Interplasmidic recombination following irradiation of the radioresistant bacterium Deinococcus radiodurans

Abstract

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.

Transcriptome dynamics of Deinococcus radiodurans recovering from ionizing radiation

“…After a dose of 15 kGy, DEIRA recovery typically progresses through three phases (5, 9, 10, 21): (i) early phase (0-3 h), where cell growth is inhibited and recA is induced, but there is little evidence of DNA repair; (ii) mid phase (3-9 h), where growth inhibition and recA expression continue, but with progressive DNA repair; and (iii) late phase (9-24 h), where recA is repressed and cell growth is restored. Consistent with these reports (5,6,(9)(10)(11), after the exposure of DEIRA to 15 kGy, Ϸ150 DSBs per haploid genome were inflicted (data not shown). As expected, after the 9-h lag in growth, cells grew exponentially and reached stationary phase 15 h later (Fig.…”

“…We examined changes in genomic expression in DEIRA recovering from an acute exposure to 15 kGy, which is about its D 37 (irradiation dose yielding 37% survival) under our standard conditions (5,(9)(10)(11). During the early and mid phases of recovery, no cell growth was observed, but within this interval many genes of diverse functional groups were induced (Table 3).…”

“…At the indicated recovery time points (0, 0.5, 1.5, 3, 5, 9, 12, 16, and 24 h), Ϸ1 ϫ 10 9 cells were harvested, washed, and frozen in RNAlater solution. Cell viability and cell numbers were determined by plate assay and hemocytometer count, respectively, as described (5). Three independent cell cultures and irradiation treatments of the same kind were performed and served as biological replicates for gene expression experiments, as well as for determining irradiation resistance profiles.…”

“…Because most organisms, generally, can tolerate so few DSBs (8), radiationinduced DSBs and their repair have been difficult to study. In DEIRA, however, there are so many DSBs in fully viable irradiated cells after high-dose irradiation that the steps in DSB repair can be monitored directly in mass culture (5,(9)(10)(11). This characteristic has been exploited and used to examine the timing of DNA recombination (5, 10, 12) after high-dose irradiation and has revealed the sequential action of RecA-independent and -dependent pathways during repair (11).…”

“…Ionizing radiation induces DNA double-stranded breaks (DSBs) that are the most lethal form of DNA damage (3). After acute exposures to 10 kGy, early stationary phase (ESP) DEIRA can reassemble its 3.285-Mbp genome, which consists of four haploid genomic copies per cell (4), from hundreds of DNA DSB fragments without lethality or induced mutagenesis (5,6). Also remarkable is DEIRA's ability to grow at 60 Gy͞h without any discernable effect on its growth rate (7).…”