Comparison of plasmid DNA topology among mesophilic and thermophilic eubacteria and archaebacteria

Abstract

Several plasmid DNAs have been isolated from mesophilic and thermophilic archaebacteria. Their superhelical densities were estimated at their host strain's optimal growth temperature, and in some representative strains, the presence of reverse gyrase activity (positive DNA supercoiling) Covalently closed DNA molecules so far isolated from eukaryotes and eubacteria are negatively supercoiled to about the same extent. Once purified of proteins, the native DNA molecules exhibit a superhelical density in a range from -0.040 to -0.060 (4,6,48). Negatively supercoiled DNA has a linking deficit, that is, the DNA has fewer duplex turns than would be found in a nicked or linear molecule of the same length. The free energy of negative supercoiling permits all biological processes such as DNA replication, transcription, transposition, and general or site-specific recombination to take place (50). In mesophilic eubacteria, the level of supercoiling is determined essentially by the opposing actions of DNA topoisomerases, which either relax or produce negative superturns in DNA (18). In the eukaryotic nucleus, DNA is negatively supercoiled around the histone core, whereas histone-free DNA is relaxed by either DNA topoisomerase I or II (50).In thermophilic organisms, the situation is not so clear. The major DNA topoisomerase activity detected in extremely thermophilic and hyperthermophilic archaebacteria is due to reverse gyrase (6,29,30). This ATP-dependent DNA topoisomerase I is able to introduce positive superturns into DNA (23,38,40). Such DNA has an excess of links, which could help to stabilize it at high temperatures. Reverse gyrase has also been found in species of the order Thermotogales (5) We have recently found that the DNA of the plasmid pGT5, isolated from the newly identified hyperthermophilic sulfurmetabolizing archaebacterium GE5, is relaxed at 95'C, the optimal growth temperature of its host strain (9). This relaxed state is completely different from that of the negatively supercoiled DNA of extrachromosomal elements that have been previously studied in mesophilic eubacteria and eukaryotes.In order to determine whether this unusual topological situation is universal to the thermophilic organisms, we purified several plasmid DNAs from mesophilic and thermophilic eubacteria as well as archaebacteria and determined their superhelical densities at the physiological growth temperature of the host strains.The results reported here strongly suggest the existence of two groups of plasmids. The group I plasmids have a highly negatively supercoiled DNA and were all isolated from eubacteria and mesophilic archaebacteria. In contrast, group II plasmids exhibit a relaxed DNA and belong exclusively to thermophilic archaebacterial group. An additional result is that all thermophilic archaebacteria that possess a relaxed plasmid DNA show strong reverse gyrase-like activity, with the exception of a moderately thermophilic archaebacterium that possesses a relaxed plasmidic DNA but has no detectable reverse gyrase activity. ...

Methanococcus vulcanius sp. nov., a novel hyperthermophilic methanogen isolated from East Pacific Rise, and identification of Methanococcus sp. DSM 4213Tas Methanococcus fervens sp. nov.

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Widespread distribution of archaeal reverse gyrase in thermophilic bacteria suggests a complex history of vertical inheritance and lateral gene transfers

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Desulfurobacterium thermolithotrophum gen. nov., sp. nov., a novel autotrophic, sulphur-reducing bacterium isolated from a deep-sea hydrothermal vent

“…Genomic DNA of strain BSAT was isolated using a modification of the procedure described by Charbonnier & Forterre (1994). The DNA was purified on a caesium chloride gradient (Sambrook et al, 1989), and purity was checked spectrophotometrically.…”