RESEARCH LETTER
Proteome analysis at the subcellular level of the cyanobacterium
Spirulina platensis in response to low-temperature stress
conditions
Apiradee Hongsthong1, Matura Sirijuntarut2, Peerada Prommeenate1, Kanda Lertladaluck3, Kriengkrai
Porkaew3, Supapon Cheevadhanarak4 & Morakot Tanticharoen1
1
BEC Unit, National Center for Genetic Engineering and Biotechnology, Thakham, Bangkhuntien, Bangkok, Thailand; 2Pilot Plant Development and
Training Institute, Thakham, Bangkhuntien, Bangkok, Thailand; 3School of Information Technology and School of Bioresources and Technology; King
Mongkut’s University of Technology, Thakham, Bangkhuntien, Bangkok, Thailand; and 4School of Bioresources and Technology, Thakham,
Bangkhuntien, Bangkok, Thailand
Correspondence: Apiradee Hongsthong,
BEC Unit, National Center for Genetic
Engineering and Biotechnology, KMUTT, 83
Moo8, Thakham, Bangkhuntien, Bangkok
10150, Thailand. Tel.: 1662 470 7509;
fax: 1662 452 3455; e-mail:
[email protected]
Received 18 June 2008; accepted 8 August
2008.
First published online 1 September 2008.
DOI:10.1111/j.1574-6968.2008.01330.x
Editor: Karl Forchhammer
Keywords
proteome analysis; low-temperature stress;
S. platensis and differential expression
Abstract
The present study addresses the differential expression of Spirulina platensis
proteins detected during cold-induced stress, analyzed at the subcellular level. In
performing differential expression analysis, the results revealed upregulated
proteins in every subcellular fraction, including two-component response systems,
DNA repair, molecular chaperones, stress-induced proteins and proteins involved
in other biological processes such as secretion systems and nitrogen assimilation.
The chlorophyll biosynthetic proteins, protochlorophyllide oxidoreductase and
ChlI, had unique expression patterns as detected in the thylakoid membrane; the
levels of these proteins immediately decreased during the first 45 min of lowtemperature exposure. In contrast, their expression levels significantly increased
after low-temperature exposure, indicating the relevance of the chlorophyll
biosynthesis in Spirulina in response to low-temperature stress in the light
condition. In addition, this is the first report in which genome-based protein
identification in S. platensis by peptide mass fingerprinting was performed using
the database derived from the unpublished Spirulina genome sequence.
Introduction
Temperature reduction is an important environmental factor that leads to impaired protein biosynthesis, stabilization
of DNA and RNA secondary structures and, particularly, to
a reduction in membrane fluidity (Wang et al., 2005). It has
been well established in terms of membrane lipid composition that, in order to cope with a reduction in membrane
fluidity, unsaturated fatty acid levels in the membrane have
to increase to cause a phase transition of the plasma
membrane (Wang et al., 2005).
Upon temperature downshift, Spirulina plantensis can
synthesize up to 23% more g-linolenic acid (GLA;
C18:3D9,12,6) as compared with cells maintained at the ideal
growth temperature (Hongsthong et al., 2003). The elevation of unsaturated fatty acid levels in membrane lipids has
been shown to play a major role in the response to
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c
temperature change in various organisms. Substantial evidence points to the association between fatty acid desaturation and temperature stress (Wada & Murata, 1990); thus,
the expression of desaturase genes in cyanobacteria in
response to low temperature has been studied extensively
(Deshnium et al., 2000). However, transcriptome and proteome analyses of responses to several environmental
stresses, including temperature stresses, have not been
performed in Spirulina. This lack of relevant research might
be a result of the unavailability of a complete Spirulina
genome sequence. Therefore, we conducted a proteome
analysis in S. platensis to determine its response to a
temperature downshift at the subcellular level. This
study revealed the proteins involved in the biochemical
mechanisms through which these cyanobacterial cells
sense temperature changes and respond to a temperature
reduction.
FEMS Microbiol Lett 288 (2008) 92–101
93
Low-temperature stress response proteins of S. platensis
Materials and methods
Organisms and culture conditions
Axenic S. platensis strain C1 cultures were grown at 35 1C
under illumination by 100 mEm2 s1 fluorescent light with
continuous stirring in 2 L of Zarrouk’s medium (Richmond,
1986). The culture was grown to the mid-log phase until the
OD560 nm reached 0.4, at which time a cell sample was
harvested by filtration before shifting the growth temperature (t = 0 min). The growth temperature was then immediately shifted from 35 to 22 1C and the culture was incubated
for 45, 90 or 180 min. After harvesting, the cell pellets were
washed twice using 10 mM HEPES–NaOH (pH 7.0).
