The estimated half-life of σS changed from 0.7 min in pgsA+ (JU01) cells to 8.5 min in pgsA3 (JU02) cells (Fig. 3). This result indicates that degradation of σS in the mutant cells is indeed retarded, and this is likely due to the presumably reduced content of ClpXP protease, although the involvement of other factor(s) in the degradation cannot be completely ruled out. In order to further assess the contribution of clpPX repression to the extended half-life U0126 of σS in pgsA3 cells, we examined the effect of the introduction

of a clpPX plasmid (pHR718-clpPX). The highly increased content (7.9-fold over wild type) of σS in the pgsA3 cells (strain ST002) decreased to almost the level found in ST001 (pgsA+) cells after the introduction of the clpPX plasmid (Fig. 2c). This result reconfirms the conclusion that the pgsA3 mutation represses the expression of clpPX (as shown in Fig. 2a and b) Belnacasan concentration and may also indicate that other factors participating in the regulation of the activity of ClpXP protease

(Hengge-Aronis, 2002) are not involved in, or contribute less to, the long half-life of σS. These other conceivably involved factors include Rsd, which is believed to affect σS association with core RNA polymerase by a putative action as anti-σD (Jishage & Ishihama, 1999), and Crl, which is assumed to act by modulating the association with the RNA polymerase core (Pratt & Silhavy, 1998); however, neither rsd nor crl expression is reduced in the pgsA mutant cells as evidenced by microarray analysis (Nagahama et al., 2007). The activity of σS in the mutant cells is therefore not

affected by these regulatory factors. The repression of clpPX may PRKACG thus very well be the main defect in the ClpXP degradation pathway of σS in cells with acidic phospholipid deficiency. We have arrived at the conclusion that the slower degradation of σS in the mutant cells contributes considerably to the accumulation of σS and that this is caused by the repression of clpPX. However, how does the acidic phospholipid deficiency trigger the repression of clpPX in pgsA3 mutant cells? It is known that the expression of the clpPX operon involves promoters under the control of σE, σH, and σD (Li et al., 2000; Phodius et al., 2006; Regulon DB ver. 6.4, http://regulondb.ccg.unam.mx/index.jsp). The expression of rpoE, which codes for σE, is negatively regulated by the Cpx two-component signal transduction system (De Wulf et al., 2002). The expression of rpoH, which codes for σH, is controlled by σE in addition to σD. The Cpx system is activated in mutant cells lacking the zwitterionic phospholipid phosphatidylethanolamine, the third major phospholipid in E. coli membranes (Mileykovskaya & Dowhan, 1997). We observed a significant activation (8.