Ethambutol, a cell wall inhibitor of Mycobacterium tuberculosis, elicits L-glutamate efflux of Corynebacterium glutamicum Eva Radmacher, 1 3 Kathrin C. Stansen, 1 3 Gurdyal S. Besra, 2 Luke J. Alderwick, 2 William N. Maughan, 2 Gu ¨ nter Hollweg, 3 Hermann Sahm, 1 Volker F. Wendisch 1 and Lothar Eggeling 1 Correspondence Lothar Eggeling l.eggeling@fz-juelich.de 1 Institute for Biotechnology, Research Centre Ju ¨ lich, D-52425 Ju ¨ lich, Germany 2 School of Biosciences, University of Birmingham, Birmingham B15 2TT, UK 3 Pathology, University Hospital, D-52074 Aachen, Germany Received 2 December 2004 Revised 31 January 2005 Accepted 3 February 2005 Corynebacterium glutamicum is used for the large-scale production of L-glutamate, but the efflux of this amino acid is poorly understood. This study shows that addition of ethambutol (EMB) to growing cultures of C. glutamicum causes L-glutamate efflux at rates of up to 15 nmol min ”1 (mg dry wt) ”1 , whereas in the absence of EMB, no efflux occurs. EMB is used for the treatment of Mycobacterium tuberculosis, and at a molecular level it targets a series of arabinosyltransferases (EmbCAB). The single arabinosyltransferase-encoding emb gene of C. glutamicum was placed under the control of a Tet repressor (TetR). Experiments with this strain, as well as with an emb-overexpressing strain, coupled with biochemical analyses showed that: (i) emb expression was correlated with L-glutamate efflux, (ii) emb overexpression increased EMB resistance, (iii) EMB caused less arabinan deposition in cell wall arabinogalactan, and (iv) EMB caused a reduced content of cell-wall-bound mycolic acids. Thus EMB addition resulted in a marked disordering of the cell envelope, which was also discernible by examining cellular morphology. In order to further characterize the cellular response to EMB addition, genome-wide expression profiling was performed using DNA microarrays. This identified 76 differentially expressed genes, with 18 of them upregulated more than eightfold. Among these were the cell-wall-related genes ftsE and mepA (encoding a secreted metalloprotease); however, genes of central metabolism were largely absent. Given that an altered lipid composition of the plasma membrane of C. glutamicum can result in L-glutamate efflux, we speculate that major structural alterations of the cell envelope are transmitted to the membrane, which in turn activates an export system, perhaps via increased membrane tension. INTRODUCTION Corynebacterium glutamicum and Mycobacterium tuber- culosis share characteristic cell walls common to all Coryne- bacterineae (Minnikin, 1982; Stackebrandt et al., 1997). They contain an unusual lipid-rich outer layer, unique among Gram-positive bacteria, containing mycolic acids, either as organic soluble lipids, such as trehalose mono- and dimycolates (TMM and TDM) or glucose monomycolates (GMM), or bound to arabinose residues of the cell wall polymer arabinogalactan (Besra et al., 1995). The major physical properties of the cell wall and its biosynthesis are key to our understanding of cell wall permeability and resistance to front-line M. tuberculosis agents (Brennan & Nikaido, 1995). This unique lipid-rich layer represents a barrier for the access of antibiotics, and its synthesis is a target of several effective antibiotics, such as isoniazid and ethambutol (EMB). Our interest in C. glutamicum stems from the use of this bacterium for amino acid production and amino acid export. Currently, more than 1?8610 6 t of amino acids are produced annually with this bacterium, and it is clear that specific mechanisms must exist for amino acids to pass through the cell wall and its lipid layers. We succeeded in identifying specific exporters for L-lysine (Vrljic et al., 1996), L-threonine (Simic et al., 2001) and L-isoleucine (Kennerknecht et al., 2002), each representing a new functional and structural type of transporter (Vrljic et al., 1999; Eggeling & Sahm, 2003). The L-lysine exporter and its subfamilies are widely distributed in a large number of 3These authors contributed equally to this work. Abbreviations: EMB, ethambutol; GMM, glucose monomycolates; MAMES, mycolic acid methyl esters; TDM, trehalose dimycolates; TMM, trehalose mono-mycolates. 0002-7804 G 2005 SGM Printed in Great Britain 1359 Microbiology (2005), 151, 1359–1368 DOI 10.1099/mic.0.27804-0