High spontaneous mutation rate in the hyperthermophilic archaeon Sulfolobus solfataricus is mediated by transposable elements

We have isolated uracil-auxotrophic mutants of the hyperthermophilic archae on Sulfolobus solfataricus in order to explore the genomic stability and mu tational frequencies of this organism and to identify complementable recipi ents for a selectable genetic transformation system, Positive selection of spontaneous mutants resistant to 5-fluoroorotate yielded uracil auxotrophs with frequencies of between 10(-4) and 10(-5) per sensitive, viable cell. F our different, nonhomologous insertion sequences (ISs) were identified at d ifferent positions within the chromosomal pyrEF locus of these mutants. The y ranged in size from 1,058 to 1,439 bp and possessed properties typical of known transposable elements, i,e,, terminal inverted repeats, flanking dup licated target sequences, and putative transposase genes encoding motifs th at are indicative of the IS4-IS5 IS element families. Between 12 and 25 cop ies of each IS element were found in chromosomal DNAs by Southern analyses. While characteristic fingerprint patterns created by IS element-specific p robes were observed with genomic DNA of different S. solfataricus strains, no homologous sequences were identified in DNA of other well-characterized strains of the order Sulfolobales.