- Email: [email protected]
Low-temperature-induced desaturation of fatty acids and expression of desaturase genes in the cyanobacterium Synechococcus sp. PCC 7002
FEMS Microbiology Letters 152 (1997) 313^320
Low-temperature-induced desaturation of fatty acids and expression of desaturase genes in the cyanobacterium Synechococcus sp. PCC 7002 Toshio Sakamoto a , Shoichi Higashi b , Hajime Wada Donald A. Bryant a *
1;b
, Norio Murata b ,
;
a
S-234 Frear Bldg., Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA 16802, USA
b
Department of Regulation Biology, National Institute for Basic Biology, Myodaiji, Okazaki 444, Japan
Received 19 February 1997 ; revised 22 April 1997; accepted 23 April 1997
Abstract
Changes in response to temperature of lipid classes, fatty acid composition and mRNA levels for acyl-lipid desaturase genes were studied in the marine unicellular cyanobacterium, Synechococcus sp. PCC 7002. The degree of unsaturation of C18 fatty acids increased in cells grown at lower temperature for all lipid classes, and g3 desaturation occurred specifically in cells grown at low temperature. While the level of 18:1(9) fatty acids declined, desaturation at the g3 position of C18 fatty acids increased gradually during a 12-h period after a temperature shift-down to 22³C. However, the mRNA levels of the desA (v12 desaturase), desB (g3 desaturase) and desC (v9 desaturase) genes increased within 15 min after a temperature shift-down to 22³C; the desaturase gene mRNA levels also rapidly declined within 15 min after a temperature shift-up to 38³C. Therefore, the elevation of mRNA levels for the desaturase genes is not the rate-limiting event for the increased desaturation of membrane lipids after a temperature shift-down. The rapid, low-temperature-induced changes in mRNA levels occurred even when cells were grown under light-limiting conditions for which the growth rates at 22³C and 38³C were identical. These studies indicate that the ambient growth temperature, and not some other growth rate-related process, regulates the expression of acyl lipid desaturation in this cyanobacterium. Keywords :
Cyanobacterium; Gene regulation; Fatty acid desaturase; Homeoviscous adaptation; Low temperature acclimatization
* Corresponding author. Fax: +1 (814) 863 7024; E-mail: [email protected]
1. Introduction
1
Present address: Department of Biology, Faculty of Science, Kyushu University, Ropponmatsu, Fukuoka 810, Japan. DGDG, digalactosyl diacylglycerol; MGDG, monogalactosyl diacylglycerol ; PG, phosphatidylglycerol ; SQDG, sulfoquinovosyl diacylglycerol; Fatty acids are represented by `X:Y(Z)', containing X carbon atoms with Y double bonds at the position Z counted from the carboxyl terminus; 14:0, myristic acid; 16:0, palmitic acid; 16:1(9), palmitoleic acid; 18:0, stearic acid; 18:1(9), oleic acid; 18:2(9, 12), linoleic acid; 18:3(9, 12, 15), K-linolenic acid
Abbreviations :
Cyanobacteria are classi¢ed as Gram-negative bacteria, and their cell envelopes are composed of an outer membrane and a plasma (inner) membrane, which are separated by the peptidoglycan layer [1]. Nearly all cyanobacteria also possess thylakoid membranes, which are intracytoplasmic membranes that house the photosynthetic apparatus. Cyanobacterial lipids are only found in their membranes [2],
0378-1097 / 97 / $17.00 ß 1997 Federation of European Microbiological Societies. Published by Elsevier Science B.V. PII S 0 3 7 8 - 1 0 9 7 ( 9 7 ) 0 0 2 1 7 - 6
FEMSLE 7645 27-8-97
314
T. Sakamoto et al. / FEMS Microbiology Letters 152 (1997) 313^320
and the organization of the cyanobacterial membranes [1], as well as the composition of their lipids and fatty acids [2], is similar to that of the plant chloroplasts. Changes in the fatty acid composition of the membrane lipids of cyanobacterial cells to changes in the ambient growth temperature [3^6] have been regarded as an adaptive response [2,7], known as homeoviscous adaptation [8]. Cyanobacterial strains have been classi¢ed into four groups based on their patterns of fatty acid desaturation [9]. Preliminary studies of fatty acid composition have suggested that the marine, unicellular cyanobacterium Synechococcus sp. PCC 7002 should be assigned to group 2 [9]. Consistent with this assignment, three acyl lipid desaturase genes (desA, v12 desaturase; desB, g3 or v15 desaturase; and desC, v9 desaturase), encoding the enzymes required for the conversion of the C18 fatty acid stearate to K-linolenic acid, were cloned from Synechococcus sp. PCC 7002 [10,11]. An advantage of studying the temperature dependence of desaturase gene expression in cyanobacteria is that the growth rate at a given temperature can be independently regulated by the light intensity at which the cells are grown. This potentially allows e¡ects dependent upon growth rate to be distin-
