Gamblers: an Antibiotic-induced Evolvable Cell Subpopulation Differentiated by Reactive-oxygen-induced General Stress Response

Abstract

32Antibiotics can induce mutations that cause antibiotic resistance. Yet, despite their importance, 33 mechanisms of antibiotic-promoted mutagenesis remain elusive. We report that the 34 fluoroquinolone antibiotic ciprofloxacin (cipro) induces mutations that cause drug resistance by 35 triggering differentiation of a mutant-generating cell subpopulation, using reactive oxygen species 36 (ROS) to signal the sigma-S (σ S ) general-stress response. Cipro-generated DNA breaks activate 37 the SOS DNA-damage response and error-prone DNA polymerases in all cells. However, 38 mutagenesis is restricted to a cell subpopulation in which electron transfer and SOS induce ROS, 39 which activate the σ S response, allowing mutagenesis during DNA-break repair. When sorted, 40 this small σ S -response-"on" subpopulation produces most antibiotic cross-resistant mutants. An 41FDA-approved drug prevents σ S induction specifically inhibiting antibiotic-promoted mutagenesis. 42 Furthermore, SOS-inhibited cell division, causing multi-chromosome cells, is required for 43 mutagenesis. The data support a model in which within-cell chromosome cooperation together 44 with development of a "gambler" cell subpopulation promote resistance evolution without risking 45 most cells. 46 47 48 49 51 break repair, reactive oxygen species (ROS), RpoS (σ S ) stress response, SOS response, 52 starvation stress response, stress-induced mutagenesis, transient differentiation 53 54 100 once antibiotics have gone (Lewis, 2010). Persister formation can occur stochastically, leaving 101 populations ready for a stress that they have not encountered (Balaban et al., 2004), and can also 102 be induced responsively via stress-response regulons including the SOS- (Dorr et al., 2009) and 103 σ S -response (Radzikowski et al., 2016) regulons. It is unknown whether antibiotics induce 104 4 transient differentiation that could promote resistance through mutagenesis, e.g., (Frenoy and 105 Bonhoeffer, 2018). 106Here we show that low, sub-inhibitory doses of cipro induce transient differentiation of a 107 small cell subpopulation with high ROS and σ S -response activity, that generates mutants, 108 including cross-resistant mutants: a "gambler" subpopulation. We show that the ROS promote 109 mutagenesis in gamblers by activating the σ S response, which allows mutagenic repair of cipro-110 triggered DSBs-a novel signaling/differentiating role of ROS in mutagenesis. We elaborate the 111 regulatory chain from cipro to ROS to σ S response to mutant production, and also discover a 112 requirement for SOS-induced inhibition of cell division, causing multiple chromosomes per cell. 113Mathematical analysis supports a model in which multiple chromosomes allow sharing of cellular 114 resources (e.g., recombination, complementation), avoiding deleterious consequences of some 115 mutations during mutagenesis and repair. Thus, multiple chromosomes allow higher mutation 116 rates to be maintained -resulting in faster adaptation. The findings imply a highly regulated, novel 117 transie...

Growth‐dependent heterogeneity in the DNA damage response in Escherichia coli

“…The induction of the SOS response is known to have multiple consequences beyond facilitating the repair of DNA (Podlesek & Žgur Bertok, 2020), including increasing antibiotic tolerance (Dörr et al , 2009; Wu et al , 2015), modulating expression of mobile genetic elements (Beaber et al , 2004; Baharoglu et al , 2010; Fornelos et al , 2016), and increasing the rate of mutagenesis (Vaisman et al , 2012; Dapa et al , 2017; Pribis et al , 2019). Our observed growth‐dependent heterogeneity in the fraction of SOS‐induced cells suggests that care must be taken when making quantitative estimates of the mutation rate under conditions of sub‐lethal DNA damage.…”

“…recA ), inhibition of cell division ( sulA ), translesion DNA synthesis, toxin‐antitoxin modules, and the lexA gene itself (Kreuzer, 2013; Baharoglu & Mazel, 2014). In addition to enabling survival under DNA damaging conditions, SOS induction can contribute to an increased rate of mutagenesis (Vaisman et al , 2012; Dapa et al , 2017; Pribis et al , 2019), an increased prevalence of antibiotic tolerance (Dörr et al , 2009; Wu et al , 2015), and misregulation of the transfer rate of conjugative plasmids and other mobile elements (Beaber et al , 2004; Baharoglu et al , 2010; Fornelos et al , 2016).…”

Hypermutation in single-stranded DNA

“…Adaptive mutagenesis relies on error-prone DNA synthesis that is activated by stress caused by DNA break(s) and is often referred to as stress-induced mutagenesis (reviewed in [20]). Stress-induced mutagenesis in E. coli relies upon a large gene-network and may be responsible for up to 50 % of spontaneous mutagenesis in starving E. coli [21] as well as for mutagenesis in strains treated with clastogens [22].…”

Targeting evolution to inhibit antibiotic resistance

“…Edaravone, used to treat patients who have suffered strokes or have ALS, was shown to alter the frequency by which the RpoS program is activated in bacterial cells. However, these effects were indirect: Edaravone is a general antioxidant and not a direct inhibitor of RpoS [72]. Therefore, although in principle reducing ROS could be a useful strategy to reduce mutagenesis, such an approach (e.g., edaravone treatment) will likely be highly detrimental to the clearance of infections given that host defenses rely on ROS production.…”