage and recombinational repair, exploiting the unique ability of D. radiodurans to survive extraordinarily high levels of DNA damage. We report that homologous recombination among plasmids following irradiation is extensive. For example, 2% of Tcs plasmids become Tcr as a result of productive recombination within a 929-bp region of the plasmids after repair. Our results suggest that each plasmid may participate in as many as 6.7 recombinational events during repair, a value that extrapolates to > 700 events per chromosome undergoing repair simultaneously. These results indicate that the study of plasmid recombination within D. radiodurans may serve as an accurate model system for simultaneously occurring repair in the chromosome.

DNA Repair↗
PubMed1995

Genetic characterization of forty ionizing radiation-sensitive strains of Deinococcus radiodurans: linkage information from transformation.

Natural transformation was used to help define a collection of ionizing radiation-sensitive strains of Deinococcus radiodurans. Three putative rec mutations were identified, as were three pol alleles. Forty of the ionizing radiation-sensitive strains were placed into 16 linkage groups, and evidence obtained indicates that each linkage group consists of a cluster of mutations not more than 1,000 bp apart. In addition, a new class of D. radiodurans mutant was described that, although radioresistant, appears to recover from ionizing radiation-induced DNA damage slowly relative to other strains of D. radiodurans.

DNA Damage↗
PubMed1996

Expression of recA in Deinococcus radiodurans.

Deinococcus (formerly Micrococcus) radiodurans is remarkable for its extraordinary resistance to ionizing and UV irradiation and many other agents that damage DNA. This organism can repair > 100 double-strand breaks per chromosome induced by ionizing radiation without lethality or mutagenesis. We have previously observed that expression of D. radiodurans recA in Escherichia coli appears lethal. We now find that the RecA protein of D. radiodurans is ot detectable in D. radiodurans except in the setting of DNA damage and that termination of its synthesis is associated with the onset of deinococcal growth. The synthesis of Shigella flexneri RecA (protein sequence identical to that of E. coli RecA) in recA-defective D. radiodurans is described. Despite a large accumulation of the S. flexneri RecA in D. radiodurans, there is no complementation of any D. radiodurans recA phenotype, including DNA damage sensitivity, inhibition of natural transformation, or inability to support a plasmid that requires RecA for replication. To ensure that the cloned S. flexneri recA gene was not inactivated, it was rescued from D. radiodurans and was shown to function normally in E. coli. We conclude that neither D. radiodurans nor S. flexneri RecA is functional in the other species, nor are the kinetics of induction and suppression similar to each other, indicating a difference between these two proteins in their modes of action.

DNA Damage↗
PubMed1996

Conformational change of the hexagonally packed intermediate layer of Deinococcus radiodurans monitored by atomic force microscopy.

Both surfaces of the hexagonally packed intermediate (HPI) layer of Deinococcus radiodurans were imaged in buffer solution by atomic force microscopy. When adsorbed to freshly cleaved mica, the hydrophilic outer surface of the HPI layer was attached to the substrate and the hydrophobic inner surface was exposed to the stylus. The height of a single HPI layer was 7.0 nm, while overlapping edges of adjacent single layers adsorbed to mica had a height of 14.7 nm. However, double-layered stacks with inner surfaces facing each other exhibited a height of 17.4 nm. These stacks exposed the outer surface to the stylus. The different heights of overlapping layers and stacks are attributed to differences in the interaction between inner and outer surfaces. At high resolution, the inner surface revealed a protruding core with a central pore connected by six emanating arms. The pores exhibited two conformations, one with and the other without a central plug. Individual pores were observed to switch from one state to the other.

Bacterial Proteins↗
PubMed1996

An alternative pathway of recombination of chromosomal fragments precedes recA-dependent recombination in the radioresistant bacterium Deinococcus radiodurans.

Deinococcus radiodurans R1 and other members of this genus are able to repair and survive extreme DNA damage induced by ionizing radiation and many other DNA-damaging agents. The ability of R1 to repair completely > 100 double-strand breaks in its chromosome without lethality or mutagenesis is recA dependent. However, during the first 1.5 h after irradiation, recA+ and recA cells show similar increases in the average size of chromosomal fragments. In recA+ cells, DNA continues to enlarge to wild-type size within 29 h. However, in recA cells, no DNA repair is observed following the first 1.5 h postirradiation. This recA-independent effect was studied further, using two slightly different Escherichia coli plasmids forming adjacent duplication insertions in the chromosome, providing repetitive sequences suitable for circularization by non-recA-dependent pathways following irradiation. After exposure to 1.75 Mrad (17,500 Gy), circular derivatives of the integration units were detected in both recA+ and recA cells. These DNA circles were formed in the first 1.5 h postirradiation, several hours before the onset of detectable recA-dependent homologous recombination. By comparison, D. radiodurans strains containing the same E. coli plasmids as nonrepetitive direct insertions did not form circular derivatives of the integration units before or after irradiation in recA+ or recA cells. The circular derivatives of the tandemly integrated plasmids were formed before the onset of recA-dependent repair and have structures consistent with the hypothesis that DNA repair occurring immediately postirradiation is by a recA-independent single-strand annealing reaction and may be a preparatory step for further DNA repair in wild-type D. radiodurans.

