Norbert Erdmann

Activation and pathway of glucosylglycerol synthesis in the cyanobacterium Synechocystis sp. PCC 6803

The biosynthetic pathway of glucosylglycerol (GG), the osmoprotective compound of Synechocystis sp. PCC 6803, was found to proceed from ADP-glucose and glycerol 3-phosphate via glucosylglycerol phosphate in a two-step reaction. Using an in vitro assay it was shown that the GG-forming enzyme system required activation, which could be initiated in vivo by hypertonic salt concentrations and osmotic shock, or in vitro by NaCl addition at the stage of enzyme extraction or assay.

Construction of promoter probe vectors for Synechocystis sp. PCC 6803 using the light-emitting reporter systems Gfp and LuxAB

Two promoter probe vectors were constructed for the cyanobacterium Synechocystis sp. strain PCC 6803 using reporter genes, which can be easily detected and quantified in vivo by the ability of their encoded proteins to emit light. The vectors allow the transcriptional fusion of promoter sequences with the gfp and luxAB genes, respectively, and their stable integration into a neutral site of the Synechocystis chromosome. Functionality of these vectors was demonstrated by cloning the promoter of the isiAB operon into both promoter probe vectors and analyzing the stress-dependent emission of light by the obtained reporter strains. As was found before for the isiAB operon, the P(isiAB) reporter gene fusions were induced by iron starvation and high salt stress. Induction rates of mRNA of the wild type operon and the reporter gene fusions were found to be essentially the same, indicating that a promoter fragment containing all necessary regulatory elements has been cloned. However, using the gfp gene a slow increase of protein and fluorescence was found, while the luxAB reporter gene constructs led to a rapid increase in luminescence. The same was found after retransfer of cells back into control media, in which the Gfp protein disappeared slowly, while the LuxAB-based luminescence decreased rapidly. These experiments show that both reporter genes can be used in Synechocystis: the luxAB system seems to be favourable regarding reaction time, while the gfp system has the advantage of being independent from any substrate.

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.

Repression by Fur is not the main mechanism controlling the iron-inducible isiAB operon in the cyanobacterium Synechocystis sp. PCC 6803

The iron deficiency-dependent regulation of isiAB transcription in Synechocystis sp. PCC 6803 was analyzed by fusion of modified isiAB promoter fragments to gfp and in vivo quantification of Gfp fluorescence. For the putative Fur box only a slight repressing impact on promoter activity could be shown. In a heteroallelic fur mutant a corresponding incomplete repression of isiAB transcription under iron-replete conditions confirmed the role of Fur in isiAB regulation. However, a 90 bp region upstream of the putative -35 box of the isiAB promoter was essential for full promoter activity under iron-deplete conditions. This pattern indicates a dual promoter regulation by both a repressing mechanism exhibited via the Fur system and an unknown activating mechanism.

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.

Salt Acclimation of Algae and Cyanobacteria: A Comparison

Almost all cells are able to live within a certain range of enhanced salt concentrations or changing salinities, since it is generally believed that all life originated in the oceans, a highly saline environment. However, during evolution, the degree of salt resistance became very divergent among present-day organisms. Salt acclimation has generated great interest for two main reasons: (1) salt represents one of the environmental master factors of aquatics and, increasingly, of terrestrial habitats, and (2) salt resistance constitutes a real biotechnological challenge in the field of crop plants. Thus, algae and cyanobacteria have attracted considerable attention since they are inhabitants of these biotopes characterized by high and also changing salinities and can serve as model organisms for higher plants. In the second half of the 20th century, the fundamental aspects of salt adaptation were unravelled and subjected to several chapters: Kirst (1990) chaptered salinity tolerance of marine macroalgae and phytoplankton species; Galinski (1993), Da Costa et al. (1998), Kempf and Bremer (1998) as well as Poolman and Glaasker (1998) discussed compatible solutes of bacteria; Serrano et al. (1999) stressed the mechanism of ion homeostasis; Oren (1999) revised the energetic costs of salt adaptation; Wood (1999) focused on bacterial osmosensing; and Bohnert and Sheveleva (1998), Yeo (1998) and McNeil et al. (1999) reviewed the metabolic engineering of salt stress resistance. Salt acclimation of cyanobacteria was reviewed by Reed and Stewart (1988), as well as Hagemann and Erdmann (1997). Comprehensive definitions of terms are to be found in the chapter of Kirst (1990) and Wood (1999). This chapter is a comparative update of recent progress in salt-adaptation research on algae and cyanobacteria.

Synthesis of glucosylglycerol in salt-stressed cells of the cyanobacterium Microcystis firma

The unicellular cyanobacterium Microcystis furma tolerates salinity by accumulating the osmoprotective compound glucosylglycerol. After salt shock, the initial rate of glucosylglycerol synthesis is independent of the NaCl concentration used. In pulse chase experiments with NaH14CO3, synthesis of glucosylglycerol by salt-adapted cells was found to be rapid, whereas no sign of its breakdown was detected. Therefore, it is concluded that no turnover of glucosylglycerol takes place in salt-adapted cells. The specific capacity of the glucosylglycerol-forming enzyme system may be one reason for the salt resistance limit.

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.

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.