Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Cloning and Characterization of the pyrH Gene Encoding UMP-Kinase from Lactobacillus reuteri ATCC 55739

  • PARK JAE-YONG (Division of Applied Life Science, Graduate School, Gyeongsang National University) ;
  • NAM SU JIN (Division of Applied Life Science, Graduate School, Gyeongsang National University) ;
  • KIM JONG-HWAN (Division of Applied Life Science, Graduate School, Gyeongsang National University) ;
  • JEONG SEON-JU (Division of Applied Life Science, Graduate School, Gyeongsang National University) ;
  • KIM JUNG KON (Division of Applied Life Science, Graduate School, Gyeongsang National University) ;
  • HA YEONG LAE (Division of Applied Life Science, Graduate School, Gyeongsang National University) ;
  • KIM JEONG HWAN (Institute of Agriculture & Life Science, Gyeongsang National University)
  • Published : 2005.06.01
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Abstract

From a genomic library of Lactobacillus reuteri ATCC 55739, one clone, NE347, carrying a pyrH gene encoding UMP kinase, was identified. pNE347 carried a 1.88 kb EcoRI fragment and the pyrH was located in the middle of the insert. pyrH ORF was 723 bp in size and capable of encoding UMP kinase composed of 240 amino acid residues. tsf encoding an elongation factor-Ts and frr encoding a ribosomal recycling factor were present upstream and downstream of pyrH, respectively. When introduced into E. coli KUR1244, a pyrH-negative strain, pNE347 restored the ability to grow at $42^{\circ}C$, indicating that pyrH from L. reuteri synthesized functional UMP kinase in E. coli. Northern blot experiment showed that pyrH and frr were cotranscribed as a 1.4 kb single transcript. pyrH was overexpressed in E. coli by using a pET26b(+) vector, and a major 25 kDa protein band appeared on SDS-polyacrylamide gel.

Keywords

References

  1. Blank, J., S. Nock, R. Kreutzer, and M. Sprinzl. 1996. Elongation factor Ts from Thermus thermophilus overproduction in Escherichia coli, quaternary structure and interaction with elongation factor Tu. Eur. J. Biochem. 236: 222-227 https://doi.org/10.1111/j.1432-1033.1996.00222.x
  2. Bucurenci, N., L. Serina, C. Zaharia, S. Landais, A. Danchin, and O. Barzu. 1998. Mutational analysis of UMP kinase from Escherichia coli. J. Bacteriol. 180: 473-477
  3. Dower, W. J., J. F. Miller, and C. W. Ragsdale. 1988. High efficiency transformation of E. coli by high voltage electroporation. Nucleic Acids Res. 16: 6127-6145 https://doi.org/10.1093/nar/16.13.6127
  4. Ingraham, J. L. and J. Neuhard. 1972. Cold-sensitive mutants of Salmonella typhimurium defective in uridine monophosphate kinase. J. Biol. Chem. 247: 6259-6265
  5. 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 fructanotransferase (CFTase) from Bacillus polymyxa MGL21. J. Microbiol. Biotechnol. 12: 921-928
  6. Kim, T. Y., J. M. Lee, H. C. Chang, D. K. Chung, J. H. Lee, J. H. Kim, and H. J. Lee. 1999. Effect of temperature and a carbon source on the expression of ${\beta}$-galactosidase gene of Lactococcus lactis ssp. lactis ATCC 7962. J. Microbiol. Biotechnol. 9: 201-205
  7. Laemmli, U. K. 1970. Cleavage of structural protein during the assembly of the head of bacteriophage T4. Nature 227: 680-685 https://doi.org/10.1038/227680a0
  8. 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 trom kimchi and characteriaztion of its nisZ gene. J. Microbiol. Biotechnol. 12: 389-397
  9. Ohnishi, M., L. Janosi, M. Shuda, H. Matsumoto, T. Hayashi, Y. Terawaki, and A. Kaji. 1999. Molecular cloning, sequencing, purification, and characterization of Pseudomonas aeruginosa ribosome recycling factor. J. Bacteriol. 181: 1281-1291
  10. Park, J. Y., S. J. Park, S. J. Nam, Y. L. Ha, and J. H. Kim. 2002. Cloning and characterization of L-Iactate dehydrogenase gene (ldhL) trom Lactobacillus reuteri ATCC 55739. J. Microbiol. Biotechnol. 12: 716- 721
  11. 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 trom kimchi and cloning of lacZ gene trom the isolated strain. J. Microbiol. Biotechnol. 12: 157-161
  12. Saavedra, J. 2000. Probiotics and infectious diarrhea. Am. J. Gastroenterol. 95: S16-S18 https://doi.org/10.1016/S0002-9270(99)00811-4
  13. Sambrook, J. E. F. Fritsch, and J. Maniatis. 1989. Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., U.S.A
  14. Serina, L., C. Blondin, E. Krin, O. Sismeiro, A. Danchin, H. Sakamoto, A.-M. Gilles, and O. Barzu. 1995. Escherichia coli UMP-kinase, a member of the aspartokinase family, is a hexamer regulated by guanine nucleotides and UTP. Biochemistry 34: 5066-5074 https://doi.org/10.1021/bi00015a018
  15. Smallshaw, J. and R. A. Kelln. 1992. Cloning, nucleotide sequence and expression of the Escherichia coli K-12 pyrH gene encoding UMP kinase. Genetics (Life Sci. Adv.) 11: 59-65
  16. Talarico, T. L., I. A. Casas, T. C. Chung, and W. J. Dobrogosz. 1988. Production and isolation of reuterin, a growth inhibitor produced by Lactobacillus reuteri. Antimicrob. Agents Chemother. 32: 1854-1858 https://doi.org/10.1128/AAC.32.12.1854
  17. Wadskov-Hansen, S. L., J. Martinussen, and K. Hammer. 2000. The pyrH gene of Lactococcus lactis subsp. cremoris encoding UMP kinase is transcribed as part of an operon including the frr1 gene encoding ribosomal recycling factor 1. Gene 241: 157-166 https://doi.org/10.1016/S0378-1119(99)00452-7