It is noteworthy that in terms of growth at different
temperatures, according to our previous data, the growth
determined by chlorophyll a level and biomass during the
first 24 h (one generation time of Spirulina) of the cells
grown at optimal temperature and the cells exposed to
immediate temperature downshift is very similar. However,
the specific growth rate (7-day experiment) of the cells
grown at optimal temperature is about 34% higher than that
of the cells exposed to immediate temperature downshift.
was labeled with fluorescent dyes according to the manufacturer’s instructions (GE Healthcare Biosciences). The 2DDIGE experiments, protein spot picking and in-gel digestion
of proteins were carried out as described previously
(Jeamton et al., 2008).
Protein profile analyses and protein
identification using matrix-assisted laser
desorption/ionization time-of-flight (MALDITOF) MS
In order to statistically analyze the differentially expressed
proteins following electrophoresis, protein profile analyses
were performed as described previously (Jeamton et al.,
2008). For protein identification, the resulting peptide mass
fingerprints (PMFs) were analyzed by in-house software
using the unpublished S. platensis C1 database, which was
generated from in silico digestion of the S. platensis C1
completed genome sequence. The search parameters and
data filtering were carried out as described previously
(Jeamton et al., 2008). The PMF identification results of a
spot had to be reproducible in order to consider that spot as
an identified protein.
Sample preparation
Results and discussion
The harvested cells were lysed using a French press at
700 psi, after which the soluble protein fraction was collected. The soluble fraction contained cytoplasmic and
periplasmic proteins. Subsequently, the two lipid membranes, the plasma and the thylakoid, were isolated on a
sucrose gradient as described by Murata & Omata (1988).
The purity of the TM and PM fractions was tested using
scanning absorption spectra (200–800 nm) and Western blot
analysis (see supporting Fig. S1). The TM fraction contained
chlorophyll a spectrum at 678 nm and a 40-kDa D12
desaturase protein band, whereas the chlorophyll a spectrum and the protein band were absent in the PM fraction.
The washed membrane pellet was then resuspended in
500 mL of dissolving buffer containing 2 M thiourea, 8 M
urea, 20 mM Tris, 30 mM dithiothreitol, 1% (v/v) immobilized pH gradient (IPG) buffer, 0.05% (w/v) b-dodecyl
maltoside and 4% (w/v) CHAPS. The proteins of all
fractions were precipitated using a 2D-clean up kit (GE
Healthcare Biosciences). The protein pellets were dissolved
in dissolving buffer without dithiothreitol. Next, the samples were analyzed for their protein concentration using a
2D-Quant kit protein assay (GE Healthcare Biosciences).
The 2D-DIGE (see supporting Figs S2 and S3) and MS
techniques were used in this study to identify the differentially expressed proteins at the subcellular level of Spirulina
under temperature downshift, from 35 to 22 1C conditions,
where many regulatory mechanisms involved in controlling
the expression of specific genes or the activation of their
products play critical roles in cell survival.
After the expression profile analyses and the differentially
expressed protein identification, 34, 15 and 24 upregulated
proteins were found in the PM, soluble and TM fractions,
respectively, whereas six downregulated proteins were identified in each of the three subcellular fractions. We observed
that low temperature mediated an induction and a reduction in the levels of proteins involved in some biological
processes that can be divided into several groups (Tables
1–4) as discussed below.
Two-dimensional differential gel
electrophoresis (2D-DIGE)
For 2D-DIGE, the pH of the protein samples was adjusted to
8.5 and 10 mg of each sample, prepared as mentioned above,
FEMS Microbiol Lett 288 (2008) 92–101
Signal transduction
The mechanisms by which organisms sense and acclimate to
environmental changes have been reported in various prokaryotes, including cyanobacteria (Panichkin et al., 2006).
The classical two-component system is composed of a
membrane-bound sensor histidine protein kinase and a
response regulator that mediates differential gene expression
(Mascher et al., 2006). Many studies using the DNA microarray techniques have revealed that histidine kinases, especially Hik33 in Synechocystis, play a critical role in the