guished from e¡ects directly dependent upon the growth temperature. To establish the direct in£uence of temperature on the expression of three acyl lipid desaturase genes and fatty acid desaturation, the expression patterns of these genes as a function of temperature were examined using cells of the unicellular marine cyanobacterium Synechococcus sp. PCC 7002 grown at a growth-rate-limiting light intensity (50 WE m32 s31 ), under which conditions the cells grew at an identical rate at 22³C and 38³C. Changes of lipid classes and fatty acid composition in response to changes in ambient temperature, as well as the time course for changes in the latter, are also presented. 2. Material and methods
A laboratory wild-type strain of Synechococcus sp. PCC 7002 (denoted strain NIBB) was originally obtained from the Pasteur Culture Collection and maintained in the National Institute for Basic Biology, Okazaki, Japan. A second laboratory wild-type strain (denoted strain PR6000) of Synechococcus sp. PCC 7002 was obtained from Dr. S. Edward Stevens, Jr. and maintained independently at The Pennsylvania State University. Cells were grown photo-
Table 1 Fatty acid composition of lipid classes from Synechococcus sp. PCC 7002 (strain NIBB)a Lipid class Amount (Mol%) Fatty acid (Mol%) 14:0 16:0 16:1(9) 18:0 18:1(9) 18:2(9, 12) 18:3(9, 12, 15) Total 34³C 1 40 16 1 23 18 tb 22³C 1 35 19 t 10 25 10 MGDG 34³C 55 t 35 20 t 24 20 1 22³C 57 t 23 29 t 7 29 12 DGDG 34³C 12 t 30 26 1 14 27 2 22³C 9 1 34 23 t 10 19 13 SQDG 34³C 17 t 65 7 1 21 5 t 22³C 11 t 60 6 t 18 10 5 PG 34³C 16 t 54 3 1 27 15 t 22³C 23 t 50 3 1 11 27 8 The values represent the averages of two experiments. The deviation of the values was within þ 2%. a Wild-type cells of Synechococcus sp. PCC 7002 (strain NIBB) were grown under continuous illumination (70 WE m32 s31 ) at 34³C or 22³C. b t: trace amount, less than 0.5%
FEMSLE 7645 27-8-97
T. Sakamoto et al. / FEMS Microbiology Letters 152 (1997) 313^320
315
described [5]. In the experiments presented in Table 3, the lyophilized cell pellets were directly methanolyzed with 5% (w/w) HCl in methanol, and the resultant methylesters extracted with n-hexane were analyzed by a gas-liquid chromatograph. Total RNA of 5 Wg was separated on formaldehyde-agarose gels, and transferred onto nylon membrane ¢lters. The amount of RNA on the blot was normalized to the intensity of the ribosomal RNA bands for the lane, which was obtained from the ethidium bromide stained gel. RNA blots was hybridized with radiolabeled probes speci¢c for the desA (v12 desaturase), desB (g3 or v15 desaturase) and desC (v9 desaturase) genes [11]. 3. Results
3.1. Changes in lipids in response to growth temperature in Synechococcus sp. PCC7002 (strain NIBB)
Fig. 1. Changes in fatty acid composition of the total lipids after a temperature shift-down from 34³C to 22³C. Wild-type cells of Synechococcus sp. PCC 7002 (strain NIBB) grown at 34³C in medium A under continuous illumination of 70 WE m32 s31 were transferred at 22³C with equivalent illumination. The values are the means obtained in two experiments. The deviation of values was within þ 2%.
autotrophically in medium A supplemented with 1 mg ml31 NaNO3 [12] with aeration of 1% (v/v) CO2 in air under constant illumination from incandescent lamps (strain NIBB) or from cool-white £uorescent lamps (strain PR6000). The strain, light intensity and growth temperature for each experiment are described in the legends for the ¢gures and tables. For lipid analyses, cells were collected by centrifugation, and lipids were extracted from the cell pellet as described [13]. The total lipids were fractionated into lipid classes by thin-layer chromatography [14]. Total lipids and the fractionated lipid classes were subjected to methanolysis, and the resultant methylesters were analyzed with a gas-liquid chromatograph equipped with a capillary column and a hydrogen £ame-ionization detector as previously
Table 1 shows the class composition of the total lipids, as well as their associated fatty acid compositions, for the NIBB strain of Synechococcus sp. PCC 7002. MGDG represented a little more than 50% of the total glycerolipids, while the relative levels of DGDG, SQDG, and PG ranged from 9 to 23%. The PG level increased signi¢cantly in cells grown at 22³C, while the levels of SQDG and DGDG decreased slightly. All lipid classes contained 18:1(9), 18:2(9, 12) and 18:3(9, 12, 15), and the unsaturation levels of the C18 fatty acids changed in response to growth temperature. Although 18:3 fatty acids were only present in trace amounts in the lipids of cells grown at 34³C, g3- (or v15-) desaturated C18 fatty acids were subTable 2 Doubling time of wild-type cells of Synechococcus sp. PCC7002 (strain PR6000)a Light intensity Temperature 38³C 22³C 3 2 31 4 þ 0.3 h 10 þ 1 h 250 WE m s 50 WE m32 s31 20 þ 1 h 20 þ 1 h The values represent the averages of at least three experiments. a Growth was monitored by the optical density at 550 nm.
