**2. Functions of σ<sup>E</sup>**

number is maintained around 10<sup>6</sup>

change: σ<sup>E</sup> and σ<sup>S</sup>

CFU/ml by unknown mechanisms for a very long period.

suggest that these factors enable cells to

are important regulators for onset of the stationary

Interestingly, in the LTSP, viable cells show a growth advantage against parent strains [16]. The phenotype of these cells is called growth advantage in stationary phase (GASP). In addition, GASP mutants consecutively occur every 10 days in the same culture [17]. Therefore, cells in the LTSP are not static and a dramatic population change occurs for adapting to the

**Figure 1.** Transition model of viable cells until the LTSP in *E. coli*. The LTSP is created presumably by the sequential

phase and the consequent death phase. PCD and SOS-induced DNA polymerases, Pol II, Pol IV, and Pol V, are thought

What factors can lead *E. coli* cells from the stationary phase to the LTSP? The onset and course of the stationary phase have been summarized well in other reviews [15, 18]. Briefly, at the beginning of the stationary phase, the abundance of specific sigma factors is known to

adapt to environments in the stationary phase by changing expression of 10% of the genes of *E. coli*. However, it has not been clarified how the death phase and LTSP start. Since protein expression level is kept low for several days in the stationary phase [22], cells may have some activity to accomplish preparation for the coming phases. These activities are probably related to PCD mechanisms [14] as described below. Several factors have been considered for the transition to the LTSP. One of these factors is reactive oxygen species (ROS). Indeed, mutants of genes for NADH dehydrogenase in the respiratory chain, which is a primary source of ROS, exhibited no GASP phenotype [23]. In addition, GASP phenotypes are altered by vessel volume of cultures, probably affecting dissolved oxygen concentrations in the medium [24]. On the other hand, we have revealed that σE-dependent PCD is essential for

molecules are increased by fivefold and by threefold to fourfold, respec-

environmental perturbation of nutrients and conditions for survival.

tively [19–21]. The physiological roles of σ<sup>E</sup> and σ<sup>S</sup>

alteration of the expression of regulators. The sigma factors σ<sup>E</sup> and σ<sup>S</sup>

to be important factors for maintenance of the LTSP. Adapted from Finkel [14].

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

### **2.1. Mechanisms of membrane stress responses for σE activation**

Bacteria have mechanisms for rapid responses to environmental stresses, especially on the envelope because cell structure is maintained by integrity of the membrane. There have been many studies on membrane stress responses. In Gram-negative bacteria, such responses are known as envelope stress responses (ESRs). There are five known ESRs, Cpx, σE, Bae, Rcs, and Psp ESRs, that are induced by a variety of envelope stresses and alter the expression of adaptive functions to modify the envelope, rid cells of a toxic entity, and/or repair substantial damage [3]. Of these ESRs, σ<sup>E</sup> ESR, a subset first found in *E. coli*, is known to respond to stresses such as stresses from heat and alkali due to damage of the outer membrane [26]. The σE ESR detects perturbations in biogenesis of the outer membrane or lipopolysaccharide (LPS) due to protein-folding problems in the periplasmic space and outer membrane (**Figure 2**). The key protein in this response is a transmembrane protein of RseA as an anti-σE protein capturing σ<sup>E</sup> to inactivate it under nonstress conditions. Under stress conditions, σE activation is accomplished by the stepped degradation of RseA via three proteases, DegS, RseP (YaeL), and ClpXP. Senescing of the integrity of OMPs, which causes the activation of DegS by binding with unfolded OMPs, is the first key mechanism of σE activation [26]. In addition, DegS cleavage of RseA is physiologically inhibited by RseB binding to a conserved region near the C-terminus of the poorly structured RseA domain [27]. Therefore, RseB can negatively regulate the RseA degradation [27, 28]. RseB senses LPS integrity for binding with released LPS, and LPS displaces RseA from RseB due to antagonization of binding [29]. The subsequent intramembrane proteolysis of RseA by RseP is not performed when RseB is bound to RseA due to blockage through the side filtering function of the two PDZ domains of RseP [30]. Under stress conditions, the exposed periplasmic domain of RseA is cleaved by DegS between V148 and S149 [26]. Consequently, specific recognition of cleaved RseA is performed by the PDZ tandem domains of RseP [30], and specific cleavage of the transmembrane region of RseA1–148 is also executed at A108 and C109 [31]. Finally, the cleaved cytoplasmic region of RseA1–108 is recognized by SspB, and RseA1–108/σE complex is delivered to cytoplasmic AAA+ proteinases such as ClpXP [32, 33]. Destruction of the RseA fragment allows σE liberation and activation to cause the transcription of stress-responsive genes under the control of σE [28, 34].

