The Spectrum of Spontaneous Rifampicin-Resistance Mutations in the Bacillus subtilis rpoB Gene Depends on the Growth Environment

Results from previous investigations into spontaneous rifampicin-resistance (RifR) mutations in the Bacillus subtilis rpoB gene suggested that the spectrum of mutations may depend on the growth environment. However, these studies were limited by low sample numbers, allowing for the potential distortion of the data by the presence of 'jackpot' mutations which may have arisen early in the growth of a population. Here we addressed this issue by performing fluctuation analyses to assess both the rate and spectrum of RifR mutations in two distinct media: LB, a complete laboratory medium and SMMAsn, a minimal medium utilizing L-asparagine as the sole carbon source. We cultivated 60 separate populations under each growth condition and determined that the mutation rate to RifR to be slightly but significantly higher in LB cultures. We then sequenced the relevant regions of rpoB to map the spectrum of RifR mutations under each growth condition. We found a distinct spectrum of mutations in each medium; LB cultures were dominated by the H482Y mutation (27/53 or 51%) whereas SMMAsn cultures were dominated by the S487L mutation (24/51 or 47%). Furthermore, we found through competition experiments that the relative fitness of the S487L mutant was significantly higher in SMMAsn than in LB medium. We therefore conclude that both the spectrum of RifR mutations in the B. subtilis rpoB gene and the fitness of resulting mutants to be influenced by the growth environment. Importance The rpoB gene encodes the beta subunit of RNA polymerase, and mutations in rpoB are key determinants of resistance to the clinically important antibiotic rifampicin. We show here that the spectrum of mutations in Bacillus subtilis rpoB depends on the medium in which the cells are cultivated. The results show that not only does the growth environment play a role in natural selection and fitness, but it also influences the probability of mutation at particular bases within the target gene.