INTRODUCTION

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The physiological basis for the adaptation to high salinities has been studied intensively in several cyanobacterial species. It includes three main subprocesses: active extrusion of inorganic ions, leading to relatively unchanged internal salt concentrations; accumulation of large internal amounts of organic osmoprotective compounds; and expression of a set of salt stress proteins (17). The ion export is mediated by Na⁺/H⁺ antiporters, which are energized by the respiratory chain localized on the cytoplasmic membrane (27), and by H⁺- ATPases (12). On the basis of the principal osmoprotective compound accumulated, three salt tolerance groups of cyanobacteria could be distinguished (29). Slightly salt-tolerant strains accumulate sucrose or trehalose, moderately halotolerant strains synthesize glucosylglycerol (GG), and halophilic strains contain glycine betaine or glutamate betaine. The synthesis of salt stress proteins was analyzed by amino acid labelling. After comparison of stress protein synthesis in salt-shocked cells with that in cells subjected to other stresses, the occurrence of general and specific stress proteins became obvious. A first general stress protein was identified in Synechocystis sp. strain PCC 6803 as a flavodoxin (11).

The genetic processes involved in cyanobacterial salt adaptation have been investigated for only a short time. With a subtractive hybridization procedure, it was shown that about 10% of the genome of Anabaena torulosa seems to be salt inducible (5). Genes encoding enzymes involved in the ion export process (cytochrome oxidase [3]), in the synthesis of osmoprotective compounds (GG-phosphate phosphatase [18]), and in the transport of osmoprotective compounds (19) have been cloned and functionally characterized. For the identification of additional genes necessary in the process of salt adaptation, the generation of mutants represents a powerful tool, since after the characterization of such mutants processes originally not thought to be essential for salt adaptation can be identified. In 1990, several spontaneous cyanobacterial mutants were obtained that showed a defect in respiration and a salt-sensitive phenotype (21).

We have employed the method of random cartridge mutagenesis (23) to generate salt-sensitive mutants of the cyanobacterium Synechocystis. Synechocystis sp. strain PCC 6803 belongs to the group of moderately halotolerant cyanobacteria (salt resistance limit, about 1.2 M NaCl) and accumulates mainly GG after salt stress (28). Recently, the genome of this strain was completely sequenced (22). Random cartridge mutagenesis offers the advantage that the mutated site is tagged by an antibiotic resistance gene marker, which makes it easier to reclone the affected genes and guarantees a stable mutation in the multicopy genome of this strain. In a first attempt, three salt-sensitive mutants (mutants 143, 406, and 549) that had different remaining salt tolerances were obtained (15). With the antibiotic resistance gene as a probe in Southern hybridization experiments, it was found that different sites of the chromosome were affected in the three mutants. The salt-sensitive phenotype of one mutant (mutant 143) could be correlated with a defect in GG synthesis, while the other two mutants (mutants 406 and 549) were able to accumulate the same amount of osmolytes as the wild type (WT) and the defect leading to reduced salt tolerance remained unknown (15).

In this paper we describe the genetic and physiological characterization of the salt-sensitive Synechocystis mutant 549. The mutated site was cloned and used to screen for the WT genes in a gene library. After sequencing, a deletion affecting four genes was found in mutant 549. A newly generated single mutant showing a defect only in a putative glycoprotease resembled the original mutant 549. After cultivation in high-salt media, a remarkable change in pigmentation accompanied by changes in the ultrastructure was found in salt treated cells of this mutant.

	MATERIALS AND METHODS

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Strains and culture conditions. The derivative of Synechocystis sp. strain PCC 6803 with enhanced transforming capacity that was used in all experiments was obtained from S. Shestakov (Moscow State University, Russia). Axenic cells were cultivated on agar plates or in liquid culture at 30°C under constant illumination with a mineral medium (15). Salt-sensitive mutants were generated by random cartridge mutagenesis (23) using the aphII gene (aminoglycoside phosphotransferase II gene, conferring kanamycin resistance) isolated from plasmid pUC4K (35). The whole procedure, including transformation of Synechocystis, was described earlier (15). Transformants were initially selected on media containing 10 µg of kanamycin (Sigma) per ml, while the segregation of clones and the cultivation of mutants were conducted with 50 µg of kanamycin per ml. Escherichia coli JM101 (30) was used for routine DNA manipulations, while strain Q358 (30) served as a host for bacteriophage . E. coli was cultivated in Luria broth (LB) medium at 37°C.

