Escape from growth restriction in small colony variants of Salmonella typhimurium by gene amplification and mutation

Antibiotic resistance in bacteria is generally associated with fitness costs that often can be reduced by second-site compensatory mutations. Here, we examined how a protamine-resistant small colony variant of Salmonella typhimurium adapts to the growth reduction conferred by a resistance mutation in hemC (encoding a haem-biosynthesis enzyme). We show that adaptation occurs in a multi-step process where fitness is successively increased. Thus, the initial adaptive response was selection for an unstable gene amplification of the mutant hemC gene that provided a small fitness increase. Fitness was increased further by a mutation that restored HemC function in one gene copy, relaxing selection for the amplification. Subsequently, the amplification segregated back to the haploid state and even higher fitness. The end result was in most cases mutant strains with a hemC sequence different from that of the wild-type strain. These findings suggest that gene amplification facilitates adaptive evolution. A higher gene dosage increases the target size for compensatory mutations and improves fitness of the cell, thereby allowing an increase in the population size, further increasing the probability of a subsequent stable mutation. Our results provide a novel genetic basis for growth compensation in small colony variants.