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94
A. Hongsthong et al.
Table 1. Significantly upregulated proteins identified in the plasma membrane fraction
Theoretical
Spot no. ORF
Two-component systems
162 AP07670017
370 AP07670017
1382 AP07670017
2310 AP07580006
2334 AP04840005
3486 AP07670017
3988 AP07670017
Chaperones
2389 AP07920017
DNA repairing system
907 AP07800023
913 AP06960003
1760 AP07800023
2941 AP04480005
Stress-related proteins
708 AP06620002
859 AP07660015
1723 AP07990044
2236 AP07810025
2325 AP04360002
3555 AP05000001
4003 AP07640012
4585 AP06740013
Secretion systems
3536 AP07410017
Hypothetical proteins
322 AP07780003
474 AP07020011
511 AP07020011
902 AP06900027
989 AP06900027
1462 AP06620005
1688 AP07780003
2439 AP07860014
3224 AP03800003
Others
726 AP07050011
2228 AP04600003
2427 AP06530030V02
2827 AP06740016
Protein name
Two-component sensor histidine kinase
Two-component sensor histidine kinase
Two-component sensor histidine kinase
Putative two-component response regulator
Serine/threonine kinase with TPR repeat
Two-component sensor histidine kinase
Two-component sensor histidine kinase
Chaperonin GroEL
DNA polymerase III, d subunit
Putative exonuclease SbcC
DNA polymerase III, d subunit
DNA polymerase III, b subunit
Coverage pI
2.59
3.98
4.16
5.85
6.34
2.59
2.59
10.46
7.28
3.62
7.28
5.6
Glycosyl transferase domain containing protein
2.29
5-Methyltetrahydrofolate-homocysteine-methyltransferase 7.72
Putative ABC transporter
9.56
Glycosyl transferase, family 2
3.24
Pyruvate kinase
7.94
Transposase
11.92
Transposase
4.03
Ferredoxin-glutamate synthase
2.81
Type II secretory pathway, ATPase PulE
11.59
Conserved hypothetical protein
Conserved hypothetical protein
Conserved hypothetical protein
Conserved hypothetical protein sll1563
Conserved hypothetical protein sll1563
Hypothetical protein
Conserved hypothetical protein
Hypothetical protein
Hypothetical protein
7.03
2.6
2.6
3.41
3.97
6.52
6.17
6.17
4.81
Putative membrane protein
Phosphoglucomutase/phosphomannomutase
GlpX protein (Synechocystis sp. PCC 6803)
mrr restriction system protein
Experimental
5.35
16.74
8.12
15.69
4.88
4.88
4.88
5.5
8.37
4.88
4.88
5
pI
124.16
124.16
124.16
67.88
82.39
124.16
124.16
6.32 215.99 0.037
5.82
6.18 183.4 0.01
15.23
6.86 105.07 0.04
1.35
6.24 42.37 0.036 11.23
5.41 41.61 0.0012 2.15
6.3
14.52 0.049
4.81
4.92
8.02 0.042
8.73
58.19
7.07
8.74 33.84
6.3 121.62
8.74 33.84
4.83 42.75
41.3
0.024
5.7 144.96 0.014
3.22
4.54 127.86 0.048
3.86
7.06 77.54 0.0071 1.68
6.17 44.51 0.019 11.79
7.27 42.56 0.049
1.87
4.51 13.57 0.044
7.04
6.48
7.88 0.0042 2.99
6.16
5.39 0.045 30.26
39.12
4.47
4.82 68.18
5.46 249.83
5.46 249.83
5.75 99.66
5.75 99.66
6.57 128.59
4.82 68.18
9.32 28.32
9.04 64.54
5.67
5.59
5.61
5.29
4.99
7.87
4.73
5.71
6.06
7.7
5.63
5.25
4.95
6.31 131.94 0.025
6.79 47.41 0.011
5.76 37.62 0.048
7.37 25.31 0.047
83.94
48.77
37.12
34.53
1.31
5.19 123.92 0.03
3.12
6.15 122.81 0.0058 11.81
6.39 63.98 0.041
5.17
5
25.14 0.013
3.87
5.92 134.25
5.13 133.55
8.57 88.65
6.11 72.62463
5.85 63.4
10.3
21.46
10.1
58.82
5.6 169.95
5.89
MW
(kDa)
Fold
P-value change
MW
(kDa)
13.88 0.047
188.4
229.93
163.23
123.36
115.86
96.96
68.59
37.54
19.08
0.0099
0.017
0.016
0.035
0.01
0.0023
0.04
0.035
0.04
3.38
2.63
2.12
2.49
2.76
5.54
2.56
5.08
5.4
1.61
4.95
2.6
1.58
2.23
Fold change value represents volume ratio of after the temperature downshift (180 min)/before the temperature downshift. Volume ratio refers to the
ratio of the normalized volumes of a pair of spots (the same spot of before and after the temperature downshift), for example, a value of 2.0 represents
a twofold increase while 2.0 represents a twofold decrease.
perception and transduction of low-temperature signals
(Mikami et al., 2002). However, to the best of our knowledge, none of the studies on this system have been carried
out at the protein level in cyanobacteria subjected to lowtemperature stress.
