The insertion of the inverted repeat of an insertion sequence (IS) element from Lacticaseibacillus rhamnosus changes the host range and stability of pGK12, a shuttle vector for lactic acid bacteria.

Insertion sequences (ISs) are key components of most bacterial genomes and play a crucial role in bacterial mutagenesis. In this study, we observed the insertion of an IS element, ISLrh, from the Lacticaseibacillus rhamnosus M1 genome into plasmid pGK12, resulting in the generation of a new plasmid, pTRK829. This insertion enabled pTRK829 to replicate in hosts previously incompatible with pGK12, including L. rhamnosus M1, L. rhamnosus GG (LGG), Lacticaseibacillus casei ATCC 393, and Lacticaseibacillus paracasei ATCC 25598. However, the ISLrh-inserted plasmid, pTRK829, was unstable and underwent a spontaneous deletion, resulting in a smaller and more stable variant, pTRK830, which retained ISLrh. Characterization of pTRK829 and pTRK830 across several host strains showed that ISLrh insertion led to a dramatic alteration in host range and impacted plasmid stability and copy number. Sequence and functional analysis of pTRK830 revealed that the terminal inverted repeats (IRs) of the inserted ISLrh and its insertion location were essential for plasmid replication in LGG. Finally, pTRK830 was successfully used as an expression vector for heterologous β-glucuronidase expression in LGG, L. casei ATCC 393, and L. paracasei ATCC 25598. In conclusion, this study demonstrated that the insertion of the IRs from ISLrh at a specific location can directly change the host range and stability of pGK12. Furthermore, we also demonstrated the potential of pTRK830 as a new cloning and expression vector for genetically intractable lactobacilli.

Importance: This study highlights the significant impact of insertion sequence (IS) elements on plasmid replication in lactobacilli. The spontaneous integration of an IS element from the Laticaseibacillus rhamnosus genome into plasmid pGK12 not only expands its host range in previously incompatible strains but also changes plasmid stability and copy number. This expansion of the plasmid's host range is crucial for developing versatile genetic tools across diverse lactobacilli species. Additionally, the stable plasmid variant of pGK12 with the IS element insertion offers a valuable tool for cloning and gene expression in lactobacilli. These findings enhance our understanding of plasmid-IS element interactions and may provide insight into a new method to expand the host range of existing plasmids.