DNA repair in the extremely radioresistant bacterium Deinococcus radiodurans

Deinococcus radiodurans and other members of the same genus share extraordinary resistance to the lethal and mutagenic effects of ionizing and u.v. radiation and to many other agents that damage DNA. While it is known that this resistance is due to exceedingly efficient DNA repair, the molecular mechanisms responsible remain poorly understood. Following very high exposures to u.v. irradiation (e.g. 500 Jm⁻², which is non-lethal to D. radiodurans), this organism carries out extremely efficient excision repair accomplished by two separate nucleotide excision repair pathways acting simultaneously. One pathway requires the uvrA gene and appears similar to the UvrABC excinuclease pathway defined in Escherichia coli. The other excision repair pathway is specific for u.v. dimeric photoproducts, but is not mediated by a pyrimidine dimer DNA glycosylase. Instead, it is initiated by a second bona fide endonuclease that may recognize both pyrimidine dimers and pyrimidine-(6–4)pyrimidones. After high doses of ionizing-radiation (e.g. 1.5Mrad), D. radiodurans can mend >100 double-strand breaks (dsb) per chromosome without lethality or mutagenesis. Both dsb mending and survival are recA-dependent, indicating that efficient dsb mending proceeds via homologous recombination. D. radiodurans contains multiple chromosomes per cell, and it is proposed that dsb mending requires extensive recombination amongst these chromosomes, a novel phenomenon in bacteria. Thus, D. radiodurans may serve as an easily accessible model system for the double-strand-break-initiated interchromosomal recombination that occurs in eukaryotic cells during mitosis and meiosis.     

Nucleoid organization in the radioresistant bacterium Deinococcus radiodurans

Processes favoring the exceptional resistance to genotoxic stress of Deinococcus radiodurans are not yet completely characterized. It was postulated that its nucleoid and chromosome(s) organization could participate in the DNA double strand break repair process. Here, we investigated the organization of chromosome 1 by localization of three chromosomal loci including oriC, Ter and a locus located in its left arm. For this purpose, we used a ParB‐parS system to visualize the position of the loci before and after exposure to γ‐rays. By comparing the number of fluorescent foci with the number of copies of the studied loci present in the cells measured by quantitative polymerase chain reaction (qPCR), we demonstrated that the 4–10 copies of chromosome 1 per cell are dispersed within the nucleoid before irradiation, indicating that the chromosome copies are not prealigned. Chromosome segregation is progressive but not co‐ordinated, allowing each locus to be paired with its sister during part of the cell cycle. After irradiation, the nucleoid organization is modified, involving a transient alignment of the loci in the late stage of DNA repair and a delay of segregation of the Ter locus. We discuss how these events can influence DNA double strand break repair.     

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.     

RecX is involved in antioxidant mechanisms of the radioresistant bacterium Deinococcus radiodurans

Deinococcus radiodurans shows remarkable resistance to reactive oxygen species (ROS), generated by irradiation. Disruption of recX (dr1310) in D. radiodurans using targeted mutagenesis method enhanced its ROS scavenging activity, and recX overexpression in this bacterium repressed its antioxidant activity significantly. Further analyses on catalase and superoxide dismutase, two important antioxidant proteins in cells, showed that RecX could repress the induction of antioxidant enzymes, revealing that it negatively regulates the ROS scavenging activity in D. radiodurans.     

Interchromosomal recombination in the extremely radioresistant bacterium Deinococcus radiodurans.

Deinococcus radiodurans and other members of the genus Deinococcus are remarkable for their extreme resistance to ionizing radiation and many other agents that damage DNA. We have recently shown that recombinational processes participate in interplasmidic repair following in vivo irradiation. We now present direct studies on interchromosomal recombination among chromosomes irradiated in vivo during stationary phase (four chromosomes per cell). Following an exposure to 1.75 Mrad (the dose required to achieve a survival of 37%, which degrades the cells' four chromosomes into about 500 fragments), we determined that there may be as many as 175 crossovers per chromosome (700 crossovers per nucleoid) undergoing repair. In addition, these studies suggest that many of the crossovers occurring during repair are nonreciprocal.     

Radiation response mechanisms of the extremely radioresistant bacterium Deinococcus radiodurans.

Effect of microgravity on recovery of bacterial cells from radiation damage was examined in IML-2, S/MM-4 and S/MM-9 experiments using the extremely radioresistant bacterium Deinococcus radiodurans. The cells were irradiated with gamma rays before the space flight and incubated on board the Space Shuttle. The survival of the wild type cells incubated in space increased compared with the ground controls, suggesting that the recovery of this bacterium from radiation damage was enhanced under the space environment. No difference was observed between the survivals of radiosensitive mutant rec30 cells incubated in space and on the ground. The amount of DNA-repair related RecA protein induced under microgravity was similar to those of ground controls, however, induction of PprA protein, product of a unique radiation-inducible gene (designated pprA) responsible for loss of radiation resistance in repair-deficient mutant, KH311, was enhanced under microgravity compared with ground controls. Recent investigation in vitro showed that PprA preferentially bound to double-stranded DNA carrying strand breaks, inhibited Escherichia coli exonuclease III activity, and stimulated the DNA end-joining reaction catalyzed by DNA ligases. These results suggest that D. radiodurans has a radiation-induced non-homologous end-joining (NHEJ) repair mechanism in which PprA plays a critical role.     