Several proteins in the two-component signal transduction systems, signal transduction histidine kinase, Ser/Thr
protein kinase and response regulator including PAC/PAS
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domains (PAC, Period protein–Aryl hydrocarbon receptor
nuclear translocator protein–Single-minded protein; PAC,
PAS C-terminal) were found to be upregulated in the PM
and soluble fractions of Spirulina (Tables 1 and 2). It should
be pointed out that a smaller number of these proteins were
detected in the soluble fraction. This suggests that the
domains of these two-component systems that are located
in the cytoplasmic phase might be dissociated from other
FEMS Microbiol Lett 288 (2008) 92–101
95
Low-temperature stress response proteins of S. platensis
Table 2. Significantly upregulated proteins identified in the soluble protein fraction
Theoretical
Spot no.
ORF
Two component systems
282
AP06700002
384
AP07670017
592
AP07350018
1627
AP06700009
Transcription
1553
AP07880013
Stress-related proteins
361
AP08040046
378
AP07250004
509
AP07090018
1266
AP06740013
1481
AP07830017
1486
AP07770002
2657
AP05380002
Secretion systems
359
AP08010042
Hypothetical proteins
329
AP07020011
Others
3384
AP06030001
Experimental
Fold
Protein name
Coverage
pI
MW
(kDa)
pI
MW
(kDa)
P-value
change
WD-40 repeat protein
Two-component sensor histidine kinase
Hybrid sensor and regulator
Multisensor hybrid histidine kinase
2.99
2.59
2.76
10.65
5.26
4.88
5.16
5.46
183.1
124.16
157.04
166.47
5.26
5.53
6.69
5.07
233.35
211.67
175.79
56.49
0.048
0.075
0.12
0.03
2.12
1.92
2.55
2.13
DNA-directed RNA polymerase, b subunit
5.94
5.39
126.9
8.83
109.53
0.028
1.56
Sulfotransferase protein
Glycosyl transferase, family 2
SAM-dependent methyltransferases
Ferredoxin-glutamate synthase
Polyphosphate kinase
Transglutaminase-like enzyme
DEAD/DEAH box helicase domain protein
(membrane-helicase)
24.07
7.17
9.52
4.91
9.14
4.6
11.1
9.17
5.45
5.8
5.6
5.47
1.78
6.09
31.85
236.54
19.62
169.95
82.79
117.51
239.75
5.19
4.27
3.99
8.74
5.15
6.19
5.01
216.14
211.67
187.16
143.86
66.46
115.29
12.29
0.019
0.028
0.22
0.022
0.055
0.038
0.036
1.92
2.33
2.17
1.78
1.82
2.03
2.86
Type I secretion system ATPase, HlyB
4.12
5.99
110.47
6.44
216.64
0.059
3.34
Conserve with hypothetical protein
3.68
5.46
249.83
5.34
224.84
0.015
2.14
14.29
8.1
27.8
7.13
14.62
0.034
1.53
Acyl-(acyl-carrier-protein)-UDP-N-acetylglucosamine
O-acyltransferase
Fold change value represents volume ratio of after the temperature downshift (180 min)/before the temperature downshift. Volume ratio refers to the
ratio of the normalized volumes of a pair of spots (the same spot of before and after the temperature downshift), for example, a value of 2.0 represents
a twofold increase while 2.0 represents a twofold decrease.
domains during sample preparation. Taken together, the
results from this study and the Spirulina genome (unpublished) suggest three major points. First, these data are
consistent with the hypothesis that two-component transduction systems are involved in low-temperature signal
transduction mechanisms in Spirulina. Second, the components for low-temperature stress responses contain only
PAC/PAS domains as their sensory input domains, regardless of the presence of other sensory input domains (e.g.
CACHE, GAF, MCP, phytochrome and USP) (CACHE,
Ca21 channels and chemotaxis receptor; GAF, cGMP phosphodiesterase adenylate cyclase Fh1A; MCP, methyl-accepting chemotaxis protein; USP, universal stress protein) in the
Spirulina genome sequence. Finally, the two-component
systems in Spirulina that respond to low-temperature stress
should be categorized as periplasmic-sensing histidine kinases, according to Mascher et al. (2006). Moreover, it is
worth noting that the total number of genes encoded for
histidine kinases and response regulators in Spirulina genome is c. 95.
In addition to the proteins observed in the two-component signal transduction system, forkhead-associated ATPbinding cassette transporter (Table 1) was also significantly
FEMS Microbiol Lett 288 (2008) 92–101
increased in the PM fraction. This protein has an forkheadassociated (FHA) domain that can bind to the Ser/Thr
kinase and is thus important for phospho-dependent signaling pathways (Curry et al., 2005). This protein has also been
reported to take part in linear signaling pathways (Grundner
et al., 2005).
