Construction of a Novel Shuttle Vector for Tetragenococcus species based on a Cryptic Plasmid from Tetragenococcus
Min Jae Kim1, Tae Jin Kim1, Yun Ji Kang1
1Division of Applied Life Science (BK21 Four), Graduate School, Gyeongsang National University, Jinju 52828, Republic of Korea.
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.
Insights
A novel plasmid, pTH32, was identified in *Tetragenococcus halophilus* and used to create a shuttle vector, pMJ32E. This vector enables gene expression in *Tetragenococcus* and *Enterococcus* species.
Area of Science:
- Microbiology
- Molecular Biology
- Genetics
Background:
- Characterization of cryptic plasmids is essential for understanding microbial genetics.
- Tetragenococcus halophilus is a key species in Korean fermented seafood (jeotgal).
- Development of genetic tools for lactic acid bacteria (LAB) is crucial for strain improvement.
Purpose of the Study:
- To characterize the cryptic plasmid pTH32 from *Tetragenococcus halophilus*.
- To construct and evaluate a novel *Tetragenococcus-Escherichia coli* shuttle vector.
- To demonstrate the utility of the shuttle vector for gene expression in *Tetragenococcus* species.
Main Methods:
- Plasmid DNA isolation, sequencing, and bioinformatics analysis.
- Construction of the shuttle vector pMJ32E by ligating pTH32 with pBluescript II KS(+) and an erythromycin resistance gene.
- Transformation of *Enterococcus faecalis* and *T. halophilus* with pMJ32E.
- Expression of the aminopeptidase A (PepA) gene using the constructed shuttle vector.
- Plasmid stability assessment in *T. halophilus*.
Main Results:
- The cryptic plasmid pTH32 (3,198 bp) was characterized, revealing four open reading frames (ORFs) suggesting a rolling-circle replication (RCR) mechanism.
- A novel shuttle vector, pMJ32E (7.3 kb, Emr), was successfully constructed and demonstrated replication in *Enterococcus faecalis* and *T. halophilus*.
- The *pepA* gene was expressed in *T. halophilus* using pMJ32E, resulting in increased aminopeptidase A activity.
- Plasmid stability studies supported the RCR mode of replication for pTH32.
Conclusions:
- The characterized plasmid pTH32 provides a foundation for developing genetic tools in *Tetragenococcus* species.
- The novel shuttle vector pMJ32E is effective for genetic manipulation and gene expression in *Tetragenococcus* and *Enterococcus* species.
- pMJ32E holds potential for the genetic engineering of industrially relevant *Tetragenococcus* and *Enterococcus* strains.