FEMSLE 7645 27-8-97
316
T. Sakamoto et al. / FEMS Microbiology Letters 152 (1997) 313^320
stantial constituents of all lipid classes in cells grown at 22³C; the levels of 18:1(9) fatty acids decreased in all classes at 22³C. C16 fatty acids accounted for 52^ 57% of the total fatty acids in MGDG, DGDG and PG; the total C16 fatty acids were highest in SQDG (approximately 70%). The highest levels (20^29%) of 16:1(9) were found in MGDG and DGDG, while much lower levels (3^7%) were found in SQDG and PG. Growth temperature had no signi¢cant effect on the total amount of C16 fatty acids in any lipid class; however, an increase in the content of 16:1(9) was observed in MGDG at 22³C. Fig. 1 shows the time course for changes of fatty acid composition of total lipids that occur in Synechococcus sp. PCC 7002 (strain NIBB) after a temperature shift-down from 34³C to 22³C. Detectable changes in the levels of C18 fatty acids occurred about 4 h after the temperature shift-down to 22³C. The level of 18:1(9) fatty acids decreased to approximately half the initial amount, while 18:3(9, 12, 15) fatty acids increased signi¢cantly, and 18:2(9, 12) increased slightly, in the period 4^12 h after the temperature shift-down. The relative level of 16:0 fatty acids began to decrease slightly, and the level of 16:1 fatty acids began to increase correspondingly, about 10^12 h after a temperature shift-down to 22³C. However, the total level of the C16 fatty acids remained nearly constant after the temperature shift-down (Table 1). When the time of treatment at 22³C was expanded to 33 h, the levels of all fatty acids reached the steady-state levels found in cells grown continuously at 22³C.
3.2. E¡ect of light intensity and temperature on growth rate and fatty acid composition in Synechococcus sp. PCC 7002 (strain PR6000)
Table 2 shows the doubling times calculated from exponentially growing cultures of strain PR6000 of Synechococcus sp. PCC 7002. For this strain, cells growing at high light intensity (250 WE m32 s31 ) at 38³C have the maximum growth rate: a doubling time of approximately 4 h. As expected for a phototroph, cells grown under lower light intensity (50 WE m32 s31 ) have a much longer doubling time of 20 h. Importantly, however, at this light intensity the doubling time for Synechococcus sp. PCC 7002 is identical at both growth temperatures (38³C and 22³C). When cells growing at 38³C under 50 WE m32 s31 were transferred to 22³C at the same light intensity, no lag phase occurred and cells continued to grow at 22³C with the same growth rate (data not shown). These results strongly suggest that cells are light-energy limited when grown at this light intensity (50 WE m32 s31 ). Table 3 shows the fatty acid composition of total lipids in strain PR6000 of Synechococcus sp. PCC 7002 when grown under di¡erent conditions of light intensity and temperature. Desaturation at the g3 (or v15) position increased at 22³C under both light intensities tested (250 WE m32 s31 and 50 WE m32 s31 ), although the increase at low light intensity was smaller than that observed at high light intensity. Some di¡erences in fatty acid composition were noted between the NIBB strain and the PR6000
Table 3 Total fatty acid composition of wild-type cells of Synechococcus sp. PCC 7002 (strain PR6000)a Growth conditions Fatty acid (Mol%) 16:0 16:1(9) 18:0 18:1(9) 18:2(9, 12) 18:3(9, 12, 15) 3 2 31 250 WE m s 38³C 55 þ2 6þ1 1 17 þ 3 18 þ 2 1 22³C 51 þ2 7þ1 1 7þ1 15 þ 1 19þ 2 22³C (12 h)b 53 þ2 7þ1 2þ1 7þ1 12 þ 1 19þ 1 50 WE m32 s31 38³C 57 þ1 9þ1 2þ1 20 þ 1 11 þ 1 tc 22³C 46 þ1 13 þ 1 1 15 þ 1 21 þ 1 5þ1 The values represent the averages of at least two experiments. a Wild-type cells of Synechococcus sp. PCC 7002 (strain PR6000) were grown under continuous illumination (250 WE m32 s31 or 50 WE m32 s31 ) at 38³C, 22³C or 15³C. b Cells grown at 38³C were shifted to 22³C for 12 h. c t: trace amount, less than 0.5%.
