Gene-engineered Rigidification of Membrane Lipids Enhances the Cold Inducibility of Gene Expression in Synechocystis*

Masami Inabaabc, Iwane Suzukiad, Balázs Szalontaie, Yu Kanesakia, Dmitry A. Losf, Hidenori Hayashibg, and Norio Murataadh

From the a Department of Regulation Biology, National Institute for Basic Biology, Myodaiji, Okazaki 444-8585, Japan, the b Satellite Venture Business Laboratory, Ehime University, Matsuyama 790-8577, Japan, the d Department of Molecular Biomechanics, School of Life Science, Graduate School for Advanced Studies, Myodaiji, Okazaki 444-8585, Japan, the e Institute of Biophysics, Biological Research Center of the Hungarian Academy of Sciences, P. O. Box 521, Szeged H-6701, Hungary, the f Institute of Plant Physiology, Russian Academy of Sciences, Botanicheskaya St. 35, Moscow 127276, Russia, and the g Department of Chemistry, Ehime University, Matsuyama 790-8577, Japan

Received for publication, December 2, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
CONCLUSION
REFERENCES

A sudden decrease in ambient temperature induces the expression of a number of genes in poikilothermic organisms. We report here that the cold inducibility of gene expression in Synechocystis sp. PCC 6803 was enhanced by the rigidification of membrane lipids that was engineered by disruption of genes for fatty acid desaturases. DNA microarray analysis revealed that cold-inducible genes could be divided into three groups according to the effects of the rigidification of membrane lipids. The first group included genes whose expression was not induced by cold in wild-type cells but became strongly cold-inducible upon rigidification of membrane lipids. This group included certain heat-shock genes, genes for subunits of the sulfate transport system, and the hik34 gene for a histidine kinase. The second group consisted of genes whose cold inducibility was moderately enhanced by the rigidification of membrane lipids. Most genes in this group encoded proteins of as yet unknown function. The third group consisted of genes whose cold inducibility was unaffected by the rigidification of membrane lipids. This group included genes for an RNA helicase and an RNA-binding protein. DNA microarray analysis also indicated that the rigidification of membrane lipids had no effect on the heat inducibility of gene expression. Hik33, a cold-sensing histidine kinase, regulated the expression of most genes in the second and third groups but of only a small number of genes in the first group, an observation that suggests that the cold-inducible expression of genes in the first group might be regulated by a cold sensor that remains to be identified.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
CONCLUSION
REFERENCES

Poikilothermic organisms respond to changes in ambient temperature by expressing specific sets of genes. Using DNA microarrays, we have demonstrated that a downward shift in temperature induces the expression of a large number of genes (1), which include the rbp1 gene for an RNA-binding protein (2) and the crhL gene for an RNA helicase (3), in Synechocystis sp. PCC 6803 (hereafter Synechocystis). These genes are known as cold-inducible genes. Cold shock also represses the expression of another set of genes, known as cold-repressible genes, which includes genes for subunits of phycobilisomes and photosystem I (1).

It has been suggested that the membrane rigidity might be involved in the sensing of low temperatures (4, 5). We demonstrated previously that the expression in Synechocystis of the desA gene, a cold-inducible gene that encodes the Delta 12 fatty acid desaturase, is induced by the rigidification of plasma membrane that results from the Pd-catalyzed hydrogenation of membrane lipids (6). We identified a membrane-bound histidine kinase, Hik33, as a cold sensor in Synechocystis (7), and we postulated that Hik33 might detect decreases in temperature by sensing the rigidification of membrane lipids. Moreover, mutation of the hik33 gene (Delta hik33) abolished the regulation of expression upon cold shock of many, but not all, of the cold-inducible and cold-repressible genes. These findings suggest that Synechocystis might have another cold sensor(s) that has not yet been identified (1).

