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

Phase variation (PV) enables bacterial pathogens to rapidly alter their surface structures through reversible mutations in simple sequence repeats, promoting immune evasion and environmental adaptation. In Campylobacter jejuni, the stochastic nature of PV has hindered the systematic functional analysis of phase-variable genes (PVGs). Here, we introduce PV-GenShift, a genome-scale screening platform built on a genetically stabilized library of phase-locked C. jejuni variants. By fixing the ON/OFF states of 15 PVGs, PV-GenShift enables reproducible, high-resolution analysis of phasotypes, defined as unique ON/OFF combinations across multiple PVGs, under defined selective pressures. Using models of human serum exposure, murine colonization, and chicken gut passage, we identified distinct phasotypes associated with serum resistance and with enrichment during mouse colonization, particularly involving capsular polysaccharide modifications such as O-methyl phosphoramidation and methylation. In contrast, chicken gut passage resulted in heterogeneous ON/OFF shifts without a dominant phasotype. These findings highlight the combinatorial impact of PVG expression states on bacterial adaptation and establish PV-GenShift as a broadly applicable framework for dissecting PV-driven phenotypic diversity. This approach provides a scalable strategy for exploring genotype-phenotype relationships and offers insights relevant to vaccine design and targeted therapeutics. Significance StatementPhase variation generates phenotypic diversity that enables pathogens to evade immunity and adapt to changing environments; however, its random nature has long obscured functional analysis. This study introduces PV-GenShift, a genome-scale platform that stabilizes phase-variable gene expression in Campylobacter jejuni, allowing the systematic identification of gene combinations that influence survival under selective pressures. Using PV-GenShift, we identified phasotypes associated with serum resistance and enrichment during mouse colonization, while chicken passage produced diverse but non-specific shifts. These results demonstrate how combinatorial ON/OFF states of multiple genes shape bacterial adaptation and provide a generalizable strategy for studying phase variation across pathogens, with implications for vaccine design and targeted therapeutics.