FEMS Microbiology Letters 127 (1995) 263-266 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQP ELSEVIER Functional expression of the glutamate uptake system from Corynebacterium glutamicum in Escherichia coli Andreas Burkovski * , Reinhard Isomer Forschungszentrum Jiilich, Institut fir Biotechnoiogie I, Postfach 1913, D-52425 Jiilich, Germany Received 22 December 1994; revised 13 February 1995; accepted 15 February 1995 zyxwvutsrqponmlkjihgfedcbaZYXW Abstract Glutamate uptake in the Gram-positive Corynebacterium glutamicum is mediated via a binding protein-dependent transport system, which is encoded by the gluABCD gene cluster. Cloning of these genes in an expression vector and subsequent transformation of the resulting plasmid allows different strains of the Gram-negative bacterium Escherichia coli to grow on glutamate as sole carbon and nitrogen source. However, overexpression of the glutamate uptake system results in growth inhibitory effects, probably due to the particular topology of the binding protein. Keywords: Corynebacterium glutamicum; Glutamate uptake; gluABCD gene cluster; Binding protein-dependent transport system; Escherichia coli 1. Introduction The Gram-positive bacterium Corynebacterium glutamicum has long been known for the secretion and production of amino acids, e.g. lysine and gluta- mate. However, this organism also possesses amino acid uptake systems most of which are not character- ized at the molecular level. Only the structure of the lysine import carrier [l] was known until recently, when the gluABCD gene cluster from C. glutam- icum, identified by homologous complementation, was cloned and sequenced [21. Interestingly, this gene cluster encodes a binding protein-dependent transport system for glutamate uptake. Such systems are quite rare in Gram-positive organisms; this is the * Corresponding author. Tel.: +49 (2461) 61 5556; Fax: +49 (2461) 61 2710; e-mail: herick@ibt013.ibt.k-juelich.de first described for C. glutamicum. On the other hand, they are frequently found in Gram-negative bacteria (for review, see [3]). Moreover, the binding proteins of these transport systems exhibit a particular topol- ogy in Gram-positive bacteria. Since there is no outer membrane, the binding protein is not trapped in the periplasm as in Gram-negatives. To retain the binding protein, it is proposed to be anchored via a glyceryl-cysteine lipid residue in the cytoplasmic membrane [4-71. As in the case of prolipoproteins from Gram-negative bacteria [8], a signal sequence precedes the mature binding protein sequence. This leader peptide is cleaved by a special signal pepti- dase II prior to modification [9]. In the light of the described basic similarities and specific differences between binding protein-depen- dent transport systems in Gram-positive and Gram- negative organisms, it was tempting to investigate if the glutamate uptake system of C. glutamicum can 0378-1097/95/$09.50 0 1995 Federation of European Microbiological Societies. All rights reserved SSDIO378-1097(95)00071-2 Downloaded from https://academic.oup.com/femsle/article-abstract/127/3/263/601143 by guest on 14 June 2020