Alleles↗
PubMed1996

Identification and characterization of uvrA, a DNA repair gene of Deinococcus radiodurans.

Deinococcus radiodurans is extraordinarily resistant to DNA damage, because of its unusually efficient DNA repair processes. The mtcA+ and mtcB+ genes of D. radiodurans, both implicated in excision repair, have been cloned and sequenced, showing that they are a single gene, highly homologous to the uvrA+ genes of other bacteria. The Escherichia coli uvrA+ gene was expressed in mtcA and mtcB strains, and it produced a high degree of complementation of the repair defect in these strains, suggesting that the UvrA protein of D. radiodurans is necessary but not sufficient to produce extreme DNA damage resistance. Upstream of the uvrA+ gene are two large open reading frames, both of which are directionally divergent from the uvrA+ gene. Evidence is presented that the proximal of these open reading frames may be irrB+.

Adenosine Triphosphatases↗
PubMed1996

Radioresistance of Deinococcus radiodurans: functions necessary to survive ionizing radiation are also necessary to survive prolonged desiccation.

Forty-one ionizing radiation-sensitive strains of Deinococcus radiodurans were evaluated for their ability to survive 6 weeks of desiccation. All exhibited a substantial loss of viability upon rehydration compared with wild-type D. radiodurans. Examination of chromosomal DNA from desiccated cultures revealed a time-dependent increase in DNA damage, as measured by an increase in DNA double-strand breaks. The evidence presented suggests that D. radiodurans' ionizing radiation resistance is incidental, a consequence of this organism's adaptation to a common physiological stress, dehydration.

DNA Damage↗
PubMed1999

Repair of oxidized bases in the extremely radiation-resistant bacterium Deinococcus radiodurans.

Deinococcus radiodurans is able to resist and survive extreme DNA damage induced by ionizing radiation and many other DNA-damaging agents. It is believed that it possesses highly efficient DNA repair mechanisms. To characterize the repair pathway of oxidized purines in this bacteria, we have purified, from crude extracts, proteins that recognize these oxidized bases. We report here that D. radiodurans possesses two proteins excising the oxidized purines (formamidopyrimidine and 8-oxoguanine) by a DNA glycosylase-a purinic/apyrimidine lyase mechanism. Moreover, one of those proteins is endowed with a thymine glycol DNA glycosylase activity. One of these proteins could be the homolog of the Escherichia coli Fpg enzyme, which confirms the existence of a base excision repair system in this bacteria.

Animals↗
PubMed1999

Peptidoglycan fine structure of the radiotolerant bacterium Deinococcus radiodurans Sark.

Peptidoglycan from Deinococcus radiodurans was analyzed by high-performance liquid chromatography and mass spectrometry. The monomeric subunit was: N-acetylglucosamine-N-acetylmuramic acid-L-Ala-D-Glu-(gamma)-L-Orn-[(delta)Gly-Gly]-D-Ala-D-Ala. Cross-linkage was mediated by (Gly)2 bridges, and glycan strands were terminated in (1-->6)anhydro-muramic acid residues. Structural relations with the phylogenetically close Thermus thermophilus are discussed.

Amino Acid Sequence↗
PubMed1999

Targeted mutagenesis by duplication insertion in the radioresistant bacterium Deinococcus radiodurans: radiation sensitivities of catalase (katA) and superoxide dismutase (sodA) mutants.

Deinococcus radiodurans R1 is extremely resistant to both oxidative stress and ionizing radiation. A simple and general targeted mutagenesis method was developed to generate catalase (katA) and superoxide dismutase (sodA) mutants. Both mutants were shown to be more sensitive to ionizing radiation than the wild type.

Catalase↗
PubMed2001

Promoter cloning in the radioresistant bacterium Deinococcus radiodurans.

Deinococcus radiodurans is a highly radiation-resistant bacterium that is classed in a major subbranch of the bacterial domain. Since very little is known about gene expression in this bacterium, an initial study of promoters was undertaken. In order to isolate promoters and study promoter function, a series of integrative vectors for stable chromosomal insertion in D. radiodurans were developed. These vectors are based on Escherichia coli replicons that are unable to replicate autonomously in D. radiodurans and carry homologous sequences for replacement recombination in the D. radiodurans chromosome. The resulting integration vectors were used to study expression of reporter genes fused to a number of putative promoters that were amplified from the D. radiodurans R1 genome. Further analysis of these and other putative promoters was performed by Northern hybridization and primer extension experiments. In contrast to previous reports, the -10 and -35 regions of these promoters resembled the sigma(70) consensus sequence of E. coli.

Catechol 2,3-Dioxygenase↗