Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Characterization of the Catabolite Control Protein (CcpA) Gene from Leuconostoc mesenteroides SY1

  • PARK JAE-YONG (Division of Applied Life Science, Graduate School, and Gyeongsang National University) ;
  • PARK JIN-SIK (Division of Applied Life Science, Graduate School, and Gyeongsang National University) ;
  • KIM JONG-HWAN (Division of Applied Life Science, Graduate School, and Gyeongsang National University) ;
  • JEONG SEON-JU (Division of Applied Life Science, Graduate School, and Gyeongsang National University) ;
  • CHUN JIYEON (Division of Applied Life Science, Graduate School, and Gyeongsang National University) ;
  • LEE JONG-HOON (Department of Foods and Biotechnology, Kyunggi University) ;
  • KIM JEONG HWAN (Institute of Agriculture & Life Science, Gyeongsang National University)
  • Published : 2005.08.01
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Abstract

The ccpA gene encoding catabolite control protein A (CcpA) of Leuconostoc mesenteroides SYl, a strain isolated from kimchi, was cloned, sequenced, analyzed for transcript, and overexpressed in Escherichia coli. The ccpA ORF (open reading frame) is 1,011 bp in size, which can encode a protein of 336 amino acid residues with a molecular mass of 36,739 Da. The transcription start site was mapped at a position 49 nucleotides upstream of the start codon, and promoter sequences were also identified. The putative cre site overlapped with the -35 promoter sequence. The deduced amino acid sequence of the CcpA contained the helix-turn-helix motif found in many DNA-binding regulatory proteins. CcpA from 1. mesenteroides SY1 had $54.6\%$ identity with CcpA from Lactobacillus casei. The Northern blot experiment showed that ccpA was transcribed as a single 1.1 kb transcript, and transcription was repressed when grown on media containing glucose. CcpA was overproduced in E. coli BL21(DE3) cells using the pET expression vector, and purified to an apparent homogeneity. Gel Mobility Shift Assay with purified CcpA and a DNA fragment containing the ere sequence of the $\alpha$-galactosidase gene (aga) from L. mesenteroides SY1 revealed that CcpA bound specifically to the cre site of aga.

