Genome-scale dissection of phase-variable gene function in Campylobacter jejuni using a stabilized phasotype library

Phase variation (PV) allows bacterial pathogens to rapidly modify surface structures through reversible mutations-often driven by simple sequence repeats (SSRs)-thereby facilitating immune evasion and environmental adaptation. In Campylobacter jejuni, SSR-mediated PV regulates numerous surface-exposed molecules, including capsular polysaccharides and lipooligosaccharides, which play key roles in host interaction and immune resistance. However, the stochastic nature of PV has made it difficult to systematically analyze the functions of individual phase-variable genes (PVGs). Here, we introduce PV-GenShift (Phase Variation Genomic Shift), a genome-scale screening platform built on a genetically stabilized library of phase-locked C. jejuni variants. Using multiplex genome editing via natural transformation, we fixed the ON/OFF states of 15 SSR-containing PVGs, enabling reproducible, high-resolution analysis of phasotypes, defined as unique combinations of ON/OFF states across multiple PVGs, under defined selective pressures. Applying PV-GenShift to models of human serum exposure, murine colonization, and chicken gut passage, we identified distinct phasotypes linked to serum resistance and enrichment during mouse colonization-particularly those involving capsular polysaccharide modifications such as O-methyl phosphoramidation and methylation. In contrast, chicken passage produced heterogeneous ON/OFF patterns without a dominant phasotype. These findings underscore the combinatorial influence of SSR-mediated PVG expression states on bacterial adaptation and establish PV-GenShift as a versatile framework for dissecting PV-driven phenotypic diversity. This approach offers a scalable strategy for mapping genotype-phenotype relationships and provides insights with implications for vaccine development, diagnostic design, and targeted therapeutics.

Phase variation allows bacteria to rapidly generate phenotypic diversity, but its randomness has long limited efforts to define how specific gene combinations influence adaptation. In Campylobacter jejuni, phase-variable genes control numerous surface structures that affect host interactions and immune resistance, yet their coordinated contributions remain poorly understood. PV-GenShift (Phase Variation Genomic Shift) overcomes this challenge by genetically stabilizing the ON/OFF states of multiple phase-variable genes, enabling systematic, reproducible analysis of phasotypes under defined selective pressures. Using this approach, we identified combinations of gene states that enhance serum resistance and promote colonization in mice, particularly those involving modifications to capsular polysaccharides. In contrast, chicken passage produced heterogeneous patterns consistent with weak or non-specific selection. These findings demonstrate that the interplay of multiple phase-variable loci shapes host adaptation and highlight PV-GenShift as a broadly applicable strategy for dissecting phase variation in diverse pathogens, with implications for vaccine design and targeted therapies.