Persistence Increases in the Absence of the Alarmone Guanosine Tetraphosphate by Reducing Cell Growth.

Nityananda Chowdhury,Thomas K Wood,Brian W Kwan

doi:10.1038/srep20519

Abstract

Most bacterial cells are stressed, and as a result, some become tolerant to antibiotics by entering a dormant state known as persistence. The key intracellular metabolite that has been linked to this persister state is guanosine tetraphosphate (ppGpp), the alarmone that was first linked to nutrient stress. In Escherichia coli, ppGpp redirects protein production during nutrient stress by interacting with RNA polymerase directly and by inhibiting several proteins. Consistently, increased levels of ppGpp lead to increased persistence; but, the mechanism by which elevated ppGpp translates into persistence has not been determined. Hence, we explored persistence in the absence of ppGpp so that the underlying mechanism of persister cell formation could be explored. We found that persister cells still form, although at lower levels, in the absence of ppGpp. Additionally, the toxin/antitoxin systems that we investigated (MqsR, MazF, GhoT, and YafQ) remain able to increase persistence dramatically in the absence of ppGpp. By overproducing each E. coli protein from the 4287 plasmid vectors of the ASKA library and selecting for increased persistence in the absence of ppGpp (via a relA spoT mutant), we identified five new proteins, YihS, PntA, YqjE, FocA, and Zur, that increase persistence simply by reducing cell growth.

Highlights

The mechanism of persister cell formation has been elusive in that deletion studies have yielded little insight to date[12], but two common elements are dependence on guanosine tetraphosphate and on toxins of toxin/ antitoxin (TA) systems
PpGpp was linked to toxin/antitoxins in 199624 since ppGpp is required for MazF toxicity (MazF is an endonuclease toxin of the type 2 MazF/MazE TA system), and ppGpp was shown in 200318 to be required for persistence of the HipA7 variant
It has been proposed that production of polyphosphate, due to elevated ppGpp levels resulting from stochastic events, activates Lon protease and that activated Lon degrades antitoxins, which would result in activated toxins[25]

Summary

Introduction

The mechanism of persister cell formation has been elusive in that deletion studies have yielded little insight to date[12], but two common elements are dependence on guanosine tetraphosphate (ppGpp) and on toxins of toxin/ antitoxin (TA) systems. For the YoeB/YefM TA system, one of the TA systems upon which the polyphosphate model is based[25], degradation of YefM antitoxin has been shown by the same lab in a subsequent publication to be independent of ppGpp and polyphosphate[27] This model[25] neglects persister formation in response to environmental stress since it relies solely on stochastic generation of persister cells[28]. PpGpp is required, Lon protease and polyphosphate are not related to persistence that stems from activation of TA systems in which the antitoxin is an antisense RNA rather than a protein[28]. The link between polyphosphate and toxin activation via Lon protease is controversial, and the underlying mechanisms for persister cell formation via ppGpp have not been fully elucidated. There are several distinct ways to trigger persister cell formation but the consistent feature is that cells that are less fit have increased persistence[30]

Methods

Results

Conclusion