1 Institute of Plant Physiology, Russian Academy of Sciences, Botanicheskaya Street 35, 127276 Moscow, Russia
2 Department of Regulation Biology, National Institute for Basic Biology, Myodaiji, Okazaki 444-8585, Japan
3 Institute of Basic Biological Problems RAS, Pushchino, Moscow Region 142292, Russia
4 Department of Molecular Biomechanics, School of Life Science, The Graduate University of Advanced Studies, Myodaiji, Okazaki 444-8585, Japan
Correspondence
Dmitry A. Los
losda{at}ippras.ru
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ABSTRACT |
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INTRODUCTION |
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Examination of Escherichia coli cells by freeze-fracture electron microscopy demonstrated that hyperosmotic stress, caused by 0·6 M sucrose, induced a twofold decrease in the cell volume over 1590 s. Mutation of the aqpZ gene, encoding aquaporin, prevented shrinkage of cells in a hyperosmotic environment (Delamarche et al., 1999). Previously we demonstrated that the aquaporin blocker p-chloromercuriphenylsulphonic acid prevented cells of the cyanobacterium Synechococcus from shrinkage under hyperosmotic stress (Allakhverdiev et al., 2000a
, b
). These observations suggest that aquaporins are involved in the cells' response to a hyperosmotic environment by mediating the water efflux.
In the present study we investigated the hyperosmotic stress responses of the cyanobacterium Synechocystis PCC 6803 (hereafter, Synechocystis), which has a single copy of the aqpZ gene for aquaporin, and its AqpZ-null mutant in terms of changes in cell volume and genome-wide gene expression. Here we demonstrate that aquaporins are responsible for the rapid water efflux from Synechocystis cells under hyperosmotic stress conditions, and that the osmo-induced changes in cytoplasmic volume might trigger osmostress-dependent gene expression.
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METHODS |
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The mutant of Synechocystis deficient in AqpZ.
A single gene that encodes a homologue of aquaporin AqpZ in Synechocystis was identified by homology search in CyanoBase (http://www.kazusa.or.jp/cyano/Synechocystis/index.html) using the amino acid sequence of AqpZ of E. coli (Calamita et al., 1995) as a query sequence. The DNA fragment that contained the aqpZ gene and its flanking regions was amplified from the genomic DNA of Synechocystis by PCR with the following primers: wchF, 5'-ACCGTTGCCGCTGTAAAGGC-3'; and wchR, 5'-CAGATAGTTGCGGATAGTGC-3'. The amplified DNA fragment was cloned in pT7-Blue vector (Novagen). The aqpZ gene of Synechocystis (slr2057; classification in accordance with the definitions of genes provided by CyanoBase) was mutated by ligating the spectinomycin-resistance (SpR) cassette (Prentki et al., 1991
) into the Eco47III sites, thus replacing part of the coding sequence in the aqpZ gene (Fig. 1
a). The AqpZ-null mutant was produced by transformation of Synechocystis cells as described by Williams (1988)
. Complete segregation of the recombinant chromosomes in the mutant strain was confirmed by PCR with the above-listed primers (Fig. 1b
). Mutant cells were cultured in BG11 medium supplemented with 25 µg spectinomycin ml1.
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Profiles of gene expression.
Cells were grown to OD750 0·4, then 5 ml 5 M sorbitol in BG11 was added to 45 ml cell suspension to give a final concentration of 0·5 M. As a control for a possible effect of increased light intensity due to dilution of the cell suspension by the addition of sorbitol solution, 5 ml BG11 medium was added to 45 ml control cell suspension. Cells that had been exposed to 0·5 M sorbitol for designated times were immediately fixed by addition of 50 ml phenol/ethanol mixture (1 : 10, w/v) to 50 ml of the cell suspension. Total RNA was isolated from the cells as described previously (Kiseleva et al., 2000) and treated with DNase I (Nippon Gene) to remove contaminating DNA.
