**3. σE-dependent PCD**

### **3.1. PCD in** *E. coli*

releasing from SurA or misfolding in BAM [28]. LPS is also transported by the aid of Ltp proteins, and LptA is a key component of the transenvelope complex to shuttle LPS to the outer membrane [36]. LptA less efficiently binds to LPS against RseB at 45°C [29], suggesting that LPS is easily caught by RseB under heat shock conditions. In addition, RseB can sense many mislocalized LPS species [29]. Therefore, both DegS activation and RseB detachment are essential for the initiation of RseA proteolysis for σ<sup>E</sup> liberation. However, σE activity increases when either OMP or LPS mutations have accumulated [26, 29, 37], suggesting that a crosstalk between OMP and LPS biogeneses might be an additional regulation that can

elimination of OMPs in σE-dependent cell lysis. Adapted from Lima et al. [29].

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

**Figure 2.** Schematic diagram of the σ<sup>E</sup> signaling pathway and the σ<sup>E</sup> regulon cascade. RseA is a key protein for σ<sup>E</sup> activation. RseA, which is an antisigma factor, captures σE and neutralizes its activity. Two types of signaling molecules, OMPs and LPSs, are key activators of the proteinase DegS because the binding of the C-terminus of an OMP is required for DegS activation and the binding of LPS to RseB is required for deblocking of the RseA cleavage by DegS. Consequently, RseA is sequentially digested by RseP, and the RseA-N terminus is degraded by AAA+ proteinase, by which σ<sup>E</sup> is released and activated to form a holo-RNA polymerase complex. Expressed σE-regulon members consist of LPS- and OMP-related proteins. sRNAs play key roles in the prevention of overproduction of LPSs and OMPs and in

This kind of proteolytic signal transduction and regulator-activating mechanism provides distinctive features for σ<sup>E</sup> regulon as a transient expression. In the σE ESR, the initial signal-sensing cleavage of RseA is a rate-limiting step but the degradation of cytoplasmically fragmented RseA by AAA+ proteinase is relatively fast. Whereas, RseA is in excess over σE under normal conditions and the expression level of *rseA* is higher than that of *rpoE* [38]. Consequently, activated σE is rapidly deactivated, resulting in a short-period response to envelope stresses [33].

Activated σE forms a holo-RNA polymerase with the core RNA polymerase complex to initiate transcription by recognizing consensus sequences located upstream from coding genes

induce σE activation [28, 29].

**2.2. σE regulon genes**

PCD in *E. coli* is also closely associated with the strategy for sensing damage in DNA and the envelope structure. Three PCD mechanisms, a TA system, apoptosis-like death (ALD) and σEdependent cell lysis, have been found in *E. coli* (**Figure 3**). Of these, the most intensively investigated PCD is *mazEF*, a TA system in which *mazF* encodes a stable toxin, sequence-specific endoribonuclease, and *mazE* encodes a labile MazF-antitoxin that is degraded *in vivo* by ATPdependent ClpPA serine protease [46–48]. Toxicity of MazF is attributable to its endoribonuclease activity, specific for the trinucleotide sequence of ACA in mRNA, including the 3′-end fragment of 16S rRNA, to block protein synthesis and to synthesize specific proteins [49]. Specifically expressed proteins are classified into "survival proteins" and "death proteins" including SlyD, YfiD, YgcR, and ClpX [50]. Death proteins induce the DNA fragmentation and membrane depolarization [48]. In addition, *mazEF*-mediated PCD is regulated by a

in 2000 [19]. This PCD occurs as an autolysis mechanism, which is a growth phase-specific cell lysis [19], and removes only viable but nonculturable (VBNC) cells [19]. The molecular mechanism of the PCD is described below in detail. Interestingly, *rpoE* coding σE is an essential gene [55] because the absence of σE causes a cell death-signaling pathway including *hicB*


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 elimina-

(*ydcQ*) that encodes for an antitoxin of the HicA toxin proteinase [56, 57].

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

tion of OMPs after the degradation of OMP mRNAs by sRNAs.

rity of the outer membrane and finally lysis of cells.

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 integ-

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

**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 connected to each other [46, 48].

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

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 [48].

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 in 2000 [19]. This PCD occurs as an autolysis mechanism, which is a growth phase-specific cell lysis [19], and removes only viable but nonculturable (VBNC) cells [19]. The molecular mechanism of the PCD is described below in detail. Interestingly, *rpoE* coding σE is an essential gene [55] because the absence of σE causes a cell death-signaling pathway including *hicB* (*ydcQ*) that encodes for an antitoxin of the HicA toxin proteinase [56, 57].
