Reverse gyrase from archaebacteria

Abstract

Reverse gyrase, an enzyme that positively supercoils DNA, was examined with regard to its distribution in thermophilic and mesophilic bacteria. It was discovered that, among the bacterial species tested, reverse gyrase activity was only present in the sulphur metabolizing archaebacteria. While screening for reverse gyrase, endonuclease and relaxing topoisomerase activities were also detected in some bacterial species. Two archaebacteria, Sulfolobus solfataricus and the Thermococcus-like isolate AN1, were chosen as representatives of the major branches of archaebacteria which exhibit the greatest phylogenetic diversity among bacteria known to contain reverse gyrase. Reverse gyrase from these organisms was further investigated following the purification and characterization of the enzyme. The results suggested that reverse gyrase was not significantly different between S. solfataricus and AN1 in physical properties or in the catalytic conditions under which activity is maximal. Furthermore this similarity extends to the reverse gyrase from Sulfolobus acidocaldarius (Forterre et al., 1985; Nadal et al., 1988; Nakasu and Kikuchi, 1985) and Desulfurococcus amylolyticus (Slesarev, 1988). The role of reverse gyrase in sulphur metabolizing archaebacteria was examined by attempting to elucidate the supercoiled state of their genomic DNA. The technique used for these experiments followed that of Worcel and Burgi, 1972 who extracted nucleoids from E. coli and ran them on sucrose gradients under different concentrations of ethidium bromide. The procedures necessary to succeed in using this technique were successfully developed using E. coli which was confirmed as containing a negatively supercoiled genome. However attempts to isolate nucleoids from S. solfataricus and AN1 were not successful and the supercoiled state of chromosomal DNA in these cells could not be determined.