Chaperones
Upregulation of a molecular chaperone, GroEL, was found
in both membrane fractions after exposure to reduced
temperature, whereas DnaJ and ClpB were only detected in
the TM fraction (Table 3). The major molecular chaperones,
such as DnaK/DnaJ, GroES/GroEL and ClpB, have complementary functions in de novo protein folding of newly
synthesized polypeptides under various stress conditions,
including low-temperature stress (Ullers et al., 2007). Moreover, it has been reported that ClpB plays a crucial role in
cold acclimation in Synechococcus (Porankiewicz & Clarke,
1997), and is also defined as a low-temperature responsive
protein in Listeria sp. (Liu et al., 2002).
Drastic increases in the levels of DnaJ, GroEL and ClpB in
S. platensis C1 were detected in the membrane fractions,
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96
A. Hongsthong et al.
Table 3. Significantly upregulated proteins identified in the thylakoid membrane fraction
Theoretical
Spot no.
ORF
Photosynthesis related proteins
448
AP07570014
458
AP07570014
1696
AP07200014
2378
AP06360002
3080
AP06900013
Chaperones
206
AP04730007
1961
AP07710033
2313
AP08040017
3168
AP07870035
Stress-related proteins
202
AP04100005V02
405
AP06740013
459
AP07790010
1076
AP08040007
1184
AP07850015
2253
AP06510002
2581
AP04600003
3716
AP05790003
Channeling
2549
AP04170001
N-assimilation
2110
AP07870030
2111
AP07870030
Hypothetical proteins
2493
AP05200009
Others
1802
AP04960006
2023
2727
AP02580003
AP08030034
Experimental
Protein name
Coverage
pI
MW
(kDa)
pI
MW
(kDa)
P-value
Fold
change
Magnesium chelatase H subunit
Magnesium chelatase H subunit
Magnesium chelatase
Light-independent
protochlorophyllide-reductase subunit N
Uroporphyrinogen decarboxylase
8.52
11.61
4.23
7.71
5.06
5.06
4.95
5.21
148.28
148.28
71.74
52.6
6.85
6.65
6.76
6.69
176.35
176.35
80.34
51.88
0.0003
0.016
0.022
0.029
6.63
6.16
8.73
5.07
11.3
6
34.99
6.55
30.86
0.018
4.96
12.27
5.4
98.73
5.93
200.51
0.032
3.19
6.37
5.95
61.04
4.26
68.16
0.027
8.94
6.27
21.68
4.89
5.18
58.72
15.4
6.77
6.63
53.68
27.86
0.034
0.083
5.05
3.46
Transposase
Ferredoxin-glutamate synthase
L-Asparaginase
Putative transglutaminase-like enzymes
Glycosyltransferase
Radical_SAM domain containing protein
S-Adenosyl-L-homocysteine hydrolase
Cyanate ABC transporter
ATP-binding-component
7.73
3.57
12.1
4.49
14.29
19.05
15.85
13.15
9.92
5.6
4.92
9.16
8.52
5.85
5.63
6.16
42.89
169.95
33.29
88.82
35.95
42.19
48.77
32.31
4.34
6.78
6.73
4.5
6.94
6.16
6.24
6.26
200.51
182.1
175.99
119.27
109.41
50.46
36.48
16.08
0.013
0.022
0.0094
0.035
0.018
0.04
0.023
0.058
3.23
3.54
4.28
2.12
2.66
1.99
2.58
3.14
Arsenite-translocating ATPase
10.95
5.03
30.22
5.81
46.56
0.027
3.16
Nitrate reductase
Nitrate reductase
8.15
10.46
8.29
8.29
82.17
82.17
4.04
4.08
62.02
62.02
0.049
0.053
4.37
4.06
Hypothetical protein
11.52
7.16
63.2
6.95
48.08
0.0076
4.84
Putative sugar nucleotide
epimerase-dehydratase protein
Putative membrane-fusion protein
Predicted oxidoreductases
13.9
5
36.96
4.07
75.34
0.0054
6.41
13.61
9.2
5.08
5.22
55.22
38.85
4.24
6.52
65.08
40.14
0.0067
0.059
3.57
3.23
ATPases with chaperone activity,
ATP-binding subunit
Zn-dependent protease with
chaperone function
Chaperonin GroEL
Chaperone protein DnaJ
Fold change value represents volume ratio of after the temperature downshift (180 min)/before the temperature downshift. Volume ratio refers to the
ratio of the normalized volumes of a pair of spots (the same spot of before and after the temperature downshift), for example, a value of 2.0 represents
a twofold increase while 2.0 represents a twofold decrease.
although these proteins have generally been reported as
soluble proteins. Membrane-associated chaperones have
been found in many eukaryotes and prokaryotes, including
cyanobacteria. These chaperones have been proposed to be
involved in protein translocation mechanisms, translational
machinery associated with the surface of the thylakoid
membrane and enhancement of membrane fluidity in
response to heat shock by association with membrane lipids
(Katano et al., 2006). The physical state of the plasma
membrane, the first target of cellular damage during stress,
has been shown to be involved in the sensing and signaling
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of temperature stress (Los & Murata, 2000). Thus, these
membrane-associated chaperones could play a role in membrane stabilization and aid the refolding of those membrane
proteins under stress conditions (Coucheney et al., 2005).
