In the present study, changes in protein synthesis patterns after salt shock visualized by 35S-methionine labeling and the changed protein composition in salt-acclimated cells of the cyanobacterium Synechocystis sp. strain PCC 6803 were analyzed by a combination of 2-DE for protein separation and PMF for protein identification. As a basis for the differential analysis, a proteome map with 500 identified protein spots comprising 337 different protein species was established. Fifty-five proteins were found, which are induced by salt shock or accumulated after long-term salt acclimation. Some of the proteins are salt stress-specific, such as enzymes involved in the synthesis of the compatible solute glucosylglycerol, while most of them are involved in general stress acclimation. Particularly, heat-shock proteins and proteins acting against lesions by reactive oxygen species were found. Moreover, changes in enzymes involved in basic carbohydrate metabolism were detected. The dynamic of the proteome of salt-stressed Synechocystis cells was compared to previous data concerning transcriptome analysis revealing that 89% of the proteins induced shortly after salt shock were also found to be induced at the RNA level. However, 42% of the stably up-regulated proteins in salt-acclimated cells were not detected previously using DNA microarrays. The comparison of transcriptomic and proteomic analyses shows the significance of post-transcriptional regulatory mechanisms in acclimation of Synechocystis to high salt concentrations.

In the complete genome sequence of the cyanobacterium Synechocystis sp. strain PCC 6803 [Kaneko et al. (1996). DNA Res 3, 109-136] genes were identified encoding putative group 3 σ-factors SigH (Sll-0856), SigG (Slr-1545) and SigF (Slr-1564) and the regulatory protein RsbU (Slr-2031). Mutations in these genes were generated by interposon mutagenesis to study their importance in stress acclimation. For the genes sigH, sigF and rsbU, the loci segregated completely. However, attempts to mutagenize the sigG locus resulted in merodiploids. Under standard growth conditions only minor differences were detected between the mutants and wild-type. However, cells of the RsbU mutant showed a clear defect in regenerating growth after a nitrogen-and sulphur-starvation-induced stationary phase. After applying salt, heat and high-light shocks, stress protein synthesis was analysed by means of one-and two-dimensional electrophoresis. Cells of the SigF mutant showed a severe defect in the induction of salt stress proteins. Although the acclimation to moderate salt stress up to 684 mM NaCl was not significantly changed in this mutant, its ability to acclimate to higher concentrations of NaCl was reduced. Northern blot experiments showed a constitutive expression of the rsbU and sigF genes. The expression of the sigH gene was found to be stressstimulated, particularly in heat-shocked cells, whilst that of sigG was transiently decreased under stress conditions. Possible functions of these regulatory proteins in stress acclimation of Synechocystis cells are discussed.

The cyanobacterium Synechocystis sp. strain PCC 6803 is able to acclimate to levels of salinity ranging from freshwater to twice the seawater concentrations of salt by accumulating the compatible solute glucosylglycerol (GG). Expression of the ggpS gene coding for the key enzyme (glucosylglycerol-phosphate synthase) in GG synthesis was examined in detail. Under control conditions, the GgpS protein is stable, so that weak constitutive transcription of the ggpS gene resulted in a significant protein content. However, the enzyme activity was biochemically switched off, and no GG was detectable. After a salt shock, an immediate increase in mRNA content proportional to the salt content occurred, while the GgpS protein and GG contents rose in a linear manner. Furthermore, the stability of the ggpS mRNA increased transiently. In salt-acclimated cells expression of the ggpS gene, the GgpS protein content, and the amount of accumulated GG depended linearly on the external salt concentration. Mapping of the 5 end of the ggpS transcript revealed a long nontranslated 5 sequence and a putative typical cyanobacterial promoter, which did not show any obvious salt-regulatory element. The alternative factor F was found to be involved in salt-dependent regulation of ggpS, since in a F mutant induction of this gene was strongly reduced. The present study demonstrated that in addition to biochemical regulation of GgpS activity, alterations of ggpS expression are involved in regulation of GG synthesis in Synechocystis sp. strain PCC 6803. A model showing the interaction of the two regulatory levels is presented.The strategies used by bacteria to survive in environments with high and changing salinities have received much attention in the last few years. The general physiological response after an upshift of the salt concentration in a medium includes three phases. First, inorganic ions (usually Na ϩ and Cl Ϫ ) enter the cell after the turgor collapse resulting from the large difference in water potentials across the cytoplasmic membrane. Second, Na ϩ is exchanged for K ϩ , which saves the cell metabolism from the toxic influence of high Na ϩ concentrations. Stabilization of turgor by accumulation of ions as a long-term acclimation strategy is possible only in halophilic archaea and some halophilic bacteria. Therefore, in the third phase so-called compatible solutes are usually accumulated, which allows exclusion of the inorganic ions without a further change of turgor (1).For some model organisms, like the gram-negative enteric bacterium Escherichia coli, the gram-positive soil bacterium Bacillus subtilis, and the cyanobacterial freshwater isolate Synechocystis sp. strain PCC 6803, the acclimation processes have been investigated in detail. In minimal media these strains produce by de novo synthesis amounts of the compatible solutes trehalose (E. coli) (11), proline (B. subtilis) (26), and glucosylglycerol (GG) (Synechocystis) that are proportional to the stress (20,5). Mutants defective in trehalose, proline, or GG production exhib...

