ArcD1 and ArcD2 arginine/ornithine exchangers encoded in the arginine deiminase pathway gene cluster of Lactococcus lactis

Abstract

The arginine deiminase (ADI) pathway gene cluster in Lactococcus lactis contains two copies of a gene encoding an L-arginine/Lornithine exchanger, the arcD1 and arcD2 genes. The physiological function of ArcD1 and ArcD2 was studied by deleting the two genes. Deletion of arcD1 resulted in loss of the growth advantage observed in the presence of high L-arginine in different growth media. Uptake of L-arginine and L-ornithine by resting cells was reduced to the low level observed for an ArcD1/ArcD2 double deletion mutant. Deletion of the arcD2 gene did not affect the growth enhancement, and uptake activities were slightly reduced. Nevertheless, recombinant expression of ArcD2 in the ArcD1/ArcD2 double mutant did recover the growth advantage. Kinetic characterization of ArcD1 and ArcD2 showed high affinities for both L-arginine and L-ornithine (Km in the micromolar range). A difference between the two transporters was the significantly lower affinity of ArcD2 for the cationic amino acids L-ornithine, L-lysine, and L-histidine. In contrast, the affinity of ArcD2 was higher for the neutral amino acid L-alanine. Moreover, ArcD2 efficiently translocated L-alanine, while ArcD1 did not. Both transporters revealed affinities in themMrange for agmatine, cadaverine, histamine, and putrescine. These amines bind but are not translocated. It is concluded that ArcD1 is the main L-arginine/L-ornithine exchanger in the ADI pathway and that ArcD2 is not functionally expressed in the media used. ArcD2 is proposed to function together with the arcT gene that encodes a putative transaminase and is found adjacent to the arcD2 gene.