FEMSLE 7645 27-8-97
T. Sakamoto et al. / FEMS Microbiology Letters 152 (1997) 313^320
317
Fig. 2. Temperature-dependent expression of the acyl-lipid desaturase genes of Synechococcus sp. PCC 7002 (strain PR6000). RNA was isolated from cells grown at 38³C (lane 1) or at 22³C (lane 2) under low light intensity (50 WE m32 s31 ). To investigate temperature-induced changes in the mRNA levels for the acyl-lipid desaturase genes, RNA was isolated from cells grown at 38³C under 50 WE m32 s31 (lane 3; untreated control), from cells grown at 38³C and treated at 22³C for 15 min (lane 4), or from cells returned to 38³C for 15 min after a 15-min treatment at 22³C (lane 5). The temperature treatments were performed under a constant light intensity of 50 WE m32 s31 . RNA blots was hybridized with radiolabeled probes speci¢c for the desA (v12 desaturase), desB (g3 or v15 desaturase) and desC (v9 desaturase) genes.
strain (compare Tables 1 and 3). Notably, the total content of C16 fatty acids was somewhat higher in the PR6000 strain (V60%) compared to the NIBB strain (V55%), but the content of 16:1(9) fatty acids was signi¢cantly greater in the NIBB strain. Nevertheless, these results indicate that temperature, but not growth rate, regulates the g3 desaturation in membrane lipids in this cyanobacterium. 3.3. Responses to shifts in temperature for mRNAs of three desaturase genes in Synechococcus sp. PCC 7002 (strain PR6000)
Fig. 2 shows the steady-state mRNA levels for the ( 12 desaturase), desB (g3 or v15 desaturase) and ( 9 desaturase) genes, as well as transient changes in mRNA levels of the desA, desB and desC genes induced by a temperature-shift treatment, in cells of Synechococcus sp. PCC 7002 (strain PR6000) grown under 50 WE m32 s31 . When the desA hybridization probe was used, 1.2-kb transcripts were detected in RNA extracted from cells grown at both temperatures (38³C and 22³C). The signal intensity of the hybridization showed that the desA mRNA level was about two-fold higher in cells grown at 22³C than in those grown at 38³C (Fig. 2, lanes 1 and 2, desA panel). When a desB hybridization probe was used to probe the RNA blot, 1.2-kb desA v desC v
transcripts were detected in the total RNA of cells grown at 22³C but not in the RNA of cells grown at 38³C (Fig. 2, lanes 1 and 2, desB panel), indicating that desB transcripts only accumulate in cells grown at 22³C. When the desC hybridization probe was used, transcripts of two size classes, 0.95 and 1.2 kb, were detected in total RNA isolated from cells grown at both temperatures (Fig. 2, lanes 1 and 2, desC panel). In order to investigate transient changes in mRNA levels of the desA, desB, and desC genes induced by a temperature-shift treatment at low light intensity, strain PR6000 cells grown at 38³C under 50 WE m32 s31 were transferred to 22³C at the same light intensity for 15 min. The relative abundance of the transcripts for all three desaturase genes increased signi¢cantly within 15 min after a temperature shift-down to 22³C under 50 WE m32 s31 (Fig. 2, lanes 4). These transcript levels quickly returned to the initial levels found in 38³C-grown cells when cells were subsequently shifted back to 38³C for 15 min (Fig. 2, lanes 5). These results indicate that mRNA abundances for the desaturase genes change very rapidly in response to ambient temperature, even under conditions where the growth rate is not altered. In contrast, the fatty acid composition of glycerolipids changes very slowly over a period of hours when cells are shifted to 22³C (see Fig. 1).
FEMSLE 7645 27-8-97
T. Sakamoto et al. / FEMS Microbiology Letters 152 (1997) 313^320
318
strain M3 [3,4], and
4. Discussion
[5]. In
A. nidulans,
Synechocystis
sp. PCC 6803
a unicellular group 1 strain that
In this study the fatty acid compositions of two
cannot synthesize polyunsaturated fatty acids [9],
laboratory wild-type strains (NIBB and PR6000) of
conversion of 16 :0 to 16 :1(9) fatty acids occurs dur-
Synechococ-
ing a 10^40-h period after a temperature shift-down ;
sp. PCC 7002 were compared. Both strains con-
subsequently, the proportion of shorter chain-length
the unicellular marine cyanobacterium
cus
A. variabilis
tained 16 :0, 16 :1(9), 18 :1(9), 18 :2(9, 12) and 18 :3(9,
fatty acids also increases [6]. In
12, 15) fatty acids (Tables 1 and 3). These character-
M3, a ¢lamentous strain of group 2 [9], a rapid but
istics, along with the presence of 16 :1(9) fatty acids,
transient exchange of 16 :0 to 16 :1(9) fatty acids at
demonstrate that this organism is a member of group
the
2 [11]. However, unlike some other group 2 members
after a temperature shift-down, and polyunsaturated
Anabaena
sn-2
strain
position of MGDG took place within 10 h
sp.), no doubly unsaturated 16 :2(9,
C16 fatty acids (16 :2(9, 12)) were also produced in
12) fatty acids were detected. Although the types of
cells at 22³C [3]. The low-temperature-induced con-
fatty acids synthesized by the two strains were iden-
version of 16 :0 to 16 :1(9) fatty acids at the
tical, the proportions of speci¢c fatty acids di¡ered
position of MGDG occurred in the dark as well as
(e.g.
