1887

Abstract

is used for the large-scale production of -glutamate, but the efflux of this amino acid is poorly understood. This study shows that addition of ethambutol (EMB) to growing cultures of causes -glutamate efflux at rates of up to 15 nmol min (mg dry wt), whereas in the absence of EMB, no efflux occurs. EMB is used for the treatment of , and at a molecular level it targets a series of arabinosyltransferases (EmbCAB). The single arabinosyltransferase-encoding gene of was placed under the control of a Tet repressor (TetR). Experiments with this strain, as well as with an -overexpressing strain, coupled with biochemical analyses showed that: (i) expression was correlated with -glutamate efflux, (ii) 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 and (encoding a secreted metalloprotease); however, genes of central metabolism were largely absent. Given that an altered lipid composition of the plasma membrane of can result in -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.

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2005-05-01
2019-11-12
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References

  1. Belanger, A. E., Besra, G. S., Ford, M. E., Mikusova, K., Belisle, J. T., Brennan, P. J. & Inamine, J. M. ( 1996; ). The embAB genes of Mycobacterium avium encode an arabinosyl transferase involved in cell wall arabinan biosynthesis that is the target for the antimycobacterial drug ethambutol. Proc Natl Acad Sci U S A 93, 11919–11924.[CrossRef]
    [Google Scholar]
  2. Bellmann, A., Vrljić, M., Pátek, M., Sahm, H., Krämer, R. & Eggeling, L. ( 2001; ). Expression control and specificity of the basic amino acid exporter LysE of Corynebacterium glutamicum. Microbiology 147, 1765–1774.
    [Google Scholar]
  3. Besra, G. S., Khoo, K., McNeil, M., Dell, A., Morris, H. & Brennan, P. J. ( 1995; ). A new interpretation of the structure of the mycolyl-arabinogalactan complex of Mycobacterium tuberculosis revealed through characterization of oliglycosylalditol framents by fast-atom bombardment mass spectrometry and 1-N nuclear magnetic resonance spectroscopy. Biochemistry 34, 4257–4266.[CrossRef]
    [Google Scholar]
  4. Brand, S., Niehaus, K., Puhler, A. & Kalinowski, J. ( 2003; ). Identification and functional analysis of six mycolyltransferase genes of Corynebacterium glutamicum ATCC 13032: the genes cop1, cmt1, and cmt2 can replace each other in the synthesis of trehalose dicorynomycolate, a component of the mycolic acid layer of the cell envelope. Arch Microbiol 180, 33–44.[CrossRef]
    [Google Scholar]
  5. Brennan, P. J. & Nikaido, H. ( 1995; ). The envelope of mycobacteria. Annu Rev Biochem 64, 29–63.[CrossRef]
    [Google Scholar]
  6. Cremer, J., Eggeling, L. & Sahm, H. ( 1990; ). Cloning the dapA dapB cluster of the lysine-secreting bacterium Corynebacterium glutamicum. Mol Gen Genet 220, 478–480.[CrossRef]
    [Google Scholar]
  7. De Sousa-D'Auria, C., Kacem, R., Puech, V., Tropis, M., Leblon, G., Houssin, C. & Daffe, M. ( 2003; ). New insights into the biogenesis of the cell envelope of corynebacteria: identification and functional characterization of five new mycoloyltransferase genes in Corynebacterium glutamicum. FEMS Microbiol Lett 224, 35–44.[CrossRef]
    [Google Scholar]
  8. Eggeling, L. & Sahm, H. ( 2001; ). The cell wall barrier of Corynebacterium glutamicum and amino acid efflux. J Biosci Bioeng 92, 201–213.[CrossRef]
    [Google Scholar]
  9. Eggeling, L. & Sahm, H. ( 2003; ). New ubiquitous translocators: amino acid export by Corynebacterium glutamicum and Escherichia coli. Arch Microbiol 180, 155–160.[CrossRef]
    [Google Scholar]
  10. Eggeling, L., Krumbach, K. & Sahm, H. ( 2001; ). l-Glutamate efflux with Corynebacterium glutamicum: why is penicillin treatment or tween addition doing the same? J Mol Microbiol Biotechnol 3, 67–68.
    [Google Scholar]
  11. 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.[CrossRef]
    [Google Scholar]
  12. Eisen, M. B., Spellman, P. T., Brown, P. O. & Botstein, D. ( 1998; ). Cluster analysis and display of genome-wide expression patterns. Proc Natl Acad Sci U S A 95, 14863–14868.[CrossRef]
    [Google Scholar]
  13. Escuyer, V. E., Lety, M. A., Torrelles, J. B. & 7 other authors ( 2001; ). The role of the embA and embB gene products in the biosynthesis of the terminal hexaarabinofuranosyl motif of Mycobacterium smegmatis arabinogalactan. J Biol Chem 276, 48854–48862.[CrossRef]
    [Google Scholar]