Keywords

References

  1. Chyun, J. H. and H. S. Rhee. 1976. Studies on the volatile fatty acids and carbon dioxide produced in different kimchis. Kor. J. Food Sci. Technol. 8: 90-94
  2. Deutscher, J., E. KUster, U. Bergstedt, V. Charrier, and W. Hillen. 1995. Protein kinase-dependent HPr/CcpA interaction links glycolytic activity to carbon catabolite repression in gram-positive bacteria. Mol. Microbial. 15: 1049-1053 https://doi.org/10.1111/j.1365-2958.1995.tb02280.x
  3. Egeter, O. and Y. Miwa. 1994. Catabolite repression mediated by the catabolite control protein CcpA protein. J. Bacterial. 176: 511-513 https://doi.org/10.1128/jb.176.2.511-513.1994
  4. Faires, N., S. Tobisch, S. Bachem, J. Martin-Verstraete, M. Hecker, and J. Sti.ilke. 1999. The catabolite control protein CcpA controls ammonium assimilation in Bacillus subtilis. J. Mol. Microbial. Biotechnol. 1: 141-148
  5. Fujita, Y., Y. Miwa, A. Galinier, and J. Deutscher. 1995. Specific recognition of the Bacillus subtilis gnt cis-acting catabolite-responsive element by a protein complex formed between CcpA and seryl-phosphorylated HPr. Mol. Microbial. 17: 953-960 https://doi.org/10.1111/j.1365-2958.1995.mmi_17050953.x
  6. Galinier, A., J. Haiech, M.-C. Kilhofer, M. Jaquinod, J. Sti.ilke, J. Deutscher, and J. Martin-Verstraete. 1997. The Bacillus subtilis crh gene encodes an HPr-like protein involved in carbon catabolite repression. Proc. Natl. Acad. Sci. USA 94: 8439-8444
  7. Gill, S. C. and P. H. von Hippel 1989. Calculation of protein extinction coefficients from amino acid sequence data. Anal. Biochem. 182: 319-326 https://doi.org/10.1016/0003-2697(89)90602-7
  8. Gosseringer, R., E. KUster, A. Galinier, J. Deutscher, and W. Hillen. 1997. Cooperative and non-cooperative DNA binding modes of catabolite control protein CcpA from Bacillus megaterium result from sensing two different signals. J. Mol. Biol. 266: 665-676 https://doi.org/10.1006/jmbi.1996.0820
  9. Grundy, F. J., D. A. Waters, T. Y. Takova, and T. M. Henkin. 1993. Identification of genes involved in utilization of acetate and acetoin in Bacillus subtilis. Mol. Microbiol. 10: 259-271 https://doi.org/10.1111/j.1365-2958.1993.tb01952.x
  10. Hall, T. A. 1999. BioEdit: A user-friendly biological sequence alignment editor and analysis program for Windows 95/98/ NT. Nucl. Acids Symp. Ser. 41: 95-98
  11. Han, H. U., C. R. Lim, and H. K. Park. 1990. Determination of microbial community as an indicator ofkimchi fermentation. Kor. J. Food Sci. Technol. 22: 26-32
  12. Henkin, T. M., F. J. Grundy, W. L. Nicholson, and G H. Chambliss. 1991. Catabolite repression of a-amylase gene expression in Bacillus subtilis involves a trans-acting gene product homologous to the Escherichia coli lacl and galR repressors. Mol. Microbiol. 5: 575-584 https://doi.org/10.1111/j.1365-2958.1991.tb00728.x
  13. Higgins D., J. Thompson, T. Gibson, J. D. Thompson, D. G Higgins, and T. J. Gibson. 1994. CLUSTAL W: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22: 4673-4680 https://doi.org/10.1093/nar/22.22.4673
  14. Hueck, C. J. and W. Hillen. 1995. Catabolite repression in Bacillus subilis: A global regulatory mechanism for the gram-positive bacteria. Mol. Microbiol. 15: 395-401 https://doi.org/10.1111/j.1365-2958.1995.tb02252.x
  15. Inacio, J. M., C. Costa, and I. de Sa-Nogueira. 2003. Distinct molecular mechanisms involved in carbon catabolite repression of the arabinose regulon in Bacillus subtilis. Microbiology 149: 2345-2355 https://doi.org/10.1099/mic.0.26326-0
  16. Jeong, S. J., D. J. You, H. J. Kwon, S. Kanaya, N. Kunihiro, K. H. Kim, Y. H. Kim, and B. W. Kim. 2002. Cloning and characterization of cycloinulooligosaccharide fiuctanotransferase . (CFTase) from Bacillus polymyxa MGL21. J. Microbiol. Biotechnol. 12: 921-928
