J. Mol. Microbiol. Biotechnol. (1999) 1(2): 327-336. Topology of LysE and aJMMB Novel Superfamily Research of Exporters Article 327 The LysE Superfamily: Topology of the Lysine Exporter LysE of Corynebacterium glutamicum, a Paradyme for a Novel Superfamily of Transmembrane Solute Translocators M. Vrljic1, J. Garg2, A. Bellmann1, S. Wachi2, thereby regulating the intracellular concentration of this R. Freudl1, M. J. Malecki2, H. Sahm1, amino acid. The need for this function is surprising as the V. J. Kozina2, L. Eggeling1*, and M. H. Saier Jr.2 L-lysine biosynthetic pathway in C. glutamicum, as in other bacteria, is strictly regulated (Nakayama, 1985; Eggeling, 1Institut für Biotechnologie, 1994). However, L-lysine export is essential in Forschungszentrum Jülich GmbH, environments containing peptides: in the presence of low 52428 Jülich, Germany concentrations of lysine-containing peptides and upon 2Department of Biology, deletion of the export carrier gene lysE, exceptionally high University of California at San Diego, cytoplasmic concentrations (>1M) of L-lysine build up La Jolla, CA 92093-0116, USA leading to bacteriostasis. The specific export function provided by LysE is also a prerequisite for the production of L-lysine with C. glutamicum, mutants of which are used Abstract for the production of about 3.5x105 tons of this amino acid per year (Leuchtenberger, 1996). LysE therefore provides In Corynebacterium glutamicum the LysE carrier a new function in regulating a cytoplasmic amino acid con- protein exhibits the unique function of exporting L- centration, and it is the first example of export as a target lysine. We here analyze the membrane topology of for the improvement of industrial amino acid production. LysE, a protein of 236 amino acyl residues, using In addition to the unique physiological function of LysE, PhoA- and LacZ-fusions. The amino-terminal end of its structure is unusual. The carrier consists of 236 amino LysE is located in the cytoplasm whereas the carboxy- acyl residues (Vrljic et al., 1996), and it exhibits six terminal end is found in the periplasm. Although 6 hydrophobic domains that could correspond to six hydrophobic domains were identified based on transmembrane helical spanners typical of many polytopic hydropathy analyses, only five transmembrane membrane transport proteins. LysE lacks significant spanning helices appear to be present. The additional sequence similarity to known export translocators which hydrophobic segment may dip into the membrane or are constituent members of the identified 12 families be surface localized. We show that LysE is a member catalyzing efflux of organic molecules and cations (Saier, of a family of proteins found, for example, in 1994; Saier et al., 1994; Paulsen et al., 1996). It therefore Escherichia coli, Bacillus subtilis, Mycobacterium represents a novel family of proteins distinct from all other tuberculosis and Helicobacter pylori. This family, which established families of transporters (Vrljic et al., 1996). The we have designated the LysE family, is distantly related current veritable flood of genome sequencing activities has to two additional protein families which we have resulted in the realization that numerous putative LysE designated the YahN and CadD families. These three homologues are encoded on bacterial chromosomes and families, the members of which exhibit similar sizes, plasmids. LysE evidently represents the tip of an iceberg hydropathy profiles, and sequence motifs comprise of a novel class of transporters which may exhibit unique the LysE superfamily. Functionally characterized physiological functions analogous to that of LysE. members of the LysE superfamily export L-lysine, In the present paper we report investigations into the cadmium and possibly quarternary amines. We membrane topology of LysE using the well established suggest that LysE superfamily members will prove to methodology of alkaline phosphatase and ß-galactosidase catalyze export of a variety of biologically important fusion constructions. Additionally, we searched the DNA solutes. and protein databases for the presence of LysE homologues. A thorough analysis revealed that LysE is a Introduction member of a novel superfamily of carriers that consists of three discrete families. The functionally characterized An unusual bacterial transport protein was recently members of this superfamily exclusively catalyze solute characterized biochemically, physiologically, and export. molecularly (Broer and Krämer, 1991; Vrljic et al., 1995; 1996). This carrier is LysE from the Gram-positive Results bacterium Corynebacterium glutamicum. It exports L-lysine, The LysE Family Currently sequenced members of the LysE family are Received January 29, 1999; revised April 27, 