**1. Introduction**

### **1.1. Brief introduction of σ<sup>E</sup>**

Living *Escherichia coli* cells are constantly suffering from various stresses. The bacterium thus possesses mechanisms to sense stresses and deal with them by changing gene expression

levels and metabolisms. σE, one of the seven sigma factors existing in *E. coli* [1], is associated with the core RNA polymerase complex and initiates transcription by directly recognizing a promoter consisting of specific elements. σE was found for the time as a heat shock sigma factor due to reduction in the expression level of heat shock proteins at 50 °C in mutants of *rpoE*, encoding σE [2] and it is thought to be one of heat shock sigmas like σ<sup>H</sup>. σE has been recognized as an envelope stress-responsive sigma factor [3] that senses an abnormality of the outer membrane integrity. Under the control of this sigma, several important genes are governed, for example, σH and genes for protein folding and degradation [4]. In addition, σ<sup>E</sup> represses the synthesis of outer membrane proteins (OMPs). We show ingenious mechanisms of σ<sup>E</sup> management of its molecule and activity and functions of genes under the control of σ<sup>E</sup> in Section 2.

into the biofilm matrix and serves as an adherence molecule [10]. For the development of sporulation, sporulating cells produce a killing factor for nonsporulating cells, from which released nutrients support sporulation. Moreover, the mother cell in the sporulating population undergoes PCD to release the mature spore via its autolysis [11]. As other mechanisms, *Fratricide* behavior during its genetic transformation, autolysis in *Myxococcus xanthus* development, developmental cycle in *Streptomyces*, and coccoid formation in *Helicobacter pylori* also

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

Many bacterial PCDs are induced through the toxin-antitoxin (TA) system. Five types of TA systems have been found and characterized [9, 12]. Type I has an antisense RNA that pairs with its corresponding toxin mRNA. The difference in transcription between toxin RNA and antitoxin RNA controls the toxin activity. Type II has a protein antitoxin that detoxifies its corresponding toxin protein by their protein-protein interaction. This type of TA system is most abundant. Type III has an antitoxin RNA that interacts directly with the target toxin protein to form an antitoxin RNA-toxin inactive complex. Type IV has a protein antitoxin that stabilizes the target of the toxin by direct binding. Type V has an endoribonuclease that cleaves the target toxin mRNA. These TA systems play important roles in several cellular processes such as plasmid stabilization, formation of persistent cells, peptidoglycan synthesis, resistance to bacteriophages and antibiotics, and inhibition of macromolecule and biofilm formation [9, 12]. PCD via a TA system is executed by the role of toxin proteins. *mazEF* in the type II TA system in *E. coli* has been the most intensively investigated and it has been shown

It has been suggested that the bacterial strategy for survival against DNA damage resembles the PCD mechanisms in eukaryotes [13]. The PCD mechanisms characterized in both prokaryotic and eukaryotic cells indicate that DNA damage leads to cell death when the damage is irreparable. Bayles reported that the death pathway also leads to apoptosis-like processes or autolysis [13]. The similarity of cell death systems in eukaryotes and bacteria suggests that the common origin of this system is derived from endosymbiotic bacteria [9]. Therefore, PCD is a basic mechanism for organisms in all kingdoms for the maintenance of communities, and this system has been acquired at a very early stage of appearance of life on earth. In Section 3, we summarize PCDs in *E. coli* and show σE-dependent cell lysis as one of the PCDs and its physi-

In nutrient-sufficient media in the laboratory, *E. coli* exhibits a typical growth curve consisting of five phases: lag phase, log phase, stationary phase, death phase, and LTSP (**Figure 1**) [14, 15]. Among these phases, characteristics of first three phases have been described in detail elsewhere [15]. It has been thought that cells in the death phase gradually die, and it is known that 99.9% of the cells are not viable in that phase. However, Finkel reported that *E. coli* is able to survive for 5 years in LB medium without any additional nutrients if the volume and osmolarity of the medium are maintained [14]. The transition of viable (colony-forming) cell

plished by a rapid decrease in an exponential fashion in the death phase. Then the viable cell

CFU/ml at the LTSP [14] is accom-


cells/ml at the stationary phase to 10<sup>6</sup>

include PCD in their processes [9].

to play a key role in the PCD process.

ological roles, which our group has discovered.

