PUB - Publikationen an der Universität Bielefeld

By complementation analysis of an isoleucine-uptake-deficient Escherichia coli strain, it was shown that a 1.6-kb HindIII-StuI fragment of Corynebacterium glutamicum ATCC 13032, located downstream of the aecD gene, encodes an isoleucine uptake system. Sequence analysis revealed that the complementing fragment carried an open reading frame, termed brnQ, that encodes a protein with sequence similarities to branched-chain amino acid carriers of gram-positive and gram-negative bacteria. The brnQ gene specifies a predominantly hydrophobic protein of 426 amino acid residues with a calculated molecular mass of 44.9 kDa. A topology prediction by neural network computer analysis suggests the existence of 12 hydrophobic segments that most probably form transmembrane alpha-helices. A C. glutamicum mutant strain harboring a defined deletion of br ne in the chromosome showed a considerably lower isoleucine uptake rate of 0.04 nmol min(-1) mg (dry mass)(-1) as compared to the wild-type strain rate of 1.2 nmol min(-1) mg (dry mass)(-1). Overexpression of brnQ by means of a tac promotor resulted in an elevated uptake rate for isoleucine of 11.3 nmol min(-1) mg (dry mass)(-1). Evidently, the brnQ gene encodes the only transport system in C. glutamicum directing isoleucine uptake.