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Few antibiotics targeting members of the archaeal domain are currently available for genetic studies. Since bacterial antibiotics are frequently directed against competing and related organisms, archaea by analogy might produce effective antiarchaeal antibiotics. Peptide antibiotic (halocin) preparations from euryarchaeal halophilic strains S8a, GN101, and TuA4 were found to be toxic for members of the hyperthermophilic crenarchaeal genus Sulfolobus. No toxicity was evident against representative bacteria or eukarya. Halocin S8 (strain S8a) and halocin R1 (strain GN101) preparations were cytostatic, while halocin A4 (strain TuA4) preparations were cytocidal. Subsequent studies focused on the use of halocin A4 preparations and Sulfolobus solfataricus. Strain TuA4 cell lysates were not toxic for S. solfataricus, and protease (but not nuclease) treatment of the halocin A4 preparation inactivated toxicity, indicating that the A4 toxic factor must be a secreted protein. Potassium chloride supplementation of the Sulfolobus assay medium potentiated toxicity, implicating use of a salt-dependent mechanism. The utility of halocin A4 preparations for genetic manipulation of S. solfataricus was assessed through the isolation of UV-induced resistant mutants. The mutants exhibited stable phenotypes and were placed into distinct classes based on their levels of resistance.

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.

Halobacterium halobium (salinarium) is able to grow fermentatively via the arginine deiminase pathway, which is mediated by three enzymes and one membrane-bound arginine-ornithine antiporter. One of the enzymes, catabolic ornithine transcarbamylase (cOTCase), was purified from fermentatively grown cultures by gel filtration and ammonium sulfate-mediated hydrophobic chromatography. It consists of a single type of subunit with an apparent molecular mass of 41 kDa. As is common for proteins of halophilic Archaea, the cOTCase is unstable below 1 M salt. In contrast to the cOTCase from Pseudomonas aeruginosa, the halophilic enzyme exhibits Michaelis-Menten kinetics with both carbamylphosphate and ornithine as substrates with Km values of 0.4 and 8 mM, respectively. The N-terminal sequences of the protein and four peptides were determined, comprising about 30% of the polypeptide. The sequence information was used to clone and sequence the corresponding gene, argB. It codes for a polypeptide of 295 amino acids with a calculated molecular mass of 32 kDa and an amino acid composition which is typical of halophilic proteins. The native molecular mass was determined to be 200 kDa, and therefore the cOTCase is a hexamer of identical subunits. The deduced protein sequence was compared to the cOTCase of P. aeruginosa and 14 anabolic OTCases, and a phylogenetic tree was constructed. The halobacterial cOTCase is more distantly related to the cOTCase than to the anabolic OTCase of P. aeruginosa. It is found in a group with the anabolic OTCases of Bacillus subtilis, P. aeruginosa, and Mycobacterium bovis.