A single point mutation in the Listeria monocytogenes ribosomal gene rpsU enables SigB activation independently of the stressosome and the anti-sigma factor antagonist RsbV

Microbial population heterogeneity leads to different stress responses and growth behavior of individual cells in a population. Previously, a point mutation in the rpsU gene (rpsU(G50C)) encoding ribosomal protein S21 was identified in a Listeria monocytogenes LO28 variant, which leads to increased multi-stress resistance and a reduced maximum specific growth rate. However, the underlying mechanisms of these phenotypic changes remain unknown. In L. monocytogenes, the alternative sigma factor SigB regulates the general stress response, with its activation controlled by a series of Rsb proteins, including RsbR1 and anti-sigma factor RsbW and its antagonist RsbV. We combined a phenotype and proteomics approach to investigate the acid and heat stress resistance, growth rate, and SigB activation of L. monocytogenes EGDe wild type and the Delta sigB, Delta rsbV, and Delta rsbR1 mutant strains. While the introduction of rpsU(G50C) in the Delta sigB mutant did not induce a SigB-mediated increase in robustness, the presence of rpsU(G50C) in the Delta rsbV and the Delta rsbR1 mutants led to SigB activation and concomitant increased robustness, indicating an alternative signaling pathway for the SigB activation in rpsU(G50C) mutants. Interestingly, all these rpsU(G50C) mutants exhibited reduced maximum specific growth rates, independent of SigB activation, possibly attributed to compromised ribosomal functioning. In summary, the increased stress resistance in the L. monocytogenes EGDe rpsU(G50C) mutant results from SigB activation through an unknown mechanism distinct from the classical stressosome and RsbV/RsbW partner switching model. Moreover, the reduced maximum specific growth rate of the EGDe rpsU(G50C) mutant is likely unrelated to SigB activation and potentially linked to impaired ribosomal function.