The 14-3-3 protein family plays a role in a wide variety of cell signaling processes including monoamine synthesis, exocytosis, and cell cycle regulation, but the structural requirements for the activity of this protein family are not known. We have previously shown that the 14-3-3 protein binds with and activates phosphorylated tryptophan hydroxylase (TPH, the rate-limiting enzyme in the biosynthesis of neurotransmitter serotonin) and proposed that this activity might be mediated through the COOH-terminal acidic region of the 14-3-3 molecules. In this report we demonstrate, using a series of truncation mutants of the 14-3-3 isoform expressed in Escherichia coli, that the COOH-terminal region, especially restricted in amino acids 171-213, binds indeed with the phosphorylated TPH. This restricted region, which we termed 14-3-3 box I, is one of the structural regions whose sequence is highly conserved beyond species, allowing that the plant 14-3-3 isoform (GF14) could also activate rat brain TPH. The 14-3-3 box I is the first functional region whose activity has directly been defined in the 14-3-3 sequence and may represent a common structural element whereby 14-3-3 interacts with other target proteins such as Raf-1 kinase. The result is consistent with the recently published crystal structure of this protein family, which suggests the importance of the negatively charged groove-like structure in the ligand binding.

Experimental evolution is a powerful tool for clarifying phenotypic and genotypic changes responsible for adaptive evolution. In this study, we isolated acid-adapted Synechocystis sp. PCC 6803 (Synechocystis 6803) strains to identify genes involved in acid tolerance. Synechocystis 6803 is rarely found in habitants with pH < 5.75. The parent (P) strain was cultured in BG-11 at pH 6.0. We gradually lowered the pH of the medium from pH 6.0 to pH 5.5 over 3 months. Our adapted cells could grow in acid stress conditions at pH 5.5, whereas the parent cells could not. We performed whole-genome sequencing and compared the acid-adapted and P strains, thereby identifying 11 SNPs in the acid-adapted strains, including in Fo F1-ATPase. To determine whether the SNP genes responded to acid stress, we examined gene expression in the adapted strains using quantitative reverse-transcription polymerase chain reaction. sll0914, sll1496, sll0528, and sll1144 expressions increased under acid stress in the P strain, whereas sll0162, sll0163, slr0623, and slr0529 expressions decreased. There were no differences in the SNP genes expression levels between the P strain and two adapted strains, except for sll0528. These results suggest that SNPs in certain genes are involved in acid stress tolerance in Synechocystis 6803.

Escherichia coli pgsA mutations, which cause acidic phospholipid deficiency, repress transcription of the flagellar master operon flhDC, and thus impair flagellar formation and motility. The molecular mechanism of the strong repression of flhDC transcription in the mutant cells, however, has not yet been clarified. In order to shed light on this mechanism we isolated genes which, when supplied in multicopy, suppress the repression of flhD, and found that three genes, gadW, metE and yeaB, were capable of suppression. Taking into account a previous report that gadW represses s S production, the level of s S in the pgsA3 mutant was examined. We found that pgsA3 cells had a high level of s S and that introduction of a gadW plasmid into pgsA3 cells did reduce the s S level. The pgsA3 cells exhibited a sharp increase in s S levels that can only be partially attributed to the slight increase in rpoS transcription; the largest part of the effect is due to a post-transcriptional accumulation of s S . GadW in multicopy exerts its effect by posttranscriptionally downregulating s S . YeaB and MetE in multicopy also exert their effect via s S .Disruption of rpoS caused an increase of the flhD mRNA level, and induction from P trc -rpoS repressed the flhD mRNA level. The strong repression of flhD transcription in pgsA3 mutant cells is thus suggested to be caused by the accumulated s S . INTRODUCTIONEscherichia coli membranes contain only three major phospholipids. Molecular genetic approaches to correlate mutationally modified lipid compositions with altered membrane functions have been successfully used to understand the biological roles of the individual phospholipids (for reviews, see Shibuya, 1992;Cronan, 2003;Dowhan et al., 2004). Lack of phosphatidylethanolamine, a zwitterionic phospholipid, which accounts for about 70 % of total phospholipids, is lethal, but the lethality is suppressed by supplementation with high concentrations of divalent cations (DeChavigny et al., 1991;Saha et al., 1996). The lack of phosphatidylethanolamine causes a defect in motility (Shi et al., 1993), an activation of the Cpx two-component signal transduction pathway (Mileykovskaya & Dowhan, 1997), a defect in division site selection, probably due to the concomitant increase in cardiolipin content that affects the localization of MinD (Mileykovskaya & Dowhan, 2005), and a defect in the native folding of lactose permease LacY, which causes the loss of active transport of the substrate (Dowhan et al., 2004).Lack of, or deficiency in, the major acidic phospholipid phosphatidylglycerol, brought about by the mutations pgsA30 : : kan or pgsA3, respectively, is lethal, due to the absence of, or the missense defect in, phosphatidylglycerophosphate synthase, which catalyses the committed step in the biosynthetic pathway for this acidic phospholipid (Shibuya, 1992;Usui et al., 1994). However, simultaneous lack of the major outer membrane lipoprotein (Braun's lipoprotein), the most abundant protein, encoded by lpp, suppresses the lethality (Shibuya, 1992;K...

ATP-binding cassette (ABC) transporter proteins mediate energy-dependent transport of substrates across cell membranes. Numerous ABC transporter-related genes have been found in the Synechocystis sp. PCC6803 genome by genome sequence analysis including H(+), iron, phosphate, polysaccharide, and CO(2) transport-related genes. The substrates of many other ABC transporters are still unknown. To identify ABC transporters involved in acid tolerance, deletion mutants of ABC transporter genes with unknown substrates were screened for acid stress sensitivities in low pH medium. It was found that cells expressing the deletion mutant of slr1045 were more sensitive to acid stress than the wild-type cells. Moreover, slr1045 expression in the wild-type cells was increased under acid stress. These results indicate that slr1045 is an essential gene for survival under acid stress. The mutant displayed high osmotic stress resistance and high/low temperature stress sensitivity. Considering the temperature-sensitive phenotype and homology to the organic solvent-resistant ABC system, we subsequently compared the lipid profiles of slr1045 mutant and wild-type cells by thin-layer chromatography. In acid stress conditions, the phosphatidylglycerol (PG) content in the slr1045 mutant cells was approximately 40% of that in the wild-type cells. Moreover, the addition of PG to the medium compensated for the growth deficiency of the slr1045 mutant cells under acid stress conditions. These data suggest that slr1045 plays a role in the stabilization of cell membranes in challenging environmental conditions. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.

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