DNA manipulations. Total DNA was isolated from Synechocystis by lysozyme treatment and phenol-chloroform purification (5). All other DNA techniques, such as plasmid isolation, transformation of E. coli, ligations, restriction analysis (restriction enzymes were obtained from Life Technologies), Southern hybridization analysis, plaque hybridization, isolation of  DNA, and DNA labelling by random priming using [-³²P]dATP (Amersham Buchler), were standard methods (30). The WT genes were cloned from a library of Synechocystis DNA fragments in the  vector EMBL3, kindly provided by H. D. Osiewacz, Ruhr Universität, Bochum, Germany. DNA fragments for sequencing were generated by subcloning defined restriction fragments into pUC18, pUC19 (37), pUCBM20, pUCBM21 (Boehringer Mannheim), or pGEM7 (Promega). Additionally, partially deleted clones were obtained by double-stranded nested deletion (kit from Pharmacia). DNA sequencing was performed by the dideoxy chain termination method using [-³⁵S]dATP (Amersham Buchler) and the Sequenase 2.0 kit (USB). For sequencing, double-stranded plasmid DNA was isolated by means of the QIAprep plasmid kit (Qiagen). The site of integration of the aphII gene into the chromosomal DNA was characterized by partial sequencing using the following synthetic primers specific for the aphII gene: kan 5', CAGGCCTGGTATGAGTCAGC; kan 3', ATTTTTATCTTGTGCAATGT (custom oligonucleotide synthesis; Pharmacia). Computer analysis of the DNA sequence was done by means of the DNASIS/PROSIS software package (Pharmacia).

Analysis of mutant phenotype. Salt-shock experiments were performed in CO₂-gassed (2%) batch cultures of mutant and WT cells after addition of solid NaCl (usually to give a final concentration of 684 mM NaCl) to the standard medium (containing 2 mM NaCl). Photosynthesis and respiration were measured with a Clark-type oxygen electrode (9). The growth of cells was monitored by estimation of the extinction at 750 nm (A₇₅₀) at appropriate dilutions employing a regression to calculate cell number and biomass. The cyanophycin (multi-L-arginyl-poly[L-aspartic acid]) content was estimated after isolation of cyanophycin granules, their solubilization by 0.1 N HCl, and chemical estimation of arginine exactly as described previously (4). The protein content was also estimated as described previously (24).

Pigment analyses. The chlorophyll a (Chl a) and carotenoid concentrations of the samples were determined by high-performance liquid chromatography (HPLC) according to a modified method described previously (32) and with previously published extinction coefficients (25). Due to difficulties encountered in quantitative extraction of phycobiliproteins, the pigment content was routinely estimated from measurements of whole-sample absorption. Absorption was measured with samples placed at the entrance of the integrating sphere accessory (Labsphere; Perkin Elmer) of a spectrophotometer (Lambda 2; Perkin Elmer) to minimize scattering. The pigment-specific absorption coefficient for phycocyanin, A*₆₃₀, determined for a subsample of WT cells was found to be relevant as well for the mutant cells, indicating that differences in pigment "packaging" were low over the range of absorbances encountered in the different cultures.

Electron microscopy. Cells used for analyses of ultrastructure were taken directly from cultures, immediately fixed with glutaraldehyde (4% in 0.1 M Na phosphate buffer), and additionally fixed with 1% osmium tetroxide, and water was removed with acetone. These cells were embedded in an epoxy resin (Araldite; Fluka) and cut into ultrathin sections. Sections of cells on the grids were treated with 2% uranyl acetate and lead citrate. The micrographs were obtained with an electron microscope, model EM902 (Zeiss).