We have produced a series of mutants of Synechocystis in which the extent of unsaturation of fatty acids is modified in a stepwise manner (8). In one of these mutants, the desA and desD genes, which encode the Delta 12 and Delta 6 fatty acid desaturases, respectively, are both inactive as a result of targeted mutagenesis. Cells of the desA-/desD- double mutant synthesize only a saturated C16 fatty acid and a mono-unsaturated C18 fatty acid, regardless of growth temperature, whereas wild-type cells synthesize di-unsaturated and tri-unsaturated C18 fatty acids in addition to the mono-unsaturated C18 fatty acid (8). Differential scanning calorimetry revealed that the replacement of polyunsaturated fatty acids by the mono-unsaturated fatty acid in desA-/desD- cells raised the temperature at which the phase separation of lipids is initiated in thylakoid membranes (8). However, in the cited studies, we did not examine effects of the double mutation of the desA and desD genes on the fluidity of membrane lipids at physiological temperatures.

Fourier transform infrared (FTIR)1 spectrometry is useful for studies of the organization of acyl lipids in both model membranes (9, 10) and biological membranes (11, 12). Lipid disorganization and changes in membrane dynamics are characterized by shifts in the frequency of the nu symCH2 stretching mode at around 2852 cm-1. In the nu symCH2 band, the contributions of the trans and gauche segments of fatty-acyl chains can be separated. Thus, the actual frequency of the nu symCH2 stretching mode can be interpreted in terms of thermally induced changes in the dynamics of membrane lipids and of protein-lipid interactions (13).

In the present study, FTIR spectrometry revealed that the double mutation of the desA and desD genes rigidified the plasma membrane of Synechocystis at physiological temperatures. We performed DNA microarray analysis to examine the effects of the membrane rigidification on the regulation of gene expression upon exposure of cells to cold and heat stress. We observed that the rigidification of membrane lipids enhanced the cold inducibility of a number of genes, whereas the rigidification did not affect the heat inducibility of gene expression. These findings suggest that the rigidification of membrane lipids might be a signal of cold perception that leads to the enhanced expression of certain genes.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
CONCLUSION
REFERENCES

Cyanobacterial Strains and Culture Conditions-- The wild-type and desA-/desD- strains of Synechocystis were obtained as described previously (8). The desA-/desD-/hik33- strain was generated by inactivating the hik33 gene in desA-/desD- cells as described previously (7). Cells were grown photoautotrophically at 34 °C under illumination from incandescent lamps at 70 µmol photons m-2 s-1 in BG-11 medium, with aeration by air that contained 1% CO2 (14). At the exponential phase of growth (when the optical density at 750 nm of the culture was 0.3), 50-ml aliquots were withdrawn and incubated under standard growth conditions, with the exception of the growth temperature or with the addition of benzylalcohol to a final concentration of 30 mM. The duration of incubation was 30 min for cold shock, 10 min for heat shock, and 20 min for benzylalcohol shock.

Examination of Membrane Rigidity by Fourier Transform Infrared Spectrometry-- FTIR spectra were recorded at a spectral resolution of 2 cm-1 with an FTIR spectrometer (Magna 560; Nicolet, Madison, WI) as described previously (15). Plasma membranes and thylakoid membranes were isolated from Synechocystis cells that had been grown at 34 °C as described previously (16). Membranes, suspended in 10 mM TES-NaOH (pH 7.0) that contained 10 mM NaCl, were collected by centrifugation at 500,000 × g for 20 min at 4 °C and then resuspended in the identical but D2O-based buffer. The difference of 0.4 pH units between pH and pD was taken into account. We analyzed FTIR spectra using the SPSERV© software package (version 3.20, BCS Software; Dr. Csaba Bagyinka, Institute of Biophysics, Biological Research Center of the Hungarian Academy of Sciences, Szeged, Hungary). The accuracy of determinations of frequencies of component bands was better than 0.1 cm-1 in the C-H stretching region.

Isolation of mRNA-- 50-ml aliquots of cell cultures, after incubation under designated conditions, were mixed, immediately after withdrawal from the main cultures, with an equal volume of ice-cold ethanol that contained 5% (w/v) phenol for rapid killing of cells and to prevent degradation of mRNA. After collection of the killed cells by centrifugation at 1000 × g for 5 min at 4 °C, total RNA was isolated by the hot-phenol method, as described previously (17). The RNA was treated with DNase I (Nippon Gene, Tokyo, Japan) to remove contaminating DNA.

