**3.2. Mechanism of σE-dependent PCD**

quorum-sensing factor as a linear pentapeptide Asn-Asn-Trp-Asn-Asn (NNWNN), called an extracellular death factor (EDF) [51]. The EDF directly binds to MazF dimers to release MazF from the MazF–MazE complex, leading to cell death [52]. Moreover, *mazEF*-mediated PCD is activated under various stressful conditions including extreme amino acid starvation, inhibition of transcription and/or translation by antibiotics including rifampicin, chloramphenicol, and spectinomycin, an inhibitor protein of translation, DNA damage caused by thymine starvation as well as by mitomycin C, nalidixic acid and UV irradiation, and oxidative stress [47]. Notably, 28 other putative TA systems including DinJ-DafQ, DinP-YafN, RelB-RelE, and

**Figure 3.** Activation pathways of PCD in *E. coli*. Several stresses affect cellular components including envelopes, DNA, and proteins, and these damaged materials become a signal for each stress response directly or indirectly. If the damage is excessive, PCD is triggered by several mechanisms. In *E. coli*, three mechanisms for PCD including TA systems, SOSresponse–dependent cell lysis and σE-dependent cell lysis have been reported. SOS mainly responds to DNA damage and σE mainly responds to envelope damage. These three responses can directly induce PCD, but they are weakly

390 *Escherichia coli* Escherichia coli - Recent Advances on Physiology, Pathogenesis and Biotechnological Applications - Recent Advances on Physiology, Pathogenesis and Biotechnological Applications

An SOS response-mediated PCD pathway was recently identified in *E. coli* is called apoptosislike death (ALD) pathway [48]. The ALD pathway is activated by an extreme SOS response under severe DNA damage conditions [53] and follows apoptosis-like characteristics including rRNA degradation by the endoribonuclease YbeY [54], upregulation of a unique set of extensive damage-induced genes, decrease in respiration activity, and formation of high levels of OH⁻, resulting in cell death [53]. Analysis of the relationship between *mazEF*-EDF and ALD revealed that the ALD pathway is inhibited by the *mazEF*-EDF–mediated PCD pathway

In addition to DNA damage, envelope damage has been shown to be a trigger of PCD in *E. coli*. Envelope damage is caused by various factors including antibiotics, toxic metabolites, bacteriocins, osmotic, pH, and salt. In Gram-negative bacteria, the damage is sensed and transduced via ESRs. The ESRs alter the expression of specific genes related to functions that modify the envelope, rid cells of the toxic entity and/or repair the envelope damage [3]. σ<sup>E</sup>-dependent PCD, which is one of envelope damage related PCDs, was first reported

ChpS-ChpB have been identified in the *E. coli* K12 genome [12].

[48].

connected to each other [46, 48].

At the early stationary phase, *E. coli* cells undergo a decrease in viable cell number and almost all of the cells become VBNC cells [16, 58]. Elevation of the activate intracellular σE level, due to disruption of *rseA* for anti-σE or *rpoE*-increased expression, causes cell lysis at the beginning of the stationary phase, and this lysis occurs in wild-type cells at a low level [19, 59]. This mechanism may contribute to the removal of VBNC cells that have accumulated at a specific phase probably due to the accumulation of intracellular oxidative stress, and it is called σE-dependent PCD. Murata et al. showed that σE-dependent PCD is mediated by MicA, RybB, and PpiD [59]. MicA and RybB are transencoded sRNAs, and their expression is positively regulated by σE [60–62]. When misfolded OMPs or periplasmic proteins have accumulated, the expression of their sRNAs is induced by active σE, and MicA and RybB cause reduction in the levels of mRNAs of *ompA* and both *ompC* and *ompW*, respectively, via interaction between the sRNAs and the corresponding mRNA by assisting Hfq as an RNA chaperon and degradation of the mRNAs by ribonucleases [63]. Some OMPs are known to be physiologically and structurally crucial for cell activity [64]. OmpA as a structure protein is involved in the maintenance of cell shape and the passage of hydrophilic compounds through the outer membrane [65]. OmpC is the major porin protein that functions as a cation-selective porin [66]. However, no physiological function of OmpW has yet been determined [67]. These OMPs are greatly decreased in σE-activated cells [4, 19], and *micA*- or *rybB*-disrupted mutants and *micA*- or *rybB*-overexpressed cells repress and induce σE-dependent PCD, respectively [59]. Therefore, σE-dependent PCD is caused by the reduction of OMPs via posttranscriptional regulation including MicA and RybB. Recently, MicL has been found as the third σE-dependent sRNA that targets an mRNA for lipoprotein Lpp [45]. Since Lpp is the most abundant protein in the outer membrane [64], MicL may also be involved in σE-dependent PCD.