Analysis of codon usage pattern in the radioresistant bacterium Deinococcus radiodurans

The main factors shaping codon usage bias in the Deinococcus radiodurans genome were reported. Correspondence analysis (COA) was carried out to analyze synonymous codon usage bias. The results showed that the main trend was strongly correlated with gene expression level assessed by the "Codon Adaptation Index" (CAI) values, a result that was confirmed by the distribution of genes along the first axis. The results of correlation analysis, variance analysis and neutrality plot indicated that gene nucleotide composition was clearly contributed to codon bias. CDS length was also key factor in dictating codon usage variation. A general tendency of more biased codon usage of genes with longer CDS length to higher expression level was found. Further, the hydrophobicity of each protein also played a role in shaping codon usage in this organism, which could be confirmed by the significant correlation between the positions of genes placed on the first axis and the hydrophobicity values (r=-0.100, P<0.01). In summary, gene expression level played a crucial role, nucleotide mutational bias, CDS length and the hydrophobicity of each protein just in a minor way in shaping the codon usage pattern of D. radiodurans. Notably, 19 codons firstly defined as "optimal codons" may provide useful clues for molecular genetic engineering and evolutionary studying.     

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.     

Vectors for regulated gene expression in the radioresistant bacterium Deinococcus radiodurans

Deinococcus radiodurans possesses an exceptional capacity to withstand the lethal and mutagenic effects of most form of DNA damage and has received considerable interest for use in both fundamental and applied research. Here we describe vectors that allow regulated expression of Deinococcal genes for functional analysis. The vectors contain the IPTG-regulated Spac system (Pspac promoter and lacI repressor gene), originally designed for Bacillus subtilis, that we have adapted to be functional in D. radiodurans. We show that the Spac system can control the expression of a lacZ reporter gene over two orders of magnitude depending on the inducer concentration and the copy number of the lacI regulatory gene. Furthermore, we demonstrate that the Spac system can be used to regulate the synthesis of a critical repair protein, such as RecA, resulting in a conditional mitomycin-resistant cell phenotype. We have also developed tools for the construction of conditional mutants where the expression of the target gene is regulated by an inducible promoter. The utility of these conditional gene inactivation systems is exemplified by the conditional lethal phenotype of a mutant expressing gyrA from the Pspac promoter.     

Characterization and distribution of IS8301 in the radioresistant bacterium Deinococcus radiodurans

The insertion sequence element IS8301 isolated from the radiation resistant bacterium Deinococcus radiodurans strain KD8301 was characterized. IS8301 is comprised of 1,736-bp, lacks terminal inverted repeats and does not duplicate target DNA upon its insertion. The amino acid sequence homology of two open reading frames encoded in IS8301 indicates that this insertion sequence element belongs to the IS200/IS605 group. There were seven loci completely identical with the IS8301 sequence in the published D. radiodurans R(1) genome sequence. The genome distribution profiles of IS8301 in strain KD8301 as well as in the three different laboratory isolates (KR(1), MR(1), and R(1)) of wild-type D. radiodurans were investigated using genomic hybridization analysis. At least 21 strong hybridization signals were detected in strain KD8301 while only one hybridization signal was detected in strain KR(1), the parent strain of KD8301. In strain MR1, a different wild-type isolate, six strong hybridization signals were detected. In spite of the identification of seven copies of IS8301 in the published D. radiodurans R(1) genome sequence, only one hybridization signal was detected in strain R(1) purchased from American Type Culture Collection. Using inverse PCR and sequencing analyses, total 13 different insertion loci of IS8301 in the D. radiodurans genome were identified. Sequence comparison of the flanking region of insertion sites indicated that the sequence 5'-TTGAT-3' preceded the left end of IS8301 in all cases.     

Characterization of the DdrD protein from the extremely radioresistant bacterium Deinococcus radiodurans

Extremophiles - Here, we report the in vitro and in vivo characterization of the DdrD protein from the extraordinary stress-resistant bacterium, D. radiodurans. DdrD is one of the most highly...     

Gene fusions with lacZ by duplication insertion in the radioresistant bacterium Deinococcus radiodurans.

