Adaptive Processes Change as Multiple Functions Evolve.

Epistasis influences the gene-environment interactions that shape bacterial fitness through antibiotic exposure, which can ultimately affect the availability of certain resistance phenotypes to bacteria. The substitutions present within confer both cephalosporin and β-lactamase inhibitor resistance. We wanted to compare the evolution of with that of another variant, , which differs in that contains only substitutions that contribute to cephalosporin resistance. Differences between the landscapes and epistatic interactions of these TEM variants are important for understanding their separate evolutionary responses to antibiotics. We hypothesized the substitutions within would result in more epistatic interactions than for As expected, we found more epistatic interactions between the substitutions present in than in Our results suggest that selection from many cephalosporins is required to achieve the full potential resistance to cephalosporins but that a single β-lactam and inhibitor combination will drive evolution of the full potential resistance phenotype. Surprisingly, we also found significantly positive increases in growth rates as antibiotic concentration increased for some of the strains expressing precursor genotypes but not the variants. This result further suggests that additive interactions more effectively optimize phenotypes than epistatic interactions, which means that exposure to numerous cephalosporins actually increases the ability of a TEM enzyme to confer resistance to any single cephalosporin.