Tobacco plants can be used to express recombinant proteins that cannot be produced in a soluble and active form using traditional platforms such as Escherichia coli. We therefore expressed the human glycoprotein interleukin 6 (IL6) in two commercial tobacco cultivars (Nicotiana tabacum cv. Virginia and cv. Geudertheimer) as well as the model host N. benthamiana to compare different transformation strategies (stable vs. transient expression) and subcellular targeting (apoplast, endoplasmic reticulum (ER) and vacuole). In T0 transgenic plants, the highest expression levels were achieved by ER targeting but the overall yields of IL6 were still low in the leaves (0.005% TSP in the ER, 0.0008% in the vacuole and 0.0005% in the apoplast). The apoplast variant accumulated to similar levels in leaves and seeds, whereas the ER-targeted variant was 1.2-fold more abundant in seeds and the vacuolar variant was 6-fold more abundant in seeds. The yields improved in subsequent generations, with the best-performing T2 plants producing the ER-targeted IL6 at 0.14% TSP in both leaves and seeds. Transient expression of ER-targeted IL6 in leaves using the MagnICON system resulted in yields of up to 7% TSP in N. benthamiana, but only 1% in N. tabacum cv. Virginia and 0.5% in cv. Geudertheimer. Although the commercial tobacco cultivars produced up to threefold more biomass than N. benthamiana, this was not enough to compensate for the lower overall yields. The recombinant IL6 produced by transient and stable expression in plants was biologically active and presented as two alternative bands matching the corresponding native protein.

In this study, we compared basic expression approaches for the efficient expression of bioactive recombinant human interleukin-6 (IL6), as an example for a difficult-to-express protein. We tested these approaches in a laboratory scale in order to pioneer the commercial production of this protein in Escherichia coli (E. coli). Among the various strategies, which were tested under Research and Development (R&D) conditions, aggregation-prone IL6 was solubilized most effectively by co-expressing cytoplasmic chaperones. Expression of a Glutathion-S-Transferase (GST) fusion protein was not efficient to increase IL6 solubility. Alteration of the cultivation temperature significantly increased the solubility in both cases, whereas reduced concentrations of IPTG to induce expression of the T7lac-promotor only had a positive effect on chaperone-assisted expression. The biological activity was comparable to that of commercial IL6. Targeting the expressed protein to an oxidizing environment was not effective in the generation of soluble IL6. Taken together, the presence of chaperones and a lowered cultivation temperature seem effective to isolate large quantities of soluble IL6. This approach led to in vivo soluble, functional protein fractions and reduces purification and refolding requirements caused by downstream purification procedures. The final yield of soluble recombinant protein averaged approximately 2.6 mg IL6/liter of cell culture. These findings might be beneficial for the development of the large-scale production of IL6 under the conditions of current good manufacturing practice (cGMP).