  14. Gande, R., Gibson, K. J., Brown, A. K. & 7 other authors ( 2004; ). Acyl-CoA carboxylases (accD2 and accD3) together with a unique polyketide synthase (Cg-pks) are key to mycolic acid biosynthesis in Corynebacterianeae like Corynebacterium glutamicum and Mycobacterium tuberculosis. J Biol Chem 279, 44847–44857.[CrossRef]
    [Google Scholar]
  15. Gibson, K. J., Eggeling, L., Maughan, W. N., Krumbach, K., Gurcha, S. S., Nigou, J., Puzo, G., Sahm, H. & Besra, G. S. ( 2003; ). Disruption of Cg-Ppm1, a polyprenyl monophosphomannose synthase, and the generation of lipoglycan-less mutants in Corynebacterium glutamicum. J Biol Chem 278, 40842–40850.[CrossRef]
    [Google Scholar]
  16. Hirasawa, T., Wachi, M. & Nagai, K. ( 2000; ). A mutation in the Corynebacterium glutamicum ltsA gene causes susceptibility to lysozyme, temperature-sensitive growth, and l-glutamate production. J Bacteriol 182, 2696–2701.[CrossRef]
    [Google Scholar]
  17. Hoischen, C. & Krämer, R. ( 1990; ). Membrane alteration is necessary but not sufficient for effective glutamate secretion in Corynebacterium glutamicum. J Bacteriol 172, 3409–3416.
    [Google Scholar]
  18. Ishige, T., Krause, M., Bott, M., Wendisch, V. F. & Sahm, H. ( 2003; ). The phosphate starvation stimulon of Corynebacterium glutamicum determined by DNA microarray analyses. J Bacteriol 185, 4519–4529.[CrossRef]
    [Google Scholar]
  19. 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.
    [Google Scholar]
  20. Kennerknecht, N., Sahm, H., Yen, M.-R., Patek, M., Saier, M. H., Jr & Eggeling, L. ( 2002; ). Export of l-isoleucine from Corynebacterium glutamicum: a two-gene-encoded member of a new translocator family. J Bacteriol 184, 3947–3956.[CrossRef]
    [Google Scholar]
  21. Kimura, E. ( 2003; ). Metabolic engineering of glutamate production. Adv Biochem Eng Biotechnol 79, 37–57.
    [Google Scholar]
  22. Kimura, E., Abe, C., Kawahara, Y., Nakamatsu, T. & Tokuda, G. ( 1997; ). A dtsR gene-disrupted mutant of Brevibacterium lactofermentum requires fatty acids for growth and efficiently produces l-glutamate in the presence of an excess of biotin. Biochem Biophys Res Commun 234, 157–161.[CrossRef]
    [Google Scholar]
  23. Manganelli, R., Dubnau, E., Tyagi, S., Kramer, F. R. & Smith, I. ( 1999; ). Differential expression of 10 sigma factor genes in Mycobacterium tuberculosis. Mol Microbiol 31, 715–724.[CrossRef]
    [Google Scholar]
  24. Minnikin, D. E. ( 1982; ). Lipids: complex lipids, their chemistry, biosynthesis and roles. In The Biology of Mycobacteria, pp. 95–184. Edited by C. Ratledge & J. Stanford. London: Academic Press.
  25. Möker, N., Brocker, M., Schaffer, S., Krämer, R., Morbach, S. & Bott, M. ( 2004; ). Deletion of the genes encoding the MtrA–MtrB two-component system of Corynebacterium glutamicum has a strong influence on cell morphology, antibiotics susceptibility and expression of genes involved in osmoprotection. Mol Microbiol 54, 420–438.[CrossRef]
    [Google Scholar]
  26. Morita, R. ( 1997; ). In Bacteria in Oligotrophic Environments, Starvation Survival Lifestyle. New York: Chapman & Hall.
  27. Nakamura, Y., Nishio, Y., Ikeo, K. & Gojobori, T. ( 2003; ). The genome stability in Corynebacterium species due to lack of the recombinational repair system. Gene 317, 149–155.[CrossRef]
    [Google Scholar]
  28. Nampoothiri, K. M., Hoischen, C., Bathe, B., Mockel, B., Pfefferle, W., Krumbach, K., Sahm, H. & Eggeling, L. ( 2002; ). Expression of genes of lipid synthesis and altered lipid composition modulates l-glutamate efflux of Corynebacterium glutamicum. Appl Microbiol Biotechnol 58, 89–96.[CrossRef]
    [Google Scholar]
  29. Nottebrock, D., Meyer, U., Kramer, R. & Morbach, S. ( 2003; ). Molecular and biochemical characterization of mechanosensitive channels in Corynebacterium glutamicum. FEMS Microbiol Lett 218, 305–309.[CrossRef]
    [Google Scholar]
  30. Polen, T. & Wendisch, V. F. ( 2004; ). Genomewide expression analysis in amino acid-producing bacteria using DNA microarrays. Appl Biochem Biotechnol 118, 215–232.[CrossRef]
    [Google Scholar]
  31. Puech, V., Bayan, N., Salim, K., Leblon, G. & Daffe, M. ( 2000; ). Characterization of the in vivo acceptors of the mycoloyl residues transferred by the corynebacterial PS1 and the related mycobacterial antigens 85. Mol Microbiol 35, 1026–1041.[CrossRef]
    [Google Scholar]
  32. Puech, V., Chami, M., Lemassu, A., Laneelle, M. A., Schiffler, B., Gounon, P., Bayan, N., Benz, R. & Daffé, M. ( 2001; ). Structure of the cell envelope of corynebacteria: importance of the non-covalently bound lipids in the formation of the cell wall permeability barrier and fracture plane. Microbiology 147, 1365–1382.
