Abstract

Both eukaryotes and archaebacteria use 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase to synthesize mevalonate, which eukaryotes employ in the production of sterols and archaebacteria need for the isoprenoid side chains of their unique and characteristic lipids. The drug mevinolin inhibits HMG-CoA reductase in eukaryotes and in the halophilic archaebacteria, and we have used a spontaneous mutation to mevinolin resistance in the construction of a selectable shuttle vector for Haloferax volcanii. Sequence analysis shows that this resistance determinant encodes an HMG-CoA reductase very like its eukaryotic homologs, but sharing with the one sequenced eubacterial HMG-CoA reductase (that of Pseudomonas mevalonii) few residues other than those common to all HMG-CoA reductases. Characterization of several spontaneous mevinolin-resistant mutants reveals that they are of two sorts: amplifications of the HMG-CoA reductase gene with varying amounts of flanking sequence, and point mutants upstream of the HMG-CoA reductase coding region. We compared sequence and expression of a mutant gene of the latter class to those of the wild-type gene. The point mutation found affects the TATA box-like "distal promoter element," results (like gene amplification) in resistance through the synthesis of excess gene product, and provides the first true genetic definition of an archaebacterial promoter.