Ribosomal mutations enable a switch between high fitness and high stress resistance in Listeria monocytogenes

Multiple stress resistant variants of with mutations in encoding ribosomal protein RpsU have previously been isolated after a single exposure to acid stress. These variants, including LO28 variant V14 with a complete deletion of the gene, showed upregulation of the general stress sigma factor Sigma B-mediated stress resistance genes and had a lower maximum specific growth rate than the LO28 WT, signifying a trade-off between stress resistance and fitness. In the current work V14 has been subjected to an experimental evolution regime, selecting for higher fitness in two parallel evolving cultures. This resulted in two evolved variants with WT-like fitness: 14EV1 and 14EV2. Comparative analysis of growth performance, acid and heat stress resistance, in combination with proteomics and RNA-sequencing, indicated that in both lines reversion to WT-like fitness also resulted in WT-like stress sensitivity, due to lack of Sigma B-activated stress defense. Notably, genotyping of 14EV1 and 14EV2 provided evidence for unique point-mutations in the ribosomal gene causing amino acid substitutions at the same position in RpsB, resulting in RpsB and RpsB , respectively. Combined with data obtained with constructed RpsB and RpsB mutants in the V14 background, we provide evidence that loss of function of RpsU resulting in the multiple stress resistant and reduced fitness phenotype, can be reversed by single point mutations in leading to arginine substitutions in RpsB at position 22 into histidine or serine, resulting in a WT-like high fitness and low stress resistance phenotype. This demonstrates the impact of genetic changes in ' ribosomes on fitness and stress resistance.