lon Deletion Impairs Persister Cell Resuscitation in Escherichia coli

Bacterial persisters are nongrowing cells highly tolerant to bactericidal antibiotics. However, this tolerance is reversible and not mediated by heritable genetic changes. Lon, an ATP-dependent protease, has repeatedly been shown to play a critical role in fluoroquinolone persistence in Escherichia coli. Although lon deletion (Δlon) is thought to eliminate persister cells via accumulation of the cell division inhibitor protein SulA, the exact mechanism underlying this phenomenon is not yet elucidated. Here, we show that Lon is an important regulatory protein for the resuscitation of the fluoroquinolone persisters in E. coli, and lon deletion impairs the ability of persister cells to form colonies during recovery through a sulA- and ftsZ-dependent mechanism. Notably, this observed "viable but nonculturable" state of antibiotic-tolerant Δlon cells is transient, as environmental conditions, such as starvation, can restore their culturability. Our data further indicate that starvation-induced SulA degradation or expression of Lon during recovery facilitates Z-ring formation in Δlon persisters, and Z-ring architecture is important for persister resuscitation in both wild-type and Δlon strains. Our in-depth image analysis clearly shows that the ratio of cell length to number of FtsZ rings for each intact ofloxacin-treated cell predicts the probability of resuscitation and, hence, can be used as a potential biomarker for persisters. IMPORTANCE The ATP-dependent Lon protease is one of the most studied bacterial proteases. Although deletion of lon has been frequently shown to reduce fluoroquinolone persistence, the proposed mechanisms underlying this phenomenon are highly controversial. Here, we have shown that lon deletion in Escherichia coli impairs the ability of persister cells to form colonies during recovery and that this reduction of persister levels in lon-deficient cells can be transient. We also found that altered Z-ring architecture is a key biomarker in both wild-type and lon-deficient persister cells transitioning to a normal cell state. Collectively, our findings highlight the importance of differentiating persister formation mechanisms from resuscitation mechanisms and underscore the critical role of the nonculturable cell state in antibiotic tolerance.