L-threonine export: use of peptides to identify a new translocator from Corynebacterium glutamicum

Abstract

Bacterial mechanisms for the uptake of peptides and their hydrolysis to amino acids are known in great detail, whereas much less is known about the fates of the peptide-derived amino acids. We show that the addition of L-threonine-containing di- or tripeptides results in reduction of the growth of Corynebacterium glutamicum, with concomitant high intracellular accumulation of L-threonine to up to 130 mM. Using transposon mutagenesis and isolation of mutants with increased Thr peptide sensitivity, nine open reading frames (ORFs) were identified, almost all encoding hypothetical proteins of unknown function. Three ORFs encode membrane proteins. Their individual functional characterizations in the wild-type background led to the identification of thrE. Upon thrE overexpression, growth is no longer sensitive to the presence of the Thr peptide, and L-threonine is exported at a rate of 3.8 nmol min(-1) mg of dry weight(-1), whereas the rate of export of a thrE inactivation mutant is reduced to 1.1 nmol min(-1) mg of dry weight(-1). In addition to L-threonine, L-serine is also a substrate for the exporter. The exporter exhibits nine predicted transmembrane-spanning helices with long charged C and N termini and with an amphipathic helix present within the N terminus. All these data suggest that the carrier encoded by thrE serves to export small molecules such as L-threonine and that the carrier is a prototype of a new translocator family. Homologues of ThrE are present in Mycobacterium tuberculosis and Streptomyces coelicolor.

FIG. 1

Consequences of l-threonine peptide addition to C. glutamicum for growth and the intracellular l-threonine concentration. (A) Growth without peptide addition (▪) and in response to the addition of 1 mM Ala-Thr (●), Thr-Ala (▵), or Thr-Thr-Thr (×). (B) Time course for the intracellular l-threonine concentration within the first 5 h. Symbols are as described for panel A.

FIG. 2

(A) Growth of the wild type (circles), of strain 13032::ORF22 (triangles), and of strain 13032::ORF81 (squares) without (open symbols) and with (solid symbols) 2 mM Thr-Thr-Thr. (B) Growth of C. glutamicum 13032(pZ1thrE) (squares) and 13032::thrE (triangles) compared to that of the control strain 13032(pZ1) (circles) without (open symbols) and with (solid symbols) the addition of 2 mM threonine tripeptide.

FIG. 3

Overview of the thrE locus of C. glutamicum and structural properties of ThrE. (A) DNA fragments used for thrE overexpression and inactivation as well as adjacent genes. (B) Average local hydrophobicity at each residue according to the algorithm of Kyte and Doolittle (22) using a window of 13 amino acids, as plotted on the vertical axis, versus the residue number on the horizontal axis. The transmembrane-spanning helices predicted by use of the neuronal network program PHD.htm (41) are highlighted as black squares and numbered I to IX. The amphipathic helix at the beginning of the protein is highlighted as an open box. (C) Part of a sequence alignment of ThrE of C. glutamicum (Cg) with putative proteins of M. tuberculosis (Mt) and S. coelicolor (Sc) in the region of the amphipathic helix, which is indicated by the thick lines. The numbers specify the amino acid positions at the start of the peptide stretches shown. Identical amino acid residues (black background) and conserved amino acid residues (gray shading) are indicated.

FIG. 4

Mapping of the transcriptional start site of thrE by primer extension analysis. The primer extension product was run in lane 1. The sequencing ladder (ACGT) of the coding strand was generated using the same primer as that used for primer extension. The transcriptional start site is indicated by the arrow.

FIG. 5

Intracellular l-threonine concentration and export in recombinant C. glutamicum strains. The internal and external l-threonine concentrations are shown. The strains are C. glutamicum 13032(pZ1thrE) (▪), 13032::thrE (▴), and 13032(pZ1) (control) (●).

FIG. 6

Effect of the proton ionophore CCCP on l-threonine export in C. glutamicum. Extracellular l-threonine accumulation by 13032::thrE (circles) is compared to that of control strain 13032(pZ1) (squares) without (open symbols) and with (solid symbols) the addition of 20 μM CCCP.