Equip Fusobacterium nucleatum genetic tool kits with compatible shuttle vectors and engineered intermediatory E. coli strains for enhanced transformation efficiency

Fusobacterium nucleatum, an oral microbe, is implicated in various human diseases, including oral-related diseases and tumors. However, efficient transformation was only achieved in limited strains of this bacterium. The challenges in conducting molecular level investigations of most strains due to their genetic intractability have hindered the biological studies of F. nucleatum. The restriction-modification (RM) systems is one of the known obstacles for efficient DNA transformation. Here, we used single molecule real time sequencing to elucidate the RM recognition sites and the corresponding methyltransferases (MTases) in two F. nucleatum strains. Based on the identified MTases, we engineered intermediatory E. coli host strains to bypass the RM systems, and showed that the plasmids harbored by these intermediatory strains can be efficiently electro-transformed, reaching 5000 transformants per microgram plasmids, paving the way for the development of efficient genetic modification tools. Furthermore, we successfully demonstrated that the conjugation-based DNA delivery to F. nucleatum can bypass the requirement of MTase methylations. By exploring the native plasmids from F. nucleatum, we identified new backbones for construction of shuttle vectors and established a dual-plasmid system for the first time, offering new avenues for genetic manipulation in this bacterium. Additionally, we evaluate promoters with variable strengths with a luciferase-based reporter system in F. nucleatum, providing valuable insights for future gene editing studies in bacterium and contributing to our understanding of its pathogenesis. All the tools developed in this study was shared via the WeKwikgene (https://wekwikgene.wllsb.edu.cn/). Impact StatementFusobacterium nucleatum, a key opportunistic pathogen implicated in periodontal diseases, rheumatoid arthritis, and tumors, presents significant challenges due to its limited transformation efficiency and lack of gene-editing tools. In this study, we present an advancement -a streamlined and robust pipeline that enhances transformation efficiency by approximately 103-fold in F. nucleatum, reaching 5000 CFU per microgram plasmids. This represents a significant breakthrough, marking the first report to achieve such a remarkable improvement in transformation efficiency in this pathogen. This improvement paves the way for the genome-wide level mutagenesis study in this bacterium.