	RESULTS

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Gene cloning. Integration of the aphII gene into the genome of the salt-sensitive Synechocystis mutant 549 was first characterized in Southern blot experiments, in which an EcoRI fragment of about 5.0 kb hybridized with the aphII gene probe (15). This 5.0-kb EcoRI fragment of chromosomal DNA from mutant 549 was cloned into the plasmid pBR329 (8). Km^r clones of E. coli were selected, and the recombinant plasmids were isolated and characterized by restriction analysis (Fig. 1). The aphII gene cartridge (about 1.3 kb) was obtained together with about 3.7 kb of flanking cyanobacterial DNA. The resulting plasmid, pBRM549, was used to transform the Synechocystis WT. Two hundred Km^r clones were selected and, after complete segregation, tested for salt and chloramphenicol resistance. All transformants tested were sensitive to both high salt concentrations (600 mM NaCl) and chloramphenicol (5 µg/ml; pBR329 harbors a chloramphenicol resistance marker). These results indicate that the salt-sensitive phenotype is coupled to the integration of the aphII gene at the site cloned in pBRM549. The absence of chloramphenicol resistance after transformation with pBRM549 demonstrates that only the EcoRI fragment, not the vector molecule, had been integrated in a double-crossover event because single recombinants should carry all the markers of the plasmid.

View larger version (27K): [in this window] [in a new window]   FIG. 1.   Schematic drawing showing the genetic organization, restriction map, and protein-encoding regions of the chromosomal site affected in the Synechocystis mutant 549 (A), the corresponding site of the Synechocystis WT (B), and the insertion sites of the aphII gene in selected sites of the different genes to obtain mutants with single mutations (C). Empty arrows, protein-encoding regions in Synechocystis; darkly shaded arrows, inserted aphII gene cassettes; black arrows, regions sequenced in this work; *, partial deleted genes; lightly stippled arrow, deletion that occurred during integration of an aphII gene in mutant 549.

Sequence analysis. After subcloning of a 3.2-kb SmaI/EcoRI fragment and generation of overlapping deletions by digestion with exonuclease III, the nucleotide sequence of this fragment was determined for both strands starting from the 5' and 3' ends (Fig. 1). During sequencing, similarities to previously published sequences from Synechocystis were recognized (7, 36). Therefore, the central part of the 3.2-kb SmaI/EcoRI fragment was not sequenced again. For precise determination of the integration site of the aphII gene during the generation of mutant 549, the cyanobacterial DNA flanking the Km^r cartridge on pBRM549 was partly sequenced with primers specific to the 5' and 3' ends of the aphII gene. Sequences identical to the WT fragment (for the 5' sequence before position 263 and for the 3' sequence after position 2092) were found (not shown). Therefore, about 1.8 kb of the WT region was deleted during integration of the aphII gene cassette into the genome of mutant 549 (Fig. 1).

Construction of mutants with mutations affecting single genes. In order to clarify the role of the four ORFs deleted in mutant 549 in salt tolerance in more detail, several insertion mutants with mutations affecting single genes were constructed (Fig. 1) (see Table 1 for nomenclature of the mutants) and physiologically characterized. The psaF and psaJ genes were mutated together by integration of an aphII gene into the BalI site present inside psaF, since both genes are transcribed as an operon (7). A gcp (ORF1) mutant was constructed by cloning the aphII gene cartridge into the internal EcoRV site of the gcp gene homolog. Finally, ORF2 was mutated by insertion of the resistance marker into the BamHI site close to the 3' end of its coding sequence (Fig. 1). Plasmids showing correct integration of the aphII gene were transformed into Synechocystis, from which Km^r clones originating from homologous recombination with the chromosomal DNA were selected (Table 1).

View larger version (99K): [in this window] [in a new window]   FIG. 2.   Southern blot experiments for characterization of complete segregation of the gcp mutant. The 32P-labelled internal BclI/KpnI fragment of the gcp gene (A) or the 32P-labelled aphII gene (B) was used as a probe for the hybridization to EcoRI-BamHI- (lanes 1 and 2) and to HindIII-BamHI-digested (lanes 3 and 4) chromosomal DNA from the WT (lanes 1 and 3) and the gcp mutant (lanes 2 and 4), respectively. The molecular masses of HindIII-digested  DNA size standards were drawn by using the positions from the photo of the ethidium bromide-stained gel at the same magnification.

