Abstract Salt-sensitive mutants of Synechocystis were obtained by random cartridge mutagenesis, and one mu- tant (mutant 4) was characterized in detail. The salt toler- ance 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 glucosylglyc- erol (GG). Salt-treated cells of mutant 4 accumulated the intermediate glucosylglycerol-phosphate (GG-P). Only low levels of phosphate-free GG were detected. The phos- phorylated 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 mu- tant 4, while GG-P-synthase remained unchanged. The in- tegration site of the aphII cartridge in mutant 4 and the corresponding wild-type region was cloned and se- quenced. 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. Re- lated 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. Key words Cyanobacteria · Glucosylglycerol- phosphate · Osmoprotective compounds · Random cartridge mutagenesis · Salt adaptation · StpA protein · Synechocystis Abbreviations GG Glucosylglycerol · GG-P Glucosylglycerol-phosphate · Km Kanamycin · Km R Kanamycin-resistant Introduction Salinity is an important environmental factor for aquatic organisms. An increase in salt concentration represents a combination of two stress conditions for living cells. The reduction in the water potential of the surrounding me- dium causes the cells to lose water. In contrast to the purely osmotic stress caused by high concentrations of nonpermeable organic agents, an increase in salinity also means a dramatic increase in inorganic ions (especially Na + and Cl – ), entering the cell along electrochemical gra- dients. The physiological basis of salt adaptation has been studied extensively in the past and has been found to in- clude two main processes: (1) synthesis and accumulation of osmoprotective compounds (compatible solutes); and (2) maintenance of low internal concentrations of inor- ganic ions by active export mechanisms. In cyanobacteria, sucrose, trehalose, glucosylglycerol, glycine betaine, and glutamate betaine serve as osmopro- tective compounds (Reed and Stewart 1988). The accu- mulation of the compound glucosylglycerol (GG; 2-O-(α- D-glucopyranosyl)-glycerol) is characteristic of moder- ately halotolerant cyanobacteria tolerating up to 1.3 M NaCl. This group includes Synechocystis sp. strain PCC 6803 (Reed and Stewart 1985). GG has also been found recently in salt-stressed pseudomonads (Pocard et al. 1994), and in higher plants (Bianchi et al. 1993). The biosynthetic pathway of GG has been elucidated in Synechocystis (Hagemann and Erdmann 1994). It con- sists of the glucosyltransferase reaction, ADP-glucose + glycerol-3-phosphate→glucosylglycerol-phosphate (GG- P) + ADP, catalyzed by GG-P-synthase and the phospho- hydrolase reaction, GG-P→GG + P i , catalyzed by GG-P- phosphatase. The enzymes for GG synthesis are probably constitutively present in the cells in a nonactive form un- der low-salt conditions. Under salt stress, their activation Martin Hagemann · Stefan Richter · Ellen Zuther · Arne Schoor Characterization of a glucosylglycerol-phosphate-accumulating, salt-sensitive mutant of the cyanobacterium Synechocystis sp. strain PCC 6803 Arch Microbiol (1996) 166 : 83–91 © Springer-Verlag 1996 Received: 12 January 1996 / Accepted: 28 May 1996 ORIGINAL PAPER M. Hagemann () · E. Zuther · A. Schoor Universität Rostock, Fachbereich Biologie, Doberaner Strasse 143, D-18051 Rostock, Germany Tel. +49-381-4942076; Fax +49-381-4942079 e-mail mh@boserv.bio4.uni-rostock.de S. Richter Max-Planck-Institut für Molekulare Genetik, Ihnestrasse 73, D-14195 Berlin, Germany