Synechocystis DNA microarrays (CyanoCHIP version 1.2) were obtained from Takara. The microarray carried 3079 of the 3168 ORFs of Synechocystis (Kaneko et al., 1996). Cy3 dye- and Cy5 dye-labelled cDNAs used for hybridization were synthesized by reverse transcription of 20 µg of the total RNA (Kanesaki et al., 2002
; Yamaguchi et al., 2002
). Hybridization was conducted at 65 °C for 16 h. After incubation the microarrays were rinsed with 2x SSC (1x SSC is 150 mM NaCl and 15 mM sodium citrate) at room temperature, with 2x SSC at 60 °C for 10 min, with 0·2x SSC, 0·1 % SDS at 60 °C for 10 min, and finally with distilled water at room temperature for 2 min. Moisture was removed with an air spray prior to analysis in the array scanner (GMS418; Affimetrix). Each signal was quantified with the ImaGene version 4.0 software (BioDiscovery). To calculate the level of the transcript of each gene, its signal on the microarray was normalized by reference to the total intensity of signals from all genes with the exception of genes for rRNAs. Two and three independent experiments at each time point were performed with wild-type and AqpZ-deficient cells, respectively.
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RESULTS |
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We used EPR spectrometry to estimate the extent of cellular shrinkage under hyperosmotic stress conditions (Blumwald et al., 1983). This method makes it possible to register relative changes in the cytoplasmic volume of cyanobacterial cells, which avoids artefacts caused by such treatments as fixation for electron microscopy. The presence of a thick and rigid cell wall does not interfere with EPR-based assessment of cytoplasmic volume.
The results in Fig. 2 indicate that 510 min after the addition of sorbitol, the cytoplasmic volume of wild-type cells was reduced to half the original level due to the efflux of water, whereas almost no shrinkage was detected in AqpZ-mutant cells. This suggests that aquaporins mediate the rapid outflow of water from the cells under hyperosmotic stress conditions. The contribution of the lipid bilayer to water conductivity appears to be insignificant at the early stage of hyperosmotic stress.
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Changes in osmo-dependent gene expression
Hyperosmotic stress is known to alter the expression of a number of genes in Synechocystis (Kanesaki et al., 2002). In order to examine whether the mutation in the aqpZ gene would affect the expression of hyperosmotic stress response genes, we used DNA microarrays to analyse overall gene expression patterns (at the level of mRNA) in wild-type and AqpZ-mutant cells.
Comparison of gene expression profiles in these cells grown under standard conditions revealed that no gene expression was altered in the mutant cells except the aqpZ gene (Fig. 3a). However, the scatter plot illustrates the greater extent of gene up-regulation in wild-type cells after 15 min of hyperosmotic treatment when compared to AqpZ-mutant cells (Fig. 3b, c
).
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Another pattern of gene expression implied a late long-term induction, apparently related to the acclimation of cells to the new environment. This process became evident after 60 min exposure to hyperosmotic stress (Table 2).
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Hyperosmotic stress repressed the expression of many genes, including petF (for ferrdoxin), chlL (for an enzyme involved in chlorophyll synthesis), trxA (for thioredoxin), sigG (for a sigma subunit of RNA polymerase), two pilT genes (for chemotaxis), and other genes (Table 3, Fig. 5
). In wild-type cells, repression was usually observed after 15 min exposure to hyperosmotic stress, and registered for more than 2 h of incubation. The level of repression and its duration were strongly reduced in AqpZ-mutant cells (Fig. 5
).
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DISCUSSION |
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AqpZ-mutant cells demonstrate an abnormal response to hyperosmotic stress and can be basically characterized by a lesser range of reaction compared to wild-type cells. The extent of changes in gene expression caused by hyperosmotic stress is markedly depressed in an AqpZ mutant. Apparently, the observed difference is related to the inability of AqpZ-mutant cells to rapidly release water under hyperosmotic stress. Thus, the above-mentioned effects of hyperosmotic stress are much less marked in the AqpZ-mutant than in wild-type cells. This could impair activation of cell sensory systems and account for the reduced stress response that is observed in the AqpZ mutant.
