PURINE SALVAGE IN 2 HALOPHILIC ARCHAEA - CHARACTERIZATION OF SALVAGE PATHWAYS AND ISOLATION OF MUTANTS RESISTANT TO PURINE ANALOGS

In exponentially growing cultures of the extreme halophile Halobacteri um halobium and the moderate halophile Haloferax volcanii, growth char acteristics including intracellular protein levels, RNA content, and n ucleotide pool sizes were analyzed, This is the first report on pool s izes of nucleoside triphosphates, NAD, and PRPP (5-phosphoribosyl-alph a-1-pyrophosphate) in archaea, The presence of a number of salvage and interconversion enzymes was determined by enzymatic assays, The level s varied significantly between the two organisms, The most significant difference was the absence of GMP reductase activity in H, halobium, The metabolism of exogenous purines was investigated in growing cultur es, Both purine bases and nucleosides were readily taken up and were i ncorporated into nucleic acids, Growth of both organisms was affected by a number of inhibitors of nucleotide synthesis, H, volcanii was mor e sensitive than H. halobium, and purine base analogs were more toxic than nucleoside analogs, Growth of H, volcanii was inhibited by trimet hoprim and sulfathiazole, while these compounds had no effect on the g rowth of H, halobium, Spontaneous mutants resistant to purine analogs were isolated, The most frequent cause of resistance was a defect in p urine phosphoribosyltransferase activity coupled with reduced purine u ptake, A single phosphoribosyltransferase seemed to convert guanine as well as hypoxanthine to nucleoside monophosphates, and another phosph oribosyltransferase had specificity towards adenine, The differences i n the metabolism of purine bases and nucleosides and the sensitivity t o purine analogs between the two halobacteria were reflected in differ ences in purine enzyme levels, Based on our results, we conclude that purine salvage and interconversion pathways differ just as much betwee n the two archaeal species as among archaea, bacteria, and eukarya.