Stabilizing genetically unstable simple sequence repeats in the Campylobacter jejuni genome by multiplex genome editing: a reliable approach for delineating multiple phase-variable genes

Hypermutability of simple sequence repeats (SSR) through DNA slippage is a major mechanism of phase variation in Campylobacter jejuni . The presence of multiple SSR-mediated phase-variable genes encoding enzymes that modify surface structures, including capsular polysaccharide (CPS) and lipooligosaccharide (LOS), generates high levels of structural variants within bacterial populations, thereby promoting adaptation to selective pressures in host environments. Therefore, the phenotypic diversity generated by phase variation can limit the reproducibility of results with C. jejuni ; therefore, researchers need to genetically control the mutability of multiple SSRs. Here, we show that natural “cotransformation” is an effective method for C. jejuni genome editing. Cotransformation is a trait of naturally competent bacteria that causes uptake and integration of multiple different DNA fragments, which has been recently adapted to multiplex genome editing by natural transformation (MuGENT), a method for introducing multiple scarless mutations into the genomes of these bacteria. We found that the cotransformation frequencies of antibiotic resistance gene-marked DNA fragments and unmarked DNA fragments reached ~40% in C. jejuni . To examine the feasibility of MuGENT in C. jejuni , we “locked” either different polyG SSR tracts in strain NCTC11168 (which are located in the biosynthetic CPS and LOS gene clusters) into either the ON or OFF configurations by interrupting the continuous runs of G residues without changing the encoded amino acids. This approach, termed “MuGENT-SSR,” enabled the generation of all eight edits within 2 weeks and the identification of a phase-locked strain with a highly stable type of Penner serotyping, a CPS-based serotyping scheme. Furthermore, extensive genome editing of this strain by MuGENT-SSR identified a phase-variable gene that determines the Penner serotype of NCTC11168. Thus, MuGENT-SSR provides a platform for genetic and phenotypic engineering of genetically unstable C. jejuni , making it a reliable approach for elucidating the mechanisms underlying phase-variable expression of specific phenotypes.