A single nucleotide polymorphism collapses phase variation and drives short-sighted evolution of O-antigen in Salmonella

The O-antigen of Salmonella is a key virulence determinant and serotype-defining structure, yet the regulatory mechanisms underlying its dynamic modification and evolutionary trade-offs remain incompletely understood. Salmonella Pullorum is a pathogen that displays both O-antigen unstable phase variation and stable antigenic conversion between standard (O123) and variant (O122) antigenic types. In this study, we identified the glycosyltransferase encoded by gtrC III as responsible for the variant-specific glucosylation of the O-antigen. The expression of gtrC III is co-regulated by the transcriptional repressor OxyR and methyltransferase Dam, generating stochastic ON‒OFF switching of the O-antigen modification, which is a classic phase variation and promotes immune evasion. Remarkably, a single C-to-A point substitution at position –34 of the gtrABC III promoter disrupts OxyR binding and simultaneously enhances intrinsic promoter activity, resulting in constitutive expression of the variant antigenic type. This monophasic variant strain exhibits enhanced resistance to standard-type antibody-mediated killing, conferring a short-term immune evasion advantage. However, in long-term colonization, it is outcompeted by the phase-variable standard type strain, exemplifying “short-sighted evolution”, where an adaptive mutation trades off persistence for transient immune escape. Our findings reveal how a single nucleotide change can subvert a complex epigenetic switch to drive sub-serotype conversion, and provide insights into the evolutionary constraints shaping O-antigen diversity.