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Characterization of pFMBL1, a small cryptic plasmid isolated from Leuconostoc mesenteroides SY2

Seon-Ju Jeong1, Jae-Yong Park, Hyong Joo Lee

  • 1Division of Applied Life Science, Graduate School, Gyeongsang National University, Jinju, Republic of Korea.

Plasmid
|November 7, 2006
PubMed

Insights

A novel cryptic plasmid, pFMBL1, was identified in Leuconostoc mesenteroides from Kimchi. This plasmid, essential for replication, was engineered into a shuttle vector, pSJ33E, enabling successful transformation of related bacteria.

Area of Science:

  • Microbiology
  • Molecular Biology
  • Genetics

Background:

  • Cryptic plasmids are extrachromosomal DNA elements found in bacteria, often lacking obvious functions.
  • Leuconostoc mesenteroides is a lactic acid bacterium commonly found in fermented foods like Kimchi.
  • Understanding plasmid replication mechanisms is crucial for genetic manipulation and strain improvement.

Purpose of the Study:

  • To isolate and characterize a cryptic plasmid (pFMBL1) from Leuconostoc mesenteroides SY2.
  • To identify genes involved in plasmid replication and construct a functional shuttle vector.
  • To assess the transformation efficiency and stability of the constructed shuttle vector in various lactic acid bacteria.

Main Methods:

  • Nucleotide sequencing and open reading frame (ORF) analysis of the isolated plasmid.
  • RNA transcript analysis to identify expressed genes.
  • Construction of an Escherichia coli-Leuconostoc shuttle vector (pSJ33E) using a minimal replication fragment.
  • Transformation of Leuconostoc and Lactobacillus species with pSJ33E.
  • Stability assays in the absence of selective pressure.

Main Results:

  • The 4661bp cryptic plasmid pFMBL1 was sequenced, revealing two ORFs (orf1 and orf2).
  • orf2 showed homology to proteins involved in plasmid replication; its transcript was detected.
  • A 3.5kb fragment containing both ORFs was sufficient for replication and used to create the shuttle vector pSJ33E.
  • pSJ33E successfully transformed L. mesenteroides and Lactobacillus brevis with high efficiencies.
  • The plasmid likely replicates via a theta mechanism and demonstrated stable maintenance in L. mesenteroides without antibiotics.

Conclusions:

  • pFMBL1 possesses functional elements for replication and can be utilized for genetic engineering in Leuconostoc species.
  • The developed shuttle vector pSJ33E is a valuable tool for the genetic manipulation of L. mesenteroides and related bacteria.
  • The findings contribute to the understanding of cryptic plasmid biology and their potential applications in food microbiology.

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