Experimental evolution reveals bifunctional genetic solutions to loss of trpF in Salmonella enterica.

How new gene functions arise while maintaining ancestral biological roles remains a central question in evolutionary genetics. To investigate genetic solutions to the disruption of a biosynthetic pathway without prior genetic bias, we used experimental evolution to study restoration of tryptophan biosynthesis in Salmonella enterica strains lacking the trpF gene. Populations were founded from bacteria carrying wild-type alleles of all relevant genes in their native genomic and regulatory contexts and evolved under conditions selecting for growth without exogenous tryptophan. Across independent populations, mutations in either hisA or trpA enabled growth of ΔtrpF strains in the absence of added tryptophan while retaining sufficient ancestral function to support growth under the same conditions. Whole-genome sequencing and genetic reconstruction showed that these mutant alleles were sufficient to confer the growth-rescue phenotype. Duplication of the target gene was detected in only a single population and showed no evidence of functional divergence. Mutational paths differed between genes: hisA-based solutions arose primarily in mutator backgrounds and were associated with stronger trade-offs with ancestral function, whereas trpA-based solutions were more frequent and often retained native function. Together, these results demonstrate that bifunctional genetic solutions can arise through point mutations in multiple genes during experimental adaptation, illustrating how physiologically relevant multifunctionality can evolve at the gene level without gene duplication.