Preparation of Fluorescent cDNA Probes for DNA Microarray Analysis-- The mRNA in each preparation of total RNA was reverse-transcribed in the presence of Cy3-dUTP or Cy5-dUTP (Amersham Biosciences, Uppsala, Sweden). Reverse transcription was performed in a 40-µl reaction mixture that contained 10 µg of total RNA, 50 mM Tris-HCl (pH 8.3), 10 mM KCl, 4 mM dithiothreitol, 10 mM MgCl2, 0.5 mM dATP, 0.5 mM dCTP, 0.5 mM dGTP, 0.2 mM dTTP, 300 pmol of random hexamers, 0.1 mM Cy3-dUTP or Cy5-dUTP, 100 units of RNase inhibitor, and 100 units of Moloney murine leukemia virus reverse transcriptase (Takara Shuzo, Tokyo, Japan). The reaction mixture was incubated at 42 °C for 1 h. Cy3- and Cy5-labeled cDNAs were purified on a CENTRI-SEP spin column (Princeton Separations, Adelphia, NJ) and concentrated, prior to hybridization, by precipitation in ethanol.

Hybridization to DNA Microarrays-- Synechocystis DNA microarrays (CyanoCHIP) were obtained from Takara Shuzo. The microarray covered 3079 genes (94% of the total genes, including 89 insertion sequences of transposons in Synechocystis) (18). The microarrays were incubated first at 65 °C for 1 h in a solution that contained 4× SSC (19), 0.2% SDS, 5× Denhardt's solution (19), and 100 ng µl-1 salmon sperm DNA. The microarrays were then rinsed once in 2× SSC and once in 0.2× SSC at room temperature. For hybridization, 25 µl of a mixture that contained Cy3- and Cy5-labeled cDNAs, 4× SSC, 0.2% SDS, 5× Denhardt's solution, and 100 ng µl-1 salmon sperm DNA were heated at 95 °C for 2 min and placed on each microarray. Then the microarrays were incubated overnight at 65 °C. After hybridization, microarrays were washed for 10 min in 2× SSC at 60 °C and for 10 min in 0.2× SSC plus 0.1% SDS at 60 °C. Finally, they were rinsed in 0.2× SSC at room temperature. Fluorescence from microarrays was examined with a microarray reader (GMS 418 Array Scanner; Affymetrix, Santa Clara, CA). Intensities of individual signals were determined with ImaGeneTM version 4.1 software (BioDiscovery, Los Angeles, CA).

Northern Blotting Analysis-- DNA fragments for use as probes in Northern blotting analysis of the expression of the hspA, dnaK2, crhL, and rbp1 genes were prepared by amplification of fragments that corresponded to the complete coding sequences of the individual genes by the polymerase chain reaction. These sequences were downloaded from the Cyanobase (available at www.kazusa.or.jp). The DNA probe for 16 S rRNA was obtained by excision of a 600-bp XbaI/KpnI fragment that included the gene for 16 S rRNA of Anabaena variabilis from plasmid pAN4 (kindly provided by Dr. A. Glatz, Institute of Biochemistry, Biological Research Center, Szeged, Hungary). Probes were labeled with [alpha -32P]dCTP using a BcaBEST labeling kit (Takara Shuzo).

Total RNA was subjected to electrophoresis in an agarose gel prepared with formaldehyde. Bands of RNA were transferred to a nylon membrane (Gene Screen Plus; PerkinElmer Life Sciences, Boston, MA) and allowed to hybridize with specific DNA probes. After hybridization, probes were removed and membranes were allowed to hybridize with the probe for 16 S rRNA (17). Results were quantified with an image analyzer (BAS Station 2000 Image Analyzer; Fuji Photo Film, Tokyo, Japan).

    RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
CONCLUSION
REFERENCES

Gene-engineered Rigidification of Membrane Lipids-- It has been proposed that inactivation of fatty acid desaturases in Synechocystis rigidifies the cell membranes (8). We employed FTIR spectrometry for direct measurements of the rigidity of plasma membranes from wild-type and desA-/desD- cells that had been grown at 34 °C. Fig. 1 shows the shift in the frequency of the nu symCH2 stretching mode as a function of temperature. The concordant data were calculated from FTIR spectra that had been recorded as the temperature was increased in steps of 2-3 °C. In FTIR spectra, higher frequencies represent higher proportions of gauche segments in fatty-acyl chains of membrane lipids, which might originate from the thermally induced disorder of fatty-acyl chains and/or from protein-lipid interactions (13). Because the ratio of proteins to lipids in thylakoid membranes and plasma membranes is unaffected by the inactivation of fatty acid desaturases (8, 15), the temperature-dependent changes in the nu symCH2 stretching vibrations of plasma membranes from wild-type and desA-/desD- cells, as shown in Fig. 1, should have corresponded, to a good approximation, to changes in the disorder of fatty-acyl chains in the membrane lipids. A lower frequency of the nu symCH2 stretching mode at a given temperature should correspond to a more ordered (rigidified) state. Thus, our results indicate that the plasma membranes from desA-/desD- cells were more rigid than those from wild-type cells, in particular at 22 °C, the temperature at which we examined the expression of cold-regulated genes (see below). This rigidification of plasma membranes by the mutation of fatty acid desaturases reflected the lack of polyunsaturated fatty-acyl chains in desA-/desD- cells. The difference between wild-type and mutant cells was greater at lower temperatures and was smaller at higher temperatures. Our FTIR analysis indicated that lipids in plasma membranes were rigidified with decreases in temperature and that the rigidification was enhanced by the saturation of fatty acids that occurred after the inactivation of fatty acid desaturases.


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Fig. 1.   FTIR analysis of the fluidity of lipids in plasma membranes from wild-type and desA-/desD- cells of Synechocystis. Plasma membranes were prepared from wild-type and desA-/desD- cells of Synechocystis that had been grown at 34 °C. Higher values for nu symCH2 frequencies correspond more disordered structures of fatty-acyl chains of membrane lipids. The vertical solid line indicates the growth temperature (34 °C), and vertical dashed lines represent the cold-shock temperature (22 °C) and the heat-shock temperature (42 °C), as indicated. open circle , wild-type cells; , desA-/desD- cells.

Modulation of Cold-responsive Expression of Genes by Membrane Rigidification-- We used DNA microarrays to examine the effects of membrane rigidification on the expression of genes in response to cold in Synechocystis. Fig. 2 shows the changes in gene expression in wild-type and desA-/desD- cells after incubation at 22 °C for 30 min that followed growth at 34 °C. Data points above the upper reference line and below the lower reference line correspond, respectively, to genes whose expression was significantly enhanced or repressed by cold. In wild-type cells, cold shock enhanced the expression of 168 genes and repressed that of 89 genes (Fig. 2A). In desA-/desD- cells, in addition to the same set of 168 cold-inducible genes, 96 additional genes appeared above the upper reference line (Fig. 2B). The results indicate that the rigidification of membrane lipids enhanced the response of gene expression to cold in Synechocystis. The expression of the cold-repressible genes was not significantly affected by the double mutation of the desA and desD genes. Under isothermal conditions, the double mutation had no significant effect on gene expression (data not shown).


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Fig. 2.   DNA microarray analysis of the cold-induced expression of genes in wild-type and desA-/desD- cells. A, wild-type (WT) cells that had been grown at 34 °C (control cells) were compared with wild-type cells that had been grown at 34 °C and then incubated at 22 °C for 30 min (treated cells). B, desA-/desD- (A-D-) cells that had been grown at 34 °C (control cells) were compared with desA-/desD- cells that had been grown at 34 °C and then incubated at 22 °C for 30 min (treated cells). RNA extracted from control cells and treated cells was used for synthesis of Cy5- and Cy3-labeled cDNAs, respectively. The Cy5- and Cy3-labeled cDNAs were mixed and applied to DNA microarrays. Dashed lines correspond to reference lines that indicate the limits of experimental deviations. Red circles correspond to genes whose expression was induced by cold in wild-type cells. Blue circles correspond to genes whose expression was induced by cold only in desA-/desD- cells.

