Genotoxic Agents Produce Stressor-Specific Spectra of Spectinomycin Resistance Mutations Based on Mechanism of Action and Selection in Bacillus subtilis

Abstract

Mutagenesis is integral for bacterial evolution and the development of antibiotic resistance. Environmental toxins and stressors are known to elevate the rate of mutagenesis through direct DNA toxicity, known as stress-associated mutagenesis, or via a more general stress-induced process that relies on intrinsic bacterial pathways. Here, we characterize the spectra of mutations induced by an array of different stressors using high-throughput sequencing to profile thousands of spectinomycin-resistant colonies of Bacillus subtilis. We found 69 unique mutations in the rpsE and rpsB genes, and that each stressor leads to a unique and specific spectrum of antibiotic-resistance mutations. While some mutations clearly reflected the DNA damage mechanism of the stress, others were likely the result of a more general stress-induced mechanism. To determine the relative fitness of these mutants under a range of antibiotic selection pressures, we used multistrain competitive fitness experiments and found an additional landscape of fitness and resistance. The data presented here support the idea that the environment in which the selection is applied (mutagenic stressors that are present), as well as changes in local drug concentration, can significantly alter the path to spectinomycin resistance in B. subtilis.

Keywords: Bacillus subtilis; DNA damage; antibiotic resistance; competition; drug resistance evolution; environmental stressors; mutagenesis; mutational spectrum; selection; spectinomycin.

FIG 1

Genotoxic agents induce a spectrum of unique, treatment-specific resistance mutations. (A) Mutation frequency of spectinomycin resistance in WT B. subtilis treated with various stressors during logarithmic- or stationary-phase growth. Mutation frequency was calculated by dividing the number of spectinomycin-resistant colonies by the total number of cells. Significant differences between untreated and treated cells were determined using the Mann-Whitney U test (***, P ≤ 0.0001). Horizontal LOD line represents the limit of detection. (B) Spectrum of nucleotide mutations in the rpsE gene of the spectinomycin-resistant mutant colonies of WT B. subtilis. Notably, there were no mutations identified in the no-treatment condition during logarithmic phase, as this condition produced very few or no resistant colonies. In lieu of the resistant colonies, logarithmic-phase cells were plated on nonselective medium and 500 of these nonresistant isolates were sequenced. (C) The relative abundance of nucleotide mutations in the rpsB gene from 90 spectinomycin-resistant mutant colonies compared to the relative abundance of mutations from the rpsE gene from 480 colonies (including the 90 colonies displaying the rpsB mutations on the left), all of which were from UV-treated cells. For B and C, each treatment represents ∼500 colonies collected from 3 to 12 biological replicates.

FIG 2

Impact of DNA repair genes on the frequency and spectrum of mitomycin C-induced mutants. (A) Mutation frequency of spectinomycin resistance in DNA repair-deficient B. subtilis treated with 100 ng/ml mitomycin C. Mutation frequency was calculated by dividing the number of spectinomycin-resistant colonies by the total number of cells. Bars represent mean ± standard error of the mean (SEM) (n = 3 minimum). Significance between mitomycin C-treated and untreated WT, and between mitomycin C-treated WT and mitomycin C-treated DNA knockouts was determined using the Mann-Whitney U Test (***, P ≤ 0.0001). Horizontal LOD line represents the limit of detection. (B) Spectrum of nucleotide mutations in the rpsE gene of the mutant colonies from WT and DNA repair-deficient strains of B. subtilis treated with mitomycin C. Each treatment represents ∼500 colonies collected from 3 to 12 biological replicates.

FIG 3

Competition of base substitution mutants over a gradient of selection. (A) Stacked bar plots of the relative abundance of each strain within a mixed community at times 0, 3, 12, 24, 48, and 72 h under spectinomycin selection concentrations of 0, 62.5, 250, and 1,000 μg/ml. Cultures were passaged to an OD₆₀₀ of 0.01 immediately after sampling of the 24- and 48-h time points. Plots represent the average relative abundance ± SEM (n = 4). Determination of statistical differences is described in the Materials and Methods section. (B) Growth rates of the 12 strains used in the competition experiments performed at each of the concentrations shown above in panel A (0, 62.5, 250, and 1,000 μg/ml spectinomycin). Points represent mean ± SEM (n = 6 minimum). Cell growth over time was determined by measuring OD₆₀₀ at 30-min time intervals. (A and B) The relative abundance plots in (A) are separated by drug concentration, and the line plots (B) correspond to the drug concentrations in the relative abundance plots shown directly above each plot.

FIG 4

Stress-associated mutagenesis leads to spectinomycin resistance through a spectrum of mutations and a gradient of selection in B. subtilis. A graphical representation of the process by which stress-associated mutagenesis in B. subtilis leads to spectinomycin resistance through the generation of base substitution mutations and subsequent selection. The initial stress causes a stress-specific pattern of mutations but, of all these mutations, most are deleterious or do not impact antibiotic susceptibility (represented by the gray portion of the triangle). Deleterious or growth-inhibiting mutants are lost as they are unable to produce viable cells. Of the viable mutations (denoted by the rainbow in the bottom portion of the pyramid), only a portion of the possible mutations will be able to manifest a resistance phenotype based on fundamental selection criteria, such as that mutation being in the mutational repertoire of the stressor and not impacting cell viability. Transparent cells represent those that were unable to meet the selection requirements for resistance. Finally, the level of antibiotic selection pressure will ultimately determine the final spectrum of base substitution mutations in a mixed population.