, 2004). In pgsA mutant cells, the deficiency in the acidic phospholipids, phosphatidylglycerol and cardiolipin, causes retarded translocation of newly synthesized proteins across the inner membrane due to impaired activation of SecA in the translocation machinery (Dowhan et al., 2004), impairment in the production of OmpF protein and flagellin (Inoue et al.,
1997), and activation of the Rcs phosphorelay regulatory system (Shiba et al., 2004; Nagahama et al., 2006). The impairment of flagellin production in pgsA3 mutant cells is due to the transcription repression of the flagellar master operon flhDC (Kitamura et al., 1994). Our recent studies have shown that accumulation of σS is involved in the repression of the master operon. The transcriptional activity, as monitored via rpoS′-lacZ
transcriptional fusion Panobinostat Selleckchem Pirfenidone and real-time PCR, in pgsA3 mutant cells is 2.6 times as high as in pgsA+ cells (Uchiyama et al., in press). While the enhanced transcription could conceivably be solely responsible for the accumulation, post-transcriptional accumulation has also been suggested to play an important role in the mutant cells, because the σS content in the mutant cells is significantly higher even if the same level of rpoS mRNA is expressed from a regulatable promoter (Uchiyama et al., in press). It is well known that σS is the master regulator that controls the genes expressed upon entry into the stationary phase and against general stress, including starvation (Tanaka et al., 1993; Pratt & Silhavy, 1998; Hengge-Aronis, 2002). Various levels SPTLC1 of σS regulation are affected by various stress signals; an increased content of σS might be obtained by rpoS transcription or rpoS mRNA translation, or by inhibition of σS proteolysis (which, under nonstress conditions in logarithmic growth, is quite rapid via the ClpXP protease) (Pratt & Silhavy,
1998; Hengge-Aronis, 2002; Majdalani et al., 2002; Bougdour et al., 2006; Peterson et al., 2006). In the present study, we focus on the mechanisms for post-transcriptional σS accumulation, that is, we investigate the translation of rpoS mRNA and the proteolytic degradation of the sigma factor in mutant cells with acidic phospholipid deficiency. The E. coli K-12 strains and plasmids used in this study are listed in Table 1. New strains were constructed by P1 phage transduction and the methods described below, and their genotypes were verified by drug resistance tests, PCR amplification, nucleotide sequencing, and determination of phospholipid composition, as applicable. Strain BW25113ΔclpPX was constructed using the λ Red system (Datsenko & Wanner, 2000).
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