Table I shows the numerical results of DNA microarray analysis for genes that were strongly induced by cold in either wild-type or desA-/desD- cells (namely, when the ratio of levels of expression in treated cells and control cells was greater than three). These cold-inducible genes could be divided into three groups according to the effects of the double mutation. The first group consisted of genes that were not induced by cold in wild-type cells but were strongly cold-inducible in desA-/desD- cells. These genes included certain heat-shock genes, such as hspA, dnaK2, and clpB2; the sbpA, cysA, cysT, and cysW genes for the sulfate-transport system; the sigB gene for an RNA polymerase sigma factor; the hik34 gene for a histidine kinase; and the pbp gene for a putative penicillin-binding protein. The second group consisted of genes whose cold inducibility was moderately enhanced by the double mutation, and it included hli genes for high light-inducible proteins and several genes for proteins of as yet unknown function. The third group consisted of genes whose cold inducibility was unaffected by the double mutation and included genes known as cold-shock genes, such as rbp1 (2) and crhL (3). Our results suggest that the cold-responsive expression of the genes in these three groups might be regulated by different mechanisms with respect to membrane rigidity.


                              
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Table I
Results of DNA microarray analysis of changes in the stress-induced expression of genes due to rigidification of membrane lipids
Cells that had been grown at 34 °C under standard growth conditions were incubated at 22 °C for 30 min or at 42 °C for 10 min. Each value indicates the ratio of the level of expression in treated cells to that in control cells. Values shown are averages and ranges of deviations of results of two independent experiments. WT, wild-type cells; A-D-, desA-/desD- cells; A-D-33; desA-/desD-/hik33- cells. Entire results obtained by the microarray are available at www.nibb.ac.jp/%7Ecelres/arraydata.

The induction of genes in the first group might require a higher degree of rigidification of membrane lipids than the cold responses of genes in the second and third groups. The cold induction of heat-shock proteins has been observed in wild-type cells of another strain of cyanobacterium, Synechococcus sp. PCC 7942 (hereafter Synechococcus). Porankiewicz and Clarke (20) demonstrated that, at low temperatures, Synechococcus synthesizes a homolog of ClpB, a member of the Clp/HSP100 family of heat-shock proteins. Synechococcus synthesizes only saturated and mono-unsaturated fatty acids but no polyunsaturated fatty acids (21), resembling the desA-/desD- mutant of Synechocystis. It is likely, therefore, that the cold inducibility of certain heat-shock genes is related to a lack of polyunsaturated fatty acids in membrane lipids. The rigidification of membrane lipids did not enhance the cold inducibility of genes in the third group, perhaps because the membrane rigidity in wild-type cells is sufficiently high at low temperatures for the maximum induction of these genes.

To verify the results in Fig. 2 and Table I, we examined the levels of expression of the hspA, dnaK2, rbp1, and crhL genes by Northern blot analysis (Figs. 3 and 4). The expression of the hspA and dnaK2 genes was induced by cold in desA-/desD- cells, with maximum induction at 24 °C, and such induction was not observed in wild-type cells (Fig. 3). These results are consistent with the results of microarray analysis. However, the difference between the two types of cell was more apparent after Northern blot analysis than after microarray analysis. By contrast, the cold inducibility of the rbp1 and crhL genes was unaffected by the double mutation (Fig. 4). Maximum induction of the rbp1 gene was observed at 25 °C, whereas that of the crhL gene was observed at ~20 °C. The reason for the difference between the rbp1 and crhL genes in terms of the temperature for maximum induction is unclear.


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Fig. 3.   Northern blotting analysis of the cold-induced expression of hspA and dnaK2 genes in wild-type (WT) and desA-/desD- (A-D-) cells. Cells that had been grown at 34 °C were incubated at indicated temperatures for 30 min. A, expression of the hspA gene. B, expression of the dnaK2 gene. Signals obtained after hybridization with the hspA and dnaK2 probes were normalized by reference to results for 16 S RNA. Upper panels, Northern blots; lower panels, quantification of results on Northern blots. open circle , wild-type cells; , desA-/desD- cells. Similar results were obtained in four independent experiments, and the figure shows the results of one of these experiments.


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Fig. 4.   Northern blotting analysis of the cold-induced expression of the rbp1 and crhL genes in wild-type (WT) and desA-/desD- (A-D-) cells. Cells that had been grown at 34 °C were incubated at indicated temperatures for 30 min. A, expression of the rbp1 gene. B, expression of the crhL gene. Signals obtained after hybridization with the rbp1 and crhL probes were normalized by reference to results for 16 S RNA. See legend to Fig. 3 for other details.

