The proline permease gene PUT4 has been cloned. Nitrogen-source regulation ('ammonia sensitivity') of this and at least two other amino-acid permeases is believed to occur at two distinct levels, i.e. permease synthesis and permease activity. Therefore, PUT4 transcription/messenger stability was examined in the ammonia-and prolinegrown wild type as well as in mutant strains supposedly affected at only one or at both of these levels. We report transcript-level repression of proline permease synthesis in ammonia-grown cells. Repression is lifted at this level in gdhCR, glnltS and gdhA mutants which exhlbit pleiotropically derepressed permease and catabolic enzyme activities.On the other hand, the npil and npi2 mutations, formerly called mut2 and mut4, relieve an inactivation process which seems only to affect permeases. These mutations do not affect the detected PUT4 RNA level. The only known positive factor in proline permease regulation, the nitrogen permease reactivator protein Nprl , is believed to counteract the inactivation process on derepressing media. This protein appears to have an additional, indirect effect on PUT4 transcription/messenger stability: it would actually mediate repression via its activating effect on ammonia uptake.In Saccharomyces cerevisiae, proline uptake is mediated by two distinct permeases. This organism possesses a general amino-acid permease [l] which can take up a wide variety of amino acids, including proline [2]. The affinity of proline uptake via this permease is very low, however. At low proline concentrations or in the presence of competing substrates, proline uptake requires the activity of a second permease [l -31 which apparently transports only imino acids [4] and structural analogues of proline, such as 4-aminobutyric acid [5]. The genes believed to encode these permeases are the GAP1 [l, 6, 71 and PUT4 [4] genes, respectively. These permeases share with the ureidosuccinate-allantoate permease a common pattern of nitrogen-source regulation, for which a mutant-based model has been proposed [8, 91. Growth on a 'good' nitrogen source, such as ammonia, glutamine or asparagine, prevents the development of these permease activities. At least two control mechanisms seem to be involved. Studies of the 'ammonia sensitivity' of these permeases in the wild type and in mutants have led to the conclusion that growth on ammonia causes both repression and inactivation of all three permeases. Ammonia repression appears to be relieved by mutations in the GDHCR [lo], (= URE2 [I 11 or USU [12]) locus and by glnl'" mutations which cause partial

The reaction catalyzed by ornithine carbamoyltransferase can participate in either the anabolism or the catabolism of arginine. The carbamoylation of ornithine, yielding citrulline, is involved in the biosynthetic sequence; the reverse reaction, the phosphorolysis of citrulline, is the second step of the arginine deiminase pathway.The ornithine carbamoyltransferases of a number of microorganisms which can fulfil both of these functions have been studied in this work. This group of organisms was found to possess two distinct ornithine carbamoyltransferases. The functions of these enzymes were surmised by determining the type of genetic regulation to which they were subjected.The kinetic properties of these various enzymes have been determined. All of them, regardless of the role they play in the cell, catalyze both the synthesis and arsenolysis of citrulline. The anabolic transferase of Pseudomonas is the only enzyme which displays functional irreversibility. A comparison of the quaternary structure of these transferases was performed and reveals interesting features in relation to the metabolic function of these enzymes. All well-characterized anabolic enzymes have low molecular weights (from 150000-105000) and are likely to be trimers. Catabolic enzymes, with the exception of those of Bacillus licheniformis and Halobacterium salinarium, display much higher molecular weights and more elaborate quaternary structure.The properties of these two groups of transferases are discussed in relation to their metabolic role in the cells.Ornithine carbamoyltransferase is an enzyme of particular metabolic interest, since it can fulfil two different functions : the carbamoylation of ornithine participating in the anabolism of arginine, and the thermodynamically less favored phosphorolysis of citrulline, which is involved in the catabolic arginine deiminase pathway.We have shown previously that these two functions exist in Pseudomonas and are fulfilled by distinct enzymes, which differ in their kinetic, regulatory and structural properties. They also differ in their ability to catalyze the two directions of the reaction [l-51. Starting from such observations we have undertaken a comparative study of the ornithine carbamoyltransferases of various organisms. We expected such investigations to provide information concerning the origin and the evolution of these enzymes.The microorganisms were chosen so as to present the widest range of distinct situations with respect to

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Bacillus licheniformis has two pathways of arginine catabolism. In well-aerated cultures, the arginase route is present, and levels of catabolic ornithine carbamoyltransferase were low. An arginase pathway-deficient mutant, BL196, failed to grow on arginine as a nitrogen source under these conditions. In anaerobiosis, the wild type contained very low levels of arginase and ornithine transaminase. BL196 grew normally on glucose plus arginine in anaerobiosis and, like the wild type, had appreciable levels of catabolic transferase. Nitrate, like oxygen, repressed ornithine carbamoyltransferase and stimulated arginase synthesis. In aerobic cultures, arginase was repressed by glutamine in the presence of glucose, but not when the carbon-energy source was poor. In anaerobic cultures, ammonia repressed catabolic ornithine carbamoyltransferase, but glutamate and glutamine stimulated its synthesis. A second mutant, derived from BL196, retained the low arginase and ornithine transaminase levels of BL196 but produced high levels of deiminase pathway enzymes in the presence of oxygen.