Obg and Membrane Depolarization Are Part of a Microbial Bet-Hedging Strategy that Leads to Antibiotic Tolerance

Abstract

Within bacterial populations, a small fraction of persister cells is transiently capable of surviving exposure to lethal doses of antibiotics. As a bet-hedging strategy, persistence levels are determined both by stochastic induction and by environmental stimuli called responsive diversification. Little is known about the mechanisms that link the low frequency of persisters to environmental signals. Our results support a central role for the conserved GTPase Obg in determining persistence in Escherichia coli in response to nutrient starvation. Obg-mediated persistence requires the stringent response alarmone (p)ppGpp and proceeds through transcriptional control of the hokB-sokB type I toxin-antitoxin module. In individual cells, increased Obg levels induce HokB expression, which in turn results in a collapse of the membrane potential, leading to dormancy. Obg also controls persistence in Pseudomonas aeruginosa and thus constitutes a conserved regulator of antibiotic tolerance. Combined, our findings signify an important step toward unraveling shared genetic mechanisms underlying persistence.

Substrate Recognition and Activity Regulation of the Escherichia coli mRNA Endonuclease MazF

“…These phenotypically distinct cells encompass only a very small fraction of the total population and are in a metabolically dormant state that makes them tolerant to a wide variety of antibiotics without having acquired resistance. Discovered almost 70 years ago, the molecular mechanisms behind persistence are becoming clear only recently (3)(4)(5)(6). The activities of toxin-antitoxin (TA) 5 modules have been tightly linked with the establishment of the persister phenotype (7)(8)(9).…”

Hypothesis: type I toxin–antitoxin genes enter the persistence field—a feedback mechanism explaining membrane homoeostasis

“…Thus, reduced or increased levels of Obg reduced or increased the persistence level, respectively. Transcriptomic analysis revealed that Obg, in the presence of ( p)ppGpp, stimulates hokB transcription [15]. Jan Michels's group also elegantly showed that modest levels of HokB induced a dramatic decrease in the membrane potential and simultaneously induced cell stasis and persistence but not cell killing [15].…”

“…Transcriptomic analysis revealed that Obg, in the presence of ( p)ppGpp, stimulates hokB transcription [15]. Jan Michels's group also elegantly showed that modest levels of HokB induced a dramatic decrease in the membrane potential and simultaneously induced cell stasis and persistence but not cell killing [15]. Cell growth could be restored by the expression of proteorhodopsin that provides an active proton pump able to increase the membrane potential.…”

“…However, when a cell encounters stressful conditions, increased levels of ( p)ppGpp, together with Obg, stimulate hokB transcription. The increased hokB mRNA level overrides inhibition by SokB-RNA, thus leading to HokB production and reduction of the membrane potential [15]. This change in membrane potential is 'sensed' by RNase E, leading to its detachment from the membrane and reduction of its enzymatic activity.…”

“…The known TA gene families can be divided into six different types, depending on how the antitoxins inhibit cognate toxins [12,13]. Here I focus on type I and II TAs that, remarkably, can mediate persistence by different mechanisms that are both controlled by ( p)ppGpp [14,15]. In type I TAs, the antitoxins are antisense RNAs inhibiting toxin mRNA translation [16], whereas in type II TAs, the antitoxins are proteins sequestering cognate toxin activity by direct protein::protein contact [12,17].…”