  17. Kravanja, M., R. Engelmann, V. Dossonnet, M. Bluggel, H. E. Meyer, R. Frank, A. Galinier, J. Deutscher, N. Schnell, and W. Hengstenberg. 1999. The hprK gene of Enterococcus faecalis encodes a novel bifunctional enzyme: The HPr kinase/phosphatase. Mol. Microbiol. 31: 59-66 https://doi.org/10.1046/j.1365-2958.1999.01146.x
  18. Koster, E., T. Hilbich, M. K. Dahl, and W. Hillen. 1999. Mutations in catabolite control protein CcpA separating growth effects from catabolite repression. J. Bacterial. 181: 4125-4128
  19. Lee, K. H., G. S. Moon, J. Y. An, H. J. Lee, H. C. Cahng, D. K. Chung, J. H. Lee, and J. H. Kim. 2002. Isolation of a nisin-producing Lactococcus lactis strain from kimchi and characteriaztion of its nisZ gene. J. Microbiol. Biotechnol. 12: 389-397
  20. Luesink, E. J., R. E. van Herpen, B. P. Grossiord, O. P. Kuipers, and W. M. de Vos. 1998. Transcriptional activation of the glycolytic las operon and catabolite repression of the gal operon in Lactococcus lactis are mediated by the catabolite control protein CcpA. Mol. Microbiol. 30: 789-798 https://doi.org/10.1046/j.1365-2958.1998.01111.x
  21. Mahr, K., W. Hillen, and F. Titgemeyer. 2000. Carbon catabolite repression in Lactobacillus pentosus: Analysis of the ccpA region. Appl. Environ. Microbiol. 66: 277-283 https://doi.org/10.1128/AEM.66.1.277-283.2000
  22. Monedero, V., M. J. Gosalbes, and G. Perez-Martinez. 1997. Catabolite repression in Lactobacillus casei ATCC 393 is mediated by CopA. J. Bacteriol. 179: 6657-6664 https://doi.org/10.1128/jb.179.21.6657-6664.1997
  23. Muscariello, L., R. Marasco, M. de Felice, and M. Sacco. 2001 The functional ccpA gene is required for carbon catabolite repression in Lactobacillus plantarum. Appl. Environ. Microbiol. 67: 2903-2907 https://doi.org/10.1128/AEM.67.7.2903-2907.2001
  24. Nam, S. J., J. Y. Park, J. K. Kim, Y. L. Hae, H. D. Yun, and J. H. Kim. 2004. Cloning of pdh genes encoding subunits of pyruvate dehydrogenase complex from Lactobacillus reuteri ATCC 55739. J. Microbiol. Biotechnol. 14: 197-201
  25. Park, J. Y., S. J. Park, S. J. Nam, Y. L. Ha, and J. H. Kim. 2002. Cloning and characterization of the L-Iactate dehydrogenase gene (ldhL) from Lactobacillus reuteri ATCC 55739. J. Microbiol. Biotechnol. 12: 716-721
  26. Park, R.-J., K.-H. Lee, S.-J. Kim, J.-Y. Park, S.-J. Nam, H.-D. Yun, H.-J. Lee, H. C. Chang, D. K. Chung, J.-H. Lee, Y H. Park, and J. H. Kim. 2002. Isolation of Lactococcus lactis strain with ${\beta}$-galactosidase activity from kimchi and cloning of lacZ gene from the isolated strain. J. Microbiol. Biotechnol. 12: 157-161
  27. Reizer, J., C. Hoischen, F. Titgemeyer, C. Rivolta, R. Rabus, J. Stlilke, D. Karamata, M. H. Saier, Jr., and W. Hillen. 1998. A novel protein kinase that controls carbon catabolite repression in bacteria. Mol. Microbiol. 27: 1157-1169 https://doi.org/10.1046/j.1365-2958.1998.00747.x
  28. Renna, M. C., N. Najimudin, L. R. Winik, and S. A. Zahler. 1993. Regulation of the Bacillus subtilis alsS, alsD, and alsR genes involved in post-exponential-phase production of acetoin. J. Bacteriol. 175: 3863 - 3875 https://doi.org/10.1128/jb.175.12.3863-3875.1993
  29. Ryu, J. Y., H. S. Lee, and H. S. Rhee. 1984. Changes of organic acids and volatile flavor compounds in kimchi fermented with different ingredients. Kor. J. Food Sci. Technol. 16: 169-173
  30. Shin, B. S., S. K. Choi, and S. H. Park. 1999. Regulation of the Bacillus subtilis phosphotransacetylase gene. J. Biochem. (Tokyo) 126: 333-339 https://doi.org/10.1093/oxfordjournals.jbchem.a022454
  31. Stlilke, J. and W. Hillen. 1999. Carbon catabolite repression in bacteria. Curr. Opin. Microbiol. 2: 195-201 https://doi.org/10.1016/S1369-5274(99)80034-4
  32. Zhang, J. and T. L Madden. 1997. PowerBLAST: A new network BLAST application for interactive or automated sequence analysis and annotation. Genome Res. 7: 649-656 https://doi.org/10.1101/gr.7.6.649