SummaryMost chromosomal mutations that cause antibiotic resistance impose fitness costs on the bacteria. This biological cost can often be reduced by compensatory mutations. In Salmonella typhimurium , the nucleotide substitution AAA 42 AE AE AE AE AAC in the rpsL gene confers resistance to streptomycin. The resulting amino acid substitution (K42N) in ribosomal protein S12 causes an increased rate of ribosomal proofreading and, as a result, the rate of protein synthesis, bacterial growth and virulence are decreased. Eightyone independent lineages of the low-fitness, K42N mutant were evolved in the absence of antibiotic to ameliorate the costs. From the rate of fixation of compensated mutants and their fitness, the rate of compensatory mutations was estimated to be ≥ ≥ ≥ ≥ 10 ----7per cell per generation. The size of the population bottleneck during evolution affected fitness of the adapted mutants: a larger bottleneck resulted in higher average fitness. Only four of the evolved lineages contained streptomycin-sensitive revertants. The remaining 77 lineages contained mutants that were still fully streptomycin resistant, had retained the original resistance mutation and also acquired compensatory mutations. Most of the compensatory mutations, resulting in at least 35 different amino acid substitutions, were novel single-nucleotide substitutions in the rpsD , rpsE , rpsL or rplS genes encoding the ribosomal proteins S4, S5, S12 and L19 respectively. Our results show that the deleterious effects of a resistance mutation can be compensated by an unexpected variety of mutations.

Compensatory mutations, due to their ability to mask the deleterious effects of another mutation, are important for the adaptation and evolution of most organisms. Resistance to antibiotics, antivirals, antifungals, herbicides and insecticides is usually associated with a fitness cost. As a result of compensatory evolution, the initial fitness costs conferred by resistance mutations (or other deleterious mutations) can often be rapidly and efficiently reduced. Such compensatory evolution is potentially of importance for (i) the long-term persistence of drug resistance, (ii) reducing the rate of fitness loss associated with the accumulation of deleterious mutations in small asexual populations, and (iii) the evolution of complexity of cellular processes.  2004 Elsevier SAS. All rights reserved.

The relationship between the number of randomly accumulated mutations in a genome and fitness is a key parameter in evolutionary biology. Mutations may interact such that their combined effect on fitness is additive (no epistasis), reinforced (synergistic epistasis) or mitigated (antagonistic epistasis). We measured the decrease in fitness caused by increasing mutation number in the bacterium Salmonella typhimurium using a regulated, error-prone DNA polymerase (polymerase IV, DinB). As mutations accumulated, fitness costs increased at a diminishing rate. This suggests that random mutations interact such that their combined effect on fitness is mitigated and that the genome is buffered against the fitness reduction caused by accumulated mutations. Levels of the heat shock chaperones DnaK and GroEL increased in lineages that had accumulated many mutations, and experimental overproduction of GroEL further increased the fitness of lineages containing deleterious mutations. These findings suggest that overexpression of chaperones contributes to antagonistic epistasis.