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 K m 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.Fermentative arginine degradation via the arginine deiminase pathway is found in different groups of Bacteria (for reviews see references 1 and 8). The pathway consists of an arginine-ornithine antiporter and the enzymes arginine deiminase (ADI; EC 3.5.3.6), catabolic ornithine transcarbamylase (cOTCase; EC 2.1.3.3), and carbamate kinase (CK; EC 2.7.2.2). Arginine is degraded to ornithine, ammonia, and CO 2 , concomitantly generating 1 mol of ATP per mol of arginine degraded. An OTCase activity is also involved in arginine biosynthesis. Organisms capable of both arginine biosynthesis and fermentative arginine degradation possess two OTCases which are differentially regulated. The ADI pathway has been most extensively studied in Pseudomonas aeruginosa (2-5, 16, 17, 21, 29, 32, 58). In this organism, the genes are situated in an operon and are cotranscribed.Halobacteria are the only Archaea for which anaerobic growth using arginine has been reported (22). As arginine consumption was coupled to the equimolar occurrence of ornithine in the medium, it was proposed that Halobacterium halobium (salinarium) (H. halobium has recently been renamed H. salinarium, which is used for the rest of this article.) degrades arginine via the ADI pathway (22).To study this pathway and its regulation in an archaeon, the cOTCase from H. salinarium was isolated and characterized. The corresponding gene was cloned and sequenced, and the deduced protein...