Evidence for an arginine exporter encoded by yggA (argO) that is regulated by the LysR-type transcriptional regulator ArgP in Escherichia coli

Abstract

The anonymous open reading frame yggA of Escherichia coli was identified in this study as a gene that is under the transcriptional control of argP (previously called iciA), which encodes a LysR-type transcriptional regulator protein. Strains with null mutations in either yggA or argP were supersensitive to the arginine analog canavanine, and yggA-lac expression in vivo exhibited argP(+)-dependent induction by arginine. Lysine supplementation phenocopied the argP null mutation in that it virtually abolished yggA expression, even in the argP+ strain. The dipeptides arginylalanine and lysylalanine behaved much like arginine and lysine, respectively, to induce and to turn off yggA transcription. Dominant missense mutations in argP (argPd) that conferred canavanine resistance and rendered yggA-lac expression constitutive were obtained. The protein deduced to be encoded by yggA shares similarity with a basic amino acid exporter (LysE) of Corynebacterium glutamicum, and we obtained evidence for increased arginine efflux from E. coli strains with either the argPd mutation or multicopy yggA+. The null yggA mutation abolished the increased arginine efflux from the argPd strain. Our results suggest that yggA encodes an ArgP-regulated arginine exporter, and we have accordingly renamed it argO (for "arginine outward transport"). We propose that the physiological function of argO may be either to prevent the accumulation to toxic levels of canavanine (which is a plant-derived antimetabolite) or arginine or to maintain an appropriate balance between the intracellular lysine and arginine concentrations.

FIG. 1.

Physical map of serA-yggB region of the E. coli genome at 69.5 min (clockwise going from left to right), as annotated in the work of Blattner et al. (5). Positions and transcriptional orientations of individual open reading frames are marked beneath the kilobase scale.

FIG. 2.

Expression of yggA-lac in pHYD956 transformants of the wild-type (WT) strain MC4100 and the following mutant derivatives: argR, GJ4748; argR yggA, GJ4894; argR argP, GJ4895; and argP^d, MC4100/pHYD926. Cultures were grown to exponential phase in glucose-minimal medium supplemented with trimethoprim and additives (each at 1 mM, except for CAN, which was added to 40 μg/ml), as indicated in the inset key, for β-galactosidase assays. Enzyme specific activity values are given in Miller units (33). Cit, citrulline.

FIG. 3.

Test for cross-feeding of Arg-auxotrophic strain SK2226/pBR329 by the following strains: argR/vector, GJ4748/pBR329; argR/argP^d, GJ4748/pHYD953; argR/m.c. (multicopy) yggA⁺, GJ4748/pHYD952; and argR yggA/argP^d, GJ4894/pHYD953. Cross-feeding was scored after 12 h of incubation.