Institut für Biotechnologie, Forschungszentrum Jülich GmbH, D-52425 Jülich, Germany1
Institute of Microbiology, Academy of Sciences, CZ-14220 Prague, Czech Republic2
Institut für Biochemie, Universität zu Köln, Zülpicher Strasse 47, D-57674 Köln, Germany3
Author for correspondence: L. Eggeling. Tel: +49 2461 61 5132. Fax: +49 2461 61 2710. e-mail: l.eggeling{at}fz-juelich.de
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Keywords: carrier, transcriptional regulator, LTTR, basic amino acid export, peptide hydrolysis
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Studies on the physiological function of lysE showed that, in the absence of the carrier, L-lysine can reach an intracellular concentration of more than 1100 mM, which prevents cell growth. Therefore, in addition to the synthesis of this amino acid, its export apparently also represents a possible means of regulating its intracellular concentration. Such a mechanism, not previously known in bacteria, is necessary in the wild-type of C. glutamicum if lysine-containing peptides are present in the environment (Erdmann et al., 1993 ). Thus, LysE serves as a valve for exporting excess L-lysine that might be harmful to the cell. Nothing is yet known about the specificity of LysE and its expression control.
In addition to its novel function, the carrier LysE also represents a novel type of structure within the membrane proteins (Vrlji et al., 1999
). It is a rather short polypeptide of 233 amino acyl residues which might span the membrane five times. The carrier is therefore a prototype of a new group of translocators, termed the LysE superfamily (Genome analysis: comparison of the transport capabilities of several bacteria. M. Saiers transport classification page; http://www.biology.ucsd.edu/~msaier/transport/titlepage.html); members of this large superfamily are present in eubacteria and archaea (Aleshin et al., 1999
). They all have a very similar structure, and are possibly all involved in the export of small solutes. Most interestingly, in Escherichia coli there are five paralogues present which might also be related to amino acid export. One of the paralogues confers resistance to L-threonine upon overexpression of the corresponding gene (rhtC), and another one (rhtB) confers resistance to L-homoserine and L-homoserine lactone (Zakataeva et al., 1999
). Recently, another exporter of E. coli has been identified (ydeD), related to the export of cysteine or cysteine-related compounds, but not belonging to the LysE superfamily (Daßler et al., 2000
).
Since export mediated by carriers belonging to the LysE superfamily is obviously of relevance both to an understanding of basic microbial functions and to biotechnological applications, here we discuss expression control of lysE and the substrate specificity of the carrier.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Plasmid pEM1dppc with the lysGE'lacZ fusion cassette was made by inserting lacZ with its ribosome-binding site obtained as an Asp718XbaI fragment (3078 bp) from piWiT10 (Wilcken-Bergmann et al., 1986 ) into the RsrII site of pJClysGE (Vrlji
et al., 1996
). The resulting lysGE'lacZ cassette (the BamHI cassette) with a lysE'lacZ transcriptional fusion at nt 62 of lysE was excised as a BamHI fragment and ligated with BamHI-digested pEM1dppc (Va
icová et al., 1998
) to give pEM1dppclysGE'lacZ. Plasmid pEM1dppclysGNarIE'lacZ was made by deletion of the 739 bp NarI fragment of lysG in pJClysGE'lacZ, re-ligation, and further processing of the resulting BamHI cassette as described above. By XhoIStuI digestion of pJClysGE'lacZ, Klenow treatment, and blunt-end ligation, a BamHI cassette was made to generate pEM1dppclysGStuIXhoIE'lacZ deleted of 567 nt within lysG. To construct pK18mobsacB
lysA, the lysA gene was obtained as a 1·3 kb PstI fragment from pCT4-1, which was ligated with pUC18. The resulting vector was restricted with DraIII and EcoRV (deletion of the central 881 bp fragment) and re-ligated. The remaining 560 bp fragment of
lysA was obtained by restriction with SalI and PstI and ligated with SalIPstI-restricted pK18mobsacB. Plasmid pK18mobargFint was made by amplifying a 551 nt internal fragment of argF by using primers to which EcoRI and PstI restriction sites were attached. The resulting fragment was ligated with EcoRIPstI-restricted pUC18, excised by restriction with EcoRI and HindIII, and ligated with EcoRIHindIII-treated pK18mob. To construct plasmid pEC7lysE, the primers 5'-CTCGAGAGCGGATCCGCGCTGACTCAC-3' and 5'-GGAGAGTACGGCCCATCCACCGTGACC-3' were used to amplify lysE as a 1·0 kb fragment with attached BamHI sites from pJClysGE, before ligating it with pEC7 (Eikmanns et al., 1991
). Overexpression of lysE was verified by increased L-lysine export activity with strain 13032
GE.
