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 halophileHalobacterium halobium and the moderate halophileHaloferax 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-α-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.

Dynamic Metabolite Profiling in an Archaeon Connects Transcriptional Regulation to Metabolic Consequences

“…Metabolite levels obtained with the two methods were strongly and significantly correlated ( Fig 1 , R 2 > 0.55, p < 10 −16 ), suggesting good reproducibility between different cultures, extraction solvents, and detection methods. Based on the regression fit of the untargeted GC-MS to the targeted data ( Fig 1 ), previously quantified gamma-glutamyl cysteine levels [ 20 ], and previously quantified NAD levels [ 21 ], metabolites with intracellular concentrations in or below the single millimolar range were detected by all three of the untargeted assays ( S1 Table ). Taken together, these data suggest that metabolomics profiling in H .…”

Purine and pyrimidine salvage pathway in thermophiles: a valuable source of biocatalysts for the industrial production of nucleic acid derivatives

“…The authors suggested that in both halobacteria, adenine is converted into adenosine by adenosine phosphorylase, which later undergoes deamination to inosine by adenosine DA. Finally, the guanine derived from guanosine catabolism is transformed into xanthine by guanine deaminase (Stuer-Lauridsen and Nygaard 1998). Hermann et al (2007) predicted the deaminase activity of an unknown protein (Tm0936) from Thermotoga maritima using theoretical chemistry methods.…”

“…Purine salvage pathway in Haloferax volcanii(Stuer-Lauridsen and Nygaard 1998). NK, nucleoside kinase; MTAP, S-methyl-5′thioadenosine phosphorylase; Gua-DA, guanine deaminase; Ado-DA, adenosine deaminase; Gua-DA, guanine deaminase; APRT, adenine phosphoribosyltransferase; HGPRT, hypoxanthine-guanine phosphoribosyltransferase; S-AMP lyase, succinyladenylate lyase; S-AMP synth, succinyladenylate synthetase; GMP reductase, guanosine-5′-monophosphate reductase; IMP dehyd, inosine-5′-monophosphate dehydrogenase; GMP synth, guanosine-5′-monophosphate synthetase…”

Expression of the Methanobacterium thermoautotrophicum hpt Gene, Encoding Hypoxanthine (Guanine) Phosphoribosyltransferase, in Escherichia coli

“…The most abundant enzyme (HGPRTase) reacts with hypoxanthine and guanine only (1,12,23,35). Another enzyme reacts with hypoxanthine, guanine, and xanthine (27,32,43,46). Finally, an enzyme exists in Bacillus subtilis that reacts with xanthine only (7).…”

“…From our data, it appears that methanogens possess only a single purine PRTase, namely HGPRTase. In contrast, halophilic archaea (32) and Sulfolobus species (23a) contain both an APRTase and an HGPRTase.…”