pIH01, a small cryptic plasmid from Leuconostoc citreum IH3

Area of Science:

• Microbiology
• Molecular Biology
• Genetics

Background:

• Cryptic plasmids are extrachromosomal DNA elements that do not confer an obvious phenotype.
• Understanding cryptic plasmids is crucial for developing genetic tools in various bacterial species.
• Leuconostoc citreum is a bacterium relevant in food fermentation and probiotic applications.

Purpose of the Study:

• To characterize the cryptic plasmid pIH01 from Leuconostoc citreum IH3.
• To assess the potential of pIH01 as a basis for developing a shuttle vector for bacterial genetic manipulation.

Main Methods:

• Plasmid DNA extraction and characterization.
• Sequence analysis of the open reading frame and replication origin.
• Construction of a shuttle vector by integrating antibiotic resistance and replication origins.
• Transformation experiments in Leuconostoc, Lactobacillus plantarum, and Lactococcus lactis.

Main Results:

• The 1.8-kb cryptic plasmid pIH01 was fully characterized.
• pIH01 encodes a RepC class protein with a pT181-type double-strand origin, indicating rolling circle replication.
• The derived shuttle vector successfully transformed Leuconostoc strains, Lactobacillus plantarum, and Lactococcus lactis.

Conclusions:

• The pIH01 cryptic plasmid possesses characteristics suitable for replication via a rolling circle mechanism.
• A functional shuttle vector was successfully constructed using pIH01, enabling genetic manipulation in multiple lactic acid bacteria.
• pIH01-based vectors offer a promising tool for the genetic engineering of Leuconostoc species.