    [Google Scholar]
  33. Ramaswamy, S. V., Amin, A. G., Goksel, S., Stager, C. E., Dou, S. J., El Sahly, H., Moghazeh, S. L., Kreiswirth, B. N. & Musser, J. M. ( 2000; ). Molecular genetic analysis of nucleotide polymorphisms associated with ethambutol resistance in human isolates of Mycobacterium tuberculosis. Antimicrob Agents Chemother 44, 326–336.[CrossRef]
    [Google Scholar]
  34. 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.[CrossRef]
    [Google Scholar]
  35. Shaw, K. J., Miller, N., Liu, X., Lerner, D., Wan, J., Bittner, A. & Morrow, B. J. ( 2003; ). Comparison of the changes in global gene expression of Escherichia coli induced by four bactericidal agents. J Mol Microbiol Biotechnol 5, 105–122.[CrossRef]
    [Google Scholar]
  36. Simic, P., Sahm, H. & Eggeling, L. ( 2001; ). l-Threonine export: use of peptides to identify a new translocator from Corynebacterium glutamicum. J Bacteriol 183, 5317–5324.[CrossRef]
    [Google Scholar]
  37. Stackebrandt, E., Rainey, F. A. & Ward-Rainey, N. L. ( 1997; ). Proposal for a new hierachic classification system, Actinobacteria classis nov. Int J Syst Bacteriol 47, 479–491.[CrossRef]
    [Google Scholar]
  38. Tillman, T. S. & Cascio, M. ( 2003; ). Effects of membrane lipids on ion channel structure and function. Cell Biochem Biophys 38, 161–190.[CrossRef]
    [Google Scholar]
  39. Uy, D., Delaunay, S., Germain, P., Engasser, J. M. & Goergen, J. L. ( 2003; ). Instability of glutamate production by Corynebacterium glutamicum 2262 in continuous culture using the temperature-triggered process. J Biotechnol 104, 173–184.[CrossRef]
    [Google Scholar]
  40. Vrljic, M., Eggeling, L. & Sahm, H. ( 1996; ). A new type of transporter with a new type of cellular function: l-lysine export from Corynebacterium glutamicum. Mol Microbiol 22, 815–826.[CrossRef]
    [Google Scholar]
  41. Vrljic, M. J., Garg, A., Bellmann, S. & 7 other authors ( 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.
    [Google Scholar]
  42. Waddell, S. J., Stabler, R. A., Laing, K., Kremer, L., Reynolds, R. C. & Besra, G. S. ( 2004; ). The use of microarray analysis to determine the gene expression profiles of Mycobacterium tuberculosis in response to anti-bacterial compounds. Tuberculosis (Edinb) 84, 263–274.[CrossRef]
    [Google Scholar]
  43. Wendisch, V. F., Zimmer, D. P., Khodursky, A., Peter, B., Cozzarelli, N. & Kustu, S. ( 2001; ). Isolation of Escherichia coli mRNA and comparison of expression using mRNA and total RNA on DNA microarrays. Anal Biochem 290, 205–213.[CrossRef]
    [Google Scholar]
  44. Westphal, O. & Jann, K. ( 1965; ). Bacterial lipopolysaccharides: extraction with phenol-water and further applications of the procedure. Methods Carbohydr Chem 5, 83–91.
    [Google Scholar]
  45. Wissmann, A., Baumeister, R., Muller, G., Hecht, B., Helbl, V., Pfleiderer, K. & Hillen, W. ( 1991; ). Amino acids determining operator binding specificity in the helix-turn-helix motif of Tn10 Tet repressor. EMBO J 10, 4145–4152.
    [Google Scholar]
  46. Zhang, N., Torrelles, J. B., McNeil, M. R., Escuyer, V. E., Khoo, K. H., Brennan, P. J. & Chatterjee, D. ( 2003; ). The Emb proteins of mycobacteria direct arabinosylation of lipoarabinomannan and arabinogalactan via an N-terminal recognition region and a C-terminal synthetic region. Mol Microbiol 50, 69–76.[CrossRef]
    [Google Scholar]
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Text files showing microarray data for the entire genome analysed are included here. Experiment series 1 contains the data where mRNA was prepared from ethambutol-treated/untreated cells harvested at constant optical density. Experiment series 2 contains the data where mRNA was prepared from ethambutol-treated/untreated cells harvested at different times. The generation of whole-genome DNA microarrays, synthesis of fluorescently labelled cDNA from total RNA, microarray hybridization, washing and data analysis were performed as described previously (Ishige , 2003; Polen & Wendisch 2004). Experiment series 1Experiment series 2_0hExperiment series 2_2hExperiment series 2_4hExperiment series 2_6hExperiment series 2_8hExperiment series 2_10hExperiment series 2_15hExperiment series 2_25h The phosphate starvation stimulon of determined by DNA microarray analyses. , 4519-4529. Genomewide expression analysis in amino acid-producing bacteria using DNA microarrays. , 215-232.