sn-2
signi¢cantly in the two strains. The content of C16
in the light [4]. The amount of 16 :1(9) fatty acids
fatty acids was greater in strain PR6000, but the
recovered to the steady-state level as an increase in
proportion of 16 :1(9) fatty acids was generally lower
the proportion of 18 :3(9, 12, 15) fatty acids occurred
in this strain than in the NIBB strain (compare Ta-
at the
bles 1 and 3). Two possible explanations for this
sn-1 position of all lipid chocystis sp. strain PCC6803, a
di¡erence can be proposed : (1) a spontaneous muta-
group 4 [9], desaturation at the
tion(s) (e.g. a mutation in a gene for the C16 fatty
duced after a temperature shift-down, and 18 :3(9,
acyl
desaturase) has occurred in strain PR6000
12, 15) and 18 :4(6, 9, 12, 15) fatty acids were syn-
that causes a reduced level of 16 :1(9) fatty acids ;
thesized in cells grown at 22³C [5]. No signi¢cant
v9
or (2) uncontrolled, minor di¡erences in the growth conditions a¡ect the expression of the C16
v9
desat-
sn-2
position in
compositions were detected in the two strains, it is
Synechocystis sp. PCC echococcus sp. PCC 7002
desC
position was in-
alter their fatty acid com-
desA,
positions in response to growth temperature (Tables
genes are identical for both strains
1 and 3) and synthesize 18 :3(9, 12, 15) fatty acids
species. The local restriction maps for the and
g3
Synechocystis sp. PCC 6803 A. variabilis [3,4] and 6803 [5], both strains of Syn-
[5]. As is the case for both in
desB
Syne-
unicellular strain of
change was observed in the C16 fatty acids bound at the
urase. Although signi¢cant di¡erences in fatty acid
highly unlikely that these two strains are di¡erent
classes [3]. In
(data not shown). It is perhaps noteworthy that the
after a temperature shift-down. However,
percentage of 16 :1(9) fatty acids in strain PR6000
coccus
Synecho-
sp. PCC 7002 does not synthesize 16 :2(9, 12)
A. variabilis,
roughly corresponds to the molar excess of C16 fatty
fatty acids, which are characteristic of
acids over C18 fatty acids. This may indicate that the
and transient desaturation of 16 :0 fatty acids after
low level of unsaturated C16 fatty acids is produced
temperature shift-down was likewise not observed in
by the activity of the DesC desaturase acting on C16 fatty acids attached at the erolipids. The
desE
sn-1
position of the glyc-
gene encoding the putative C16
v9/sn-2 acyl lipid desaturase has recently been cloned and partially characterized [15]. The
desE
Synechococcus
sp. PCC 7002 (Fig. 1).
The expression of three desaturase genes (desA,
v12
desaturase ;
desC, v9
desB, g3
or
v15
desaturase ; and
desaturase) and fatty acid composition
gene is
were studied as a function of growth temperature
expressed at much lower levels in strain PR6000
(38³C and 22³C) in cells maintained at identical
than the other three desaturase genes, and it will be interesting to determine whether this gene is ex-
growth rates (doubling time=20 h) under light-limit2 1 s ) in strain PR6000. ing conditions (50 E m
pressed at higher levels in the NIBB strain.
The overall fatty acid compositions of cells grown
Desaturation of fatty acids following a temperature shift-down has also been studied in the cyanobacteria
Anacystis nidulans
[6],
Anabaena variabilis
W
3
3
at high or low light intensity at 38³C were similar, and
g3
desaturation increased signi¢cantly in cells
grown at 22³C under both light intensity conditions
FEMSLE 7645 27-8-97
T. Sakamoto et al. / FEMS Microbiology Letters 152 (1997) 313^320 (Table 3). Since transcripts from the
desB
gene are
predominantly by ambient growth temperature with
only observed in 22³C-grown cells (Fig. 2), it can be
relatively
concluded that fatty acid desaturation at the
growth rate.
g3 po-
sition is regulated by the temperature-dependent expression of the
desB
gene.
