Evolution of Bacillus subtilis: A Novel Phenotype and its Challenges

Abstract

The enzyme LeuB from the last common ancestor of the firmicutes has been statistically recreated using several different methods. This project deals with two versions, Rec/LG and Rec/EX. To better understand these resurrected enzymes, the proteins were crystallised. Despite extensive efforts to produce crystals of diffraction quality, none of the crystals developed during this project were large enough to be of any use in X-Ray crystallography. Strains of Bacillus subtilis were genetically modified to each have one of either Rec/LG or Rec/Rec/EX in place of the native leuB gene, and then were allowed to evolve alongside an unmodified strain for 500 generations. Attempts were made to extract DNA from the evolved strains at different points throughout the evolution experiment for whole genome sequencing, and to amplify the leuB gene via PCR for direct comparisons of changes that may have occurred to the leuB gene over the course of the evolution experiment. These attempts were ultimately unsuccessful. In lieu of this genetic data, phenotypic changes to the evolved strains were characterised. A new phenotype appeared extremely early on in the experiment (by generation 54), and in less than 300 generations had swept to fixation in 9 independent strains. In all cases, this phenotype included poor growth on LB agar plates, small colonies, and small cells. Similar changes have been observed before in laboratory experiments of B. subtilis, but none occurred so quickly or so uniformly across independent samples.