The Fourth Arginine Catabolic Pathway of Pseudomonas aeruginosa

Abstract

D-Arginine dehydrogenase activity was discovered in Pseudomonas aeruginosa. This enzyme was inducible by its substrate, D-arginine, as well as by its product, 2-ketoarginine, but not by L-arginine. The enzyme activity was measured in uitro, in the presence of artificial electron acceptors (phenazine methosulphate and iodonitrotetrazolium chloride). 2-Ketoarginine was catabolized further to 4-guanidinobutyraldehyde, 4-guanidinobutyrate and 4-aminobutyrate. Two enzymes involved, 4-guanidinobutyraldehyde dehydrogenase and guanidinobutyrase, were inducible by 2-ketoarginine ; the latter enzyme was also strongly induced by 4-guanidinobutyrate. An arginine racemase activity was detected by an in vivo test. D-Arginine had the potential to be catabolized via the D-arginine dehydrogenase pathway and, after racemization, via the three L-arginine catabolic pathways previously demonstrated in P. aeruginosa. In mutants blocked in the L-arginine succinyltransferase pathway, but not in the wild-type, L-arginine was channelled partially into the D-arginine dehydrogenase pathway. Mutations in the kauB locus abolished growth of P. aeruginosa on 2-ketoarginine, agmatine and putrescine, and led to loss of 4-guanidino bu t yralde h yde de h ydrogenase and 4-amino bu tyralde hyde de hydrogenase activities.Thus, these two activities appear to be due to one enzyme in P. aeruginosa. The kauB locus was mapped on the chromosome between 1ysA and argB and was not linked to known genes involved in the three L-arginine catabolic pathways. The existence of four arginine catabolic pathways illustrates the metabolic versatility of P. aeruginosa. I N T R O D U C T I O NThree arginine catabolic pathways are known in Pseudomonas aeruginosa P A 0 (Fig. 1). (I) The arginine deiminase pathway converts L-arginine to L-ornithine, with the formation of ATP. The enzymes of this pathway are inducible by energy depletion and by the presence of L-arginine. Mutants which are defective in one of the three enzymes involved (arcA, B, C mutants) cannot grow anaerobically with L-arginine as the energy source (Mercenier et al., 19806,1982;Vander Wauven et al., 1984). (11) The arginine succinyltransferase pathway is used for aerobic L-arginine degradation. The products are L-glutamate and succinate, and L-arginine induces all enzymes of this route. Mutants blocked in the arginine succinyltransferase pathway (aru mutants) grow very poorly if at all on L-arginine as the only carbon and nitrogen source (Jann et al., 1986). (111) The arginine decarboxylase pathway produces putrescine, which can be converted to spermidine, and permits good growth on agmatine and putrescine. The first enzyme, L-arginine decarboxylase (enzyme 12 in Fig. l), is induced by L-arginine. The synthesis of agmatine deiminase and N-carbamoylputrescine hydrolase (enzymes 13 and 14 in Fig. 1) is switched on by agmatine. Putrescine is further catabolized to succinate and ammonia (Jakoby & Fredericks, 1959;Voellmy & Leisinger, 1976; Mercenier et al., 1980a

Arginine racemization by coupled catabolic and anabolic dehydrogenases

“…For the measurements of ␤-galactosidase activity, o-nitrophenyl-␤-D-galactopyranoside was used as substrate. D-Arginine dehydrogenase activity was assayed with the addition of artificial electron acceptors, PMS and INT, as described previously (6). One unit of D-arginine dehydrogenase activity was defined as the amount of enzyme that led to the reduction of 1 nmol iodonitrotetrazolium chloride per minute under the standard assay conditions.…”

“…Pseudomonas aeruginosa, an opportunistic human pathogen with an enormous catabolic capacity, is capable of growing on D-arginine as the sole source of carbon and nitrogen (5). The presence of an inducible D-arginine dehydrogenase activity in this organism was initially reported by Haas and coworkers (6), and 2-ketoarginine derived from this reaction could be converged into the arginine transaminase (ATA) pathway (7,8), 1 of the 4 pathways for L-arginine catabolism in pseudomonads (Fig. 1).…”

“…In the case of D-arginine, 2-ketoarginine has been identified as a product of an inducible D-arginine dehydrogenase in PAO1 (6). Therefore, one would expect that genes in the ATA pathway (group III of Promoter Induction by D-Arginine.…”

“…In fact, it has been proposed that L-arginine might be converted into D-arginine via racemization (6), reminiscent of L-alanine utilization through a catabolic alanine racemase and D-alanine dehydrogenase in Escherichia coli and many bacteria (9,10). Existence of an arginine racemase in P. aeruginosa was supported by growth complementation of arginine auxotrophs with D-arginine (6). However, the activity of P. aeruginosa arginine racemase has never been demonstrated in vitro, presumably because of the instant decomposition of both L-and D-arginine in extracts.…”

“…Genes of the ATA pathway are composed of several modules that are subjected to sequential induction by different intermediate compounds in the pathway (6). Expression of the aruHI genes encoding the first 2 enzymes of the ATA pathway is controlled by the AruRS 2-component systems (7) in response to L-arginine, and the gbuA gene encoding 4-guanidinobutyrase is controlled by GbuR and 4-guanidinobutyrate (14).…”