Osmotic shrinkage of cells of Synechocystis sp. PCC 6803 by water efflux via aquaporins regulates osmostress-inducible gene expression

Alexey Shapiguzov1, Alexander A. Lyukevich1,2, Suleyman I. Allakhverdiev2,3, Tatiana V. Sergeyenko1, Iwane Suzuki2,4, Norio Murata2,4 and Dmitry A. Los1

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


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Osmotic stress causes water molecules to efflux from cells through the cytoplasmic membrane. This study reveals that targeted mutation of the aqpZ gene, encoding an aquaporin water channel protein, in the cyanobacterium Synechocystis sp. PCC 6803 prevents the osmotic shrinkage of cells, suggesting that it is the water channel rather than the lipid bilayer that is primarily responsible for water transition through the membrane of this organism. The observations suggest that the aquaporin-mediated shrinkage of the Synechocystis cells plays an important role in changes of gene expression in response to hyperosmotic stress.


Abbreviations: EPR, electron paramagnetic resonance


   INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Water conductivity of cell membranes was formerly attributed to diffusion through the lipid bilayer, until the family of aquaporin channel proteins was discovered. Members of this family conduct bidirectional passive transport of water and some other compounds across membranes. Aquaporins are usually highly substrate-specific and impermeable to ions (Borgnia et al., 1999; Heymann & Engel, 1999, 2000; Johansson et al., 2000; Zardoya & Villalba, 2001). They have been found in all major groups of living organisms, in both prokaryotes and eukaryotes.

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 15–90 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.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Cells and culture conditions.
The wild-type strain of Synechocystis sp. PCC 6803 (a glucose-tolerant strain) was originally obtained from Dr J. G. K. Williams (Du Pont de Nemours & Company, Inc., Wilmington, DE, USA). Cells were grown at 32 °C under continuous illumination by incandescent lamps at 70 µE m–2 s–1 and aerated with air containing 2 % CO2 in BG11 medium (Stanier et al., 1971) buffered with 20 mM HEPES/NaOH pH 7·5. To provide the hyperosmotic stress, a solution of 5 M sorbitol in BG11 was added to the cell suspension to a final concentration of 0·5 M.

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. 1a). 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 ml–1.



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Fig. 1. (a) Construction of AqpZ-null mutant of Synechocystis. The aqpZ gene was disrupted by insertion of the SpR cassette into the Eco47III sites. Arrows indicate the primers for amplification of the corresponding region. (b) PCR with specific primers (indicated by arrows in a) to examine the extent of segregation of wild-type chromosomes. Lane 1, genomic DNA from wild-type cells; lane 2, genomic DNA from AqpZ-null cells; lane 3, DNA molecular mass markers.

 
Measurement of cytoplasmic volume.
The cytoplasmic volume was determined by electron paramagnetic resonance (EPR) spectrometry as described by Blumwald et al. (1983). For measurements, cells were harvested by centrifugation at 30 °C at 8000 g for 4 min and resuspended in fresh BG-11 medium at 400 µg chlorophyll ml–1. Cells were incubated for 30 min with shaking every 2–3 min under standard conditions, and then sorbitol was added at final concentration of 0·5 M. At designated times, aliquots of wild-type and AqpZ-deficient mutant cell suspensions were withdrawn. Cell volume was examined in a solution that contained 1·0 mM 2,2,6,6-tetramethyl-4-oxopiperidinooxy free radical (TEMPO; a spin probe), 20 mM K3[Fe(CN)6]3 and 75 mM Na2MnEDTA. Being oxidized by K3[Fe(CN)6]3, TEMPO rapidly passes through the plasma membrane and reaches equilibrium in all phases of the cell suspension. The addition of the paramagnetic quencher Na2MnEDTA, which cannot penetrate the plasma membrane, broadened the EPR signal from everywhere except the space bounded by the plasma membrane. The changes in cytoplasmic volume of cells were calculated based on the difference between the EPR spectra of sorbitol-treated and untreated cells. The suspension of cells was enclosed in a sealed glass capillary tube (i.d. 0·02 cm) in a final volume of 40 µl, and spectra were recorded at room temperature in an EPR spectrometer (model ESP 300E; Bruker). The EPR signal from a 40 µl capillary tube filled with 1·0 mM TEMPO alone was used as a blank control. It took approximately 2 min from sampling to measurement. Measurements were made in darkness under the following conditions: 100 kHz field modulation at a microwave frequency of 11·72 GHz, a modulation amplitude of 0·4 mT, microwave power of 10 mW, a time constant of 80 ms, and a scan rate of 0·4 G s–1. The cell volume that corresponded to 100 % was 0·31±0·08 fl.

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.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Changes in cytoplasmic volume
To investigate the physiological role of aquaporin in Synechocystis, the aqpZ gene was inactivated by targeted mutagenesis. PCR was conducted to confirm that all chromosomes of AqpZ-mutant cells had the disrupted copy of the gene (Fig. 1). The impacts of osmotic stress caused by the addition of 0·5 M sorbitol to the culture media were examined on wild-type and AqpZ-mutant cells.

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 5–10 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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Fig. 2. Changes in cell volume in the wild-type ({blacktriangleup}) and AqpZ-null cells ({bullet}) after addition of 0·5 M sorbitol, as measured by EPR.

