The ΔybeX strain is sensitive to ribosome-targeting antibiotics
ybeX disruption has been reported to cause decreased survival in the presence of chloramphenicol (Smith et al. , 2007). Therefore, we explored the effects of various antibiotics on the ΔybeXcells. First, we determined the minimal inhibitory concentrations (MICs) in LB for WT and ΔybeX strains. The MICs of chloramphenicol, tetracycline, erythromycin, clindamycin, and fusidic acid were two times lower for ΔybeX (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-MIC concentrations of indicated antibiotics (see Materials and Methods). The ΔybeX strain exhibited severe sensitivity to sub-MIC concentrations of chloramphenicol, tetracycline, erythromycin, clindamycin, and fusidic acid (Fig. 3A ). The presence ofybeX single-copy plasmid in the absence of inducer fully rescued the described antibiotic sensitivities (Fig. 3B ). In contrast, protein synthesis-targeting antibiotics that do not induce the cold shock response (amikacin, streptomycin, kanamycin, tobramycin and mupirocin), exhibit similar inhibitory effect on ΔybeX and WT strains. The RNA synthesis inhibitor rifampicin also revealed no differential effect on the ΔybeX strain (Fig. 3B, Fig. S4b ).
To exclude strain-specific effects, we tested two isogenic wild-type strains, MG1655 and BW25113 and the corresponding deletion strainsΔybeX::kanMG andΔybeX::kanBW under sub-inhibitory antibiotic concentrations (Fig. S4b ). Both genetic backgrounds exhibited similar antibiotic sensitivities, and removal of the kanamycin resistance cassette (in strains ΔybeX/-kanMGand ΔybeX/-kanBW ) had no effect.