Physiological characterization of the gcp (ORF1) mutant. Among the mutants with single mutations constructed in the genes affected in mutant 549, only the gcp mutant showed a significant reduction in its salt tolerance level and it was therefore chosen for further analyses. The growth and photosynthesis of the gcp mutant was compared to those of the original mutant 549 and the WT after applying a salt shock of 684 mM NaCl. In all cases an immediate decrease in photosynthesis was observed after the addition of high salt concentrations (Fig. 3). During the first 8 h after shock, photosynthesis recovered in the WT as well as in the mutants. While the WT cells were able to adapt photosynthesis to the demands of high salt concentrations completely after only 48 h, in cells of the mutant 549 and in the gcp mutant, respectively, it decreased continuously until 96 h, when photosynthesis was completely inhibited (Fig. 3). Growth of the mutants stopped by 48 h after the salt shock, while growth of the WT had already recovered (Table 2).

View larger version (15K): [in this window] [in a new window]   FIG. 3.   Alterations of photosynthetic oxygen evolution after a salt shock of 684 mM NaCl in WT cells (), mutant 549 cells (), and the gcp mutant (). The diagram represents means from three independent experiments.

View larger version (14K): [in this window] [in a new window]   FIG. 4.   Comparison of in vivo absorption spectra obtained from WT cells and from the gcp mutant grown in basal medium or after a salt shock of 684 mM NaCl for 48 and 96 h. All spectra were normalized at the absorption maximum of Chl a at 680 nm. WT: control cells, ; 48 h, ; 96 h, . gcp mutant: control cells, ; 48 h, - - -; 96 h, - - -.

View larger version (139K): [in this window] [in a new window]   FIG. 5.   Comparison by electron microscopy of the ultrastructures of WT cells (A and B) and of the gcp mutant (C and D) grown in basal medium (A and C) or after a salt shock of 684 mM NaCl for 96 h (B and D). The bars represent 0.25 µm. CG, cyanophycin granules; G, glycogen; PP, polyphosphate.

	DISCUSSION

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During the characterization of Synechocystis mutant 549, a deletion of about 1.8 kb was found. The occurrence of deletions with random cartridge mutagenesis was also found in previous studies, in which 1.3 to 50 kb has been lost, leading to very stable mutants (6, 16). These deletions make it difficult to decide which of the affected ORFs is responsible for the observed phenotype. In order to identify the gene responsible for the reduced salt tolerance of mutant 549, mutants with single mutations were generated and characterized regarding salt tolerance. The psaFJ genes, which were completely deleted in mutant 549, seem to be nonessential for salt adaptation in Synechocystis, since an insertion mutant impaired in psaF could grow on 684 mM NaCl. Nevertheless, in the kinetics of adaptation to high salt concentrations, differences compared to the WT were detectable (unpublished data). These subunits of photosystem I are of minor importance for its function, because psaFJ mutants showed no significant alterations in photosynthetic activity (7). Recently, it was found that the PsaF protein might be involved in electron transfer from plastocyanin to P700 in higher plants (20). orfII, encoding a protein of completely unknown function, was partly deleted in mutant 549, but an insertion mutant specific for this gene showed no alterations after growth in high-salt and basal medium compared with the WT.

The reduction in salt tolerance found for mutant 549 could be reproduced in experiments with an insertion mutation in orfI or gcp. Both the mutant 549 and the gcp mutant showed a reduction of remaining salt tolerance to the same extent (limit less than 550 mM NaCl) and the same kinetic behavior after a lethal salt shock of 684 mM NaCl (Fig. 3). Therefore, it could be concluded that the partial deletion of this gene in mutant 549 was responsible for the mutant's reduced salt tolerance. Compared with mutants of Synechocystis defective in the synthesis of the osmoprotective compound GG, the salt tolerance of the gcp mutant remained relatively high. GG mutants were also not able to restore the immediate decrease in photosynthesis after a salt shock of 684 mM NaCl. Photosynthesis became completely inhibited by about 30 h, and their salt tolerance limit was reduced to less than 350 mM NaCl (15, 16). Therefore, the gcp gene product seems not to be directly involved in basic processes of salt adaptation and its defect might lead only indirectly to a reduction in salt tolerance.

