Diverse mutational routes to the restoration of motility in a soil bacterium

Abstract

Restoration of a phenotype following insertion of a crippling mutation gives us insights into the adaptability of an organism. Mutational routes to the restoration of phenotypes can be diverse. Deletion of the master regulator (fleQ) for flagellar synthesis genes renders Pseudomonas fluorescens immotile, but after being put under strong selection to swim, it reliably re-evolves motility. In order to uncover how the immotile bacteria regain their ability to swim they are inoculated into swimming agar plates and sampled from the leading edge of the motile zone. These samples are sequenced at a range single locus sites, predicted to be where causative mutations for the restoration of motility will be found (as indicated from previous research). In the engineered immotile strain Pf0-2x, a huge variety of mutations, consisting of SNPs, multi-codon deletions, and frameshifts are observed across all candidate loci. In a complex environment (LB) 50% of initial restorative mutations occur in glnA, a glutamine synthetase gene. In a minimal media environment (M9) 45% of these mutations occur in ntrB, a gene for a kinase that forms part of a two component system. These findings contrast strongly with similar work in the related P. fluorescens strain SBW25, where the same SNP is repeatedly found across all environments. Interestingly, this SNP has never been seen in Pf0-2x. These results highlight how preferential mutational routes can vary across environments, and that strain-to-strain differences can have a major impact types of mutations that are uncovered and selected for by evolution.