Sabine Fulda

Effects of Adaptation to Different Salt Concentrations on Photosynthesis and Pigmentation of the Cyanobacterium Synechocystis sp. PCC 6803

Summary After adaptation of Synechocystis sp. PCC 6803 to different salt concentrations the growth optimum was found to be around 342 mM NaCl. At this salinity photosynthesis per cell-volume and the contents of chlorophyll and phycocyanin showed the highest values. Based on pigment contents maximal photosynthesis was measured in cells adapted to the highest salt concentration of 1026 mM NaCl. Therefore, it was concluded that in less pigmented cells the absorbed light could be more efficiently used for photosynthesis than in cells with excess pigment contents. In fluorescence emission spectra, inefficient energy transfer between phycobilisomes and chlorophyll was measured in cells rich in phycocyanin growing at 342 mM NaCl. Adaptation to higher salt concentrations also led to changes in the content and composition of the carotenoids. In cells adapted to 1026 mM NaCl the highest contents of carotenoid especially echinenone and glycosidic carotenoids, were detected.

Glucosylglycerol accumulation during salt acclimation of two unicellular cyanobacteria

SUMMARY: A turbidostat culture technique was used to study the effects of different salt shocks on the freshwater cyanobacteria Synechocystis sp. strain PCC 6803 and Microcystis firma. Shocks were performed either suddenly or gradually, on both unacclimated cultures and those pre-acclimated to 0·77 M-NaCl. All suddenly shocked cultures exhibited a decline in growth after a few hours, characterized by severely decreased metabolic activities (e.g. photosynthesis, respiration, glucose-6-phosphate dehydrogenase activity) and a time course of restoration which coincided with the accumulation of glucosylglycerol. Additionally, all untreated cultures had a late (after a few days) growth depression, distinguished by the stagnation of cell division. This was overcome by physiological adaptation of the whole cells or selection of cells with superior salt tolerance. The different types of growth depressions and the unique pattern of glucosylglycerol accumulation led to the conclusion that glucosylglycerol was necessary to maintain metabolic processes, but that this alone cannot account for successful salt acclimation.

Analysis of Stress Responses in the Cyanobacterial Strains Synechococcus sp. PCC 7942, Synechocystis sp. PCC 6803, and Synechococcus sp. PCC 7418: Osmolyte Accumulation and Stress Protein Synthesis

Summary The influence of salt, heat and light shock treatments on physiological processes was compared in the cyanobacteria Synechococcus sp. PCC 7942, Synechocystis sp. PCC 6803, and Synechococcus sp. PCC 7418, which differ regarding their salt tolerance. The accumulation of the osmolytes sucrose and glucosylglycerol started after salt shocks without a lag in the strains 7942 and 6803. In strain 7418 the synthesis of glycinebetaine showed a lag phase of several hours. During this time glucosylglycerol and proline were accumulated. Light shocks led in all strains to the highest reduction of carbon fixation rate followed by salt shock, while heat shocks decreased it only slightly in the strains 6803 and 7418. Protein synthesis rates measured as 35S-methionine incorporation were reduced after a salt shock, remained almost unchanged in lightshocked cells and increased after heat shocks. Comparisons of protein synthesis patterns showed that most of the detected stress proteins seem to be strain-specific and belong to the group of general stress proteins, since they were induced under heat, salt and light stress treatments, respectively. Furthermore, some proteins specific for salt and heat stress were also found. Among the general stress proteins the chaperone DnaK was identified using cross reactions with a specific antibody.

Salt Treatment Induces Accumulation of Flavodoxin in the Cyanobacterium Synechocystis sp. PCC 6803

Summary Salt adaptation of Synechocystis sp. PCC 6803 leads to a changed protein composition of cells. A major salt-stress-induced protein was isolated in a preparative scale by nondenaturating gelelectrophoresis. Eighteen amino acid residues of the NH 2 -terminus were estimated by partial protein sequencing. This peptide resembles the NH 2 -terminal amino acid sequences of flavodoxins from several cyanobacterial strains. Beside sequence similarities, further biochemical and immunological properties indicate that flavodoxin is accumulated in high amounts in salt-adapted cells of Synechocystis . The synthesis of f lavodoxin is also induced in iron-deficient and heat-shocked Synechocystis cells. A comparison of the protein composition of cells and kinetics of flavodoxin accumulation revealed that the induction of flavodoxin synthesis by salt and iron deficiency seems to be differently regulated.