Involvement of Hik33 and Other Cold Sensors in the Regulation of Cold-responsive Gene Expression-- In a previous study, we identified a membrane-bound histidine kinase, Hik33, as a cold sensor in Synechocystis (1). The structure of this protein, predicted from its amino acid sequence, suggests that Hik33 might span the plasma membrane twice and form a homodimer (1). The dimeric structure might be influenced by the physical properties of the membrane, such as rigidity, that are regulated by temperature and the extent of unsaturation of fatty acids. Therefore, we postulated that Hik33 might detect decreases in temperature by sensing the rigidification of membrane lipids.

To examine whether Hik33 might regulate the cold-responsive gene expression that depends on membrane rigidity, we used DNA microarrays to examine gene expression in desA-/desD-/hik33- cells. The results are shown in Table I. The mutation of Hik33 abolished or reduced the cold inducibility of 10 of the 17 genes in the second group and of 7 of 25 genes in the third group. By contrast, the mutation did not significantly affect the cold inducibility of genes in the first group, and only two genes, hspA and cysA, among the 15 genes, were affected. These results indicate that Hik33 regulates the expression of many genes in the second and third group, whereas the expression of very few genes in the first group is under the control of Hik33. These results also suggest that the activity of Hik33 is moderately sensitive to membrane rigidity and that one or more as yet unidentified cold sensors exists that are more sensitive to membrane rigidity. Consistent with these observations, our previous study demonstrated that mutation of the hik33 gene (hik33-) abolished the regulation of expression by cold of many, but not all, of the cold-inducible and cold-repressible genes, suggesting the presence of one or more other cold sensors (7).

Membrane Rigidification Does Not Affect Heat-responsive Gene Expression-- It has been postulated that the fluidity of membrane lipids might be involved in the perception of heat stress (22). In Saccharomyces cerevisiae, the heat-induced expression of some heat-shock genes depends on the extent of saturation or unsaturation of membrane lipids, suggesting that the perception of increases in temperature in yeast might also involve changes in membrane fluidity (23). To examine this hypothesis in Synechocystis, we investigated the effects of membrane rigidification, caused by the double mutation of the desA and desD genes, on the regulation of gene expression upon heat shock (Table I). DNA microarray analysis revealed that heat shock at 42 °C for 10 min induced the expression of a large number of genes in wild-type Synechocystis cells. These heat-inducible genes were also induced in desA-/desD- cells to a similar extent. When we examined, in addition, the heat-inducible expression of genes that are not listed in Table I, we found that the double mutation of the desA and desD genes had no effect on the heat inducibility (data not shown). Thus, it is likely that, in Synechocystis at least, membrane rigidity is not involved in the regulation of gene expression in response to heat stress and, thus, in the perception of such stress.

Effects of Heat and Benzylalcohol on Gene Expression-- Horváth et al. (24) reported that the heat inducibility of the hspA gene and other heat-shock genes in Synechocystis was enhanced in the presence of 30 mM benzylalcohol. They explained this observation as being the result of the enhanced fluidity of membranes and postulated that heat shock was also perceived via the fluidization of membranes. To evaluate this hypothesis, we compared the effects of a temperature shift from 34 °C to 42 °C with the effects of exposure to 30 mM benzylalcohol on gene expression in wild-type cells of Synechocystis. Fig. 5 shows that heat shock and incubation with benzylalcohol enhanced the expression of a large number of genes: expression of 119 genes was enhanced by heat shock and that of 177 genes was enhanced by the incubation with benzylalcohol. Moreover, 53 genes were induced by both kinds of stress. Table II lists the genes for which the ratio between the levels of expression in either heat-treated or benzylalcohol-treated cells to that in control cells was higher than four. The group of genes that were induced by heat shock, but not by incubation with benzylalcohol, included the clpB2 gene for a heat-shock protease and the ama gene for N-acyl-L-amino acid amidohydrolase. The group of genes that was induced by benzylalcohol, but not by heat shock, included the ndhD2 gene for a subunit of NADH dehydrogenase, the gifA and gifB genes for glutamine synthetase-inactiviting factors, and the hliA gene for a high light-inducible protein. The group of genes that was induced both by heat shock and by benzylalcohol included most of the so-called heat-shock genes, such as hspA, clpB1, htpG, dnaJ, dnak2, groEL2, and groESL, as well as the sigB gene for a sigma factor and the sodB gene for superoxide dismutase. These results indicate that heat shock and benzylalcohol induced different sets of genes, suggesting that Synechocystis might perceive these kinds of stress as different signals. Our observations suggest, moreover, that the changes in heat inducibility of heat-shock genes that Horváth et al. (24) observed might have been a result simply of the additive effects of heat and benzylalcohol and might not have reflected a cooperative effect of these signals that occurred as a consequence of fluidization of membrane lipids. Therefore, it seems unlikely that membrane fluidity is involved in the perception and transduction of the heat signal.


