Most antibiotics are natural compounds or their derivatives, and bacteria have evolved defensive mechanisms to resist them. Many of these mechanisms are still poorly understood or unknown. This study reveals that in Bacillus subtilis, the transcription factor HelD increases resistance to rifampicin by protecting its target, RNA polymerase (RNAP). This protection is mediated by the HelD N-terminal domain that penetrates into RNAP to the close vicinity of the rifampicin binding pocket. Importantly, the bacterium detects low rifampicin levels using a unique regulatory system involving two convergent promoters with finely tuned kinetic properties. In the absence of rifampicin, the stronger antisense promoter inhibits transcription from the sense promoter. In the presence of subinhibitory rifampicin concentration, the antisense promoter is more likely to encounter rifampicin-bound RNAP. This relieves the repression from the sense promoter, increasing its transcription by almost two orders of magnitude, boosting helD expression. A similar two-promoter arrangement also controls the pps gene, which encodes a rifampicin-modifying enzyme. These findings define a widespread bacterial response system sensitive to rifampicin, as this dual-promoter architecture is conserved across many bacterial species and found upstream of genes potentially involved in rifampicin resistance, such as those for hydrolases, transporters, and transferases.

Department of Genetics and Microbiology Faculty of Science Charles University Viničná 5 128 44 Prague Czech Republic

Department of Mass Spectrometry of Biopolymers Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences Flemingovo náměstí 542 2 160 00 Prague Czech Republic

Institute of Physics Faculty of Mathematics and Physics Charles University Ke Karlovu 5 121 16 Prague 2 Czech Republic

Laboratory of Bioinformatics Institute of Microbiology of the Czech Academy of Sciences Vídeňská 1083 142 20 Prague Czech Republic

Laboratory of Microbial Genetics and Gene Expression Institute of Microbiology of the Czech Academy of Sciences Vídeňská 1083 142 20 Prague Czech Republic

Laboratory of Structural Biology and Cell Signaling Institute of Microbiology of the Czech Academy of Sciences Vídeňská 1083 142 20 Prague Czech Republic

Laboratory of Structure and Function of Biomolecules Institute of Biotechnology of the Czech Academy of Sciences Průmyslová 595 252 50 Vestec Czech Republic

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