Amino acid substitutions in ribosomal protein RpsU enable switching between high fitness and multiple-stress resistance in Listeria monocytogenes

On the role of ribosomal proteins in stress resistance and fitness of Listeria monocytogenes : a laboratory evolution approach

“…We linked stress resistance in variants 14 and 15 with a complete gene deletion, or point mutation in rpsU respectively, to induction of the SigB regulon, and showed the correlation between increased stress resistance and reduced fitness. By using experimental evolution to select for increased fitness in variant 15 in two parallel lines, we were previously able to show that this trade-off was reversible (although not fully) via point mutations in RpsU at the same location of the initial mutation: RpsU 17Pro-His and RpsU 17Pro-Thr , respectively (Koomen et al, 2021). Here, we applied a similar experimental evolution approach using L. monocytogenes LO28 variant 14, that has a complete deletion of RpsU, and by selecting for higher fitness in two parallel lines, we were able to select two evolved variants of variant 14 (14EV1, and 14EV2).…”

“…Experimental evolution was performed as described in Koomen et al (2021). Briefly, we inoculated two parallel lines with 1% (v/v) of ON culture of L. monocytogenes LO28 variant 14 in 20 ml BHI broth in 100 ml Erlenmeyer flasks.…”

“…The maximum specific growth rate μmax (h -1 ) was determined at 30°C for the two evolved strains (14EV1 and 14EV2), variant 14 and the Listeria monocytogenes LO28 WT, following the procedure as described previously by , and Koomen et al (2021). This method is based on the time-to-detection (TTD) of five serially diluted cultures of which the initial bacterial concentration is known.…”

“…Ancestor variant 14 and evolved variants 14EV1 and 14EV2 obtained in the evolution experiment were sequenced using Illumina chemistry as described before (Koomen et al, 2021). Briefly, cells were pelleted, and resuspended in 450 µl DNA/RNA Shield (Zymo Research) at 4°C until DNA extraction.…”

“…In a follow-up study (Koomen et al, 2021), we subjected L. monocytogenes LO28 variant 15, with its single RpsU 17Arg-Pro point mutation, to an experimental evolution protocol where we selected for increased fitness, defined as a higher maximum specific growth rate (µmax) compared to variant 15. Both evolved variants fixed mutations in rpsU (resulting in RpsU 17Pro-His and RpsU 17Pro-Thr ), and reverted back to WT-like high maximum specific growth rate and relative low stress resistance.…”