Pseudomonas cepacia NCTC 10743 utilizes arginine as the sole source of carbon and nitrogen for growth. Arginine is degraded to glutamate via succinyl derivatives. The catabolic sequence in this pathway is L-arginine -N2-succinylarginine -) N2-succinylornithine -+ N2-succinylglutamate semialdehyde --N2-succinylglutamate glutamate + succinate. The formation of the enzymes responsible for arginine degradation is regulated not only by induction but also by both carbon and nitrogen catabolite

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... ion. Several Pseudomonas species use L-arginine as the sole carbon and nitrogen source. Various pathways for arginine utilization in Pseudomonas species and their importance and distribution have been discussed (1, 22, 23) . The arginine deiminase pathway serves to generate ATP under energy depletion conditions (16, 23, 28) . The arginine decarboxylase-agmatine deiminase pathway provides a source of polyamine when arginine is abundant (15, 23). The arginine oxidase route is the main arginine catabolic pathway during growth of P. putida on arginine (18, 23). Although most of the Pseudomonas strains are able to use the product of the oxidase reaction, 2-ketoarginine, the enzyme seems to be absent in other Pseudomonas strains (23). Nevertheless, arginine catabolism remains poorly understood in other Pseudomonas species. Indeed, a triple mutant of P. aeruginosa, which is blocked (i) in the first two enzymes of the arginine deiminase pathway, (ii) in agmatine deiminase, the second enzyme of the arginine decarboxylase route, and (iii) in guanidinobutyrase, the third enzyme involved in 2-ketoarginine catabolism, still retained the ability to grow on arginine as the sole carbon and nitrogen source (9). Other species such as P. cepacia grow on arginine but do not display any of the catabolic pathway known for arginine catabolism (23). We have recently shown that some mutants of P. aeruginosa, unable to utilize arginine as a carbon source, accumulate N2-succinylarginine when grown in the presence of arginine and another carbon source (29). The possibility of this compound being present as an intermediate in a novel arginine catabolic pathway for Pseudomonas strains was investigated. P. cepacia was used to examine this hypothesis since it is devoid of the arginine dissimilation activities present in other Pseudomonas strains. We observed that a pathway involving succinyl derivatives does indeed allow the conversion of arginine to glutamate in P. cepacia. Evidence that this pathway accounts for the utilization of arginine as the growth substrate is discussed. MATERIALS AND METHODS Bacterial strains and growth of the organism. P. cepacia NCTC 10743 was routinely grown at 30°C on minimal medium 154 (24). Carbon and nitrogen sources were added at 20 mM each after sterilization by filtration. Cultures were grown on a rotary shaker in flasks fitted with side arms. The volume of * Corresponding author. the culture fluid was 10o of the flask volume. Cells from exponential-phase culture (about 5 x 108 cells ml-') were harvested by centrifugation, washed with 0.9% (wt/vol) NaCl at 0°C, and if not used immediately, stored frozen as a pellet. Preparation of ceil extracts. Extracts were prepared by suspending the cells in 2 ml of 50 mM phosphate buffer (pH 7.5) supplemented with 1 mM P-mercaptoethanol, subjecting the cell suspension to 3 min disruption in a Mullard sonic oscillator, and removing the cell debris by centrifugation at 20,000 x g for 10 min at 40C. High-voltage electrophoresis. Substrates and products of the arginine succinyltransferase pathway were identified and separated by high-voltage paper electrophoresis. Proteins were removed from the samples after precipitation with acetic acid (0.5 M [final concentration]). Two to ten microliters of the supernatant was brought onto Whatman 3MM paper sheets and submitted to electrophoresis at 5,000 V for 10 min in pH 2.0 buffer (acetic acid-formic acid-H20 [28.2:59:922.5]). The papers were air dried. Amino derivatives were identified with ninhydrin reagent (19). For guanidino derivative identification, papers were dipped into a solution of o-phenanthrene quinone and immediately air dried. The spots were located by viewing under UV light (33) . Acyl derivatives were visualized with o-tolidine reagent (26). Synthesis of N-acyl derivatives of arginine and arginine catabolites. N-2-acyl derivatives were prepared by acylation of the amino compound with the corresponding acyl anhydride. Specifically, for 100 mmol of amino acid dissolved in a minimum of water (ca. 50 ml), 120 mmol of the acyl anhydride was added in small portions to the solution. The reaction mixture was maintained at pH 8 to 9 by the addition of NaOH (40% [wt/vol]) with stirring. The yield was routinely more than 98%. N2-succinylornithine was synthesized from N-b-(tert-butoxycarbonyl)ornithine. At the end of the reaction, indicated by the absence of reactivity with ninhydrin, the N-B-(tert-butoxycarbonyl) group was eliminated by the addition of HCl to a final concentration of 1 M. Residual unreacted amino acids were separated from succinyl derivatives by chromatography on a Dowex 50 W X 8 (H+ form) column (2.5 by 30 cm). Succinylcitrulline was retarded just sufficiently to be separated from succinate. Succinylarginine or succinylornithine was eluted with 1 N HCl. Amino acids were desorbed by elution with 1 N NaOH. Succinylglutamate was eluted with water and was not purified further. However, in this preparation, succinate never represented 882 on May 4, 2020 by guest