Insight into the cellular involvement of the two reverse gyrases from the hyperthermophilic archaeon Sulfolobus solfataricus

Abstract

BackgroundReverse gyrases are DNA topoisomerases characterized by their unique DNA positive-supercoiling activity. Sulfolobus solfataricus, like most Crenarchaeota, contains two genes each encoding a reverse gyrase. We showed previously that the two genes are differently regulated according to temperature and that the corresponding purified recombinant reverse gyrases have different enzymatic characteristics. These observations suggest a specialization of functions of the two reverse gyrases. As no mutants of the TopR genes could be obtained in Sulfolobales, we used immunodetection techniques to study the function(s) of these proteins in S. solfataricus in vivo. In particular, we investigated whether one or both reverse gyrases are required for the hyperthermophilic lifestyle.ResultsFor the first time the two reverse gyrases of S. solfataricus have been discriminated at the protein level and their respective amounts have been determined in vivo. Actively dividing S. solfataricus cells contain only small amounts of both reverse gyrases, approximately 50 TopR1 and 125 TopR2 molecules per cell at 80°C. S. solfataricus cells are resistant at 45°C for several weeks, but there is neither cell division nor replication initiation; these processes are fully restored upon a return to 80°C. TopR1 is not found after three weeks at 45°C whereas the amount of TopR2 remains constant. Enzymatic assays in vitro indicate that TopR1 is not active at 45°C but that TopR2 exhibits highly positive DNA supercoiling activity at 45°C.ConclusionsThe two reverse gyrases of S. solfataricus are differently regulated, in terms of protein abundance, in vivo at 80°C and 45°C. TopR2 is present both at high and low temperatures and is therefore presumably required whether cells are dividing or not. By contrast, TopR1 is present only at high temperature where the cell division occurs, suggesting that TopR1 is required for controlling DNA topology associated with cell division activity and/or life at high temperature. Our findings in vitro that TopR1 is able to positively supercoil DNA only at high temperature, and TopR2 is active at both temperatures are consistent with them having different functions within the cells.

The reverse gyrase TopR1 is responsible for the homeostatic control of DNA supercoiling in the hyperthermophilic archaeon Sulfolobus solfataricus

“…It is in agreement with an extremely low level of such protein, as previously reported in Sulfolobus islandicus (Li et al, 2011). At 80°C, the two reverse gyrases of S. solfataricus are present at a low level: about 52 TopR1 per cell and 125 TopR2 per cell (Table 1) as previously described (Couturier et al, 2014). At 88°C, the number of TopR1 per cell, about 28, is reduced twice compared to 80°C, while the number of TopR2 per cell is not significantly modified (Table 1).…”

“…Indeed, resting cells undergoing less topological fluctuations than in actively dividing cells, do not need to maintain the distributive reverse gyrase responsible for the fine‐tuning of DNA topology. The needs for controlling the residual topological constraints could be easily assumed by the second remaining reverse gyrase, TopR2, as previously proposed (Couturier et al, ). Indeed, the processivity of TopR2 is largely reduced by the low temperature and it could thus catalyze some functions requiring a more distributive property.…”

“…This adaptative regulatory pathway could be extended in response to other parameters as the DNA topological state is very sensitive to numerous environmental parameters, such as pH or ionic strength (Balke & Gralla, 1987;Duguet, 1993;Goldstein & Drlica, 1984;Higgins et al, 1988;Wang & Syvanen, 1992). We previously reported that S. solfataricus cells survive to a long exposure at 45°C, during which no replication and no cell division occurred (Couturier et al, 2014). Interestingly, the regulation of the reverse gyrase content was targeting only TopR1 by depleting it, TopR2 remaining constant and active in low temperature adapted cells.…”

“…Primary antibodies used for specifically detecting the two reverse gyrases of S. solfataricus were the same as previously described (Couturier et al, 2014).…”

“…Both reverse gyrases, TopR1 and TopR2, introduce positive supercoils into DNA (Bizard, Garnier, & Nadal, ). However, we have previously shown that the two reverse gyrases of S. solfataricus exhibit specific enzymatic properties in regards of temperature, ATP and salt dependence (Bizard et al, ; Couturier, Bizard, Garnier, & Nadal, ). In addition, TopR2 was reported to be a processive enzyme, while TopR1 is a distributive one (Bizard et al, ).…”