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

from cells cultured for 10 days in LB medium, and its mutation, which was identified in

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>

ity and the GASP phenotype has not been clarified yet. The effect of the attenuation might be

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

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

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

regulon. σ<sup>S</sup>

activity [16]. In addition, such σ<sup>S</sup>

activity-attenuated

activity may change the

competes with other sigma factors to

activ-

*rpoS* coding σ<sup>S</sup>

also been reported [79].

the LTSP.

, causes reduction of σ<sup>S</sup>

balance in the competition among sigma factors [16].

bind to the core RNA polymerase complex, and the attenuation of σ<sup>S</sup>

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

due to the misregulation of members of σ<sup>S</sup>

*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 involved in the mechanism [25].

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 maintenance of new populations in the LTSP.

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 are removed.

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, active metabolisms should be maintained in fractions of the cell population. These active metabolisms are thought to lead to the selection of a dominant mutant and generate genetic diversity.

**Author details**

Yamaguchi, Japan

**References**

03.001.

2005;**199**:169-173.

cddis.2014.570.

Tomoyuki Kosaka1,2, Masayuki Murata1

\*Address all correspondence to: m-yamada@yamaguchi-u.ac.jp

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

sigma factor of *Escherichia coli*. EMBO J. 1995;**14**:1043-1055.

*coli*. Microbiology. 2005;**151**:2721-2735. DOI: 10.1099/mic.0.28004-0.

ranging implications in tissue kinetics. Br J Cancer. 1972;**26**:239-257.

Microbiol. 2007;**5**:721-726. DOI: 10.1038/nrmicro1743.

2003;**301**:510-513. DOI: 10.1126/science.1086462.

and Mamoru Yamada1,2\*


1 Department of Biological Chemistry, Faculty of Agriculture and Life Science, Graduate School of Science and Technology for Innovation, Yamaguchi University, Yamaguchi, Japan 2 Research Center for Thermotolerant Microbial Resources, Yamaguchi University,

[1] Ishihama A. Modulation of the nucleoid, the transcription apparatus, and the translation machinery in bacteria for stationary phase survival. Genes Cells. 1999;**4**:135-143.

[2] Raina S, Missiakas D, Georgopoulos C. The *rpoE* gene encoding the σ<sup>E</sup> (σ24) heat shock

[3] Guest RL, Raivio TL. Role of the Gram-negative envelope stress response in the presence of antimicrobial agents. Trends Microbiol. 2016;**24**:377-390. DOI: 10.1016/j.tim.2016.

[4] Kabir MS, Yamashita D, Koyama S, Oshima T, Kurokawa K, Maeda M, Tsunedomi R, Murata M, Wada C, Mori H, Yamada M. Cell lysis directed by σE in early stationary phase and effect of induction of the *rpoE* gene on global gene expression in *Escherichia* 

[5] Lockshin RA. Programmed cell death: history and future of a concept. J Soc Biol.

[6] Kerr JF, Wyllie AH, Currie AR. Apoptosis: a basic biological phenomenon with wide-

[8] Kabir MS, Yamada M. σE-dependent programmed cell death in *Escherichia coli*. In: Yamada M, editor. Survival and death in bacteria. Kerala, India: Research Signpost; 2005. pp. 1-13.

[9] Allocati N, Masulli M, Di Ilio C, De Laurenzi V. Die for the community: an overview of programmed cell death in bacteria. Cell Death Dis. 2015;**6**:e1609. DOI: 10.1038/

[10] Bayles KW. The biological role of death and lysis in biofilm development. Nat Rev

[11] González-Pastor JE, Hobbs EC, Losick R. Cannibalism by sporulating bacteria. Science.

[7] Danial NN, Korsmeyer SJ. Cell death: critical control points. Cell. 2004;**116**:205-219.

Further analysis of the LTSP *in vitro* seems to be important for understanding the life cycles of bacterial flora or biofilms and for elucidating the mechanisms of bacterial evolution. In addition, fundamental mechanisms for LTSP formation might be targets for drug design.