RNA analysis.
Total RNA was isolated from C. glutamicum clones harbouring the plasmids pET2PlysE or pET2PlysG as described by Börmann et al. (1992) . For primer extension experiments, 30 µg RNA was hybridized to 0·5 pmol fluorescein-labelled primer (5'-GAAAATCTCGTCGAAGCTCG-3') complementary to vector sequences (Va
icová et al., 1998
). Denaturation, annealing and reverse transcription were as described by Peters-Wendisch et al. (1998)
, and the products were analysed by using an automated laser fluorescence DNA sequencer with sequencing reactions carried out in parallel.
Assay of amino acid export.
For the determination of export rates in short-term experiments, pre-grown cells were washed twice with ice-cold CGXII and used to inoculate new CGXII. After growth overnight, the cells were harvested by centrifugation (5000 g, 10 min), washed again (twice with ice-cold CGXII), then amino acid excretion was initiated by resuspending the cells in pre-warmed CGXII containing 2 mM of the appropriate dipeptide (see Results). The resulting cell density (OD600) was 810, corresponding to 2·43·0 mg dry weight ml-1. The cells were stirred and incubated at 30 °C. Samples for silica oil centrifugation (Klingenberg & Pfaff, 1977 ) were taken after 1·5 min and then every 15 min for a period of 1·25 h. The procedures for deriving cellular and extracellular fractions and for the quantification of the amino acids as their o-phthaldialdehyde derivatives via HPLC were as described by Bröer & Krämer (1991)
.
Determination of the specific ß-galactosidase activity.
For the determination of the specific ß-galactosidase activity, pre-grown cells (BHI; Difco) were transferred into BHI medium and cultivated for 4 h at 30 °C. After the cells had been harvested by centrifugation (5000 g, 10 min), they were washed with ice-cold 0·1 M potassium phosphate buffer, pH 7·0, centrifuged (5000 g, 10 min), resuspended in 1 ml ß-galactosidase reaction buffer (5 mM Tris, pH 7·5; 5% glycerol; 10 mM KCl) and disrupted by sonication in the same buffer. After pelleting of the cellular debris by centrifugation (5000 g, 10 min, 4 °C) the supernatant was immediately used for the enzyme assay. The specific activity is given in U (mg protein)-1.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Transcript initiation sites of lysE and lysG
To define the transcript initiation sites of the regulatory gene and its target gene, a fragment of 198 bp carrying the 70 bp intergenic lysGlysE region together with the 5' ends of both genes was amplified and cloned into the promoter-probe vector pET2 (Vaicová et al., 1998
). As expected, this fragment conferred chloramphenicol resistance on C. glutamicum in both orientations through expression of the cat reporter gene. The position of the lysE and lysG transcript initiation sites was localized in several primer-extension experiments, using fluorescein-labelled primers which were hybridized to 30 µg RNA isolated from C. glutamicum clones harbouring plasmid pET2PlysE or plasmid pET2PlysG. Two fluorograms of these experiments, together with the sequencing reactions carried out in parallel, are shown in Fig. 2(a
, b
). This defines C1024 and G969 (on the opposite strand) in sequence X96471 as the transcript initiation sites for lysE and lysG, respectively. The two promoters overlap and share a palindromic sequence of 10 bp, labelled ABS (for activating binding site) in Fig. 2(c)
, which could be an activation binding site of LysG. The transcript initiation site of lysE is in close proximity to the deduced translation initiation site, as inferred from sequence comparisons (Vrlji
et al., 1999
). Apparently lysE belongs to those genes of C. glutamicum for which the transcript does not provide a ribosome-binding site (Morbach et al., 2000
). This seems to be a more general feature of the Actinomyces subdivision of Gram-positive bacteria to which C. glutamicum belongs (Strohl, 1992
).