To the best of our knowledge, this is the first time that
membrane-associated chaperones have been found in response to low-temperature stress. Therefore, the induction
of these chaperone levels in S. platensis C1 supports the
hypotheses that these proteins may function in retaining
proper protein conformation and aid in protein transport
across the membranes at a low temperature.
FEMS Microbiol Lett 288 (2008) 92–101
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Low-temperature stress response proteins of S. platensis
Table 4. Significantly downregulated proteins identified in the three subcellular fractions
Theoretical
Spot no. ORF
Protein name
Coverage pI
PM fraction
Channeling and secretion systems
2137 AP05710002 Sec-independent protein secretion pathway components
3754 AP05710002 Sec-independent protein secretion pathway components
4198 AP05710002 Sec-independent protein secretion pathway components
DNA damage
2224 AP06440006 Putative DNA modification methyltransferase
2768 AP05970009 DNA gyrase subunit A
Stress-related proteins
4438 AP07530034 Cyanoglobin
Soluble fraction
Secretion system
543 AP06100009 GTP-binding domain of ferrous iron transport protein B
1049 AP05960003 Preprotein translocase SecA subunit
Stress-related proteins
1174 AP07920028 Predicted ATPase of the PP-loop superfamilyimplicated in cell cycle control
Others
1275 AP06350002 Phosphoenolpyruvate synthase
1341 AP07770002 Uncharacterized protein conserved in bacteria
(Microbulbifer degradans 2–40)
1475 AP08030057 Phosphoenolpyruvate carboxylase
TM fraction
Chaperone
1675 AP08030020 Heat shock protein 90
Stress-related proteins
132 AP05090002 Ferredoxin-NADP reductase (FNR)
354 AP04300002 HEAT:Peptidase M1,
membrane alanine aminopeptidase:PBS lyase HEAT-like repeat
2518 AP07300010 (p)ppGpp synthetase I (GTP pyrophosphokinase), SpoT/RelA
Secretion system
2729 AP08010042 Type I secretion system ATPase, HlyB
3110 AP07680021 Putative HlyD family secretion protein
MW
(kDa)
Experimental
pI
MW
(kDa)
Fold
P-value change
20
20
20
5.03
5.03
5.03
8.37 6.06
8.37 6.72
8.37 5.71
48.79 0.014
10.49 0.042
6.78 0.04
3.59
3.84
5.17
5.04
10.49
6.33
5.47
45.09 6.62
36.21 6.52
45.72 0.044
28.69 0.005
10.89
10.11
22.92
5.76
11.09 6.65
5.85 0.006
2.28
13.06
7.43
6.49 24.35 4.82 181.18 0.013
5.14 105.74 5.23 107.97 0.009
1.79
2.26
11.05
7.67
39.21 4.97
95.69 0.039
2.06
7.4
2.87
6.04 90.27 5.22
5.29 117.51 4.79
85.6 0.005
78.19 0.011
1.57
2.03
6.67
5.95 117.68 7.26 117.46 0.035
1.82
5.6
4.97
80.82 0.006
3.06
6.34 45.14 4.18 209.55 0.045
5.63 101.01 5.12 184.68 0.013
1.57
1.74
7.66
47.03 0.04
2.19
39.9
26.3
2.8
2.26
5.74
10.55
7.96
13.18
5.6
75.81 5.74
77.28 6.86
5.99 110.47 5.36
5.05 55.27 5
0.006
0.001
Fold change value represents volume ratio of after the temperature downshift (180 min)/before the temperature downshift. Volume ratio refers to the
ratio of the normalized volumes of a pair of spots (the same spot of before and after the temperature downshift), for example, a value of 2.0 represents
a twofold increase while 2.0 represents a twofold decrease.
Some of the downregulated proteins cannot be visualized on the spot map shown in Fig. 1.
Proteins related to DNA modification and repair
Proteins involved in DNA repair, DNA polymerase III and
exonuclease (SbcC), were dramatically induced in the PM
fraction upon temperature downshift (Table 1). Under stress
conditions, where DNA damage possibly occurred, induction of DNA polymerase III, an error-prone DNA polymerase involved in DNA repair (Motlagh et al., 2006), is to be
expected. In the case of SbcC, this exonuclease removes
unusual DNA structure(s), such as hairpins, that are generated upon DNA damage (Darmon et al., 2007).