Last year the complete genome sequence of Synechocystis was published (22). The ORF1 protein was identified as a putative sialoglycoprotease, which might be involved in the degradation of proteins. A comparison of the Gcp protein to several other putative glycoproteases from heterotrophic bacteria showed high sequence similarities (about 40% identical amino acid residues) (Fig. 6). Most of the related sequences were obtained during genome sequencing projects. The function of these proteins was derived from sequence comparisons alone. Only for the protein from Pasteurella haemolytica has the function as a protease been biochemically proven. This protease, belonging to the group of metalloproteases, showed specificity for a glycosylated protein, the glycophorin A, with a major cleavage site at Arg-31-Asp-32 (2). A similar protein from E. coli was thought to be involved in the regulation of a macromolecular operon (26).

View larger version (119K): [in this window] [in a new window]   FIG. 6.   Amino acid sequence alignment of the gcp gene homolog (ORF1) from Synechocystis sp. strain PCC 6803 (SYNE [22]) and other putative gcp genes from several bacteria. HINF, Haemophilus influenzae (10); ECOL, Escherichia coli (26); PHAE, Pasteurella haemolytica (1); MLEP, Mycobacterium leprae (33a). Uppercase letters are used when amino acids are identical to those in the Synechocystis sequence (shaded boxes).

Besides the reduced salt tolerance, the most obvious alterations in the phenotype of the Synechocystis gcp mutant were related to changed pigmentation and a remarkable accumulation of cyanophycin and glycogen. An increase in the carotenoids and especially in echinenone and myxoxanthophyll, as well as in glycogen accumulation, was also observed for salt-adapted cells of the WT (9, 32), but these increases and the decrease in phycocyanin were much more pronounced in the salt-stressed cells of the gcp mutant. Cyanophycin, composed of polyaspartate and arginine, which is synthesized without the participation of ribosomes, has been found only as a high-molecular-weight nitrogen reserve in many cyanobacteria, mainly accumulating in cells during the transition to stationary phase (33). The enhanced content of cyanophycin could be best explained by the assumption that the putative protease encoded by the gcp gene is responsible for cyanophycin degradation in the Synechocystis WT. The enzymes involved in cyanophycin synthesis and degradation are only poorly characterized in cyanobacteria, but they were found to be constitutively expressed (33), except in Anabaena, where the level of both activities increased in heterocysts during their differentiation (14). A defect in the degradative activity would lead to uncontrolled accumulation of cyanophycin. Under our culture conditions, cyanophycin synthesis seems to be especially induced in high-salt media, since in the control cells the cyanophycin level showed no (chemical estimation), or only minor (ultrastructural studies), changes in the gcp mutant, while it is massively induced in salt-treated cells. This massive accumulation of cyanophycin, which could not be remobilized, would starve the cells for nitrogen. The observed changes in pigmentation, as well as the accumulation of large amounts of glycogen, are very characteristic of nitrogen-starved cyanobacteria (13). A reduced supply or availability of nitrogen reduces protein synthesis relative to glycogen and induces the degradation of phycobilisomes, because the phycobiliproteins serve not only as light-harvesting pigments but also as a nitrogen storage mechanism in cyanobacteria. In contrast to phycocyanin, the content of carotenoids as nitrogen-free pigments is enhanced, especially that of xanthophylls, decreasing effects of excess light absorption by the remaining pigments (13). The unfavorable situation of shortened nitrogen supply seems to lead to more dramatic effects, when a second stress factor is introduced, such as salt stress. Under the combined stresses of nitrogen starvation and salt, the cell is unable to adapt to a new steady state and lysis occurs. The gcp mutant also shows interesting possibilities for biotechnological applications, since after a salt shock the cyanophycin accumulation is dramatically enhanced and the content of several carotenoids, especially echinenone and myxoxanthophyll, which could be used as antioxidants, is also increased. In further studies, the effects of the gcp gene defect on the nitrogen metabolism and on general stress tolerance will be investigated with this mutant.