TEXT

Text files showing microarray data for the entire genome analysed are included here. Experiment series 1 contains the data where mRNA was prepared from ethambutol-treated/untreated cells harvested at constant optical density. Experiment series 2 contains the data where mRNA was prepared from ethambutol-treated/untreated cells harvested at different times. The generation of whole-genome DNA microarrays, synthesis of fluorescently labelled cDNA from total RNA, microarray hybridization, washing and data analysis were performed as described previously (Ishige , 2003; Polen & Wendisch 2004). Experiment series 1Experiment series 2_0hExperiment series 2_2hExperiment series 2_4hExperiment series 2_6hExperiment series 2_8hExperiment series 2_10hExperiment series 2_15hExperiment series 2_25h The phosphate starvation stimulon of determined by DNA microarray analyses. , 4519-4529. Genomewide expression analysis in amino acid-producing bacteria using DNA microarrays. , 215-232.

TEXT

Text files showing microarray data for the entire genome analysed are included here. Experiment series 1 contains the data where mRNA was prepared from ethambutol-treated/untreated cells harvested at constant optical density. Experiment series 2 contains the data where mRNA was prepared from ethambutol-treated/untreated cells harvested at different times. The generation of whole-genome DNA microarrays, synthesis of fluorescently labelled cDNA from total RNA, microarray hybridization, washing and data analysis were performed as described previously (Ishige , 2003; Polen & Wendisch 2004). Experiment series 1Experiment series 2_0hExperiment series 2_2hExperiment series 2_4hExperiment series 2_6hExperiment series 2_8hExperiment series 2_10hExperiment series 2_15hExperiment series 2_25h The phosphate starvation stimulon of determined by DNA microarray analyses. , 4519-4529. Genomewide expression analysis in amino acid-producing bacteria using DNA microarrays. , 215-232.

TEXT

Text files showing microarray data for the entire genome analysed are included here. Experiment series 1 contains the data where mRNA was prepared from ethambutol-treated/untreated cells harvested at constant optical density. Experiment series 2 contains the data where mRNA was prepared from ethambutol-treated/untreated cells harvested at different times. The generation of whole-genome DNA microarrays, synthesis of fluorescently labelled cDNA from total RNA, microarray hybridization, washing and data analysis were performed as described previously (Ishige , 2003; Polen & Wendisch 2004). Experiment series 1Experiment series 2_0hExperiment series 2_2hExperiment series 2_4hExperiment series 2_6hExperiment series 2_8hExperiment series 2_10hExperiment series 2_15hExperiment series 2_25h The phosphate starvation stimulon of determined by DNA microarray analyses. , 4519-4529. Genomewide expression analysis in amino acid-producing bacteria using DNA microarrays. , 215-232.

