RESEARCH HIGHLIGHTS Nature Reviews Microbiology | AOP, published online 29 June 2015; doi:10.1038/nrmicro3526
B AC T E R I A L P H Y S I O LO GY
Obg controls bacterial persistence Obg determines persistence by reducing membrane potential
Stochastic switching and environmental sensing within bacterial populations generates a small subpopulation of dormant persister cells that can survive exposure to normally-lethal doses of antibiotics. The mechanisms controlling persistence have been largely unclear; however, Verstraeten et al. now show that the GTPase Obg determines persistence by reducing membrane potential via a pathway that involves (p)ppGpp and HokB. Bacterial persistence probably occurs due to the suppression of major cellular processes, such as DNA and protein synthesis, resulting in tolerance to antibiotics. Furthermore, the generation of persisters has been linked to the stringent response, which is a bacterial adaptation to stress conditions that is mediated by the alarmone (p)ppGpp. As Obg is a conserved GTPase that is involved in important cellular processes that are targeted by antibiotics, such as DNA and protein synthesis, and has also been shown to bind (p)ppGpp, the authors investigated a potential role for Obg in regulating bacterial persistence. The authors examined the effect of Obg overexpression on the survival of Escherichia coli cultures following treatment with
NPG
antibiotics and observed a significant dose-dependent increase in persistence. Conversely, decreasing Obg using antisense RNA significantly reduced survival, demonstrating that Obg concentration controls persistence in E. coli populations. To assess the effect of Obg at a single-cell level, the authors performed fluorescence- activated cell sorting (FACS) analysis of cells expressing a fluorescent version of Obg; this analysis revealed that subpopulations expressing high levels of Obg contained a greater proportion of persisters than subpopulations expressing low levels of Obg, demonstrating that persistence in individual cells is controlled by natural fluctuations in the expression of Obg. To investigate a role for the stringent response in the generation of persisters, the authors overexpressed Obg in mutants lacking (p)ppGpp and found that survival was no longer increased, demonstrating a requirement for (p)ppGpp in Obg-mediated persistence. Microarray analysis of Obg overexpressing and underexpressing cells identified hokB, which encodes a toxin that causes membrane depolarization, as a potential candidate for involvement in the Obg-mediated persistence pathway. FACS analysis showed that expression of hokB and Obg was tightly correlated in individual cells, suggesting that Obg causes persistence via the transcriptional activation of hokB. Consistent with this, Obg did not induce persistence in a mutant lacking hokB, and HokB overexpression resulted in a significant increase in the number of persisters.
NATURE REVIEWS | MICROBIOLOGY
To determine whether persistence could be attributed to the membrane depolarization activity of HokB, the authors used a voltage-sensitive green fluorescent dye to monitor membrane potential and observed membrane depolarization following the induction of Obg or HokB expression. A direct correlation between membrane depolarization and Obg or HokB levels was also observed at the single-cell level, demonstrating that HokB induces persistence by decreasing membrane polarization. Consistent with this hypothesis, overexpression of either Obg or HokB was not able to significantly increase persistence in cells in which membrane repolarization was induced by expression of proteor hodopsin or by mannitol treatment. In summary, these experiments provide a mechanistic insight into persistence and suggest a model in which binding of (p)ppGpp to Obg or an unknown effector activates hokB transcription, which, in cells expressing high levels of Obg, results in membrane depolarization and transition to a persistent state. Importantly, although these data elucidate how persisters are generated, how cells return to the non-persistent state is currently unknown. Finally, as persistence is a major cause of failure of antibiotic treatment, this study establishes Obg as a potential drug target for the treatment of chronic bacterial infections. Denise Waldron ORIGINAL RESEARCH PAPER Verstraeten, N. et al. Obg and membrane depolarization are part of a microbial bet-hedging strategy that leads to antibiotic tolerance. Mol Cell. http://dx.doi. org/10.1016/j.molcel.2015.05.011 (2015)
