Multiple and interconnected pathways for L-lysine catabolism in Pseudomonas putida KT2440

Abstract

L-lysine catabolism in Pseudomonas putida KT2440 was generally thought to occur via the aminovalerate pathway. In this study we demonstrate the operation of the alternative aminoadipate pathway with the intermediates D-lysine, L-pipecolate, and aminoadipate. The simultaneous operation of both pathways for the use of L-lysine as the sole carbon and nitrogen source was confirmed genetically. Mutants with mutations in either pathway failed to use L-lysine as the sole carbon and nitrogen source, although they still used L-lysine as the nitrogen source, albeit at reduced growth rates. New genes were identified in both pathways, including the davB and davA genes that encode the enzymes involved in the oxidation of L-lysine to delta-aminovaleramide and the hydrolysis of the latter to delta-aminovalerate, respectively. The amaA, dkpA, and amaB genes, in contrast, encode proteins involved in the transformation of Delta1-piperidine-2-carboxylate into aminoadipate. Based on L-[U-13C, U-15N]lysine experiments, we quantified the relative use of pathways in the wild type and its isogenic mutants. The fate of 13C label of L-lysine indicates that in addition to the existing connection between the D- and L-lysine pathways at the early steps of the catabolism of L-lysine mediated by a lysine racemase, there is yet another interconnection at the lower end of the pathways in which aminoadipate is channeled to yield glutarate. This study establishes an unequivocal relationship between gene and pathway enzymes in the metabolism of L-lysine, which is of crucial importance for the successful colonization of the rhizosphere of plants by this microorganism.

FIG. 1.

Proposed catabolic pathways for the degradation of l- and d-lysine by bacteria of the genus Pseudomonas. Reactions from d-lysine to 2-aminoadipate represent the AMA pathway, those from l-lysine to glutaric acid represent the AMV pathway, and the utmost left branch of reactions from l-lysine to δ-aminovalerate via cadaverine/1-piperidine represent the cadaverine pathway. When they are known, the corresponding gene and the number of its translated product are given. In shaded ovals are the mutant strain numbers corresponding to strains deposited in the publicly available Pseudomonas putida Stock Center.

FIG. 2.

Gas chromatography analysis of the products from l-lysine accumulated intracellularly by P. putida. (A) Wild type; (B) amaB; (C) rei-2. Peaks at 7.71, 7.57, and 12.77 min correspond to pipecolate, AMV, and aminoadipate, respectively (see Fig. 3 and 4).

FIG. 3.

Mass spectra of products of aminovalerate and pipecolate in the metabolism of l-lysine by P. putida. P. putida KT2440 cells were grown on M9 minimal medium with glucose and unlabeled l-lysine or l-[U-¹⁵N, U-¹³C]lysine. The mass spectra of products corresponding to peaks at 7.57 and 7.71 are shown. (A) The fragmentation spectrum corresponds to AMV formed in cultures with unlabeled l-lysine. (B) The fragmentation spectrum corresponds to AMV derived from l-[¹⁵N, ¹³C]lysine. The two spectra at the bottom correspond to pipecolate formed from unlabeled l-lysine (C) or labeled l-lysine (D).

FIG. 4.

Mass spectra of aminoadipate in the metabolism of l-lysine by P. putida. Conditions are the same as those described for Fig. 3, except that the spectra corresponds to AMA formed from unlabeled l-lysine (top) or labeled l-lysine (bottom).