Development of a gene-editing strategy to overcome genetic intractability in <i>Lactobacillus johnsonii</i>

ABSTRACT Genetic editing in lactobacilli is often constrained by their robust defense mechanisms against foreign DNA and low transformation efficiency, resulting in pronounced species- and strain-specific variability in the performance of genetic tools. In this study, we developed a markerless gene editing system for Lactobacillus johnsonii MR1, a Jackson mouse isolate with a closed genome and well-documented host immunomodulatory properties. Initial testing revealed that existing transformation and gene-editing protocols produced low transformation efficiency, poor reproducibility, and exhibited poor plasmid stability in L. johnsonii MR1. To address these challenges, we established a counterselectable marker-based approach utilizing the upp gene, encoding uracil phosphoribosyltransferase, to enable efficient selection of recombinant colonies following plasmid curing. We generated an in-frame isogenic upp mutant strain ( L. johnsonii KR09) and constructed a temperature-sensitive counterselectable plasmid, pG+DualMarker3, derived from pG+Host9 and carrying the Lactobacillus acidophilus upp cassette. Deletion of upp conferred resistance to 5-fluorouracil (5-FU), which was reversed upon plasmid introduction. We demonstrated the efficacy of this system by creating an in-frame deletion of lacLM , encoding beta-galactosidase, and restoring the wild-type phenotype through complementation. Additionally, 5-FU resistance enabled differentiation and tracking of inoculated versus indigenous L. johnsonii populations in vivo in mouse models. This robust gene-replacement strategy enhances genetic accessibility in L. johnsonii MR1 and establishes a foundation for mechanistic studies of host colonization and host-microbe interactions in mouse models. IMPORTANCE Understanding the complex interplay between beneficial gut microbes and their hosts demands tools that enable precise gene editing of the microbes. Lactobacillus johnsonii MR1, a rodent isolate with host health beneficial properties, has posed major obstacles to gene editing due to its high strain heterogeneity and limited compatibility with existing molecular tools. In this study, we establish a robust markerless gene replacement system employing a counterselectable marker in L. johnsonii MR1, enabling efficient and targeted genome modifications. By expanding the genetic toolbox available for L. johnsonii , our approach provides a solid foundation for future investigations into microbe-microbe and host-microbe interactions and facilitates the engineering of strains with customized health benefits.