Evolution of Escherichia coli rifampicin resistance in an antibiotic-free environment during thermal stress

Background: Beneficial mutations play an essential role in bacterial adaptation, yet little is known about their fitness effects across genetic backgrounds and environments. One prominent example of bacterial adaptation is antibiotic resistance. Until recently, the paradigm has been that antibiotic resistance is selected by the presence of antibiotics because resistant mutations confer fitness costs in antibiotic free environments. In this study we show that it is not always the case, documenting the selection and fixation of resistant mutations in populations of Escherichia coli B that had never been exposed to antibiotics but instead evolved for 2000 generations at high temperature (42.2 degrees C). Results: We found parallel mutations within the rpoB gene encoding the beta subunit of RNA polymerase. These amino acid substitutions conferred different levels of rifampicin resistance. The resistant mutations typically appeared, and were fixed, early in the evolution experiment. We confirmed the high advantage of these mutations at 42.2 degrees C in glucose-limited medium. However, the rpoB mutations had different fitness effects across three genetic backgrounds and six environments. Conclusions: We describe resistance mutations that are not necessarily costly in the absence of antibiotics or compensatory mutations but are highly beneficial at high temperature and low glucose. Their fitness effects depend on the environment and the genetic background, providing glimpses into the prevalence of epistasis and pleiotropy.