Abstract
A gene encoding a homolog of purine efflux proteins of Escherichia coli and Bacillus subtilis was identified in the genome of Corynebacterium glutamicum and designated as cepA. The gene encoded a putative protein product, containing 12 transmembrane helixes, which is a typical feature of integral membrane transport proteins. To elucidate the function of the gene, we constructed a cepA deletion mutant (ΔcepA) and a cepA-overexpressing strain and analyzed their physiological characteristics. The cepA gene could be deleted with no critical effect on cell growth. However, the cell yield of a ΔcepA strain was decreased by 10% as compared to that of a strain carrying a cepA-overexpression plasmid (P180-cepA). Further analysis identified increased resistance of the P180-cepA strain to the purine analogues 6-mercaptopurine and 6-mercaptoguanine, but not to 2-aminopurine and purine nucleoside analogues. Moreover, this strain showed increased resistance to the antibiotics nalidixic acid and ampicillin. Collectively, these data suggest that cepA is a novel multidrug resistance gene and probably functions in the efflux of toxic substances from the inside of cells to the environment, thus allowing cells to reach a higher cell yield.
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References
Alekshun, M.N. and Levy, S.B. 2007. Molecular mechanisms of antibacterial multidrug resistance. Cell 128, 1037–1050.
Cserzo, M., Wallin, E., Simon, I., von Heijne, G., and Elofsson, A. 1997. Prediction of transmembrane α-helices in prokaryotic membrane proteins: the dense alignment surface method. Prot. Eng. 10, 673–676.
Eggeling, L. and Sahm, H. 2003. New ubiquitous translocators: amino acid export by Corynebacterium glutamicum and Escherichia coli. Arch. Microbiol. 180, 155–160
Follettie, M.T., Peoples, O.P., Agoropoulou, C., and Sinskey, A.J. 1993. Gene structure and expression of the Corynebacterium flavum N13 ask-asd operon. J. Bacteriol. 175, 4096–4103.
Gronskiy, S.V., Zakataeva, N.P., Vitushkina, M.V., Ptitsyn, L.R., Altman, I.B., Novikova, A.E., and Livshits, V.A. 2005. The yicM (nepI) gene of Escherichia coli encodes a major facilitator superfamily protein involved in efflux of purine ribonucleosides. FEMS Microbiol. Lett. 250, 39–47.
Hwang, B.J., Yeom, H.J., Kim, Y., and Lee, H.S. 2002. Corynebacterium glutamicum utilizes both transsulfuration and direct sulfhydrylation pathways for methionine biosynthesis. J. Bacteriol. 184, 1277–1286.
Ikeda, M. and Nakagawa, S. 2003. The Corynebacterium glutamicum genome: features and impacts on biotechnological processes. Appl. Microbiol. Biotechnol. 62, 99–109.
Johansen, L.E., Nygaard, P., Lassen, C., Agers Y., and Saxild, H.H. 2003. Definition of a second Bacillus subtilis pur regulon comprising the pur and xpt-pbuX operons plus pbuG, nupG (yxjA), and pbuE (ydhL). J. Bacteriol. 185, 5200–5209.
Kalinowski, J., Bathe, B., Bartels, D., Bischoff, N., Bott, M., Burkovski, A., Dusch, N., Eggeling, L., Eikmanns, B.J., Gaigalat, L., and et al. 2003. The complete Corynebacterium glutamicum genome sequence and its impact on the production of L-aspartate-derived amino acids and vitamins. J. Biotechnol. 104, 5–25.
Kim, H.J., Kim, T.H., Kim, Y., and Lee, H.S. 2004. Identification and characterization of glxR, a gene involved in regulation of glyoxylate bypass in Corynebacterium glutamicum. J. Bacteriol. 186, 3453–3460.
Kim, H.J., Kim, Y., Lee, M.S., and Lee, H.S. 2001. Gene lmrB of Corynebacterium glutamicum confers efflux-mediated resistance to lincomycin. Mol. Cells 12, 112–116.
Lee, J.Y., Park, J.S., Kim, H.J., Kim, Y., and Lee, H.S. 2012. Corynebacterium glutamicum whcB, a stationary phase-specific regulatory gene. FEMS Microbiol. Lett. 327, 103–109.
Leuchtenberger, W., Huthmacher, K., and Drauz, K. 2005. Biotechnological production of amino acids and derivatives: current status and prospects. Appl. Microbiol. Biotechnol. 69, 1–8.
Li, X.Z. and Nikaido, H. 2004. Efflux-mediated drug resistance in bacteria. Drugs 64, 159–204.
Marin, K. and Krämer, R. 2007. Amino acid transport systems in biotechnologically relevant bacteria, pp. 290–325. In Wendisch, V.F. (ed). Microbiol Monogr. Springer, Heidelberg, Germany.
Nikaido, H. 1996. Multidrug efflux pumps of Gram-negative bacteria. J. Bacteriol. 178, 5853–5859.
Nygaard, P. and Saxild, H.H. 2005. The purine efflux pump PbuE in Bacillus subtilis modulates expression of the PurR and G-box (XptR) regulons by adjusting the purine base pool size. J. Bacteriol. 187, 791–794.
Park, J.S., Lee, J.Y., Kim, H.J., Kim, E.S., Kim, P., Kim, Y., and Lee, H.S. 2012. The role of Corynebacterium glutamicum spiA gene in whcA-mediated oxidative stress gene regulation. FEMS Microbiol. Lett. 331, 63–69.
Park, S.D., Lee, S.N., Park, I.H., Choi, J.S., Jeong, W.K., Kim, Y., and Lee, H.S. 2004. Isolation and characterization of transcriptional elements from Corynebacterium glutamicum. J. Microbiol. Biotechnol. 14, 789–795.
Sambrook, J. and Russell, D.W. 2001. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA.
Schäfer, A., Tauch, A., Jager, W., Kalinowski, J., Thierbach, G., and 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.
Sheremet, A.S., Gronskiy, S.V., Akhmadyshin, R.A., Novikova, A.E., Livshits, V.A., Shakulov, R.S., and Zakataeva, N.P. 2011. Enhancement of extracellular purine nucleoside accumulation by Bacillus strains through genetic modifications of genes involved in nucleoside export. J. Ind. Microbiol. Biotechnol. 38, 65–70.
von der Osten, C.H., Barbas, C.F., Wong, C.H., and Sinskey, A.J. 1989. Molecular cloning, nucleotide sequence and fine-structural analysis of the Corynebacterium glutamicum fda gene: structural comparison of C. glutamicum fructose-1,6-biphosphate aldolase to class I and class II aldolases. Mol. Microbiol. 3, 1625–1637.
Yukawa, H., Omumasaba, C.A., Nonaka, H., Kós, P., Okai, N., Suzuki, N., Suda, M., Tsuge, Y., Watanabe, J., Ikeda, Y., Vertès, A.A., and Inui, M. 2007. Comparative analysis of the Corynebacterium glutamicum group and complete genome sequence of strain R. Microbiology 153, 1042–1058.
Zakataeva, N.P., Gronskiy, S.V., Sheremet, A.S., Kutukova, E.A., Novikova, A.E., and Livshits, V.A. 2007. A new function for the Bacillus PbuE purine base efflux pump: efflux of purine nucleosides. Res. Microbiol. 158, 659–665.
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Sim, SY., Hong, EJ., Kim, Y. et al. Analysis of cepA encoding an efflux pump-like protein in Corynebacterium glutamicum . J Microbiol. 52, 278–283 (2014). https://doi.org/10.1007/s12275-014-3461-1
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DOI: https://doi.org/10.1007/s12275-014-3461-1