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

Bacterial mechanisms for the uptake of peptides and their hydrolysis to ami no acids are known in great detail, whereas much less is known about the fa tes of the peptide-derived amino acids. We show that the addition Of L-thre onine-containing di- or tripeptides results in reduction of the growth of C orynebacterium 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 frame s (ORFs) were identified, almost all encoding hypothetical proteins of unkn own function. Three ORFs encode membrane proteins. Their individual functio nal characterizations in the wild-type background led to the identification of thrE. Upon thrE overexpression, growth is no longer sensitive to the pr esence of the Thr peptide, and L-threonine is exported at a rate of 3.8 nmo l min(-1) mg of dry weight(-1), whereas the rate of export of a thrE inacti vation mutant is reduced to 1.1 nmol min(-1) mg of dry weight(-1). In addit ion to L-threonine, L-serine is also a substrate for the exporter. The expo rter exhibits nine predicted transmembrane-spanning helices with long charg ed C and N termini and with an amphipathic helix present within the N termi nus. All these data suggest that the carrier encoded by thrE serves to expo rt small molecules such as L-threonine and that the carrier is a prototype of a new translocator family. Homologues of ThrE are present in Mycobacteri um tuberculosis and Streptomyces coelicolor.