1999; accepted May 4, 1999. *For correspondence. Email [email protected]; presented in Table 1. The LysE protein of C. glutamicum is Tel. +49 2461 61 5132; Fax. +49 2461 61 2710. the only functionally characterized member of this family. © 1999 Horizon Scientific Press Further Reading Caister Academic Press is a leading academic publisher of advanced texts in microbiology, molecular biology and medical research. Full details of all our publications at caister.com • MALDI-TOF Mass Spectrometry in Microbiology Edited by: M Kostrzewa, S Schubert (2016) www.caister.com/malditof • Aspergillus and Penicillium in the Post-genomic Era Edited by: RP Vries, IB Gelber, MR Andersen (2016) www.caister.com/aspergillus2 • The Bacteriocins: Current Knowledge and Future Prospects Edited by: RL Dorit, SM Roy, MA Riley (2016) www.caister.com/bacteriocins • Omics in Plant Disease Resistance Edited by: V Bhadauria (2016) www.caister.com/opdr • Acidophiles: Life in Extremely Acidic Environments Edited by: R Quatrini, DB Johnson (2016) www.caister.com/acidophiles • Climate Change and Microbial Ecology: Current Research and Future Trends Edited by: J Marxsen (2016) www.caister.com/climate • Biofilms in Bioremediation: Current Research and Emerging Technologies • Flow Cytometry in Microbiology: Technology and Applications Edited by: G Lear (2016) www.caister.com/biorem Edited by: MG Wilkinson (2015) www.caister.com/flow • Microalgae: Current Research and Applications • Probiotics and Prebiotics: Current Research and Future Trends Edited by: MN Tsaloglou (2016) www.caister.com/microalgae Edited by: K Venema, AP Carmo (2015) www.caister.com/probiotics • Gas Plasma Sterilization in Microbiology: Theory, Applications, Pitfalls and New Perspectives • Epigenetics: Current Research and Emerging Trends Edited by: H Shintani, A Sakudo (2016) Edited by: BP Chadwick (2015) www.caister.com/gasplasma www.caister.com/epigenetics2015 • Virus Evolution: Current Research and Future Directions • Corynebacterium glutamicum: From Systems Biology to Biotechnological Applications Edited by: SC Weaver, M Denison, M Roossinck, et al. (2016) www.caister.com/virusevol Edited by: A Burkovski (2015) www.caister.com/cory2 • Arboviruses: Molecular Biology, Evolution and Control Edited by: N Vasilakis, DJ Gubler (2016) • Advanced Vaccine Research Methods for the Decade of www.caister.com/arbo Vaccines Edited by: F Bagnoli, R Rappuoli (2015) • Shigella: Molecular and Cellular Biology www.caister.com/vaccines Edited by: WD Picking, WL Picking (2016) www.caister.com/shigella • Antifungals: From Genomics to Resistance and the Development of Novel Agents • Aquatic Biofilms: Ecology, Water Quality and Wastewater Edited by: AT Coste, P Vandeputte (2015) Treatment www.caister.com/antifungals Edited by: AM Romaní, H Guasch, MD Balaguer (2016) www.caister.com/aquaticbiofilms • Bacteria-Plant Interactions: Advanced Research and Future Trends Edited by: J Murillo, BA Vinatzer, RW Jackson, et al. (2015) • Alphaviruses: Current Biology www.caister.com/bacteria-plant Edited by: S Mahalingam, L Herrero, B Herring (2016) www.caister.com/alpha • Aeromonas Edited by: J Graf (2015) • Thermophilic Microorganisms www.caister.com/aeromonas Edited by: F Li (2015) www.caister.com/thermophile • Antibiotics: Current Innovations and Future Trends Edited by: S Sánchez, AL Demain (2015) www.caister.com/antibiotics • Leishmania: Current Biology and Control Edited by: S Adak, R Datta (2015) www.caister.com/leish2 • Acanthamoeba: Biology and Pathogenesis (2nd edition) Author: NA Khan (2015) www.caister.com/acanthamoeba2 • Microarrays: Current Technology, Innovations and Applications Edited by: Z He (2014) www.caister.com/microarrays2 • Metagenomics of the Microbial Nitrogen Cycle: Theory, Methods and Applications Edited by: D Marco (2014) www.caister.com/n2 Order from caister.com/order 328 Vrljic et al. Table 1. Protein Members of the LysE Family Abbreviation Name or description in database Organism Size (no. residues) Database and accession no. LysE Cgl Lysine exporter protein Corynebacterium glutamicum 236 gbX96471 Orf1 Mtu* Hypothetical 20.9 KD protein CY20G9.14 Mycobacterium tuberculosis 212 spQ11154 Orf2 Mtu Hypothetical 20.8 KD protein CY39.33C Mycobacterium tuberculosis 199 spQ10871 YggA Eco Hypothetical 23.2 KD protein in SBM-FBA intergenic region Escherichia coli 211 spP11667 YggA Asa* Lysine export homologue Aeromonas salmonicida 206 gbU65741 YggA Ahy* YggA homologue gene product Aeromonas hydrophila 206 gbX89469 Orf Bsu Hypothetical protein YisU Bacillus subtilis 220 gbY09476 Orf Hpy Conserved hypothetical integral membrane protein Helicobacter pylori 210 gbAE000585 * Based on the DNA sequences; the amino acid sequences of these three proteins (used in the reported analyses) were modified