Antibiotic-producing streptomycetes are rich sources of resistance mechanisms against endogenous and exogenous antibiotics. An ECF sigma factor σR (SigR) is known to govern the thiol-oxidative stress response in Streptomyces coelicolor. Amplification of this response is achieved by producing an unstable isoform of σR called σR′. In this work, we present evidence that antibiotics induce the SigR regulon via a redox-independent pathway, leading to antibiotic resistance. The translation-inhibiting antibiotics enhanced the synthesis of stable σR, eliciting a prolonged response. WblC/WhiB7, a WhiB-like DNA-binding protein, is responsible for inducing sigRp1 transcripts encoding the stable σR. The amount of WblC protein and its binding to the sigRp1 promoter in vivo increased upon antibiotic treatment. A similar phenomenon appears to exist in Mycobacterium tuberculosis as well. These findings reveal a novel antibiotic-induced resistance mechanism conserved among actinomycetes, and also give an explicit example of overlap in cellular damage and defense mechanisms between thiol-oxidative and anti- translational stresses.

Bacteria in natural habitats are exposed to myriad redox-active compounds (RACs), which include producers of reactive oxygen species (ROS) and reactive electrophile species (RES) that alkylate or oxidize thiols. RACs can induce oxidative stress in cells and activate response pathways by modulating the activity of sensitive regulators. However, the effect of a certain compound on the cell has been investigated primarily with respect to a specific regulatory pathway. Since a single compound can exert multiple chemical effects in the cell, its effect can be better understood by time-course monitoring of multiple sensitive regulatory pathways that the compound induces. We investigated the effect of representative RACs by monitoring the activity of three sensor-regulators in the model actinobacterium Streptomyces coelicolor; SoxR that senses reactive compounds directly through oxidation of its [2Fe–2S] cluster, CatR/PerR that senses peroxides through bound iron, and an anti-sigma factor RsrA that senses RES via disulfide formation. The time course and magnitude of induction of their target transcripts were monitored to predict the chemical activities of each compound in S. coelicolor. Phenazine methosulfate (PMS) was found to be an effective RAC that directly activated SoxR and an effective ROS-producer that induced CatR/PerR with little thiol-perturbing activity. p-Benzoquinone was an effective RAC that directly activated SoxR, with slower ROS-producing activity, and an effective RES that induced the RsrA-SigR system. Plumbagin was an effective RAC that activated SoxR, an effective ROS-producer, and a less agile but effective RES. Diamide was an RES that effectively formed disulfides and a weak RAC that activated SoxR. Monobromobimane was a moderately effective RES and a slow producer of ROS. Interestingly, benzoquinone induced the SigR system by forming adducts on cysteine thiols in RsrA, revealing a new pathway to modulate RsrA activity. Overall, this study showed that multiple chemical activities of a reactive compound can be conveniently monitored in vivo by examining the temporal response of multiple sensitive regulators in the cell to reveal novel activities of the chemicals.

σR (SigR) is an alternative sigma factor that enables gene expression in Streptomyces coelicolor to cope with thiol oxidation and antibiotic stresses. Its activity is repressed by a zinc‐containing anti‐sigma (ZAS) factor RsrA that senses thiol oxidants and electrophiles. Inactivation of RsrA by disulfide formation has been well studied. Here we investigated another pathway of RsrA inactivation by electrophiles. Mass spectrometry revealed alkylation of RsrA in vivo by N‐ethylmaleimide (NEM) at C61 and C62 located in the C‐terminal loop. Substitution mutation (C61S/C62S) in RsrA decreased the induction of σR target genes by electrophiles and made cells more sensitive to electrophiles. In contrast to stable protein of oxidized RsrA, alkylated RsrA is subjected to degradation partly mediated by ClpP proteases. RsrA2, a redox‐sensitive homolog of RsrA in S. coelicolor lacking cysteine in the terminal loop, did not respond to electrophiles. However, redox‐sensitive RsrA homologs in other Actinobacteria also harboring terminal loop cysteines all responded to electrophiles. These results indicate that the activity of RsrA can be modulated via cysteine alkylation, apart from disulfide formation of zinc‐coordinating cysteines. This pathway expands the spectrum of signals that the σR‐RsrA system can sense and reveals another intricate regulatory layer for optimal survival of Actinobacteria.