 
These results agree with previous observations that the addition of mercuric compounds, known as aquaporin blockers (Pfeuffer et al., 1998; Tyerman et al., 1999), impaired the cytoplasmic volume decrease in Synechococcus cells.

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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Fig. 3. DNA microarray analysis of sorbitol stress-dependent gene expression. (a) Control experiment: RNA was isolated from wild-type and AqpZ-null cells of Synechocystis grown under standard conditions. Dashed lines indicate the limits of experimental deviation (twofold induction and twofold repression). (b) RNA isolated from wild-type cells that had been exposed to 0·5 M sorbitol for 15 min was compared with RNA from unstressed cells. (c) RNA isolated from AqpZ-null cells that had been exposed to 0·5 M sorbitol for 15 min was compared to RNA from the unstressed cells of the mutant.

 
We examined time-courses of gene expression (at the level of mRNA) in cells that were subjected to hyperosmotic stress for 15, 60 and 120 min. Several general scenarios of changes in gene expression were observed. The levels of mRNAs of certain genes showed a transient uplift at 15 min followed by a decrease. It is most likely that this is due to mRNA synthesis de novo, although the possibility of changes in mRNA stability cannot be ruled out. About 150 genes showed more than twofold rapid increase in mRNA levels in wild-type cells of Synechocystis (Table 1, Fig. 4).


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Table 1. Effect of mutation of the aqpZ gene on early hyperosmotic stress-induced gene expression

Genes that are induced more than twofold in the wild-type cells after 15 min hyperosmotic stress due to 0·5 M sorbitol are listed. IF, induction factor; SD, standard deviation. The total list of genes can be accessed at http://www.genome.ad.jp/kegg/expression/.

 


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Fig. 4. Dynamics of gene induction in response to hyperosmotic stress in wild-type ({circ}) and AqpZ-mutant ({bullet}) cells: examples of early-induced genes (a) and late-induced genes (b) that depend on aquaporin-mediated changes. The assays were repeated three times in independent experiments, with similar results.

 
The induction of many genes was significantly impaired in the mutant cells. This group of genes includes hspA, htpG (for chaperones), htrA, clpB, hhoA (for proteases), ggpS (for glucosylglycerolphosphate synthase), and many other genes including ones encoding proteins of unknown function (Table 1, Fig. 4a). Another group of early induced genes was slightly affected or not affected by the mutation. This group includes an rlpA homologue, sodB (for superoxide dismutase), crhR (for RNA-helicase), and some other genes (Table 1).

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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Table 2. Effect of mutation of the aqpZ gene on late hyperosmotic stress-induced gene expression

Genes that are induced more than threefold in the wild-type cells after 60 min hyperosmotic stress due to 0·5 M sorbitol are listed. IF, induction factor; SD, standard deviation. The total list of genes can be accessed at http://www.genome.ad.jp/kegg/expression/.

 
The induction observed in AqpZ-mutant cells was much less marked than that in wild-type cells, although the sets of induced genes were similar in both cell lines. Interestingly, for most of the genes demonstrating long-term induction, the difference between cell lines was not evident at the 15 min time point. The effect of the mutation on gene induction could only be registered after 60 min incubation in hyperosmotic conditions (Fig. 4b). This suggests that the expression of these genes was not directly influenced by the disruption of aqpZ. However, the induction of some genes, including ggpS encoding the osmoprotective enzyme (Engelbrecht et al., 1999), was markedly reduced by the mutation at both 15 min and 60 min time points.

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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Table 3. Genes whose repression (at 60 min) is affected by aqpZ disruption

Gene repression after 60 min hyperosmotic stress due to 0·5 M sorbitol is shown. RF, repression factor [1/(signal intensity)]; SD, standard deviation.

 


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Fig. 5. Dynamics of gene repression in response to hyperosmotic stress in wild-type ({circ}) and AqpZ-mutant ({bullet}) cells. The data are presented on a logarithmic scale. The assays were repeated three times in independent experiments, with similar results.

 

   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Hyperosmotic stress due to non-ionic solutes causes changes in gene expression in bacteria (Csonka, 1989; Poolman & Glaasker, 1998; Hecker & Volker, 2001), cyanobacteria (Kanesaki et al., 2002), yeast (Rep et al., 2000), and plants (Zhu, 2001). However, the molecular mechanisms of its sensing remain unclear. Various effects have been suggested to activate sensory systems that alter gene expression. These include alterations of hydraulic pressure (Csonka, 1989; Hoffmann & Dunham, 1995), cell volume (Wood, 1999), plasma membrane tension (Strom & Kaasen, 1993), concentrations of cytoplasmic solutes and/or activities of enzymes in the periplasmic space.

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.


   ACKNOWLEDGEMENTS
 
This work was supported in part by Grants-in-Aid for Scientific Research 13854002 and 14086207 (to N. M.), 14014257 and 14654169 (to I. S.) from the Ministry of Education, Science, Sports and Culture of Japan; and by a grant from the Russian Foundation for Basic Research (03-04-48581), a grant from the programme ‘Molecular and Cell Biology’, and a grant from the Russian Science Support Foundation to D. A. L.


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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Received 30 July 2004; revised 13 October 2004; accepted 18 October 2004.



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