|
To assay for a specific intracellular increase in amino acids, we used the addition of peptides, some of which are known to be taken up and hydrolysed by C. glutamicum (Erdmann et al., 1993 ). The dipeptides used are given in Table 2
. They contained the basic amino acids Lys, Arg, Cit (L-citrulline) and His. The Thr- and Phe-containing peptides served as controls. These peptides were added at a concentration of 0·5 mM to salt medium CGXII, and, after growth of strain 13032
GE::lysGE'lacZ for 4 h, intracellular amino acid concentrations were determined by silica-oil centrifugation. The specific ß-galactosidase activities were quantified in extracts made from the same culture. As shown in Table 2
, a specific increase in the intracellular Lys, Arg, Cit, His and Thr concentrations was obtained, whereas with Phe no strong increase was present. The Ala concentration was comparable in all strains and was not substantially increased, indicating that it is rapidly metabolized. All the other peptide-specific amino acids quantified (for instance Thr or Phe in the cells supplied with LysAla) were at concentrations below 1 mM (not shown). A high ß-galactosidase activity, elevated 1118-fold relative to the controls, was present when the concentration of Lys, Arg, Cit or His was increased. This is evidence that these basic amino acids might serve as inducers of lysE expression. With the strain carrying the NarI deletion construct of lysGE'lacZ in its chromosome (Fig. 1
) being used as a control, the specific ß-galactosidase activities in cells grown under identical conditions to those given in Table 2
were 0·20·3 U mg-1. This clearly indicates that LysG is required, together with one of the four basic amino acids, for lysE expression, and that no additional regulator, other than LysG, is present in the cell able to interact with Lys, Arg, His or Cit and capable of binding upstream of the fusion.
|
LysE exports L-arginine and L-lysine
lysE was isolated as the gene encoding the L-lysine export carrier (Vrlji et al., 1996
). In view of the finding that lysE is induced by several basic amino acids, we studied the specificity of LysE in terms of its catalytic activity, and first focused on L-arginine as a possible exported cytoplasmic solute. In a growth experiment the behaviour of wild-type strain 13032 and strain 13032
EG was studied. When 2 mM of the peptide ArgAla was added to the mutant, after an intial doubling, growth was arrested, which was not the case with the wild-type (Fig. 3
). Determination of the intracellular L-arginine concentration revealed its very high accumulation (up to 823 mM) relative to the wild-type (245 mM). These characteristics are in accord with the known growth-inhibiting effects of high intracellular L-lysine concentrations (Vrlji
et al., 1996
). It illustrates that LysE also exports L-arginine, but does not exclude induction of a second exporter during the growth experiment. However, when the export activity was quantified over a 1 h period directly after inoculation, the efflux rate for L-arginine in the deletion mutant was below 0·01 nmol min-1 (mg protein)-1 (not shown). This is of the order of the efflux mediated by passive diffusion (Vrlji
et al., 1996
) and thus shows that, under these conditions, LysE is the only relevant system for L-arginine and L-lysine export present in C. glutamicum.
|
|
|
In a similar approach using chromosomal gene inactivation, we derived a strain suitable for assaying for the capacity of LysE to accept intracellular DL-diaminopimelate as a substrate. A C. glutamicum lysA mutant preventing the decarboxylation of DL-diaminopimelate to L-lysine was constructed. This mutant accumulated up to 1600 mM DL-diaminopimelate in the cytoplasm (results not shown). However, in the medium, only concentrations of about 3 mM accumulated within 2 d, with the wild-type and with the lysE-overexpressing strain as well. This shows that LysE does not accept the basic amino acid DL-diaminopimelate as a substrate. Furthermore, it strengthens the belief that the basic amino acids have an extremely low permeability via the lipid bilayer of C. glutamicum.
