The Spectrum of Spontaneous Rifampin Resistance Mutations in the Bacillus subtilis rpoB Gene Depends on the Growth...

Results from previous investigations into spontaneous rifampin resistance (Rif^r) mutations in the Bacillus subtilis rpoB gene suggested that the spectrum of mutations depends 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 that 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 Rif^r mutations in two distinct media: LB, a complete laboratory medium, and SMM_Asn, a minimal medium utilizing l-asparagine as the sole carbon source. We cultivated 60 separate populations under each growth condition and determined the mutation rate to Rif^r to be slightly but significantly higher in LB cultures. We then sequenced the relevant regions of rpoB to map the spectrum of Rif^r 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 SMM_Asn 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 SMM_Asn than in LB medium. We therefore conclude that both the spectrum of Rif^r mutations in the B. subtilis rpoB gene and the fitness of resulting mutants are 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 rifampin. 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 the growth environment not only plays a role in natural selection and fitness but also influences the probability of mutation at particular bases within the target gene.