TEXT

Text files showing microarray data for the entire genome analysed are included here. Experiment series 1 contains the data where mRNA was prepared from ethambutol-treated/untreated cells harvested at constant optical density. Experiment series 2 contains the data where mRNA was prepared from ethambutol-treated/untreated cells harvested at different times. The generation of whole-genome DNA microarrays, synthesis of fluorescently labelled cDNA from total RNA, microarray hybridization, washing and data analysis were performed as described previously (Ishige , 2003; Polen & Wendisch 2004). Experiment series 1Experiment series 2_0hExperiment series 2_2hExperiment series 2_4hExperiment series 2_6hExperiment series 2_8hExperiment series 2_10hExperiment series 2_15hExperiment series 2_25h The phosphate starvation stimulon of determined by DNA microarray analyses. , 4519-4529. Genomewide expression analysis in amino acid-producing bacteria using DNA microarrays. , 215-232.

TEXT

Text files showing microarray data for the entire genome analysed are included here. Experiment series 1 contains the data where mRNA was prepared from ethambutol-treated/untreated cells harvested at constant optical density. Experiment series 2 contains the data where mRNA was prepared from ethambutol-treated/untreated cells harvested at different times. The generation of whole-genome DNA microarrays, synthesis of fluorescently labelled cDNA from total RNA, microarray hybridization, washing and data analysis were performed as described previously (Ishige , 2003; Polen & Wendisch 2004). Experiment series 1Experiment series 2_0hExperiment series 2_2hExperiment series 2_4hExperiment series 2_6hExperiment series 2_8hExperiment series 2_10hExperiment series 2_15hExperiment series 2_25h The phosphate starvation stimulon of determined by DNA microarray analyses. , 4519-4529. Genomewide expression analysis in amino acid-producing bacteria using DNA microarrays. , 215-232.

TEXT

Text files showing microarray data for the entire genome analysed are included here. Experiment series 1 contains the data where mRNA was prepared from ethambutol-treated/untreated cells harvested at constant optical density. Experiment series 2 contains the data where mRNA was prepared from ethambutol-treated/untreated cells harvested at different times. The generation of whole-genome DNA microarrays, synthesis of fluorescently labelled cDNA from total RNA, microarray hybridization, washing and data analysis were performed as described previously (Ishige , 2003; Polen & Wendisch 2004). Experiment series 1Experiment series 2_0hExperiment series 2_2hExperiment series 2_4hExperiment series 2_6hExperiment series 2_8hExperiment series 2_10hExperiment series 2_15hExperiment series 2_25h The phosphate starvation stimulon of determined by DNA microarray analyses. , 4519-4529. Genomewide expression analysis in amino acid-producing bacteria using DNA microarrays. , 215-232.

TEXT

Text files showing microarray data for the entire genome analysed are included here. Experiment series 1 contains the data where mRNA was prepared from ethambutol-treated/untreated cells harvested at constant optical density. Experiment series 2 contains the data where mRNA was prepared from ethambutol-treated/untreated cells harvested at different times. The generation of whole-genome DNA microarrays, synthesis of fluorescently labelled cDNA from total RNA, microarray hybridization, washing and data analysis were performed as described previously (Ishige , 2003; Polen & Wendisch 2004). Experiment series 1Experiment series 2_0hExperiment series 2_2hExperiment series 2_4hExperiment series 2_6hExperiment series 2_8hExperiment series 2_10hExperiment series 2_15hExperiment series 2_25h The phosphate starvation stimulon of determined by DNA microarray analyses. , 4519-4529. Genomewide expression analysis in amino acid-producing bacteria using DNA microarrays. , 215-232.

TEXT

Text files showing microarray data for the entire genome analysed are included here. Experiment series 1 contains the data where mRNA was prepared from ethambutol-treated/untreated cells harvested at constant optical density. Experiment series 2 contains the data where mRNA was prepared from ethambutol-treated/untreated cells harvested at different times. The generation of whole-genome DNA microarrays, synthesis of fluorescently labelled cDNA from total RNA, microarray hybridization, washing and data analysis were performed as described previously (Ishige , 2003; Polen & Wendisch 2004). Experiment series 1Experiment series 2_0hExperiment series 2_2hExperiment series 2_4hExperiment series 2_6hExperiment series 2_8hExperiment series 2_10hExperiment series 2_15hExperiment series 2_25h The phosphate starvation stimulon of determined by DNA microarray analyses. , 4519-4529. Genomewide expression analysis in amino acid-producing bacteria using DNA microarrays. , 215-232.

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