319
little
in£uence
from
light
intensity
or
As an explanation for the chilling injury of cyanobacteria, it has been proposed that a phase transition
In the studies presented here (Fig. 2), the relative
in the plasma membrane causes some irreversible
mRNA level for each desaturase gene increased sig-
damage to the cells at low temperature [2,7]. The
ni¢cantly within 15 min after a temperature shift-
critical temperature at which irreversible damage to
down under low light intensity conditions under
cells occurs shifts to lower temperature when cells
which
have
are grown at lower temperatures. This suggests that
changed signi¢cantly. Previous studies have shown
low-temperature-induced changes of membrane lipid
the
overall
should
not
composition might a¡ect the susceptibility of cells to
sp. PCC 7002 are signi¢cantly more stable in cells at
low temperature [7]. Through genetic manipulation
low temperature [11], and this property could con-
of the
tribute substantially to the accumulation of these
been shown that desaturation of the
transcripts to high levels after a temperature shift-
in membrane lipids is essential for low-temperature
and
desB
rate
Synechococcus
that the
desA
growth
transcripts of
down. Since the stability of
desC
transcripts is not
desA
v
( 12 desaturase) genes [18^20], it has
tolerance of cyanobacteria. In
v12
Synechocystis
position
sp. PCC
signi¢cantly di¡erent at 22³C and 38³C [11], the
6803, diunsaturated fatty acids facilitate the recovery
transient increase in the mRNA levels for this acyl-
of photosystem II from photodamage and thereby
lipid desaturase gene presumably re£ects enhanced
increase the tolerance of cells to photoinhibition of
transcription of this gene initially after a temperature
photosynthesis at low temperature [21,22]. Although
shift-down.
the
the signi¢cantly increased levels of triunsaturated, or
desaturase genes increased very rapidly, the fatty
even tetraunsaturated, fatty acids in low-tempera-
acid composition of membrane lipids changed only
ture-grown cyanobacterial cells is a striking phenom-
very slowly during a 10^12-h period following the
enon [3^5] (Fig. 1 in this work), the biochemical and
temperature shift-down (Fig. 1, also see Table 3).
physiological signi¢cance of
These results clearly demonstrate that the rate-limit-
temperature acclimatization remains to be discov-
ing step for increasing the desaturation of membrane
ered.
Although
the
mRNA
levels
for
g3 desaturation in low-
lipids at low temperature is post-transcriptional. In
Synechocystis
sp. PCC 6803 the steady-state
mRNA level for the
desA
gene was shown to in-
Acknowledgments
crease when cells were grown at lower temperature [16]. However, in this cyanobacterium, no increase in the
desA
This work was supported by USPHS Grant GM-
mRNA was observed 15 min after temper-
31625 to D.A.B. T.S. is the recipient of a postdoc-
ature shift-down, and little or no decline of mRNA
toral fellowship from the Yamada Foundation (Osa-
level was observed 10 min after a temperature shift-
ka, Japan).
up [16]. Chemical hydrogenation of the cytoplasmic membrane stimulated accumulation of the
desA tran-
scripts without a temperature shift, implying that a change in membrane £uidity might be the primary signal for the onset of mRNA accumulation of the
desA gene [17], although the mechanism(s) regulating the mRNA levels of the desA gene is still not established. Although the pathway(s) for cyanobacterial regulation of gene expression in response to important environmental signals such as temperature and light remain to be determined, the expression of the acyl-lipid desaturase genes appears to be controlled
References [1] Gantt, E. (1994) Supramolecular membrane organization. In : The Molecular Biology of Cyanobacteria (Bryant, D.A., Ed.), pp. 119^138. Kluwer, Dordrecht. [2] Murata, N. and Nishida, I. (1987) Lipids of blue-green algae (cyanobacteria).
In :
The
Biochemistry
of
Plants
(Stumpf,
P.K., Ed.), Vol. 9, pp. 315^347. Academic Press, San Diego, CA. [3] Sato, N. and Murata, N. (1980) Temperature shift-induced responses in lipids in the blue-green alga,
FEMSLE 7645 27-8-97
Anabaena variabilis :
T. Sakamoto et al. / FEMS Microbiology Letters 152 (1997) 313^320
320
The central role of diacylmonogalactosylglycerol in thermo-
[4] Sato, N. and Murata, N. (1981) Studies on the temperature shift induced desaturation of fatty acids in monogalactosyl diacylglycerol in the blue-green alga (cyanobacterium),
baena variabilis. [5] Wada,
H.
and
Ana-
N.
(1990)
Temperature-induced
changes in the fatty acids composition of the cyanobacterium,
Synechocystis
[15] Sakamoto, T., Stirewalt, V.L. and Bryant, D.A. (1997) Two acyl-lipid
coccus
v9 desaturase genes of the cyanobacterium Synecho-
sp. PCC 7002. In : Physiology, Biochemistry, and Mo-
lecular Biology of Plant Lipids (Williams, J.P., Khan, M.U.
Plant Cell Physiol. 22, 1043^1050. Murata,
[14] Sato, N. and Murata, N. (1988) Membrane lipids. Methods Enzymol. 167, 251^259.
adaptation. Biochim. Biophys. Acta 619, 353^366.
and Lem, N.W., Eds.), pp. 380^381. Kluwer, Dordrecht. [16] Los, D., Horvath, I., Vigh, L. and Murata, N. (1993) The temperature-dependent
PCC6803. Plant Physiol. 92, 1062^1069.
[6] Murata, N., Ono, T.-A. and Sato, N. (1979) Lipid phase of
desA
in
Synechocystis
expression
of
the
desaturase
gene
PCC 6803. FEBS Lett. 318, 57^60.