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Fig. 5.   DNA microarray analysis of the heat-induced and benzylalcohol-induced expression of genes in wild-type cells. Wild-type (WT) cells that had been grown at 34 °C (control cells) were compared with wild-type cells that had been grown at 34 °C and then incubated at 42 °C for 10 min (A) or at 34 °C for 20 min in the presence of 30 mM benzylalcohol (B). RNA extracted from control cells and from treated cells was used for synthesis of Cy5-labeled cDNAs and Cy3-labeled cDNAs, respectively. The Cy5- and Cy3-labeled cDNAs were mixed and applied to DNA microarrays. Dashed lines correspond to reference lines that indicate the limits of experimental deviations. The red circles correspond to genes whose expression was induced by heat shock, blue circles correspond to genes whose expression was induced by benzylalcohol, and yellow circles correspond to genes whose expression was induced by both kinds of stress.


                              
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Table II
Results of DNA microarray analysis of the heat- and benzylalcohol-induced expression of genes in wild-type cells of Synechocystis
Cells that had been grown at 34 °C under standard growth conditions were incubated at 42 °C for 10 min or at 34 °C in the presence of 30 mM benzylalcohol for 20 min. Values shown are averages and ranges of deviations of results of two independent experiments.


    CONCLUSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
CONCLUSION
REFERENCES

Our investigations of cold-induced gene expression in the desA-/desD- mutant of Synechocystis indicate that this microorganism experiences a decrease in membrane fluidity upon a downward shift in temperature and that transduction of this signal leads to the regulation of gene expression. By contrast, heat shock might be perceived by mechanisms that do not include a signal that results from a change in the fluidity of membrane lipids.

    FOOTNOTES

* This work was supported by grants-in-aid for scientific research (S; 13854002) and for scientific research on priority areas (14086207) (to N. M.) and grants-in-aid for scientific research on priority areas (C, Genome Biology; 13206081) and for Exploratory Research (14654169) (to I. S.) from the Ministry of Education, Science, Sports and Culture of Japan and by the Hungarian Scientific Research Foundation (Grant TO31973) (to B. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

c Present address: Botanisches Institut, Universität München, Menzingerstrasse 67, München D-80638, Germany.

h To whom correspondence should be addressed. Tel.: 81-564-55-7600; Fax: 81-564-54-4866; E-mail: murata@nibb.ac.jp.

Published, JBC Papers in Press, December 26, 2002, DOI 10.1074/jbc.M212204200

    ABBREVIATIONS

The abbreviations used are: FTIR, Fourier transform infrared; TES, 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid; ORF, open reading frame.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
CONCLUSION
REFERENCES

1. Suzuki, I., Kanesaki, Y., Mikami, K., Kanehisa, M., and Murata, N. (2001) Mol. Microbiol. 40, 235-244[CrossRef][Medline] [Order article via Infotrieve]
2. Sato, N. (1995) Nucleic Acids Res. 23, 2161-2167[Abstract]
3. Chamot, D., Magee, W. C., Yu, E., and Owttrim, G. W. (1999) J. Bacteriol. 181, 1728-1732[Abstract/Free Full Text]
4. Murata, N., and Los, D. A. (1997) Plant Physiol. 115, 875-879[Free Full Text]
5. Los, D. A., and Murata, N. (2000) Science's STKE http://stke/sciencemag.org/cig/content/full/oc_sigtrans:2000/62/pe1.
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