(58e) Lon Deletion Impairs Persister Cell Resuscitation in Escherichia coli

Bacterial persisters are a small subpopulation of phenotypic variants in isogenic cell populations that are transiently tolerant to high concentrations of antibiotics. Persisters aggravate the crisis of antibiotic failure as they can evolve to antibiotic-resistant mutant strains (1). The deletion of lon has been frequently shown to reduce fluoroquinolone persistence (2, 3), and the mechanism underlying this phenomenon is not clear. Lon was initially thought to induce persistence by degrading antitoxin molecules through a ppGpp/polyphosphate-dependent mechanism (4). Another suggested mechanism for the role of Lon in bacterial persistence depends on the cell division inhibitor protein SulA and is based on the observation that deletion of the sulA gene restores fluoroquinolone persister levels in lon-deficient strains (2, 5). However, it is unclear from these studies whether targeting Lon protease chemically or genetically eradicates persister cells or converts them to a viable but non-culturable (VBNC) state. In this study, using vector constructs that allow fine-tuning of recombinant protein expression, we have demonstrated that deletion of lon impairs the ability of persister cells to form colonies during recovery (Fig. 1AB), without killing these cells, through a sulA- and ftsZ-dependent mechanism. We have also shown that the reduced persister levels in Δlon strain can be transient, and starvation can restore their culturability (Fig. 1C). Starvation-induced SulA degradation or expression of Lon during recovery facilitates Z-ring formation in Δlon persisters. When we calculated the ratio of the cell length (L in µm) to the number of Z-rings (Z) for each ofloxacin-treated intact cell analyzed, we found a correlation between persister resuscitation and calculated L/Z values. Both WT and Δlon persisters having L/Z ≈1 with L>5 µm and Z>5 were mostly resuscitated under the conditions studied here (Fig. 1DE). Overall, our findings here suggest that Z-ring formation may be a key biomarker for persisters.

Note: This study has been published in mBio (6).

References

1. Levin-Reisman I, Ronin I, Gefen O, Braniss I, Shoresh N, Balaban NQ. 2017. Antibiotic tolerance facilitates the evolution of resistance. Science. 355:826–830.
2. Matange N. 2020. Highly contingent phenotypes of lon protease deficiency in Escherichia coli upon antibiotic challenge. J Bacteriol 202:561–580.
3. Theodore A, Lewis K, Vulić M. 2013. Tolerance of Escherichia coli to fluoroquinolone antibiotics depends on specific components of the SOS response pathway. Genetics 195:1265–1276.
4. Harms A, Fino C, Sørensen MA, Semsey S, Gerdes K. 2017. Prophages and growth dynamics confound experimental results with antibiotic-tolerant persister cells. MBio 8.
5. Shan Y, Gandt AB, Rowe SE, Deisinger JP, Conlon BP, Lewis K. 2017. ATP-Dependent persister formation in Escherichia coli. MBio 8.
6. Mohiuddin SG, Massahi A, Orman MA. 2022. lon Deletion Impairs Persister Cell Resuscitation in Escherichia coli. MBio 13:e02187-21.

Figure Legend

Fig. 1. Lon overexpression during recovery or PBS starvation rescue Δlon persisters. (A-B) E. coli WT and Δlon cells containing an inducible lon overexpression plasmid (pUA66-lon) or empty vector control (pUA66-EV) were treated with ofloxacin (5 μg/ml) for 6 h and transferred to LB agar plates supplemented with isopropyl β-D-1-thiogalactopyranoside (IPTG) at the indicated concentrations (0 and 1 mM). Colony formation units (CFU) were measured for each strain before and after ofloxacin treatment. The number of independent biological replicates, N = 4. (C) WT and Δlon cells with or without pUA66-lon or pUA66-EV were treated with ofloxacin for 6 h, transferred to sterile PBS solution with or without 1 mM IPTG, and incubated at 37°C in a shaker for 7 days. At the designated time points starved cells were collected and spotted onto LB agar plates to enumerate surviving cells. n = 4. (D-E) WT and Δlon strains harboring the IPTG-inducible FtsZ-GFP expression plasmid (pUA66-ftsZ-gfp) were transferred to PBS solution after 6 h of ofloxacin treatment and cultured at 37°C with shaking. IPTG (1 mM) was added to cultures before and during ofloxacin treatment to express FtsZ-GFP. After 2 days of incubation in PBS solution, cells were then collected and spread on LB agarose pads containing 1 mM IPTG. Fluorescence microscope with an onstage incubator was used to monitor the cellular growth on the pads for 24 h. Cell lengths, as well as Z-ring structures and numbers, were determined for both resuscitating (persisters) and nonresuscitating (VBNC) cells to generate plots of the number of Z-rings versus cell length N=4. One-way analysis of variance (ANOVA) with Dunnett’s post hoc test was used to determine statistical significance. *, P<0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, nonsignificant.