VOLUME 13 | AUGUST 2015 © 2015 Macmillan Publishers Limited. All rights reserved
B AC T E R I A L P H Y S I O LO GY
Obg controls bacterial persistence Obg determines persistence by reducing membrane potential
Stochastic switching and environmental sensing within bacterial populations generates a small subpopulation of dormant persister cells that can survive exposure to normally-lethal doses of antibiotics. The mechanisms controlling persistence have been largely unclear; however, Verstraeten et al. now show that the GTPase Obg determines persistence by reducing membrane potential via a pathway that involves (p)ppGpp and HokB. Bacterial persistence probably occurs due to the suppression of major cellular processes, such as DNA and protein synthesis, resulting in tolerance to antibiotics. Furthermore, the generation of persisters has been linked to the stringent response, which is a bacterial adaptation to stress conditions that is mediated by the alarmone (p)ppGpp. As Obg is a conserved GTPase that is involved in important cellular processes that are targeted by antibiotics, such as DNA and protein synthesis, and has also been shown to bind (p)ppGpp, the authors investigated a potential role for Obg in regulating bacterial persistence. The authors examined the effect of Obg overexpression on the survival of Escherichia coli cultures following treatment with
NPG
antibiotics and observed a significant dose-dependent increase in persistence. Conversely, decreasing Obg using antisense RNA significantly reduced survival, demonstrating that Obg concentration controls persistence in E. coli populations. To assess the effect of Obg at a single-cell level, the authors performed fluorescence- activated cell sorting (FACS) analysis of cells expressing a fluorescent version of Obg; this analysis revealed that subpopulations expressing high levels of Obg contained a greater proportion of persisters than subpopulations expressing low levels of Obg, demonstrating that persistence in individual cells is controlled by natural fluctuations in the expression of Obg. To investigate a role for the stringent response in the generation of persisters, the authors overexpressed Obg in mutants lacking (p)ppGpp and found that survival was no longer increased, demonstrating a requirement for (p)ppGpp in Obg-mediated persistence. Microarray analysis of Obg overexpressing and underexpressing cells identified hokB, which encodes a toxin that causes membrane depolarization, as a potential candidate for involvement in the Obg-mediated persistence pathway. FACS analysis showed that expression of hokB and Obg was tightly correlated in individual cells, suggesting that Obg causes persistence via the transcriptional activation of hokB. Consistent with this, Obg did not induce persistence in a mutant lacking hokB, and HokB overexpression resulted in a significant increase in the number of persisters.
NATURE REVIEWS | MICROBIOLOGY
To determine whether persistence could be attributed to the membrane depolarization activity of HokB, the authors used a voltage-sensitive green fluorescent dye to monitor membrane potential and observed membrane depolarization following the induction of Obg or HokB expression. A direct correlation between membrane depolarization and Obg or HokB levels was also observed at the single-cell level, demonstrating that HokB induces persistence by decreasing membrane polarization. Consistent with this hypothesis, overexpression of either Obg or HokB was not able to significantly increase persistence in cells in which membrane repolarization was induced by expression of proteor hodopsin or by mannitol treatment. In summary, these experiments provide a mechanistic insight into persistence and suggest a model in which binding of (p)ppGpp to Obg or an unknown effector activates hokB transcription, which, in cells expressing high levels of Obg, results in membrane depolarization and transition to a persistent state. Importantly, although these data elucidate how persisters are generated, how cells return to the non-persistent state is currently unknown. Finally, as persistence is a major cause of failure of antibiotic treatment, this study establishes Obg as a potential drug target for the treatment of chronic bacterial infections. Denise Waldron ORIGINAL RESEARCH PAPER Verstraeten, N. et al. Obg and membrane depolarization are part of a microbial bet-hedging strategy that leads to antibiotic tolerance. Mol Cell. http://dx.doi. org/10.1016/j.molcel.2015.05.011 (2015)
VOLUME 13 | AUGUST 2015 © 2015 Macmillan Publishers Limited. All rights reserved
