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
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ABSTRACT |
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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.
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 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 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 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.
Cyanobacterial Strains and Culture Conditions--
The wild-type
and desA Examination of Membrane Rigidity by Fourier Transform Infrared
Spectrometry--
FTIR spectra were recorded at a spectral resolution
of 2 cm 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 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 [
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).
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 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
Table I shows the numerical results of
DNA microarray analysis for genes that were strongly induced by cold in
either wild-type or
desA
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
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 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 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 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.
Our investigations of cold-induced gene expression in the
desA
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
CONCLUSION
REFERENCES
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
(
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).
12 and
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.
symCH2
stretching mode at around 2852 cm
1. In the
symCH2 band, the contributions of the
trans and gauche segments of fatty-acyl chains
can be separated. Thus, the actual frequency of the
symCH2 stretching mode can be interpreted in terms of thermally induced changes in the dynamics of membrane lipids
and of protein-lipid interactions (13).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
CONCLUSION
REFERENCES
/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.
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.
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).
-32P]dCTP using a
BcaBEST labeling kit (Takara Shuzo).
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
CONCLUSION
REFERENCES
/desD
cells that
had been grown at 34 °C. Fig. 1 shows
the shift in the frequency of the
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
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
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
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.
, wild-type cells;
,
desA
/desD
cells.
/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.
/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.
Results of DNA microarray analysis of changes in the stress-induced
expression of genes due to rigidification of membrane lipids
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.
/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.
/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.
, 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.
/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).
/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.
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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.
Results of DNA microarray analysis of the heat- and
benzylalcohol-induced expression of genes in wild-type cells of
Synechocystis
CONCLUSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
CONCLUSION
REFERENCES
/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.
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FOOTNOTES |
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* 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
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ABBREVIATIONS |
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The abbreviations used are: FTIR, Fourier transform infrared; TES, 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid; ORF, open reading frame.
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