It should be noted that the early gene induction seems to depend on physical changes that are caused by a hyperosmotic environment and can be influenced by the mutation in the aqpZ gene (Fig. 4a). On the other hand, the late gene induction (Fig. 4b
) and gene repression (Fig. 5
) are likely to develop in a secondary manner in response to cellular regulatory cascades rather than being directly due to physical changes.
Five sensory histidine kinases responsible for the perception of hyperosmotic stress (Hik2, Hik10, Hik16, Hik33 and Hik34) have recently been identified in Synechocystis (Paithoonrangsarid et al., 2004). These sensors control the osmo-induced expression of distinct groups of genes. We found that the mutation in the aqpZ gene affects gene induction in all these groups. This suggests that the activity of the above-mentioned sensory histidine kinases depends on the AqpZ-mediated changes accompanied by a decrease in cell volume. These changes might, therefore, be the primary trigger for the hyperosmotic stress-induced gene expression.
Conclusion
Aquaporins are responsible for rapid efflux of water in a hyperosmotic environment. This efflux leads to a decrease in the cytoplasmic volume of cells. The latter is likely to be the key event to trigger the stress signal cascade and to regulate stress-induced gene expression.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Allakhverdiev, S. I., Sakamoto, A., Nishiyama, Y., Inaba, M. & Murata, N. (2000b). Ionic and osmotic effects of NaCl-induced inactivation of photosystems I and II in Synechococcus sp. Plant Physiol 123, 10471056.
Blumwald, E., Mehlhorn, R. J. & Packer, L. (1983). Studies of osmoregulation in salt adaptation of cyanobacteria with ESR spin-probe techniques. Proc Natl Acad Sci U S A 80, 25992602.[Abstract]
Borgnia, M., Nielsen, S., Engel, A. & Agre, P. (1999). Cellular and molecular biology of the aquaporin water channels. Annu Rev Biochem 68, 425458.[CrossRef][Medline]
Calamita, G., Bishai, W. R., Preston, G. M., Guggino, W. B. & Agre, P. (1995). Molecular cloning and characterization of AqpZ, a water channel from Escherichia coli. J Biol Chem 270, 2906329066.
Csonka, L. N. (1989). Physiological and genetic responses of bacteria to osmotic stress. Microbiol Rev 53, 121147.[Medline]
Delamarche, C., Thomas, D., Rolland, J. P., Froger, A., Gouranton, J., Svelto, M., Agre, P. & Calamita, G. (1999). Visualization of AqpZ-mediated water permeability in Escherichia coli by cryoelectron microscopy. J Bacteriol 181, 41934197.
Engelbrecht, F., Marin, K. & Hagemann, M. (1999). Expression of the ggpS gene, involved in osmolyte synthesis in the marine cyanobacterium Synechococcus sp. strain PCC 7002, revealed regulatory differences between this strain and the freshwater strain Synechocystis sp. strain PCC 6803. Appl Environ Microbiol 65, 48224829.
Hecker, M. & Volker, U. (2001). General stress response of Bacillus subtilis and other bacteria. Adv Microb Physiol 44, 3591.[Medline]
Heymann, J. B. & Engel, A. (1999). Aquaporins: phylogeny, structure, and physiology of water channels. News Physiol Sci 14, 187193.[Medline]
Heymann, J. B. & Engel, A. (2000). Structural clues in the sequences of the aquaporins. J Mol Biol 295, 10391053.[CrossRef][Medline]
Hoffmann, E. K. & Dunham, P. B. (1995). Membrane mechanisms and intracellular signalling in cell volume regulation. Int Rev Cytol 161, 173262.[Medline]
Johansson, I., Karlsson, M., Johanson, U., Larsson, C. & Kjellbom, P. (2000). The role of aquaporins in cellular and whole plant water balance. Biochim Biophys Acta 1465, 324342.[Medline]
Kaneko, T., Sato, S., Kotani, H. & 21 other authors (1996). Sequence analysis of the genome of the unicellular cyanobacterium Synechocystis sp. strain PCC6803. II. Sequence determination of the entire genome and assignment of potential protein-coding regions. DNA Res Suppl 3, 109136.