**1.3. Brief introduction of LTSP**

number from 10<sup>9</sup>

### **1.2. Brief introduction of PCD**

PCD is conserved for all genetically encoded processes that lead to cell suicide. This conceptual word was first proposed in 1964 [5]. PCD that is observed in development, aging, and pathology in eukaryotic multicellular organisms is classified into three categories based on morphological characteristics such as apoptosis, autophagy, and necrosis. Among these, apoptosis, first described in 1972 [6], is the most well-characterized PCD. The morphological manifestations associated with apoptosis include chromatin condensation, chromosomal DNA fragmentation, membrane blebbing, cell shrinkage, and disassembly of the cell into membrane-enclosed vesicles. Apoptosis is highly regulated, and proteases called caspases play key roles in the induction of DNA fragmentation in the activation cascade [7]. Autophagy is the process by which a vesicle called an autophagosome is constructed for atrophy of the nucleus but with no DNA fragmentation [7]. Necrosis is triggered by activation of various receptors for loss of cell membrane integrity and uncontrollable release of intracellular contents into the extracellular space [7]. The physiological importance of these PCDs in the development of an animal has been well defined. For example, during embryonic development, the earliest form of the human hand resembles a paddle due to the elimination of excess cells by apoptosis. PCD mechanisms are also responsible for the homeostasis of multicellular organisms by the elimination of damaged cells that may become a source of cancer cells in the body.

It was thought that PCD only exists in eukaryotic cells, but several scientists have considered the possibility of the existence of bacterial PCD resembling eukaryotic PCD mechanisms. Indeed, a growing body of evidence has shown that PCD is indispensable for bacterial development and is closely associated with bacterial survival mechanisms [8, 9]. Bacterial communities utilize PCD for survival of their population when suffering from oxidative stress, nutrient deprivation, phage infections, or other problems. The cell survival mechanism is a response to stresses outside cells and inside cells, but excessive damage turns on the PCD mechanism of some cells to help sibling cells. In the development processes of bacteria, PCD provides nutrients to sibling cells, releases components, and promotes special aspects. Indeed, biofilm formation, sporulation, and other multiple cell-like developments have been shown to bear PCD mechanisms in these processes. In biofilm development, cell death and lysis are required for the release of genomic DNA (known as extracellular DNA), which becomes incorporated into the biofilm matrix and serves as an adherence molecule [10]. For the development of sporulation, sporulating cells produce a killing factor for nonsporulating cells, from which released nutrients support sporulation. Moreover, the mother cell in the sporulating population undergoes PCD to release the mature spore via its autolysis [11]. As other mechanisms, *Fratricide* behavior during its genetic transformation, autolysis in *Myxococcus xanthus* development, developmental cycle in *Streptomyces*, and coccoid formation in *Helicobacter pylori* also include PCD in their processes [9].

Many bacterial PCDs are induced through the toxin-antitoxin (TA) system. Five types of TA systems have been found and characterized [9, 12]. Type I has an antisense RNA that pairs with its corresponding toxin mRNA. The difference in transcription between toxin RNA and antitoxin RNA controls the toxin activity. Type II has a protein antitoxin that detoxifies its corresponding toxin protein by their protein-protein interaction. This type of TA system is most abundant. Type III has an antitoxin RNA that interacts directly with the target toxin protein to form an antitoxin RNA-toxin inactive complex. Type IV has a protein antitoxin that stabilizes the target of the toxin by direct binding. Type V has an endoribonuclease that cleaves the target toxin mRNA. These TA systems play important roles in several cellular processes such as plasmid stabilization, formation of persistent cells, peptidoglycan synthesis, resistance to bacteriophages and antibiotics, and inhibition of macromolecule and biofilm formation [9, 12]. PCD via a TA system is executed by the role of toxin proteins. *mazEF* in the type II TA system in *E. coli* has been the most intensively investigated and it has been shown to play a key role in the PCD process.

It has been suggested that the bacterial strategy for survival against DNA damage resembles the PCD mechanisms in eukaryotes [13]. The PCD mechanisms characterized in both prokaryotic and eukaryotic cells indicate that DNA damage leads to cell death when the damage is irreparable. Bayles reported that the death pathway also leads to apoptosis-like processes or autolysis [13]. The similarity of cell death systems in eukaryotes and bacteria suggests that the common origin of this system is derived from endosymbiotic bacteria [9]. Therefore, PCD is a basic mechanism for organisms in all kingdoms for the maintenance of communities, and this system has been acquired at a very early stage of appearance of life on earth. In Section 3, we summarize PCDs in *E. coli* and show σE-dependent cell lysis as one of the PCDs and its physiological roles, which our group has discovered.

