**4. Contribution of PCD for LTSP**

### **4.1. Survival mechanisms in LTSP**

**3.3. Function of σE-dependent PCD**

PCD. Adapted from Murata et al. [59].

*rpoS* for σ<sup>S</sup>

phase [14, 18]. Since σ<sup>S</sup>

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

**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

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

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

with this, extracellular stress like toxic materials increases in a medium at the early stationary

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 mem-

enhanced σE-dependent cell lysis at the early stationary phase [72]. Consistent

functions as a general stress-response sigma factor to protect cells from

stress causes damage of OMPs by a modification such as carbonylation [71].

brane proteins, which activates σ<sup>E</sup>, resulting in σE-dependent cell lysis.

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 from cells cultured for 10 days in LB medium, and its mutation, which was identified in *rpoS* coding σ<sup>S</sup> , causes reduction of σ<sup>S</sup> activity [16]. In addition, such σ<sup>S</sup> activity-attenuated GASP mutants frequently appeared in nonbuffered media and basic media, but not so many appeared in acid and neutral media [76]. The relationship between the attenuation of σ<sup>S</sup> activity and the GASP phenotype has not been clarified yet. The effect of the attenuation might be due to the misregulation of members of σ<sup>S</sup> regulon. σ<sup>S</sup> competes with other sigma factors to bind to the core RNA polymerase complex, and the attenuation of σ<sup>S</sup> activity may change the balance in the competition among sigma factors [16].

genes for Pol IV and Pol V, are induced during the stationary phase [83]. These facts suggest that there are some mechanisms for the expression of these alternative polymerases and that


*E. coli* can maintain living cells to some extent for several years (LTSP) in the same medium without supplementation of any nutrients during the cultivation. On the basis of results of recent studies and the discovery of mutants that had gained growth advantages in the beginning of the LTSP [16], it has been proposed that the LTSP consists of a number of distinct populations that continuously appear one after another as shown in **Figure 1** [14]. One of the big questions is how nutrients are supplied to support the formation of each new population in such a closed environment. One possible answer is a simple mechanism by which nutrients are supplied from existing cells. Nagamitsu et al. suggested that σE-dependent PCD is

σ<sup>E</sup>-dependent PCD lyses damaged cells but not undamaged cells or cells with little damage and thus has no influence on viable and culturable (VAC) cells [19]. This PCD is responsible for major cell lysis under general cultivation conditions and is enhanced in the stationary phase due to accumulation of stresses including oxidative stress as described above, and forms ghost cells that discharge cytosolic contents to the outside [59]. This lysis thus appears to be different from explosive cell lysis for the biogenesis of membrane vesicles [84]. As in the stationary phase, it is assumed that cells in the LTSP are exposed to metabolically accumulated stresses including oxidative stress, which trigger σE-dependent PCD. Therefore, σE-dependent PCD may provide nutrients that are indispensable for the formation and main-

As mentioned in the previous section, disrupted mutations of *micA* and *rybB*, which are essential factors for σE-dependent PCD, caused serious problems such that they were unable to keep VAC cells at the very early period in the LTSP. These mutations give rise to a sudden increase in the mutation rate just before the disappearance of VAC cells [25]. σE-dependent PCD thus seems to play an important role in the elimination of DNA-damaged cells in the LTSP in addition to the provision of nutrients. Its role appears to resemble that of PCD, socalled apoptosis in multicellular organisms, by which abnormal cells or DNA-damaged cells

Although we still have no evidence that dynamic cell population changes continuously occur in the LTSP, results of studies [14, 16, 17] and results of preliminary experiments in its early phase suggest that cells acquiring mutations for GASP become dominant to form a new population and that new GASP mutations constantly appear and displace the preexisting population. σE-dependent PCD may contribute to the alteration of populations by the lysis of preexisting populations and the provision of nutrients during the LTSP. For the emergence of GASP mutations, a large number of mutations should be present in addition to them under such nutrient-limited conditions. A hypermutable state might exist in the LTSP as mentioned above [14]. In order for hypermutation and σE-dependent PCD to take place,

the mechanisms contribute to the relative high mutation rate in the LTSP.

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

**4.2. Importance of σE-dependent PCD for survival in the LTSP**

involved in the mechanism [25].

tenance of new populations in the LTSP.

are removed.

Using the *rpoS* mutant as a starting strain, subsequent mutants with GASP phenotypes have been isolated. The additional mutations to the *rpoS* mutation have been mapped to *lrp*, coding the leucine-responsive regulator protein as a global regulator [77], or to the *ybeJ–gltJKL* cluster, encoding a high-affinity aspartate, and glutamate transporter [78]. A mutation in the DNA-binding domain of *lrp* has been shown to cause a GASP phenotype by increase in amino acid catabolism during carbon starvation, and mutants having mutation of *ybeJ–gltJKL* also show GASP phenotypes by increase in amino acid utilization [77]. Therefore, although these mutations are involved in different metabolic processes, it is likely that the enhancement of catabolic activity of amino acids for carbon and energy sources is responsible for these GASP phenotypes. Similarly, *sgaA, sgaB*, and *sgaC* mutants have been isolated as GASP mutants but have not been characterized yet [77]. Notably, non-*rpoS* mutation-related GASP mutants have also been reported [79].

The mechanism of GASP acquisition has been investigated and two interesting aspects have been shown. One is the reproducibility of GASP mutants and the other is a relatively high mutation rate in the LTSP. Since the speed of cell proliferation is very low in the LTSP, beneficial mutations for the GASP phenotype can appear only under high mutation conditions. It is thus assumed that there are some molecular mechanisms to generate genetic diversity in the LTSP.

Involvement of the methyl-directed mismatch repair (MMR) system and SOS-induced DNA polymerases has been considered for GASP mutations (**Figure 1**). It is known that when *E. coli* enters the stationary phase, the expression of MMR is reduced [80]. On the other hand, SOS DNA polymerases (Pols II, IV, and V) contribute to the generation of GASP mutations. These polymerases work during DNA replication when DNA polymerase III encounters a lesion and cannot proceed further in DNA synthesis. SOS polymerases are error-prone DNA polymerases and are thus responsible for the generation of adaptive mutations. Pol V Mut is a stand-alone DNA polymerase that is able to perform translesion synthesis, and polymerization of the polymerase is regulated by its intrinsic ATP hydrolase activity [81]. The occurrence of the GASP phenotype is highly related to the presence of SOS polymerases. Indeed, when grown in competition with the wild-type strain, mutants lacking one or more of the SOS polymerases suffer from a severe reduction in fitness to the LTSP. These mutants also fail to express the GASP phenotype as do wild-type strains, instead expressing two additional new types of GASP phenotype [82]. In addition, Pol IV and Pol V confer greater relative fitness than does Pol II during the LTSP, but Pol II can express the GASP phenotype faster than can Pol IV or Pol V [83]. Moreover, genes for the SOS polymerases and other SOS genes, especially genes for Pol IV and Pol V, are induced during the stationary phase [83]. These facts suggest that there are some mechanisms for the expression of these alternative polymerases and that the mechanisms contribute to the relative high mutation rate in the LTSP.
