| Homologous recombination repair is a primary means for the repair of double-stranded DNA breaks (DSBs). The RecA and RadA/Rad51 proteins are one of the most important contributors to the process of homologous recombination in Bacteria and Eucarya respectively and their archaeal is called RadA. The archaeal RadA was purified and studied biochemically first from the hyperthermophilic crenarchaeote Sulfolobus solfataricus. A role for an archaeal RadA protein in DNA repair via homologous recombination came from genetic analysis in Haloferax volcanii showing that deleting the radA gene decreased its growth rate, and increased its sensitivity to DNA damaging agents. However, due to the lack of the genetic system for hyperthermophilic archaeon, the research of radA is limited to the physiological and biochemical studies in vitro and an in vivo analysis of the radA functions has never been conducted.In this work, aΔpyrEFΔlacS double-deletion mutant of Sulfolobus islandicus E233s was used as the host to conduct genetic manipulation with two markers. To construct the knockout plasmid, the flanking sequences of radA were amplified and used as the homologous sequence arms, L-arm and R-arm. The promoter of radA was replaced by araS promoter in order to create a T-Gene arm for homologous recombination. The final knockout plasmid was constructed by inserting the knockout cassette of L-arm, R-arm, pyrEF+lacS marker and T-Gene arm into an E. coli vector pUC19. After transforming the linear knockout plasmid into S. islandicus E233s, the cassette recombined with the host chromosome through double crossover, yielding uracil-proficient transformants which formed colonies on the selective plates. The transformants were purified via streaking for single colonies on the selective plates for 3 consecutive times, the designed deletion mutants were selected by the pyrEF counter selection. However, the colonies grown up on the counter selection plates were all identified as the spontaneous pyrEF mutants by PCR. Then, the mutants were enriched by incubation under the pyrEF counter selection allowing only the deletion mutants to grow up as white colonies upon X-gal staining because spontaneous mutations both for pyrEF and for lacS could only occur at very low rate (<10-10 event). Intriguingly, white colonies appeared on selective plates. However, they were most likely double spontaneous mutants rather than the radA deletion mutants. Taken together, these results indicated that radA gene is most likely essential to the survival of S. islandicus. In addition, the relationship of radA gene expression and cell growth was analyzed by culturing the radA transformants in 3 media containing different carbon sources. It was observed that the different expression levels of radA in different carbon sources could have a significant effect on host growth.This work represents the first research that has demonstrated that a radA gene can be essential to the survival of the hyperthermophile S. islandicus. This raises an interesting question whether radA is also essential to other hyperthermophilic organisms. If so, homologous recombination should have more important cellular functions in hyperthermophiles. Alternatively, radA should have another important role in hyperthermophiles which remains to be unrevailed.
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