Purine salvage in two halophilic archaea: characterization of salvage pathways and isolation of mutants resistant to purine analogs

Abstract

In exponentially growing cultures of the extreme halophile Halobacterium halobium and the moderate halophile Haloferax volcanii, growth characteristics including intracellular protein levels, RNA content, and nucleotide pool sizes were analyzed. This is the first report on pool sizes of nucleoside triphosphates, NAD, and PRPP (5-phosphoribosyl-alpha-1-pyrophosphate) in archaea. The presence of a number of salvage and interconversion enzymes was determined by enzymatic assays. The levels 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 cultures. Both purine bases and nucleosides were readily taken up and were incorporated into nucleic acids. Growth of both organisms was affected by a number of inhibitors of nucleotide synthesis. H. volcanii was more sensitive than H. halobium, and purine base analogs were more toxic than nucleoside analogs. Growth of H. volcanii was inhibited by trimethoprim and sulfathiazole, while these compounds had no effect on the growth of H. halobium. Spontaneous mutants resistant to purine analogs were isolated. The most frequent cause of resistance was a defect in purine phosphoribosyltransferase activity coupled with reduced purine uptake. A single phosphoribosyltransferase seemed to convert guanine as well as hypoxanthine to nucleoside monophosphates, and another phosphoribosyltransferase had specificity towards adenine. The differences in the metabolism of purine bases and nucleosides and the sensitivity to purine analogs between the two halobacteria were reflected in differences in purine enzyme levels. Based on our results, we conclude that purine salvage and interconversion pathways differ just as much between the two archaeal species as among archaea, bacteria, and eukarya.

FIG. 1

Metabolism of exogenous purine bases and nucleosides in H. volcanii and H. halobium. The uptake, incorporation, and excretion of [¹⁴C]purine bases and nucleosides in growing cells were monitored. Samples of the culture were directly subjected to ion-exchange chromatography. The system used allowed the separation of purine bases and nucleosides in the medium from nucleotides and nucleic acids in the cells. The distribution of the labeling was calculated as described in Materials and Methods. Ade, adenine; Ado, adenosine; Hyp, hypoxanthine; Ino, inosine; Gua, guanine; Guo, guanosine.

FIG. 2

Purine salvage and interconversion pathways in H. volcanii and H. halobium. The individual reactions are identified by numbers: 1, adenosine deaminase; 2, adenosine phosphorylase; 3, guanosine phosphorylase; 4, inosine phosphorylase; 5, inosine kinase; 6, guanosine kinase; 7, adenine phosphoribosyltransferase; 8, hypoxanthine(guanine) phosphoribosyltransferase; 9, guanine deaminase; 10, adenylosuccinate synthetase; 11, adenylosuccinate lyase; 12, IMP dehydrogenase; 13, GMP synthetase; 14, GMP reductase (found only in H. volcanii).