(597e) High-Throughput Screening of a Promoter Library Reveals New Persister Mechanisms in Escherichia coli | AIChE

(597e) High-Throughput Screening of a Promoter Library Reveals New Persister Mechanisms in Escherichia coli


Mohiuddin, S. G. - Presenter, University of Houston
Massahi, A., University of Houston
Orman, M., University of Houston

Persisters are generally nongrowing cells that are transiently tolerant to bactericidal antibiotics. This transient state enables persister cells to recover and grow into an antibiotic-sensitive population. Persister cells can be formed stochastically or triggered by environmental stresses such as antibiotic treatment. Although the quinolone-induced SOS response is known to play a critical role in persistence (1, 2), other mechanisms induced by antibiotics are largely unknown. Using promoter and knockout cell libraries in Escherichia coli, we have identified several genes, such as waaG, guaA, and guaB, whose expression was considerably up-regulated in antibiotic treated cultures. These genes were found to be important for persister formation in E. coli as their deletion significantly enhanced the sensitivity of cells to various antibiotics (Fig. 1A). The GuaA and GuaB are the essential enzymes for the biosynthesis of ppGpp (a global persister molecule) and their deletion significantly reduces the ppGpp levels in the cells. The WaaG glucosyltransferase plays a vital role in the outer membrane by providing the first outer core D-glucose of lipopolysaccharides. The deletion of waaG selectively disrupted the transmembrane proton gradient component of the cellular proton motive force, impaired the intracellular ATP production, and significantly reduced the formation of type I persisters in stationary phase (Fig. 1BC). Previously, it has been shown that active respiration in the stationary phase aids in the arrest of growth in some cells, and inhibition of stationary phase respiration, which drives the proton motive force (PMF), reduces the number of non-growing cells (3). Here, our findings associated with the ΔwaaG strain further validates this notion. Overall, given that persistence is a critical survival strategy that evolved in many bacteria, our study will enhance the current molecular-level understanding of this conserved mechanism.

Note: This study has been published in “Microbiology Spectrum” (4).


  1. Völzing KG, Brynildsen MP. 2015. Stationary-phase persisters to ofloxacin sustain DNA damage and require repair systems only during recovery. MBio 6:e00731-15.
  2. Goormaghtigh F, Van Melderen L. 2019. Single-cell imaging and characterization of Escherichia coli persister cells to ofloxacin in exponential cultures. Sci Adv 5:eaav9462.
  3. Orman MA, Brynildsen MP. 2015. Inhibition of stationary phase respiration impairs persister formation in E. coli. Nat Commun 2015 61 6:1–13.
  4. Mohiuddin SG, Massahi A, Orman MA. 2022. High-Throughput Screening of a Promoter Library Reveals New Persister Mechanisms in Escherichia Coli. Microbiol Spectr 10:e02253-21.

Figure Legend

Fig. 1. Deletion of the waaG, guaA, and guaB genes reduced persister levels in E. coli. (A) Bi-phasic kill curves of WT, ΔwaaG, ΔguaA, and ΔguaB were generated by treating exponential phase cultures of these strains with ofloxacin for 5 h. Colony formation units (CFU) was determined by plating cells on the agar plates at indicated time points. (B) Late stationary phase (t=24 h) cells of WT and ΔwaaG strains were collected, washed, and stained with DiSC3 probe. Once the fluorescence levels reached equilibrium state the stained cells were treated with polymyxin B (PMB), chlorpromazine (CPZ) at the specified concentrations. The fluorescence levels were measured at the indicated time points using a plate reader. (C) The intracellular ATP levels of WT and ΔwaaG strains were measured at late stationary phase using a BacTiter-Glo Microbial Cell Viability assay kit. A two-tailed Student's t test with unequal variance was used to determine the statistical significance where, **P<0.01. The number of independent biological replicates, N=4. Data points represent mean ± standard deviation (SD).