The level of PpiD is greatly reduced in σE-activated cells, though its regulation mechanism is unknown [68]. PpiD is a peptidyl-prolyl *cis*-*trans* isomerase as a periplasmic folding catalyst that catalyzes the rapid interconversion between the *cis* and *trans* forms of the peptide bond Xaa-Pro [69]. PpiD recognizes the early OMP folding intermediates and suppresses OMP biogenesis defects. Indeed, overexpression of PpiD represses σE-dependent cell lysis probably due to the acceleration of OMP folding [68]. Thus, the reduction of PpiD ensures the elimination of OMPs after the degradation of OMP mRNAs by sRNAs.

As shown in the model in **Figure 4**, when cells are exposed to some stresses as signals, mainly oxidative stress [19, 70], unfolded proteins accumulate in the outer membrane or periplasmic space, in turn causing the elevation of active σE in the cytoplasm. Active σE induces the expression of sRNAs, leading to the reduction of OMPs including Lpp. Furthermore, the reduction of PpiD via active σE enhances the disintegration of OMPs, resulting in collapse of the integrity of the outer membrane and finally lysis of cells.

As shown in **Figure 4**, active σE determines the direction to either the repair or cell lysis pathway, presumably reflecting the level of damage of OMPs. If only a few OMPs are damaged, the number of active σE molecules may be not enough to express sRNAs such as *micA* and *rybB*, which may be insufficient to cause cell lysis but can express genes for the repair pathway. On the other hand, damage of OMPs over a certain threshold evokes the cascade of σE-dependent cell lysis. The coincidence of the fact that OMPs are monitoring proteins for cell damage and/or σ<sup>E</sup>-dependent cell lysis and the fact that σE-dependent cell lysis is induced by reduction in the amount of OMPs is highly notable. OMP-damaged cells may be much more


Survival Strategy of *Escherichia coli* in Stationary Phase: Involvement of σ<sup>E</sup>

σE-dependent cell lysis seems to eliminate some of the VBNC cells that have been damaged by some kinds of stress. The amount of cell lysis increases in parallel with increase in VBNC cells in the stationary phase, and most of the lysis was suppressed by enhanced expression of *rseA* [68]. An *rseA*-disrupted mutant that constitutively expresses active σE shows a phenotype that is characterized by decrease in cell density without a significant influence on colony-forming unit (CFU) but with protein accumulation in the medium [19]. This phenotype suggests that VBNC cells or some of the VBNC cells are subjected to σE-dependent cell lysis. This limited cell lysis might reflect the existence of a mechanism to distinguish damaged and undamaged cells. It is hypothesized that VBNC cells to be lysed have damaged OMPs to some extent and thus are susceptible to σE-dependent cell lysis. Notably, sRNAs such as MicA and RybB play crucial roles in σE-dependent cell lysis in the LTSP because mutation rate drastically increases

Taken together, the findings have shown that *E. coli* has developed an ingenious mechanism for elimination of damaged cells in order to suppress the accumulation of mutated cells, and this mechanism might contribute to the preservation of the species. Since oxidative stress causes damage to DNA molecules in addition to other macromolecules including RNA, protein and phospholipid, it is assumed that the degree of damage of OMPs is consistent with that of DNA and that an abnormality of OMPs is a signal for removal of cells that have dam-

In the LTSP, *E. coli* cells can survive for several years [14, 17]. For survival, the cells induce specific sets of genes that support maintenance of their viability and protection against environmental stresses such as an oxidative stress [74]. However, there are potentials for genetic alteration in most cells in the LTSP. It was reported that 10-day-old cells, GASP mutants, were able to compete against 1-day-old cells when they were mixed together [14, 16, 17, 75]. It has been proposed that population exchange continuously occurs in the LTSP (**Figure 1**). Interestingly, the GASP phenotype is mediated by stable genomic mutations that provide benefits to cells for survival. The first mutant exhibiting a GASP phenotype was obtained

sensitive than undamaged cells to σE-dependent cell lysis.

in *micA*- and *rybB*-disrupted mutants [25].

aged DNA molecules from the cell population.

**4. Contribution of PCD for LTSP**

**4.1. Survival mechanisms in LTSP**

**Figure 4.** A model of σ<sup>E</sup>-dependent PCD. When cells are exposed to stresses such as oxidative stress, σE is activated in response to damaged OMPs and increases and decreases the amounts of sRNAs (MicA and RybB) and PpiD as a folding catalyst protein, respectively. The expression of *ppiD* is greatly reduced under the condition of accumulation of active σ<sup>E</sup>. The relationship between σE and *ppiD*, however, has not been clarified yet. MicA and RybB repress the expression of mRNAs of *ompA* and both *ompC* and *ompW*, respectively. The biosynthesis and repair of damaged OMPs are repressed by reduction in the PpiD level. As a result, the integrity of the outer membrane collapses and cell lysis progresses and finally causes cell death. MicL sRNA, which represses the expression of *lpp* mRNA, may also participate in σE-dependent PCD. Adapted from Murata et al. [59].