These dual molecular signals (unfolded OMPs and LPS) are key factors for the σE ESR to sense outer membrane stresses [29]. For cell formation, OMPs and LPS are transported from the cytoplasm to the outer membrane in *E. coli*. The transport of OMPs as a beta-barrel structure is performed by the Sec-SurA-BAM system [35]. DegS is activated by binding of a peptide bearing a YxF motif at the C-terminus of an OMP, which is exposed by envelope stress,

called promoters. Several experiments have been carried out in *E. coli* to find consensus sequences of promoters and σ<sup>E</sup>-regulating genes, σ<sup>E</sup> regulon genes. Attempts were made to identify consensus sequences for σE by several procedures, and genomic information and a search algorithm predicted a conserved −35 motif (GGAACTTTT) and a conserved −10 motif (T/CGGTCAAAA) [39–41]. σE regulon members in *E. coli* have been found by proteomics [4, 39], genetic strategies, [39, 40] and microarray analysis [4, 41]. Results of those studies showed that σE-holo RNA polymerase transcribes two kinds of RNAs, mRNAs for several genes and antisense sRNAs that repress the expression of several genes. Analysis of σ<sup>E</sup> regulon genes showed that the regulon consists of 19 transcription units and 23 proteins. At least 60% of the regulon members are responsible for the synthesis and assembly of LPS and OMPs or regulatory proteins for these two key elements of the outer membrane [41]. The majority of σE regulon genes in *E. coli* are genes encoding periplasmic folding factors, periplasmic proteases, OMP assembly proteins, LPS translocation and assembly proteins, proteins for synthesis of phospholipids and lipid A, and a heat shock sigma factor coded by *rpoH* [39, 42]. One of most important operons under the control of σE is the *rpoE-rseABC* operon coding σ<sup>E</sup> itself, RseA as an anti-σE, RseB repressor, and a *soxR-*influencing protein, respectively [43]. This operon is induced by two σE promoters, one upstream of *rpoE* and the other upstream of *rseA*. Therefore, σE activation causes a negative feedback loop by double transcriptions from the two promoters for rapid repression of σE activity. On the other hand, it has been revealed that small RNAs (sRNAs) are controlled by σE and work as repressors for gene expression. There are two distinct σE-inducible sRNAs, MicA and RybB, that bind to Hfq, an RNA chaperone protein required for the function and/or stabilization of sRNAs, and target mRNAs from 31 genes for major porins, metabolism, ribosome biosynthesis, toxin-antitoxin, and transcriptional factor PhoP [44]. In addition, MicL (SlrA) targets only one mRNA, which encodes the outer membrane lipoprotein Lpp, the most abundant protein of the cell [45]. Taken together, MicA, RybB and MicL allow σE to prevent the synthesis of


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

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

abundant outer membrane proteins in response to stresses.

**3. σE-dependent PCD**

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

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

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 induce σE activation [28, 29].

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].

### **2.2. σE regulon genes**

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 called promoters. Several experiments have been carried out in *E. coli* to find consensus sequences of promoters and σ<sup>E</sup>-regulating genes, σ<sup>E</sup> regulon genes. Attempts were made to identify consensus sequences for σE by several procedures, and genomic information and a search algorithm predicted a conserved −35 motif (GGAACTTTT) and a conserved −10 motif (T/CGGTCAAAA) [39–41]. σE regulon members in *E. coli* have been found by proteomics [4, 39], genetic strategies, [39, 40] and microarray analysis [4, 41]. Results of those studies showed that σE-holo RNA polymerase transcribes two kinds of RNAs, mRNAs for several genes and antisense sRNAs that repress the expression of several genes. Analysis of σ<sup>E</sup> regulon genes showed that the regulon consists of 19 transcription units and 23 proteins. At least 60% of the regulon members are responsible for the synthesis and assembly of LPS and OMPs or regulatory proteins for these two key elements of the outer membrane [41]. The majority of σE regulon genes in *E. coli* are genes encoding periplasmic folding factors, periplasmic proteases, OMP assembly proteins, LPS translocation and assembly proteins, proteins for synthesis of phospholipids and lipid A, and a heat shock sigma factor coded by *rpoH* [39, 42]. One of most important operons under the control of σE is the *rpoE-rseABC* operon coding σ<sup>E</sup> itself, RseA as an anti-σE, RseB repressor, and a *soxR-*influencing protein, respectively [43]. This operon is induced by two σE promoters, one upstream of *rpoE* and the other upstream of *rseA*. Therefore, σE activation causes a negative feedback loop by double transcriptions from the two promoters for rapid repression of σE activity. On the other hand, it has been revealed that small RNAs (sRNAs) are controlled by σE and work as repressors for gene expression. There are two distinct σE-inducible sRNAs, MicA and RybB, that bind to Hfq, an RNA chaperone protein required for the function and/or stabilization of sRNAs, and target mRNAs from 31 genes for major porins, metabolism, ribosome biosynthesis, toxin-antitoxin, and transcriptional factor PhoP [44]. In addition, MicL (SlrA) targets only one mRNA, which encodes the outer membrane lipoprotein Lpp, the most abundant protein of the cell [45]. Taken together, MicA, RybB and MicL allow σE to prevent the synthesis of abundant outer membrane proteins in response to stresses.