On the contrary, the significant reduction of DNA gyrase
could lead to the same effect as that of DNA gyrase
inhibition. The inhibition caused DNA damage, followed
FEMS Microbiol Lett 288 (2008) 92–101
by differential expression of DNA damage-inducible genes
(Dwyer et al., 2007). Moreover, the level of DNA modification methytransferase was also reduced. When the decrease
in the degree of DNA methylation occurred upon lowtemperature treatment, DNA replication ceased and alteration in gene expression controlled by temperature change
was observed (Steward et al., 2002).
Stress-related proteins
Our results demonstrate that the expression levels of many
stress-related proteins are significantly increased in the three
subcellular fractions in response to low-temperature stress
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c
98
(Tables 1–3). Transposase and methyltransferase were detected
in the PM and TM, and the PM and soluble fractions,
respectively. The former was discovered in response to osmotic
dehydration in nematodes (Chen et al., 2005). It is worth noting
that the upregulated transposase enzymes found in Spirulina
can be categorized into two families: IS605 and ISSoc7.
In spite of phosphorylation, glycosylation is a posttranslational modification that is very important in the cold
stress response (Komatsu et al., 2008). Therefore, it was
expected that glycosyl transferase was the only stress-related
protein detected in every subcellular fraction.
In addition to transposase and glycosyl transferase, four
other stress-related proteins were elevated in the TM fraction: transglutaminase, asparaginase, thiamine biosynthesis
enzyme and S-adenosyl homocysteine (SAH) hydrolase.
Transglutaminase and asparaginase have been reported to
be upregulated in response to different stimuli (Ientile et al.,
2007; Woo-Cho et al., 2007), and transglutaminase was also
detected in the soluble fraction of Spirulina. According to
Woo-Cho et al. (2007), asparaginase was induced at the
transcriptional level within 3 h after exposure to low-temperature stress in plants, corresponding to the results found
in Spirulina. Moreover, the induction of SAH hydrolase,
which catalyzes homocysteine production (Sun et al., 2004),
implies that homocysteine, an autoinducer in the quorumsensing mechanism required for controlling cell population
density (Sun et al., 2004), is probably essential under lowtemperature conditions in Spirulina.
In the soluble fraction, four proteins were induced, one of
which was the extensively studied DEAD/DEAH box helicase
induced by abiotic stress (Owttrim, 2006). This helicase
unwinds the cold-stabilized secondary structure in the 5 0
UTR during cold stress, resulting in an enhancement of target
mRNA translational efficiency and contributing to the removal of a metabolic block in translation initiation induced by
low-temperature stress (Yu & Owttrim, 2000). This ATPdependent helicase is also accumulated in the cyanobacterium
Anabaena grown under low temperature (o 25 1C) (Yu &
Owttrim, 2000). Furthermore, it is interesting that polyphosphate kinase (PPK) was detected in Spirulina among the
inducible proteins in the group of stress-related proteins.
Several research groups have reported on its relationship with
various stress conditions, and its role in membrane channel
formation and inhibition of RNA degradation (Manganelli,
2007). The enhancement of mRNA stability was demonstrated
to play a crucial role in the increasing level of fatty acid
biosynthesis gene transcripts in S. platensis C1 grown under
low-temperature conditions (Jeamton et al., 2008).
Channeling and secretion
Upregulation of proteins in this group was revealed in all
subcellular fractions of S. platensis (Tables 1–3). Type I, a
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c
A. Hongsthong et al.
Sec-independent secretion system, and type II, a Sec-dependent secretion system, were detected in the soluble and PM
fractions, respectively, whereas anion-transporting ATPase
was found in the TM fraction.
Types I and II secretion systems are commonly used by
Gram-negative bacteria to translocate proteins across the
membranes in the form of unfolded and folded proteins,
respectively (Hanekop et al., 2006; Johnson et al., 2006). The
finding of a type I secretion system in the soluble fraction
might be a result of protein dissociation during sample
preparation. The increasing levels of the secretion systems
suggest that secreted proteins possibly play roles in the
induction of low-temperature responses, in conjunction
with extracellular sensing components (ESCs) that are able
to detect temperature changes (Rowbury, 2005).
In the PM, where the Sec-dependent (type II) secretion
system was induced drastically, the Sec-independent secretion system was downregulated c. 3.6-fold after temperature
reduction. This result indicates that the Sec-dependent
secretion system induced in the PM might be unique to the
low-temperature response.
Photosynthesis-related proteins
Among the inducible proteins in the TM fraction, several
photosynthesis-related proteins were identified (Table 3).
