Arne Schoor

Active transport of glucosylglycerol is involved in salt adaptation of the cyanobacterium Synechocystis sp. strain PCC 6803

An active-transport system for the osmoprotective compound glucosylglycerol (GG) was found in the cyanobacterium Synechocystis sp. strain PCC 6803. Uptake assays with 14C-labelled GG showed that the GG transport was enhanced in cells adapted to increasing concentrations of NaCl. Kinetic studies indicated a Michaelis-Menten relationship. The uptake of GG was energy dependent and occurred against a steep concentration gradient. It was inhibited by uncouplers as well as by a combination of darkness and KCN. The affinity of the transporter seems to be restricted to osmoprotective compounds of cyanobacteria; from a variety of compounds tested only sucrose and trehalose competed with GG for uptake. A salt-sensitive mutant of Synechocystis 6803 unable to synthesize GG could be complemented to salt resistance by exogenous GG. Accumulation of GG from the medium was essential for the restoration of photosynthesis and growth in mutant cells under high-salt conditions. In wild-type cells, the GG transporter probably serves to prevent GG leaking out of salt-stressed cells.

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.

Determination of the cyanobacterial osmolyte glucosylglycerol by high-performance liquid chromatography

Abstract A combination of reversed-phase chromatographic (RPC) [octadecyl silica (ODS)] and ion-modrated partition chromatographic (IMPC) (Ca 2+ ) stationary phases with water as mobile phase provides separation of the cyanobacterial osmolyte glucosylglycerol (2-O-α- d -glucopyranosylglycerol, GG) from other ubiquitous osmolytes (sucrose, trehalose, glycinebetaine) and major natural carbohydrates, also in the presence of common osmotic stressors (mannitol, sorbitol). The method allows investigations of GG biosynthesis in vitro where glucose and glycerol can be released. The separate use of RPC or IMPC columns is restricted to samples containing no significant amounts of sucrose and glucose, respectively. Amino-bonded silica and acetonitrile-water mixtures provide excellent separation of GG from disaccharides but separation from important hexoses is limited.

Biochemical Characterization of Glucosylglycerol-Phosphate Synthase of Synechocystis sp. Strain PCC 6803: Comparison of Crude, Purified, and Recombinant Enzymes

Glucosylglycerol-phosphate synthase (GGPS), the key enzyme of the glucosylglycerol biosynthesis in salt-stressed cells of Synechocystis, was biochemically analyzed in crude extracts, after partial purification by FPLC and after overexpression of the gene ggpS in Escherichia coli and purification to homogenity of the recombinant protein, respectively. These GGPS preparations behaved similarly with regard to temperature stability, pH optimum, Mg2+ dependence, inhibition by phosphates, and Km values, but differed in their dependence on NaCl concentration: crude enzyme needed activation by addition of NaCl, whereas both partially-purified and recombinant GGPS showed high activities independent of the NaCl concentration.

Response of phytoplankton communities to salinity changes: a mesocosm approach

The response of natural phytoplankton communities, originating from oligohaline brackish water systems, to salinity changes (3–12 PSU) has been studied in small-scale mesocosms. Simultaneously, their reaction on iron manipulations was tested. The experiments, each lasting 7 days, were repeated three times at different dates. Treatments were evaluated with respect to biomass development (Chl a concentration), photosynthesis behaviour, and rough taxonomic composition. The investigated phytoplankton communities were dominated by cyanobacteria. Salt addition was not effective for overall phytoplankton biomass development. Filamentous cyanobacteria, however, were promoted by NaCl enrichments. Dark yield and non-photochemical quenching of fluorescence (NPQ) analyses revealed differences for treatments in dependence on iron supplements. Iron partially seemed to dampen the effects of salt shocks, and iron addition reduced both, capacity and irradiance dependency of NPQ, irrespective of the NaCl treatment.

Salt adaptation in pseudomonads: characterization of glucosylglycerol-synthesizing isolates from brackish coastal waters and the rhizosphere.

The compatible solute glucosylglycerol (GG) is widespread among cyanobacteria, but, until now, has been reported for only two species of heterotrophic bacteria. About 120 bacterial isolates from coastal regions of the Baltic Sea were screened by HPLC for their ability to synthesize GG. Positive isolates (26) were grouped by SDS-PAGE of whole-cell proteins and representative strains of each group were investigated by sequencing their 16S rRNA genes and phenotypic characterization. All GG-synthesizing isolates were shown to belong to the genus Pseudomonas (sensu stricto) and were assigned to 4 distinct groups, although none of the GG-synthesizing isolates could be unambiguously assigned to described species. The identity of GG was verified by 13C NMR analysis and enzymatic digestion with alpha- and beta-glucosidases. Besides GG, salt adapted cultures of the aquatic isolates accumulated the dipeptide N-acetylglutaminylglutamine amide (NAGGN) and glutamate. The accumulation of noncharged compatible solutes was also tested in previously identified pseudomonads isolated from the rhizosphere of oilseed rape and potato. The majority of these strains were fluorescent species of the genus Pseudomonas and accumulated trehalose and NAGGN when grown under salt stress conditions. However, rhizosphere isolates of Stenotrophomonas maltophilia synthesized GG and trehalose or only trehalose in a strain-dependent manner. These data indicate that the ability to synthesize GG is widely distributed among slightly or moderately halotolerant pseudomonads.

