Enhancing Electroporation-Mediated Transformation Efficiency in Bacteroides uniformis BNCC368209 Through Electroporation Optimization and Targeted Gene Knockdown

Bacteroides uniformis is a promising candidate for targeted modulation of the gut microbiota. However, the functions of its genes remain to be elucidated. Electroporation-based genetic manipulation systems are powerful tools for studying gene function; However, the low efficiency of electroporation-mediated transformation of Bacteroides with plasmids isolated from Escherichia coli limits their application. In this study, we improved the transformation efficiency of B. uniformis BNCC368209 with E. coli-derived plasmids by optimizing electroporation conditions and knocking down selected genes. Under optimized electroporation conditions, transformation efficiency with E. coli-derived plasmids reached 2.35 × 104 CFU/μg DNA. Under the same optimized conditions, transformation efficiency with Bacteroides-derived plasmids reached 3.25 × 106 CFU/μg DNA. RNA sequencing (RNA-seq) was used to compare post-selection transformants obtained with B. uniformis-derived and E. coli-derived plasmids as a hypothesis-generating screen. Of the 905 differentially expressed genes, nine candidates were prioritized for subsequent CRISPRi testing based on functional annotation and RT-qPCR screening. CRISPR interference (CRISPRi) using catalytically inactive Cas9 (dCas9) was used to generate strains with targeted knockdown of these genes. Knockdown of gene0629 (mraY) and gene0737 (hupB) increased transformation efficiency with E. coli-derived plasmids to 21.9 and 41.9 times that of the control strain without gene knockdown, respectively. Phenotypic analyses suggested altered cell envelope integrity following mraY knockdown and showed increased pB006 retention following hupB knockdown. These phenotypes may be associated with improved transformation efficiency, although causal relationships remain to be established. Together, these findings provide a strategy for improving transformation efficiency in B. uniformis and may inform the development of genetic manipulation systems for other Bacteroides species.