Kanesaki, Y., Suzuki, I., Allakhverdiev, S. I., Mikami, K. & Murata, N. (2002). Salt stress and hyperosmotic stress regulate the expression of different sets of genes in Synechocystis sp. PCC 6803. Biochem Biophys Res Commun 290, 339348.[CrossRef][Medline]
Kiseleva, L. L., Serebriiskaya, T. S., Horvàth, I., Vigh, L., Lyukevich, A. A. & Los, D. A. (2000). Expression of the gene for the 9 acyl-lipid desaturase in the thermophilic cyanobacterium. J Mol Microbiol Biotechnol 2, 331338.[Medline]
Paithoonrangsarid, K., Shoumskaya, M. A., Kanesaki, Y. & 8 other authors (2004). Five histidine kinases perceive osmotic stress and regulate distinct sets of genes in Synechocystis. J Biol Chem published October 7, 2004 as doi: 10.1074/jbc.M410162200.
Pfeuffer, J., Broer, S., Broer, A., Lechte, M., Flogel, U. & Leibfritz, D. (1998). Expression of aquaporins in Xenopus laevis oocytes and glial cells as detected by diffusion-weighted 1H NMR spectroscopy and photometric swelling assay. Biochim Biophys Acta 1448, 2736.[CrossRef][Medline]
Poolman, B. & Glaasker, E. (1998). Regulation of compatible solute accumulation in bacteria. Mol Microbiol 29, 397407.[CrossRef][Medline]
Prentki, P., Binda, A. & Epstein, A. (1991). Plasmid vectors for selecting IS1-promoted deletions in cloned DNA: sequence analysis of the omega interposon. Gene 103, 1723.[CrossRef][Medline]
Rep, M., Krantz, M., Thevelein, J. M. & Hohmann, S. (2000). The transcriptional response of Saccharomyces cerevisiae to osmotic shock. Hot1p and Msn2p/Msn4p are required for the induction of subsets of high osmolarity glycerol pathway-dependent genes. J Biol Chem 275, 82908300.
Stanier, R. Y., Kunisawa, R., Mandel, M. & Cohen-Bazire, G. (1971). Purification and properties of unicellular blue-green algae (order Chroococcales). Bacteriol Rev 35, 171205.[Medline]
Strom, A. R. & Kaasen, I. (1993). Trehalose metabolism in Escherichia coli: stress protection and stress regulation of gene expression. Mol Microbiol 8, 205210.[Medline]
Tyerman, S. D., Bohnert, H. J., Maurel, C., Steudle, E. & Smith, J. A. C. (1999). Plant aquaporins: their molecular biology, biophysics and significance for plant water relations. J Exp Bot 50, 10551071.[Abstract]
Williams, J. G. K. (1988). Construction of specific mutations in photosystem II photosynthetic reaction center by genetic engineering methods in Synechocystis 6803. Methods Enzymol 167, 766778.
Wood, J. M. (1999). Osmosensing by bacteria: signals and membrane-based sensors. Microbiol Mol Biol Rev 63, 230262.
Yamaguchi, K., Suzuki, I., Yamamoto, H. & 7 other authors (2002). A two-component Mn2+-sensing system negatively regulates expression of the mntCAB operon in Synechocystis. Plant Cell 14, 29012913.
Zardoya, R. & Villalba, S. (2001). A phylogenetic framework for the aquaporin family in eukaryotes. J Mol Evol 52, 391404.[Medline]
Zhu, J. K. (2001). Cell signaling under salt, water and cold stresses. Curr Opin Plant Biol 4, 401406.[CrossRef][Medline]
Received 30 July 2004;
revised 13 October 2004;
accepted 18 October 2004.
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