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

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

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