Anacystis
[17] Vigh, L., Los, D.A., Horvath, I. and Murata, N. (1993) The
In : Low Temperature Stress in Crop Plants : The
primary signal in the biological perception of temperature :
Role of the Membrane (Lyons, J.M., Graham, D. and Radi-
Pd-catalyzed hydrogenation of membrane lipids stimulated
son, J.K., Eds.), pp. 337^345. Academic Press, New York.
the expression of the
membrane and chilling injury in the blue-green alga,
nidulans.
[7] Murata, N. (1989) Low-temperature e¡ects on cyanobacterial
desA
gene in
Synechocystis
PCC 6803.
Proc. Natl. Acad. Sci. USA 90, 9090^9094. [18] Wada, H., Gombos, Z. and Murata, N. (1990) Enhancement
membranes. J. Bioenerg. Biomembr. 21, 61^75. [8] Cossins, A.R. (1994) Homeoviscous adaptation of biological membranes and its functional signi¢cance. In : Temperature Adaptation of Biological Membranes (Cossins, A.R., Ed.),
of chilling tolerance of a cyanobacterium by genetic manipulation of fatty acid desaturation. Nature 347, 200^203. [19] Wada, H., Gombos, Z., Sakamoto, T. and Murata, N. (1992) Genetic manipulation of the extent of desaturation of fatty
pp. 63^76. Portland, London. [9] Murata, N., Wada, H. and Gombos, Z. (1992) Modes of fatty-acid desaturation in cyanobacteria. Plant Cell Physiol.
acids in membrane lipids in the cyanobacterium
Synechocystis
PCC6803. Plant Cell Physiol. 33, 535^540. [20] Wada, H., Gombos, Z. and Murata, N. (1994) Contribution
33, 933^941. [10] Sakamoto, T., Wada, H., Nishida, I., Ohmori, M. and Mu-
of membrane lipids to the ability of the photosynthetic machi-
rata, N. (1994) Identi¢cation of conserved domains in the
nery to tolerate temperature stress. Proc. Natl. Acad. Sci.USA
v12
desaturases of cyanobacteria. Plant Mol. Biol. 24, 643^650.
91, 4273^4277.
[11] Sakamoto, T. and Bryant, D.A. (1997) Temperature-regulated
[21] Gombos, Z., Wada, H. and Murata, N. (1994) The recovery
mRNA accumulation and stabilization for fatty acid desatur-
of photosynthesis from low-temperature photoinhibition is ac-
ase genes in the cyanobacterium
Synechococcus
sp. strain
celerated by the unsaturation of membrane lipids : A mechanism of chilling tolerance. Proc. Natl. Acad. Sci. USA 91,
PCC7002. Mol. Microbiol. 23, 1281^1292. [12] Stevens, Jr., S.E., Patterson, C.O.P. and Myers, J. (1973) The production of hydrogen peroxide by blue-green algae : A sur-
8787^8791. [22] Tasaka, Y., Gombos, Z., Nishiyama, Y., Mohanty, P., Ohba, T., Ohki, K. and Murata, N. (1996) Targeted mutagenesis of
vey. J. Phycol. 9, 427^430. [13] Bligh, E.G. and Dyer, W.J. (1959) A rapid method of total
acyl-lipid desaturases in
Synechocystis :
evidence for the im-
lipid extraction and puri¢cation. Can. J. Biochem. Physiol. 37,
portant roles of polyunsaturated membrane lipids in growth,
911^917.
respiration, and photosynthesis. EMBO J. 15, 6416^6425.