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
As inducers of lysE for mediating transcriptional activation by LysG, L-lysine and L-arginine, as well as L-histidine and L-citrulline, were identified. One of the very few LTTR regulators for which an attempt has been made to quantify the intracellular inducer concentration is NhaR. This regulator controls the synthesis of the Na+/H+ antiporter of E. coli. It is known that it is fully induced at an extracellular Na+ concentration of 50 mM (Dover et al., 1997 ), when the intracellular Na+ concentration is around 60 mM (Harel-Bronstein et al., 1995
). With the C. glutamicum wild-type derivative carrying the lysE'lacZ fusion, a specific ß-galactosidase activity of 5·5 U at a concentration of 42 mM (Table 2
) was obtained and an activity of 6·5 U was obtained at an intracellular L-lysine concentration of about 60 mM (Fig. 1
). This fits very well with the values obtained with C. glutamicum MH20-22B (5·8 U at 35 mM). Even higher values of 7·2 U were achieved (not shown), but these values were not given consideration since growth was already retarded at the extraordinarily high internal concentration of 225 mM L-lysine. In summary, these data indicate that a comparatively moderate L-lysine concentration of around 3040 mM is sufficient for almost full induction of lysE. The range of induction obtained is about 20-fold (see Table 2
). Such strong control might be required to prevent expression of the export carrier under conditions in which L-lysine is synthesized from glucose, otherwise the viability of the cell would be endangered. In any case, L-lysine and L-arginine, and also L-histidine and L-citrulline, act as inducers (though the latter two amino acids are not exported by LysE). This differential specificity of LysG and LysE is not unexpected, since both proteins, of course, have entirely different structures. A wide range of different inducer structures is known for the LTTR NahR of salicylate-degrading pseudomonads (Cebolla et al., 1997
).
The affinity of LysE towards L-lysine and L-arginine is comparable. The other basic amino acids assayed (L-histidine, L-citrulline, L-ornithine, DL-diaminopimelate) are not accepted as transport substrates by LysE. Basic amino acid exporters in other bacteria have not yet been identified. The uptake carrier of Penicillium chrysogenum accepts L-arginine, L-lysine and L-ornithine as substrates (Hillenga et al., 1996 ). E. coli has at least five importers for basic amino acids, which have different specificities. Three of them exhibit a high substrate affinity. Similarly, the lysine importer of C. glutamicum encoded by lysI (Seep-Feldhaus et al., 1991
) has a Km of 10 µM for lysine (Bröer & Krämer, 1991
). Obviously, a high substrate affinity is reasonable for carriers transporting amino acids into the cell, as it allows the cell to cope with low substrate concentrations in the environment. However, it would be harmful for amino acid exporters, because of the inevitable loss of these metabolites when synthesized from carbohydrates. Accordingly, the Km of LysE of C. glutamicum for L-lysine is three orders of magnitudes higher (20 mM) than that of the importer (Bröer & Krämer, 1991
).
However, it should be borne in mind that in particular metabolic situations, for instance when intracellular L-arginine is low and L-lysine high, co-export of L-arginine by the lysine exporter, which would be active under these conditions, would be disadvantageous for the cell. This scenario possibly explains why argS and lysA in C. glutamicum form an operon (Marcel et al., 1990 ). The gene argS encodes the arginyl-tRNA synthetase and lysA encodes the DL-diaminopimelate decarboxylase, which is the only specific gene of L-lysine synthesis. Both L-arginine and L-lysine control expression of this operon (Oguiza et al., 1993
); this might serve to counteract intracellular amino acid imbalances possibly arising from the action of the lysine exporter.