Characterization of a glucosylglycerol-phosphate-accumulating, salt-sensitive mutant of the cyanobacterium Synechocystis sp. strain PCC 6803

Abstract Salt-sensitive mutants of Synechocystis were obtained by random cartridge mutagenesis, and one mutant (mutant 4) was characterized in detail. The salt tolerance of mutant 4 was reduced to about 20% of that of the wild-type. This was caused by a defect in the biosynthetic pathway of the osmoprotective compound glucosylglycerol (GG). Salt-treated cells of mutant 4 accumulated the intermediate glucosylglycerol-phosphate (GG-P). Only low levels of phosphate-free GG were detected. The phosphorylated form of GG was not osmoprotective and seemed to be toxic. In vitro enzyme assays revealed that GG-P-phosphatase activity was completely absent in mutant 4, while GG-P-synthase remained unchanged. The integration site of the aphII cartridge in mutant 4 and the corresponding wild-type region was cloned and sequenced. Mutant 4 was complemented to salt resistance after transformation by the cloned wild-type region. The integration of the cartridge led to a deletion of about 1.1 kb of the chromosomal DNA. This affected two of the identified putative protein coding regions, orfII and stpA. The ORFII protein shows a high degree of similarity to the receiver domain of response regulator proteins. Related sequences were not found for StpA. We assume that in mutant 4, regulatory genes necessary for the process of salt adaptation in Synechocystis are impaired.

NaCI acts as a direct modulator in the salt adaptive response: Salt-dependent activation of glucosylglycerol synthesis in vivo and in vitro

Summary The formation of glucosylglycerol (GG) was studied in dependence on the NaCl concentration in vivo after moderate salt shocks of cells and in vitro after enzyme activation in cell extracts of Synechocystis sp. PCC 6803. After salt shock treatments between 342 and 684 mmol/L NaCl GG was accumulated with about the same rate without a lag-phase. In vitro enzyme assays showed that the GG synthesizing enzyme system was activated immediately after a shock of 342 mmol/L NaCl. The amount of these enzymes was further enhanced about 3 fold within the first 3 hours of the adaptation process. Only the latter increase of enzyme activity could be inhibited by chloramphenicol treatment indicating an immediate activation of preformed enzymes combined with a weak activation of gene expression of the GG synthesizing enzymes. In vitro a 15 fold increase of the activity of the GG synthesizing enzymes were achieved by adding NaCl in the concentration range of 171 to 342 mmol/L to protein extracts. In contrast to the remarkable high salt tolerance of the GG-phosphate synthase (GGP-S) in vitro the GG-phosphate phosphatase was fairly inhibited in the presense of higher NaCl concentrations. Their salt tolerance in vitro did not change regardless whether the enzymes were extracted from control or NaCl-adapted cells. It is assumed that the reversible modulation of the GGP-S activity by NaCl may function as a control mode in other living cells, too.

Detection of Prochlorothrix in Brackish Waters by Specific Amplification of pcb Genes

ABSTRACT Prochlorothrix hollandica is the only filamentous chlorophyll b (Chlb)-containing oxyphotobacterium that has been found in freshwater habitats to date. Chlb serves as a light-harvesting pigment which is bound to special binding proteins (Pcb). Even though Prochlorothrix was initially characterized as a highly salt-sensitive species, we detected it in a brackish water environment that is characterized by salinities of up to 12 practical salinity units. Using PCR and reverse transcription, we amplified pcb gene fragments of phytoplankton samples taken along a salinity gradient in the eutrophic Darss-Zingst estuary (southern Baltic Sea). After sequencing, high levels of homology to the pcbB and pcbC genes of P. hollandica were found. Furthermore, autofluorescence of Prochlorothrix-like filaments that indicated that Chlb was present was detected in enrichment cultures prepared from the estuarine phytoplankton. The detection of Chlb-containing filaments, as well as the pcb and 16S ribosomal DNA sequences, suggests that Prochlorothrix is an indigenous genus in the Darss-Zingst estuary and may also inhabit many other brackish water environments. The potential of using pcb gene detection to differentiate Prochlorothrix from morphologically indistinguishable species belonging to the genera Pseudanabaena and Planktothrix (Oscillatoria) in phytoplankton analyses is discussed.

Glucosylglycerol-phosphate synthase : target for ion-mediated regulation of osmolyte synthesis in the cyanobacterium Synechocystis sp. strain PCC 6803

Abstract The response of cyanobacteria to a changing osmotic environment includes the accumulation of organic osmolytes such as glucosylglycerol. The activation of the enzymes involved in glucosylglycerol synthesis [glucosylglycerol-phosphate synthase (GGPS) and glucosylglycerol-phosphate phosphatase (GGPP)] in Synechocystis sp. strain PCC 6803 by various salts and salt concentrations was investigated in vitro. GGPS seemed to be the target for salt-mediated regulation of glucosylglycerol synthesis in vitro. GGPS activation was dependent on the concentration of NaCl, and a sigmoidal plot was obtained. Sensitivity to NaCl was markedly enhanced by low Mg+2 concentrations (optimal at 4 mM), but Mg2+ was not absolutely necessary for the Na+ stimulation. As in the case of NaCl, other salts (including MgCl2) stimulated GGPS. The relative order of GGPS activation in the presence of chloride by the cations at constant ionic strength was Li+ > Na+ > K+, Mg2+ Mn2+. No absolute dependence on ionic strength was observed in Mg2+/Na+-exchange experiments. The degree of activation by ions at various concentrations was positively related to the increasing destabilizing properties of the cations according to the Hofmeister rule, where chaotropic cations are most efficient. Cations were responsible for activation since chaotropic anions counteracted the activating effect of cations.