Phosphorylation of the periplasmic binding protein in two transport systems for arginine incorporation in Escherichia coli K-12 is unrelated to the function of the transport system

Abstract

In Escherichia coli K-12, the accumulation of arginine is mediated by two distinct periplasmic binding protein-dependent transport systems, one common to arginine and ornithine (AO system) and one for lysine, arginine, and ornithine (LAO system). Each of these systems includes a specific periplasmic binding protein, the AO-binding protein for the AO system and the LAO-binding protein for the LAO system. The two systems include a common inner membrane transport protein which is able to hydrolyze ATP and also phosphorylate the two periplasmic binding proteins. Previously, a mutant resistant to the toxic effects of canavanine, with low levels of transport activities and reduced levels of phosphorylation of the two periplasmic binding proteins, was isolated and characterized (R. T. F. Celis, J. Biol. Chem. 265:1787-1793, 1990). The gene encoding the transport ATPase enzyme (argK) has been cloned and sequenced. The gene possesses an open reading frame with the capacity to encode 268 amino acids (mass of 29.370 Da). The amino acid sequence of the protein includes two short sequence motifs which constitute a well-defined nucleotide-binding fold (Walker sequences A and B) present in the ATP-binding subunits of many transporters. We report here the isolation of canavanine-sensitive derivatives of the previously characterized mutant. We describe the properties of these suppressor mutations in which the transport of arginine, ornithine, and lysine has been restored. In these mutants, the phosphorylation of the AO- and LAO-binding proteins remains at a low level. This information indicates that whereas hydrolysis of ATP by the transport ATPase is an obligatory requirement for the accumulation of these amino acids in E. coli K-12, the phosphorylation of the periplasmic binding protein is not related to the function of the transport system.

FIG. 1

Physical map of the 65.8-min chromosomal area and locations of λ phage clone 1A2 and the argK gene. (A) Chromosomal fragments produced by the eight restriction enzymes used to map the chromosome (20). Kilobase coordinates are shown at the top. (B) Physical map of the λ phage clone 1A2 (miniset 471) from the Kohara collection (20) and location of the sequenced BamHI fragment encoding the argK gene. The argK gene is located downstream of the sbm gene (24) and is transcribed in the same direction. B, BamHI; H, HindIII; E, EcoRI; V, EcoRV; Bg, BglI; K, KpmI; P, PstI; Pv; PvuI.

FIG. 2

Nucleotide sequence of the 2,140-bp BamHI fragment encoding the argK gene and its deduced amino acid sequence. The conserved sequences for nucleotide binding (Walker sequences A and B) are underlined. Indicated at nt 1008 to 1011 is the putative Shine-Dalgarno sequence. Sequences conforming to the consensus for E. coli promoters (−35 and −10) are boxed. An inverted repeat with the potential to form a hairpin structure for transcriptional termination is shown by the two arrows between nt 1909 and 1919.

FIG. 3

SDS-PAGE of a purified preparation of the ATPase enzyme. Electrophoresis was performed as described previously (14), using 12.6% polyacrylamide gels. Molecular weight standards were phosphorylase b (M_r = 94,000), bovine serum albumin (M_r = 67,000), ovalbumin (M_r = 43,000), carbonic anhydrase (M_r = 30,000), and soybean trypsin inhibitor (M_r = 20,100).