In 1979 and 1982, Payne and coworkers had already observed the formation of amino acids derived from peptides with E. coli and Streptococcus faecalis, respectively (for a review, see Payne & Smith, 1994 ). Thus, export of amino acids is a general phenomenon and there is now firm evidence that in E. coli and, in particular, in C. glutamicum, distinct transporters catalysing amino acid export are present (Krämer, 1994
). For instance, in C. glutamicum export of L-isoleucine and L-threonine (Palmieri et al., 1996
) is also a carrier-mediated process, and, as indicated in the present work, further transport systems specific for the export of at least L-citrulline, L-histidine and L-ornithine are present in C. glutamicum. In E. coli, it has been shown that overexpression of rhtB results in increased extracellular accumulation of L-homoserine, and that rhtC overexpression confers resistance to L-homoserine lactone (Zakataeva et al., 1999
). It may be assumed, however, that the physiological function of rhtB, as well as the functions of its four paralogues present in E. coli, might be to serve cell-to-cell communication. Consequently, the observed amino acid export in E. coli might be due to limited specificity. Similarly, L-cysteine export in E. coli is probably due to limited specificity of the exporter YdeD (Daßler et al., 2000
). In contrast, the function of LysE of C. glutamicum is obviously to control the intracellular L-lysine and L-arginine pools. This is in line with the fact that C. glutamicum is unable to use these amino acids for catabolic purposes. In combination with LysG, the whole system is designed to sense and export intracellular L-lysine and L-arginine. The cell is therefore able to respond to imbalances in cytosolic amino acid pools. These might occur both physiologically under particular environmental conditions (during growth in the presence of peptides) as well as during amino acid production.
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Börmann, E., Eikmanns, B. J. & Sahm, H. (1992). Molecular analysis of the Corynebacterium glutamicum gdh gene encoding glutamate dehydrogenase. Mol Microbiol 6, 317-326.[Medline]
Bröer, S. & Krämer, R. (1991). Lysine excretion by Corynebacterium glutamicum. 2. Energetics and mechanism of the transport system. Eur J Biochem 202, 137-143.[Abstract]
Bröer, S., Eggeling, L. & Krämer, R. (1993). Strains of Corynebacterium glutamicum with different lysine productivities may have different lysine excretion systems. Appl Environ Microbiol 59, 316-321.[Abstract]
Cebolla, A., Sousa, C. & de Lorenzo, V. (1997). Effector specificity mutants of the transcriptional activator NahR of naphthalene degrading Pseudomonas define protein sites involved in binding of aromatic inducers. J Biol Chem 272, 3986-3992.
Choi, D., Ryu, W., Chung, B. H., Hwang, S. & Park, Y. H. (1995). Effect of dilution rate on continuous production of L-ornithine by an arginine auxotrophic mutant. J Ferment Bioeng 80, 97-100.
Cremer, J., Eggeling, L. & Sahm, H. (1991). Control of the lysine biosynthetic sequence in Corynebacterium glutamicum as analyzed by overexpression of the individual corresponding genes. Appl Environ Microbiol 57, 1746-1752.
Daßler, T., Maier, T., Winterhalter, C. & Böck, A. (2000). Identification of a major facilitator protein from Escherichia coli involved in efflux of metabolites of the cysteine pathway. Mol Microbiol 36, 1101-1112.[Medline]
Dover, N., Higgins, C. F., Carmel, O., Pinner, A. R. & Padan, E. (1997). Na+-induced transcription of nhaA, which encodes an Na+/H+ antiporter in Escherichia coli, is positively regulated by nhaR and affected by hns. J Bacteriol 178, 6508-6517.[Abstract]
Eggeling, L., Oberle, S. & Sahm, H. (1996). Improved L-lysine yield with Corynebacterium glutamicum: use of dapA resulting in increased flux combined with growth limitation. Appl Microbiol Biotechnol 49, 24-30.