### **1.3. Brief introduction of LTSP**

levels and metabolisms. σE, one of the seven sigma factors existing in *E. coli* [1], is associated with the core RNA polymerase complex and initiates transcription by directly recognizing a promoter consisting of specific elements. σE was found for the time as a heat shock sigma factor due to reduction in the expression level of heat shock proteins at 50 °C in mutants of *rpoE*, encoding σE [2] and it is thought to be one of heat shock sigmas like σ<sup>H</sup>. σE has been recognized as an envelope stress-responsive sigma factor [3] that senses an abnormality of the outer membrane integrity. Under the control of this sigma, several important genes are governed, for example, σH and genes for protein folding and degradation [4]. In addition, σ<sup>E</sup> represses the synthesis of outer membrane proteins (OMPs). We show ingenious mechanisms of σ<sup>E</sup> management of its molecule and activity and functions of genes under the control of σ<sup>E</sup>

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

PCD is conserved for all genetically encoded processes that lead to cell suicide. This conceptual word was first proposed in 1964 [5]. PCD that is observed in development, aging, and pathology in eukaryotic multicellular organisms is classified into three categories based on morphological characteristics such as apoptosis, autophagy, and necrosis. Among these, apoptosis, first described in 1972 [6], is the most well-characterized PCD. The morphological manifestations associated with apoptosis include chromatin condensation, chromosomal DNA fragmentation, membrane blebbing, cell shrinkage, and disassembly of the cell into membrane-enclosed vesicles. Apoptosis is highly regulated, and proteases called caspases play key roles in the induction of DNA fragmentation in the activation cascade [7]. Autophagy is the process by which a vesicle called an autophagosome is constructed for atrophy of the nucleus but with no DNA fragmentation [7]. Necrosis is triggered by activation of various receptors for loss of cell membrane integrity and uncontrollable release of intracellular contents into the extracellular space [7]. The physiological importance of these PCDs in the development of an animal has been well defined. For example, during embryonic development, the earliest form of the human hand resembles a paddle due to the elimination of excess cells by apoptosis. PCD mechanisms are also responsible for the homeostasis of multicellular organisms by the elimination of damaged cells that may become a source of cancer cells in the body. It was thought that PCD only exists in eukaryotic cells, but several scientists have considered the possibility of the existence of bacterial PCD resembling eukaryotic PCD mechanisms. Indeed, a growing body of evidence has shown that PCD is indispensable for bacterial development and is closely associated with bacterial survival mechanisms [8, 9]. Bacterial communities utilize PCD for survival of their population when suffering from oxidative stress, nutrient deprivation, phage infections, or other problems. The cell survival mechanism is a response to stresses outside cells and inside cells, but excessive damage turns on the PCD mechanism of some cells to help sibling cells. In the development processes of bacteria, PCD provides nutrients to sibling cells, releases components, and promotes special aspects. Indeed, biofilm formation, sporulation, and other multiple cell-like developments have been shown to bear PCD mechanisms in these processes. In biofilm development, cell death and lysis are required for the release of genomic DNA (known as extracellular DNA), which becomes incorporated

in Section 2.

**1.2. Brief introduction of PCD**

In nutrient-sufficient media in the laboratory, *E. coli* exhibits a typical growth curve consisting of five phases: lag phase, log phase, stationary phase, death phase, and LTSP (**Figure 1**) [14, 15]. Among these phases, characteristics of first three phases have been described in detail elsewhere [15]. It has been thought that cells in the death phase gradually die, and it is known that 99.9% of the cells are not viable in that phase. However, Finkel reported that *E. coli* is able to survive for 5 years in LB medium without any additional nutrients if the volume and osmolarity of the medium are maintained [14]. The transition of viable (colony-forming) cell number from 10<sup>9</sup> cells/ml at the stationary phase to 10<sup>6</sup> CFU/ml at the LTSP [14] is accomplished by a rapid decrease in an exponential fashion in the death phase. Then the viable cell

the GASP phenotype [25], indicating the responsibility of PCDs for the LTSP transition. We describe the possible survival mechanism of *E. coli* in the LTSP and the importance of σE-


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,

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

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

dependent PCD in Section 4.

**2. Functions of σ<sup>E</sup>**

**Figure 1.** Transition model of viable cells until the LTSP in *E. coli*. The LTSP is created presumably by the sequential alteration of the expression of regulators. The sigma factors σ<sup>E</sup> and σ<sup>S</sup> are important regulators for onset of the stationary phase and the consequent death phase. PCD and SOS-induced DNA polymerases, Pol II, Pol IV, and Pol V, are thought to be important factors for maintenance of the LTSP. Adapted from Finkel [14].

number is maintained around 10<sup>6</sup> CFU/ml by unknown mechanisms for a very long period. 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 environmental perturbation of nutrients and conditions for survival.

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 change: σ<sup>E</sup> and σ<sup>S</sup> molecules are increased by fivefold and by threefold to fourfold, respectively [19–21]. The physiological roles of σ<sup>E</sup> and σ<sup>S</sup> suggest that these factors enable cells 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 the GASP phenotype [25], indicating the responsibility of PCDs for the LTSP transition. We describe the possible survival mechanism of *E. coli* in the LTSP and the importance of σEdependent PCD in Section 4.
