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

• DOI: 10.1128/aem.01908-24
• Publication Date: 2025-03-14T13:01:05Z
• Authors: Zifan Xie; Yong-Su Jin; Todd R. Klaenhammer; Michael J. Miller
• Keywords: Insertion sequence; Shuttle vector; Inverted repeat; Cloning vector

ABSTRACT Insertion sequences (ISs) are key components of most bacterial genomes and play a crucial role in mutagenesis. In this study, we observed the insertion an IS element, ISLrh, from Lacticaseibacillus rhamnosus M1 genome into plasmid pGK12, resulting generation new plasmid, pTRK829. This enabled pTRK829 to replicate hosts previously incompatible with including L. M1, GG (LGG), casei ATCC 393, paracasei 25598. However, ISLrh-inserted pTRK829, was unstable underwent spontaneous deletion, smaller more stable variant, pTRK830, which retained ISLrh. Characterization pTRK830 across several host strains showed that ISLrh led dramatic alteration range impacted stability copy number. Sequence functional analysis revealed terminal inverted repeats (IRs) inserted its location were essential for replication LGG. Finally, successfully used as expression vector heterologous β-glucuronidase LGG, conclusion, study demonstrated IRs at specific can directly change pGK12. Furthermore, also potential cloning genetically intractable lactobacilli. IMPORTANCE highlights significant impact sequence (IS) elements on The integration element Laticaseibacillus pGK12 not only expands but changes expansion plasmid's is developing versatile genetic tools diverse lactobacilli species. Additionally, variant offers valuable tool gene These findings enhance our understanding plasmid-IS interactions may provide insight method expand existing plasmids.