Mutations in a gene, stpA, had been correlated with the loss of tolerance to high NaCl concentrations in the cyanobacterium Synechocystis sp. strain PCC 6803. Genetic, biochemical, and physiological evidence shows that stpA encodes glucosylglycerol-phosphate phosphatase. stpA mutants are salt sensitive and accumulate glucosylglycerol-phosphate, the precursor of the osmoprotectant glucosylglycerol necessary for salt adaptation of Synechocystis. The consensus motif present in acid phosphatases was found in StpA; however, the homology with other sugar phosphatases is very poor. The amount of stpA mRNA was increased by growth of the cells in the presence of NaCl concentrations above 170 mM. Expression of stpA in Escherichia coli allowed the production of a 46-kDa protein which exhibited glucosylglycerol-phosphate phosphatase activity. The StpAspecific antibody revealed a protein of similar size in extracts of Synechocystis, and the amount of this protein was increased in salt-adapted cells. The protein produced in E. coli had lost the requirement for activation by NaCl that was observed for the genuine cyanobacterial enzyme.

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 uncouplen 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.

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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.

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.