Eikmanns, B. J., Follettie, M. T., Griot, M. U. & Sinskey, A. J. (1989). The phosphoenolpyruvate carboxylase gene of Corynebacterium glutamicum: molecular cloning, nucleotide sequence, and expression. Mol Gen Genet 218, 330-339.[Medline]
Eikmanns, B., Kleinertz, E., Liebl, W. & Sahm, H. (1991). A family of Corynebacterium glutamicum/Escherichia coli shuttle vectors for gene cloning, controlled gene expression, and promoter probing. Gene 102, 93-98.[Medline]
Erdmann, A., Weil, B. & Krämer, R. (1993). Lysine secretion by wild-type Corynebacterium glutamicum triggered by dipeptide uptake. J Gen Microbiol 139, 3115-3122.
Grant, S. G. N., Jessee, J., Bloom, F. R. & Hanahan, D. (1990). Differential plasmid rescue from transgenic mouse DNAs into Escherichia coli methylation-restriction mutants. Proc Natl Acad Sci USA 87, 4645-4649.[Abstract]
Harel-Bronstein, M., Dibrov, P., Olami, Y., Pinner, E., Schuldiner, S. & Padan, E. (1995). MH1, a second-site revertant of an Escherichia coli mutant lacking Na+/H+ antiporters regains Na+ resistance and a capacity to excrete Na+ in a µH+-independent fashion. J Biol Chem 270, 3816-3822.
Hillenga, D. J., Versantvoort, H. J. M., Driessen, A. J. M. & Konings, W. N. (1996). Basic amino acid transport in plasma membrane vesicles of Penicillium chrysogenum. J Bacteriol 178, 3991-3995.[Abstract]
Keilhauer, C., Eggeling, L. & Sahm, H. (1993). Isoleucine synthesis in Corynebacterium glutamicum: molecular analysis of the ilvB-ilvN-ilvC operon. J Bacteriol 175, 5595-5603.[Abstract]
Klingenberg, M. & Pfaff, E. (1977). Means of terminating reactions. Methods Enzymol 10, 680-684.
Krämer, R. (1994). Secretion of amino acids by bacteria: physiology and mechanism. FEMS Microbiol Rev 13, 75-79.
Leuchtenberger, W. (1996). Amino acids technical production and use. In Products of Primary Metabolism: Biotechnology , pp. 455-502. Edited by H. J. Rehm & G. Reeds. Weinheim:VHC.
Liebl, W., Bayerl, A., Stillner, U. & Schleifer, K. H. (1989). High efficiency electroporation of intact Corynebacterium glutamicum cells. FEMS Microbiol Lett 65, 299-304.
McFall, S. M., Chugani, S. A. & Chakrabarty, A. M. (1998). Transcriptional activation of the catechol and chlorocatechol operons: variations on a theme. Mol Microbiol 223, 257-267.
Marcel, T., Archer, J. A. C., Mengin-Lecreulx, D. & Sinskey, A. J. (1990). Nucleotide sequence and organization of the upstream region of the Corynebacterium glutamicum lysA gene. Mol Microbiol 4, 1819-1830.[Medline]
Morbach, S., Junger, C., Sahm, H. & Eggeling, L. (2000). Attenuation control of ilvBNC in Corynebacterium glutamicum: evidence of leader peptide formation without the presence of a ribosome binding site. Biosci Biotechnol Biochem 90, 501-507.
Oguiza, J. A., Malumbres, M., Eriani, G., Pisabarro, A., Mateos, L. M., Martin, F. & Martin, J. F. (1993). A gene encoding arginyl-tRNA synthetase is located in the upstream region of the lysA gene in Brevibacterium lactofermentum: regulation of argSlysA cluster expression by arginine. J Bacteriol 175, 7356-7362.[Abstract]
Palmieri, L., Berns, D., Krämer, R. & Eikmanns, M. (1996). Threonine diffusion and threonine transport in Corynebacterium glutamicum and their role in threonine production. Arch Microbiol 165, 48-54.
Payne, J. W. & Smith, M. W. (1994). Peptide transport by microorganisms. Adv Microb Physiol 36, 2-80.
