Evolutionary rescue of sphericalmreBdeletion mutants of the rod-shape bacteriumPseudomonas fluorescensSBW25

Maintenance of rod-shape in bacterial cells depends on the actin-like protein MreB. Deletion ofmreBfromPseudomonas fluorescensSBW25 results in viable spherical cells of variable volume and reduced fitness. Using a combination of time-resolved microscopy and biochemical assay of peptidoglycan synthesis we show that reduced fitness is a consequence of perturbed cell size homeostasis that arises primarily from differential growth of daughter cells. A 1,000-generation selection experiment resulted in rapid restoration of fitness with derived cells retaining spherical shape. Mutations in the peptidoglycan synthesis protein Pbp1A were identified as the main route for fitness restoration with genetic reconstructions demonstrating causality. Thepbp1Amutations targeting transpeptidase activity enhance homogeneity in cell wall synthesis on lateral surfaces, thus restoring cell size homeostasis in the population. Together our experimental approach emphasizes the new knowledge to be gained from strategies that exploit the power of natural selection to rescue fitness-compromised mutants.