Experimental evolution of enhanced growth by Bacillus subtilis at low atmospheric pressure: genomic changes revealed by whole-genome sequencing

Abstract

Knowledge of how microorganisms respond and adapt to low-pressure (LP) environments is limited. Previously, Bacillus subtilis strain WN624 was grown at the near-inhibitory LP of 5 kPa for 1,000 generations and strain WN1106, which exhibited increased relative fitness at 5 kPa, was isolated. Genomic sequence differences between ancestral strain WN624 and LP-evolved strain WN1106 were identified using whole-genome sequencing. LP-evolved strain WN1106 carried amino acid-altering mutations in the coding sequences of only seven genes (fliI, parC, ytoI, bacD, resD, walK, and yvlD) and a single 9-nucleotide in-frame deletion in the rnjB gene that encodes RNase J2, a component of the RNA degradosome. By using a collection of frozen stocks of the LP-evolved culture taken at 50-generation intervals, it was determined that (i) the fitness increase at LP occurred rapidly, while (ii) mutation acquisition exhibited complex kinetics. A knockout mutant of rnjB was shown to increase the competitive fitness of B. subtilis at both LP and standard atmospheric pressure.

FIG 1

DNA sequence of the rnjB gene and deduced amino acid sequence of RNase J2 in ancestral strain WN624 (top) and the DNA sequence of the rnjB(Δ9) mutant and deduced amino acid sequence of the resulting RNase J2Δ3 in LP-evolved strain WN1106 (bottom). Direct repeats (boxed, boldface type) are indicated. A triangle indicates deletion of 3 amino acids (AKI) from the deduced RNase J2 sequence in strain WN1106.

FIG 2

(A) Kinetics of mutational sweeps during LP evolution experiment. Data are depicted as the proportion of the mutant allele divided by the total (mutant + ancestral) allele (filled circles). For rnjB, a second allele was discovered during this experiment, i.e., an SNP resulting in a nonsense mutation at codon 177 (Table 3), which is depicted by white circles. (B) Results from competition experiments in which each frozen stock of the LP-evolving populations (Spc^r), taken at 50-generation intervals, was competed at 5 kPa against either ancestral strain WN628 (Cm^r; open circles) or LP-evolved strain WN1279 (Cm^r; filled circles). Competition at the zero-generation mark used ancestor strain WN624. Data are shown as averages ± standard deviations of results of duplicate experiments. See the text for details.

FIG 3

Competition experiments to test the effect of the rnjB::spc knockout mutation on relative fitness. (Left) Strain WN1518 (ΔrnjB::spc amyE::neo in WN624 background) was competed against congenic ancestral strain WN628 (amyE::cat). (Right) Strain WN1519 (ΔrnjB::spc amyE::neo in WN1106 background) was competed against congenic strain WN1106 [rnjB(Δ9) amyE::spc]. Competitions were performed in liquid LB medium at 27°C and either ∼101 kPa (black bars) or 5 kPa (gray bars). Data are averages ± standard deviations of results of duplicate experiments.