DNA., RNA, and Protein Synthesis in the Cyanobacterium Synechocystis sp. PCC 6803 Adapted to Different Salt Concentrations

A salt shock of 684mm NaCl reduced RNA and DNA synthesis to about 30% of the control level inSynechocystis. DNA synthesis recovered to the initial level within 4 h, while for recovery of RNA synthesis about 8 h were necessary. In cells completely adapted to different salt concentrations (from 171 to 1026mm NaCl), a continuous decrease in the RNA content with increasing salt concentrations up to 684mm NaCl was found, whereas the lowest DNA content was measured around 342mm NaCl, i.e., the salinity at which maximal growth occurred. With the uracil and thymidien incorporation technique, maxima in DNA and RNA synthesis were detected in control cells. Comparing these rates with nucleic acid synthesis rates calculated from the contents of DNA and RNA and the growth rates indicated that adaptation to 1026mm NaCl seemed to lead to an increased RNA turnover inSynechocystis. Analysis of protein synthesis with35S-methionine labeling showed alterations in salt-adapated cells ofSynechocystis. At least three proteins (20.5, 25.8, and 35.8 kDa) were synthesized with highest rates at salinities leading to maximal growth, the synthesis of nine proteins (12.5, 16.9, 19.2, 22.2, 24.7, 28.5, 30.5, 50.3, and 63.5 kDa) increased and that of several other proteins decreased with increasing salinity; but only three proteins (12.5, 22.2, and 30.5 kDa) accumulated under these conditions. The adaptation ofSynechocystis to enhanced salt concentrations led also to increased contents of glucosylglycerol, glycogen, and significant amounts of K+ as well as Na+ ions.

A membrane-bound FtsH protease is involved in osmoregulation in Synechocystis sp. PCC 6803: the compatible solute synthesizing enzyme GgpS is one of the targets for proteolysis

Protein quality control and proteolysis are involved in cell maintenance and environmental acclimatization in bacteria and eukaryotes. The AAA protease FtsH2 of the cyanobacterium Synechocystis sp. PCC 6803 was identified during a screening for mutants impaired in osmoregulation. The ftsH2– mutant was salt sensitive because of a decreased level of the osmoprotectant glucosylglycerol (GG). In spite of wild type‐like transcription of the ggpS gene in ftsH2– cells the GgpS protein content increased but only low levels of GgpS activity were observed. Consequently, salt tolerance of the ftsH2– mutant decreased while addition of external osmolyte complemented the salt sensitivity. The proteolytic degradation of the GgpS protein by FtsH2 was demonstrated by an in vitro assay using inverted membrane vesicles. The GgpS is part of a GG synthesizing complex, because yeast two‐hybrid screens identified a close interaction with the GG‐phosphate phosphatase. Besides GgpS as the first soluble substrate of a cyanobacterial FtsH protease, several other putative targets were identified by a proteomic approach. We present a novel molecular explanation for the salt‐sensitive phenotype of bacterial ftsH– mutants as the result of accumulation of inactive enzymes for compatible solute synthesis, in this case GgpS the key enzyme of GG synthesis.

Isolation of salt-induced periplasmic proteins from Synechocystis sp. strain PCC 6803

Abstract Periplasmic proteins were obtained from control cells and salt-adapted cells of the cyanobacterium Synechocystis sp. PCC 6803 using the method of cold osmotic shock. Two of these proteins (PP 1, apparent mol. mass 27.6 kDa, and PP 3, apparent mol. mass 39.9 kDa) were accumulated in high amounts in the periplasm of salt-adapted cells, while the major periplasmic protein (PP 2, apparent mol. mass 36.0 kDa) was accumulated independently from salt. After isolation from gels and partial sequencing, the proteins could be assigned to proteins deduced from the complete genome sequence of Synechocystis. Neither salt-induced periplasmic proteins (PP 1, Slr0924 and PP 3, Slr1485) exhibited sequence similarity to proteins of known function from databases. The major protein (PP 2-Slr0513) showed significant sequence similarities to iron-binding proteins. All proteins included typical leader sequences at their N-terminus.

A 2D gel electrophoresis-based snapshot of the phosphoproteome in the cyanobacterium Synechocystis sp. strain PCC 6803

Cyanobacteria are photoautotrophic prokaryotes that occur in highly variable environments. Protein phosphorylation is one of the most widespread means to adjust cell metabolism and gene expression to the demands of changing growth conditions. Using a 2D gel electrophoresis-based approach and a phosphoprotein-specific dye, we investigated the protein phosphorylation pattern in cells of the model cyanobacterium Synechocystis sp. strain PCC 6803. The comparison of gels stained for total and phosphorylated proteins revealed that approximately 5 % of the protein spots seemed to be phosphoproteins, from which 32 were identified using MALDI-TOF MS. For eight of them the phosphorylated amino acid residues were mapped by subsequent mass spectrometric investigations of isolated phosphopeptides. Among the phosphoproteins, we found regulatory proteins, mostly putative anti-sigma factor antagonists, and proteins involved in translation. Moreover, a number of enzymes catalysing steps in glycolysis or the Calvin-Benson cycle were found to be phosphorylated, implying that protein phosphorylation might represent an important mechanism for the regulation of the primary carbon metabolism in cyanobacterial cells.