These proteins, protochlorophyllide oxidoreductase (POR),
uroporphyrinogen decarboxylase (HemE) and magnesium
chelatase (ChlI), involved in chlorophyll biosynthesis of
various plant tissues, have been reported previously to be
reduced or stable in response to cold stress (Mohanty et al.,
2006). In Spirulina, the levels of POR, HemE and ChlI
increased significantly (approximately five-, five- and threefold, respectively), after 180 min of temperature downshift
as compared with those of the control bacteria grown at
35 1C. It is noteworthy, however, that the levels of POR and
ChlI decreased about twofold after 45 min of temperature
shift, and then kept increasing afterward (Fig. 1). These
results demonstrate that these proteins may play a role in the
tolerance of the chlorophyll biosynthesis in this cyanobacterium to low-temperature stress in the presence of light.
According to our previous work, when cells were exposed to
the temperature downshift in the dark, only POR was found
to be upregulated, albeit to a lesser extent (Jeamton et al.,
2008).
Conclusions
Taken together, the results suggest that when S. platensis cells
are exposed to a low temperature, the cold signal is
perceived through the two-component regulatory systems
found in the PM and soluble fractions. Only the PAC/PAS
sensory input domains of the two-component systems were
FEMS Microbiol Lett 288 (2008) 92–101
99
Low-temperature stress response proteins of S. platensis
Magnesium chelatase
1.6
1.4
1.2
1
TM 180 min
1
0.8
0.6
0.4
0.2
0
−0.2
TM 180 min
TM 90 min
TM 45 min
−0.4
TM 0 min
Standardized log abundance
1.2
detected in response to reduced temperature, suggesting an
association of this type of sensory input domain of the signal
transduction system with the low-temperature stress response. Following this association, an alteration occurs in
the expression of many genes and posttranslational modifications of many proteins. The evidence obtained in this
study indicates that the cellular response of Spirulina to low
temperature involves various processes. The processes related to DNA and RNA structures, as well as de novo protein
synthesis, are DNA damage, DNA repair, destabilization of
cold-stabilized RNA secondary structure to facilitate gene
expression under low temperatures and chaperone function
in de novo protein folding for newly synthesized
polypeptides.
Interestingly, two chlorophyll biosynthetic proteins, POR
and ChlI, detected in the TM have unique expression
patterns. When the cyanobacterial cells were initially exposed to a low temperature, these levels of these proteins
immediately decreased during the first 45 min, consistent
FEMS Microbiol Lett 288 (2008) 92–101
TM 90 min
Light-independent protochlorophyllide reductase
(b)
Fig. 1. Quantitative evaluation and regions of
2D gel images obtained from the thylakoid
membrane fraction before (0 min) and after
(45, 90 and 180 min) the temperature downshift:
(a) Magnesium chelatase (spot ID1696) and (b)
POR (spot ID2378). [A spot represents abundance
of a protein after normalization from an experiment. A line connects mean values of each
experimental group (0, 45, 90 and 180 min)].
TM 45 min
0.8
0.6
0.4
0.2
0
−0.2
TM 0 min
Standardized log abundance
(a)
with the report by Tewari & Tripathy (1998). On the
contrary, their expression levels increased dramatically after
45 min of low-temperature exposure, indicating the relevance of the greening process in Spirulina in response to
low-temperature stress in the presence of light.
The data obtained in this study are in part an attempt to
understand the changes of various cellular mechanisms in
response to temperature reduction, which is a suitable
condition for polyunsaturated fatty acid (PUFA) biosynthesis. This knowledge can be applied not only at the industrial
level of PUFA production but also in the future study of
various aspects of Spirulina.
Acknowledgements
This research was funded by a grant from the National
Center for Genetic Engineering and Biotechnology (BIOTEC),
Bangkok, Thailand.
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Published by Blackwell Publishing Ltd. All rights reserved
c
100
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Supporting Information
Additional Supporting Information may be found in the
online version of this article.
FEMS Microbiol Lett 288 (2008) 92–101
Fig. S1. A purity test of PM and TM by western blot
analysis using polyclonal antibody raised against SpirulinaD12 desaturase.
Fig. S2. Fluorescently labeled two dimensional gel maps
of S. platensis C1 cellular fractions using IPG-strips with a
pH range of 4–7 in the first dimension: (a) plasma membrane fraction, (b) soluble protein fraction, and (c) thylakoid membrane fraction.
Fig. S3. Fluorescently labeled two-dimensional gel
maps of S. platensis C1 cellular fractions using IPG-strips
with a pH range of 3–10 in the first dimension: (a) plasma
membrane fraction, (b) soluble protein fraction, and (c)
thylakoid membrane fraction.
Table S1. Ratio of matched peptide masses versus all
peptide masses obtained from MALDI-TOF mass spectrometry analysis of the proteins identified from the three
subcellular fractions.
Please note: Wiley-Blackwell is not responsible for the
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material) should be directed to the corresponding author
for the article.
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