### **3.3. Function of σE-dependent PCD**

Cell lysis in *E. coli* occurs under a general cultivation condition and remarkably increases after the early stationary phase. Most of the lysis seems to be σE-dependent because enhanced expression of *rseA* for anti-σE diminished the lysis [68]. The lysis level was significantly reduced when plasmid clones of *sodA* and *katE* for superoxide dismutase and catalase, respectively, were introduced [70]. Consistent with the level of lysis, the amounts of ROS are small in the exponential phase and large with a peak at the early stationary phase. The introduction of antioxidative stress genes eliminated about 80% of ROS. These findings suggest that oxidative stress is a trigger for the lysis [70]. The lysis is greatly enhanced in a *katE*-disrupted background, indicating that intracellular oxidative stress is involved in the lysis. Considering the signal transduction cascade to provide active σE [28], it is assumed that intracellular oxidative stress causes damage of OMPs by a modification such as carbonylation [71].

The trigger for σE-dependent cell lysis seems to be not only oxidative stress but also other stresses. The proposed signal transduction cascade for active σE [28] indicates the possibility that extracellular stress evokes σE-dependent cell lysis. Indeed, a disrupted mutation of *rpoS* for σ<sup>S</sup> enhanced σE-dependent cell lysis at the early stationary phase [72]. Consistent with this, extracellular stress like toxic materials increases in a medium at the early stationary phase [14, 18]. Since σ<sup>S</sup> functions as a general stress-response sigma factor to protect cells from various stresses [73], *rpoS* mutation results in the elevation of extracellular stress. It is known that σ<sup>E</sup> becomes active through the σE activation cascade, which is initiated by conformation change of OMPs caused by a high temperature or ethanol as an extracellular stress [26, 29, 61]. Therefore, accumulation of extracellular and/or intracellular stresses beyond the elimination capacity by the stress response mechanism may cause conformation change of outer membrane proteins, which activates σ<sup>E</sup>, resulting in σE-dependent cell lysis.

As shown in **Figure 4**, active σE determines the direction to either the repair or cell lysis pathway, presumably reflecting the level of damage of OMPs. If only a few OMPs are damaged, the number of active σE molecules may be not enough to express sRNAs such as *micA* and *rybB*, which may be insufficient to cause cell lysis but can express genes for the repair pathway. On the other hand, damage of OMPs over a certain threshold evokes the cascade of σE-dependent cell lysis. The coincidence of the fact that OMPs are monitoring proteins for cell damage and/or σ<sup>E</sup>-dependent cell lysis and the fact that σE-dependent cell lysis is induced by reduction in the amount of OMPs is highly notable. OMP-damaged cells may be much more sensitive than undamaged cells to σE-dependent cell lysis.

σE-dependent cell lysis seems to eliminate some of the VBNC cells that have been damaged by some kinds of stress. The amount of cell lysis increases in parallel with increase in VBNC cells in the stationary phase, and most of the lysis was suppressed by enhanced expression of *rseA* [68]. An *rseA*-disrupted mutant that constitutively expresses active σE shows a phenotype that is characterized by decrease in cell density without a significant influence on colony-forming unit (CFU) but with protein accumulation in the medium [19]. This phenotype suggests that VBNC cells or some of the VBNC cells are subjected to σE-dependent cell lysis. This limited cell lysis might reflect the existence of a mechanism to distinguish damaged and undamaged cells. It is hypothesized that VBNC cells to be lysed have damaged OMPs to some extent and thus are susceptible to σE-dependent cell lysis. Notably, sRNAs such as MicA and RybB play crucial roles in σE-dependent cell lysis in the LTSP because mutation rate drastically increases in *micA*- and *rybB*-disrupted mutants [25].

Taken together, the findings have shown that *E. coli* has developed an ingenious mechanism for elimination of damaged cells in order to suppress the accumulation of mutated cells, and this mechanism might contribute to the preservation of the species. Since oxidative stress causes damage to DNA molecules in addition to other macromolecules including RNA, protein and phospholipid, it is assumed that the degree of damage of OMPs is consistent with that of DNA and that an abnormality of OMPs is a signal for removal of cells that have damaged DNA molecules from the cell population.