Deinococcus radiodurans is the most-studied species of a eubacterial family characterized by extreme resistance to DNA damage. We have focused on developing molecular biological techniques to investigate the genetics of this organism. We report construction of lacZ gene fusions by a method involving both in vitro splicing and the natural transformation of D. radiodurans. Numerous fusion strains were identified by expression of beta-galactosidase. Among these fusion strains, several were inducible by exposure to the DNA-damaging agent mitomycin C, and four of the inducible fusion constructs were cloned in Escherichia coli. Hybridization studies indicate that one of the damage-inducible genes contains a sequence reiterated throughout the D. radiodurans chromosome. Survival measurements show that two of the fusion strains have increased sensitivity to mitomycin C, suggesting that the fusions within these strains inactivate repair functions.     

Duplication insertion of drug resistance determinants in the radioresistant bacterium Deinococcus radiodurans.

Escherichia coli drug resistance plasmids were introduced into Deinococcus radiodurans by cloning D. radiodurans DNA into the plasmids prior to transformation. The plasmids were integrated into the chromosome of the transformants and flanked by a direct repeat of the cloned D. radiodurans segment. The plasmid and one copy of the flanking chromosomal segment constituted a unit ("amplification unit") which was found repeated in tandem at the site of chromosomal integration. Up to 50 copies of the amplification unit were present per chromosome, accounting for approximately 10% of the genomic DNA. Circular forms of the amplification unit were also present in D. radiodurans transformants. These circles were introduced into E. coli, where they replicated as plasmids. The drug resistance determinants which have been introduced into D. radiodurans in this fashion are cat (from Tn9) and aphA (from Tn903). Transformation of D. radiodurans to drug resistance was efficient when the donor DNA was from D. radiodurans or E. coli, but was greatly reduced when the donor DNA was linearized with restriction enzymes prior to transformation. In the course of the study, a plasmid, pS16, was discovered in D. radiodurans R1, establishing that all Deinococcus strains so far examined contain plasmids.     

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.     

RecO is essential for DNA damage repair in Deinococcus radiodurans

Here we present direct evidence for the vital role of RecO in Deinococcus radiodurans's radioresistance. A recO null mutant was constructed using a deletion replacement method. The mutant exhibited a growth defect and extreme sensitivity to irradiation with gamma rays and UV light. These results suggest that DNA repair in this organism occurs mainly via the RecF pathway.     

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.     

Induction of a futile Embden-Meyerhof-Parnas pathway in Deinococcus radiodurans by Mn: possible role of the pentose phosphate pathway in cell survival

Statistical models were used to predict the effects of tryptone, glucose, yeast extract (TGY) and Mn on biomass formation of the highly radioresistant bacterium Deinococcus radiodurans. Results suggested that glucose had marginal effect on biomass buildup, but Mn was a significant factor for biomass formation. Mn also facilitated glucose interactions with other nutrient components. These predictions were verified by in vivo and in vitro experiments. TGY-grown cells metabolized glucose solely by the pentose phosphate pathway (PPP). Although only a fraction of glucose from the medium was transported into the cells, glucose was incorporated into the DNA efficiently after cells were exposed to UV light. The presence of glucose also enhanced the radioresistance of the culture. Mn could induce an Embden-Meyerhof-Parnas (EMP) pathway in D. radiodurans. The EMP pathway and the PPP of the Mn-treated cells oxidized glucose simultaneously at a 6:1 ratio. Although glucose was hydrolyzed rapidly by the Mn-treated cells, most glucose was released as CO(2). Mn-treated cultures retained less glucose per cell than cells grown without Mn, and still less glucose was incorporated into the DNA after cells were exposed to UV light. Mn-treated cells were also more sensitive to UV light. Results suggested that metabolites of glucose generated from the PPP enhanced the survival of D. radiodurans. Induction of the EMP pathway by Mn may deplete metabolites for DNA repair and may induce oxidative stress for the cell, leading to reduction of radioresistance.     

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.     

Identification and disruption analysis of the recN gene in the extremely radioresistant bacterium Deinococcus radiodurans.

We isolated a radiosensitive mutant strain, KR4128, from a wild-type strain of Deinococcus radiodurans, which is known as a extremely radioresistant bacterium. The gene that restore the defect of the mutant in DNA repair was cloned, and it turned out to be the homolog of the recN gene of Escherichia coli. The recN gene encoded a protein of 58 kDa, and, in its N-terminal region, a potential ATP binding domain was conserved as expected for a prokaryotic RecN protein. An analysis of sequence of the mutant recN gene revealed a G:C to T:A transversion near the 3' end of the coding region. This alteration causes an ochre mutation, and results in the truncation of 47 amino acids from the C-terminal region of the RecN protein. The null mutant of recN gene was constructed by insertional mutagenesis, and it showed substantial sensitivities to various types of DNA damaging agents, indicating that a single defect in the recN gene can directly affect the DNA damage resistant phenotype in D. radiodurans. The recN locus of KR4128 was also disrupted and the disruptant indicated the sensitivity that was indistinguishable from its progenitor. The result indicate that the transversion in the recN gene of KR4128 cells causes a complete loss of function of the RecN protein and thus the C-terminal region of the RecN protein includes domain essential to its function.