Peters-Wendisch, P. G., Kreutzer, C., Kalinowski, J., Pátek, M., Sahm, H. & Eikmanns, B. J. (1998). Pyruvate carboxylase from Corynebacterium glutamicum: characterization, expression and inactivation of the pyc gene. Microbiology 144, 915-927.[Abstract]
Schäfer, A., Kalinowski, J., Simon, R., Seep-Feldhaus, A. & Pühler, A. (1990). High-frequency conjugal plasmid transfer from Gram-negative Escherichia coli to various Gram-positive coryneform bacteria. J Bacteriol 172, 1663-1666.[Medline]
Schäfer, A., Tauch, A., Jäger, W., Kalinowski, J., Thierbach, G. & Pühler, A. (1994). Small mobilizable multi-purpose cloning vectors derived from the Escherichia coli plasmids pK18 and pK19: selection of defined deletions in the chromosome of Corynebacterium glutamicum. Gene 145, 69-73.[Medline]
Schell, M. A. (1993). Molecular biology of the LysR family of transcriptional regulators. Annu Rev Microbiol 47, 597-626.[Medline]
Schrumpf, B., Eggeling, L. & Sahm, H. (1992). Isolation and prominent characteristics of an L-lysine hyperproducing strain of Corynebacterium glutamicum. Appl Microbiol Biotechnol 37, 566-571.
Seep-Feldhaus, A. H., Kalinowski, J. & Pühler, A. (1991). Molecular analysis of the Corynebacterium glutamicum lysI gene involved in lysine uptake. Mol Microbiol 5, 2995-3005.[Medline]
Simon, R., Priefer, U. & Pühler, A. (1983). A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in Gram negative bacteria. Bio/Technology 11, 784-791.
Strohl, W. R. (1992). Compilation and analysis of DNA sequences associated with apparent streptomyces promoters. Nucleic Acids Res 20, 961-974.[Abstract]
Vaicová, P., Abrhámová, Z., Ne
vera, J., Pátek , M., Sahm, H. & Eikmanns, B. (1998). Integrative and autonomously replicating vectors for analysis of promoters in Corynebacterium glutamicum. Biotechnol Tech 12, 743-746.
Vrlji, M., Eggeling, L. & Sahm, H. (1995). Unbalance of L-lysine flux in Corynebacterium glutamicum and its use for the isolation of excretion defective mutants. J Bacteriol 177, 4021-4027.[Abstract]
Vrlji, M., Sahm, H. & Eggeling, L. (1996). A new type of transporter with a new type of cellular function: L-lysine export from Corynebacterium glutamicum. Mol Microbiol 22, 815-826.[Medline]
Vrlji, M., Garg, J., Bellmann, A., Wachi, S., Freudl, R., Malecki, M. J., Sahm, H., Kozina, V. J., Eggeling, L. & Saier, M. H.Jr (1999). The LysE superfamily: topology of the lysine exporter LysE of Corynebacterium glutamicum, a paradigm for a novel superfamily of transmembrane solute translocators. J Mol Microbiol Biotechnol 1, 327-336.[Medline]
Wendisch, V. (1997). Physiological investigations and studies by NMR spectroscopy on the central metabolism of different recombinant Corynebacterium glutamicum strains. PhD thesis, University of Düsseldorf.
von Wilken-Bergmann, B., Tils, D., Sartorius, J., Auerswald, E. A., Schröder, W. & Müller-Hill, B. (1986). A synthetic operon containing 14 bovine pancreatic trypsin inhibitor genes is expressed in E. coli. EMBO J 5, 3219-3225.
Wissenbach, U., Six, S., Bongaerts, J., Ternes, D., Steinwachs, S. & Unden, G. (1995). A third periplasmic transport system for L-arginine in Escherichia coli: molecular characterization of the artPIQMJ genes, arginine binding and transport. Mol Microbiol 17, 675-686.[Medline]
Zakataeva, N. P., Aleshin, V. V., Tokmakova, I. L., Troshin, P. V. & Livshits, V. A. (1999). The novel transmembrane Escherichia coli proteins involved in the amino acid efflux. FEBS Lett 452, 228-232.[Medline]
Received 16 January 2001;
revised 15 March 2001;
accepted 26 March 2001.