Control of DNA topology during thermal stress in hyperthermophilic archaea: DNA topoisomerase levels, activities and induced thermotolerance during heat and cold shock in Sulfolobus.
Plasmid topology varies transiently in hyperthermophilic archaea during thermal stress. As in mesophilic bacteria, DNA linking number (Lk) increases during heat shock and decreases during cold shock. Despite this correspondence, plasmid DNA topology and proteins presumably involved in DNA topological control in each case are different. Plasmid DNA in hyperthermophilic archaea is found in a topological form from relaxed to positively supercoiled in contrast to the negatively supercoiled state typical of bacteria, eukaryotes and mesophilic archaea. We have analysed the regulation of DNA topological changes during thermal stress in Sulfolobus islandicus (kingdom Crenarchaeota), which harbours two plasmids, pRN1 and pRN2. In parallel with plasmid topological variations, we analysed levels of reverse gyrase, topoisomerase VI (Topo VI) and the small DNA-binding protein Sis7, as well as topoisomerase activities in crude extracts during heat shock from 80 degrees C to 85-87 degrees C, and cold shock from 80 degrees C to 65 degrees C. Quantitative changes in reverse gyrase, Topo VI and Sis7 were not significant. In support of this, inhibition of protein synthesis in S. islandicus during shocks did not alter plasmid topological dynamics, suggesting that an increase in topoisomerase levels is not needed for control of DNA topology during thermal stress. A reverse gyrase activity was detected in crude extracts, which was strongly dependent on the assay temperature. It was inhibited at 65 degrees C, but was greatly enhanced at 85 degrees C. However, the intrinsic reverse gyrase activity did not vary with heat or cold shock. These results suggest that the control of DNA topology during stress in Sulfolobus relies primarily on the physical effect of temperature on topoisomerase activities and on the geometry of DNA itself. Additionally, we have detected an enhanced thermoresistance of reverse gyrase activities in cultures subject to prolonged heat shock (but not cold shock). This acquired thermotolerance at the enzymatic level is abolished when cultures are treated with puromycin, suggesting a requirement for protein synthesis.
López-García
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Institut de Génétique et Microbiologie, Université Paris-Sud, Bât. 409, 91405 Orsay Cedex, France.
Forterre
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D019843:Archaeal Proteins / Q000378:metabolism; D003080:Cold Temperature; D004264:DNA Topoisomerases, Type I / Q000378:metabolism*; D004250:DNA Topoisomerases, Type II / Q000378:metabolism; D019641:DNA, Archaeal / Q000737:chemistry*; D004278:DNA, Superhelical / Q000737:chemistry; D006358:Hot Temperature; D009690:Nucleic Acid Conformation; D010957:Plasmids / Q000737:chemistry; D014176:Protein Biosynthesis; D011691:Puromycin; D016944:Sulfolobus / Q000235:genetics* / Q000378:metabolism; D013696:Temperature
D019843:Archaeal Proteins; D019641:DNA, Archaeal; D004278:DNA, Superhelical; D011691:Puromycin; C041813:DNA reverse gyrase; C105141:DNA topoisomerase VI; D004264:DNA Topoisomerases, Type I; D004250:DNA Topoisomerases, Type II