Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Construction of a Novel Shuttle Vector for Tetragenococcus species based on a Cryptic Plasmid from Tetragenococcus halophilus

  • Min Jae Kim (Division of Applied Life Science (BK21 Four), Graduate School) ;
  • Tae Jin Kim (Division of Applied Life Science (BK21 Four), Graduate School) ;
  • Yun Ji Kang (Division of Applied Life Science (BK21 Four), Graduate School) ;
  • Ji Yeon Yoo (Division of Applied Life Science (BK21 Four), Graduate School) ;
  • Jeong Hwan Kim (Division of Applied Life Science (BK21 Four), Graduate School)
  • Received : 2022.09.15
  • Accepted : 2022.12.19
  • Published : 2023.02.28
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Abstract

A cryptic plasmid (pTH32) was characterized from Tetragenococcus halophilus 32, an isolate from jeotgal, Korean traditional fermented seafood. pTH32 is 3,198 bp in size with G+C content of 35.84%, and contains 4 open reading frames (ORFs). orf1 and orf2 are 456 bp and 273 bp in size, respectively, and their translation products showed 65.16% and 69.35% similarities with RepB family plasmid replication initiators, respectively, suggesting the rolling-circle replication (RCR) mode of pTH32. orf3 and orf4 encodes putative hypothetical protein of 186 and 76 amino acids, respectively. A novel Tetragenococcus-Escherichia coli shuttle vector, pMJ32E (7.3 kb, Emr), was constructed by ligation of pTH32 with pBluescript II KS(+) and an erythromycin resistance gene (ErmC). pMJ32E successfully replicated in Enterococcus faecalis 29212 and T. halophilus 31 but not in other LAB species. A pepA gene, encoding aminopeptidase A (PepA) from T. halophilus CY54, was successfully expressed in T. halophilus 31 using pMJ32E. The transformant (TF) showed higher PepA activity (49.8 U/mg protein) than T. halophilus 31 cell (control). When T. halophilus 31 TF was subculturd in MRS broth without antibiotic at 48 h intervals, 53.8% of cells retained pMJ32E after 96 h, and only 2.4% of cells retained pMJ32E after 14 days, supporting the RCR mode of pTH32. pMJ32E could be useful for the genetic engineering of Tetragenococcus and Enterococcus species.

Keywords

Acknowledgement

This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (No. NRF-2020R1A2C100826711). Kim MJ, Kim TJ, Kang YJ, and Yoo JY have been supported by BK21 four program from MOE, Korea. T. halophilus 31 and 32 strains were kindly provided by professor Lee, Jong-Hoon at Kyonggi University, Korea.

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