Genome-wide screen reveals cellular functions that counteract rifampicin lethality in Escherichia coli

The antibacterial activity of rifamycins specifically relies on the inhibition of transcription by directly binding to the beta-subunit of bacterial DNA-dependent RNA polymerase (RNAP). However, its killing efficacy is substantially diminished in most gram-negative bacteria. To systematically reveal the cellular functions that counteract rifamycin-mediated killing in the gram-negative model organism Escherichia coli, we performed a genome-wide Tn5 transposon-mediated screen to identify mutants with altered susceptibility to rifampicin. Combined with targeted gene knockouts, our results showed that the beta-barrel assembly machinery plays a crucial role in restricting rifampicin from entering the cell, whereas mutants deficient in other cellular permeability barriers, such as lipopolysaccharide and enterobacterial common antigen, had no such effect. At bactericidal concentrations, the killing efficacy of rifampicin was strongly influenced by cellular functions, including iron acquisition, DNA repair, aerobic respiration, and carbon metabolism. Although iron acquisition de facto has a strong impact or dependence on cellular redox, our results suggest that their effects on rifampicin efficacy do not rely on hydroxyl radical formation. We provide evidence that maintenance of DNA replication and transcription-coupled nucleotide excision repair protects E. coli cells against rifampicin killing. Moreover, our results showed that sustained aerobic respiration and carbon catabolism diminish rifampicin's killing efficacy, and this effect relies on the inhibition of transcription but not on translation. These findings suggest that the killing efficacy of rifamycins is largely determined by cellular responses upon the inhibition of RNAP and may expand our knowledge of the action mechanisms of rifamycins.

Automated summary

The antibacterial activity of rifamycins relies on the inhibition of transcription by directly binding to bacterial DNA-dependent RNA polymerase (RNAP). However, this killing efficacy is limited in most gram-negative bacteria. In this study, we identified the cellular functions that counteract rifamycin-mediated killing in Escherichia coli, a gram-negative model organism, and found that the beta-barrel assembly machinery plays a crucial role in restricting rifampicin from entering the cell. Additionally, cellular functions such as iron acquisition, DNA repair, aerobic respiration, and carbon metabolism strongly influenced the killing efficacy of rifampicin. Maintenance of DNA replication and transcription-coupled nucleotide excision repair protected E. coli cells against rifampicin killing, while sustained aerobic respiration and carbon catabolism diminished rifampicin's killing efficacy.