The ∆ybeX strain is sensitive to ribosome-targeting antibiotics
ybeX disruption has been reported to cause cell death in the presence of chloramphenicol (Smith et al. , 2007). We therefore explored the effects of various antibiotics on the ∆ybeX cells. First, we determined the minimal inhibitory concentrations (MICs) in LB for the WT and the ∆ybeX strains (see Materials and Methods). The MICs were two times lower for ∆ybeX in the presence of fusidic acid, clindamycin, chloramphenicol, tetracycline and erythromycin (Table S1 ). These structurally unrelated ribosome-targeting antibiotics have been shown to induce cold-shock proteins or block the induction of heat-shock proteins (VanBogelen and Neidhardt, 1990; Cruz-Loya et al. , 2019). We further inspected the effects of these antibiotics using the dot spot assay described above, except that the LB agar plates were supplemented with sub-inhibitory concentrations of indicated antibiotics (see Materials and Methods). The ∆ybeX strain exhibited severe sensitivity to sub-lethal concentrations of all of these antibiotics (Fig. 3A ). Expressing the YbeX protein from a single-copy plasmid in the absence of the inducer completely rescued the antibiotic sensitivity (Fig. 3B ). In contrast, protein synthesis-targeting antibiotics, for which we do not have evidence that they affect the cold shock response (amikacin, streptomycin, kanamycin, tobramycin and mupirocin), was founded not to have an effect. In addition, the transcriptional inhibitor rifampicin revealed no effect on theΔybeX strain compared to the WT (Fig. 3B, Fig. S4b ). We also measured the MICs in the MHB cation-adjusted media. The MICs for wild type and ybeZ , and ybeX deletion strains remained the same, while ybeY deletion strain showed lower MICs for all tested antibiotics (data not shown).
We also tested the survival of two isogenic wild-type strains, MG1655 and BW25113 and the corresponding deletion strains∆ybeX::kanMG and∆ybeX::kanBW under sub-inhibitory antibiotic concentrations. Both genetic backgrounds exhibited similar antibiotic sensitivities, and removal of the kanamycin resistance cassette (in strains ∆ybeX/-kanMG and∆ybeX/-kanBW ) had no effect (Fig. S4 ). In contrast, ectopic expression of ybeX in the absence of an inducer abolished the antibiotics sensitivity (Fig. 3B ).