Xie Zifan, Jin Yong-Su, Klaenhammer Todd R, Miller Michael J
Food Science and Human Nutrition, University of Illinois Urbana-Champaign, Urbana, Illinois, USA.
Carl R. Woese Institute for Genomic Biology, University of Illinois Urbana-Champaign, Urbana, Illinois, USA.
Appl Environ Microbiol. 2025 Apr 23;91(4):e0190824. doi: 10.1128/aem.01908-24. Epub 2025 Mar 14.
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 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 M1, GG (LGG), ATCC 393, and 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, ATCC 393, and 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.
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 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.
插入序列（ISs）是大多数细菌基因组的关键组成部分，在细菌诱变中起关键作用。在本研究中，我们观察到来自M1基因组的一个IS元件ISLrh插入到质粒pGK12中，产生了一个新的质粒pTRK829。这种插入使pTRK829能够在先前与pGK12不兼容的宿主中复制，包括M1、GG（LGG）、ATCC 393和ATCC 25598。然而，插入了ISLrh的质粒pTRK829不稳定，会发生自发缺失，产生一个更小且更稳定的变体pTRK830，它保留了ISLrh。对pTRK829和pTRK830在多个宿主菌株中的特性分析表明，ISLrh插入导致宿主范围发生显著改变，并影响质粒稳定性和拷贝数。对pTRK830的序列和功能分析表明，插入的ISLrh的末端反向重复序列（IRs）及其插入位置对于在LGG中质粒复制至关重要。最后，pTRK830成功用作在LGG、ATCC 393和ATCC 25598中异源β-葡萄糖醛酸酶表达的表达载体。总之，本研究表明在特定位置插入来自ISLrh的IRs可直接改变pGK12的宿主范围和稳定性。此外，我们还证明了pTRK830作为遗传上难以处理的乳酸杆菌的新型克隆和表达载体的潜力。
本研究突出了插入序列（IS）元件对乳酸杆菌中质粒复制的重大影响。来自基因组的一个IS元件自发整合到质粒pGK12中，不仅扩大了其在先前不兼容菌株中的宿主范围，还改变了质粒稳定性和拷贝数。这种质粒宿主范围的扩大对于开发适用于多种乳酸杆菌物种的通用遗传工具至关重要。此外，插入了IS元件的pGK12稳定质粒变体为乳酸杆菌中的克隆和基因表达提供了有价值的工具。这些发现增强了我们对质粒 - IS元件相互作用的理解，并可能为扩大现有质粒宿主范围的新方法提供见解。