FEMSLE 7645 27-8-97
Low-temperature-induced desaturation of fatty acids and expression of desaturase genes in the cyanobacterium Synechococcus sp. PCC 7002 Toshio Sakamoto a , Shoichi Higashi b , Hajime Wada Donald A. Bryant a *
1;b
, Norio Murata b ,
;
a
S-234 Frear Bldg., Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA 16802, USA
b
Department of Regulation Biology, National Institute for Basic Biology, Myodaiji, Okazaki 444, Japan
Received 19 February 1997 ; revised 22 April 1997; accepted 23 April 1997
Abstract
Changes in response to temperature of lipid classes, fatty acid composition and mRNA levels for acyl-lipid desaturase genes were studied in the marine unicellular cyanobacterium, Synechococcus sp. PCC 7002. The degree of unsaturation of C18 fatty acids increased in cells grown at lower temperature for all lipid classes, and g3 desaturation occurred specifically in cells grown at low temperature. While the level of 18:1(9) fatty acids declined, desaturation at the g3 position of C18 fatty acids increased gradually during a 12-h period after a temperature shift-down to 22³C. However, the mRNA levels of the desA (v12 desaturase), desB (g3 desaturase) and desC (v9 desaturase) genes increased within 15 min after a temperature shift-down to 22³C; the desaturase gene mRNA levels also rapidly declined within 15 min after a temperature shift-up to 38³C. Therefore, the elevation of mRNA levels for the desaturase genes is not the rate-limiting event for the increased desaturation of membrane lipids after a temperature shift-down. The rapid, low-temperature-induced changes in mRNA levels occurred even when cells were grown under light-limiting conditions for which the growth rates at 22³C and 38³C were identical. These studies indicate that the ambient growth temperature, and not some other growth rate-related process, regulates the expression of acyl lipid desaturation in this cyanobacterium. Keywords :
Cyanobacterium; Gene regulation; Fatty acid desaturase; Homeoviscous adaptation; Low temperature acclimatization
* Corresponding author. Fax: +1 (814) 863 7024; E-mail: [email protected]
1. Introduction
1
Present address: Department of Biology, Faculty of Science, Kyushu University, Ropponmatsu, Fukuoka 810, Japan. DGDG, digalactosyl diacylglycerol; MGDG, monogalactosyl diacylglycerol ; PG, phosphatidylglycerol ; SQDG, sulfoquinovosyl diacylglycerol; Fatty acids are represented by `X:Y(Z)', containing X carbon atoms with Y double bonds at the position Z counted from the carboxyl terminus; 14:0, myristic acid; 16:0, palmitic acid; 16:1(9), palmitoleic acid; 18:0, stearic acid; 18:1(9), oleic acid; 18:2(9, 12), linoleic acid; 18:3(9, 12, 15), K-linolenic acid
Abbreviations :
Cyanobacteria are classi¢ed as Gram-negative bacteria, and their cell envelopes are composed of an outer membrane and a plasma (inner) membrane, which are separated by the peptidoglycan layer [1]. Nearly all cyanobacteria also possess thylakoid membranes, which are intracytoplasmic membranes that house the photosynthetic apparatus. Cyanobacterial lipids are only found in their membranes [2],
0378-1097 / 97 / $17.00 ß 1997 Federation of European Microbiological Societies. Published by Elsevier Science B.V. PII S 0 3 7 8 - 1 0 9 7 ( 9 7 ) 0 0 2 1 7 - 6
FEMSLE 7645 27-8-97
314
T. Sakamoto et al. / FEMS Microbiology Letters 152 (1997) 313^320
and the organization of the cyanobacterial membranes [1], as well as the composition of their lipids and fatty acids [2], is similar to that of the plant chloroplasts. Changes in the fatty acid composition of the membrane lipids of cyanobacterial cells to changes in the ambient growth temperature [3^6] have been regarded as an adaptive response [2,7], known as homeoviscous adaptation [8]. Cyanobacterial strains have been classi¢ed into four groups based on their patterns of fatty acid desaturation [9]. Preliminary studies of fatty acid composition have suggested that the marine, unicellular cyanobacterium Synechococcus sp. PCC 7002 should be assigned to group 2 [9]. Consistent with this assignment, three acyl lipid desaturase genes (desA, v12 desaturase; desB, g3 or v15 desaturase; and desC, v9 desaturase), encoding the enzymes required for the conversion of the C18 fatty acid stearate to K-linolenic acid, were cloned from Synechococcus sp. PCC 7002 [10,11]. An advantage of studying the temperature dependence of desaturase gene expression in cyanobacteria is that the growth rate at a given temperature can be independently regulated by the light intensity at which the cells are grown. This potentially allows e¡ects dependent upon growth rate to be distin-
guished from e¡ects directly dependent upon the growth temperature. To establish the direct in£uence of temperature on the expression of three acyl lipid desaturase genes and fatty acid desaturation, the expression patterns of these genes as a function of temperature were examined using cells of the unicellular marine cyanobacterium Synechococcus sp. PCC 7002 grown at a growth-rate-limiting light intensity (50 WE m32 s31 ), under which conditions the cells grew at an identical rate at 22³C and 38³C. Changes of lipid classes and fatty acid composition in response to changes in ambient temperature, as well as the time course for changes in the latter, are also presented. 2. Material and methods
A laboratory wild-type strain of Synechococcus sp. PCC 7002 (denoted strain NIBB) was originally obtained from the Pasteur Culture Collection and maintained in the National Institute for Basic Biology, Okazaki, Japan. A second laboratory wild-type strain (denoted strain PR6000) of Synechococcus sp. PCC 7002 was obtained from Dr. S. Edward Stevens, Jr. and maintained independently at The Pennsylvania State University. Cells were grown photo-
Table 1 Fatty acid composition of lipid classes from Synechococcus sp. PCC 7002 (strain NIBB)a Lipid class Amount (Mol%) Fatty acid (Mol%) 14:0 16:0 16:1(9) 18:0 18:1(9) 18:2(9, 12) 18:3(9, 12, 15) Total 34³C 1 40 16 1 23 18 tb 22³C 1 35 19 t 10 25 10 MGDG 34³C 55 t 35 20 t 24 20 1 22³C 57 t 23 29 t 7 29 12 DGDG 34³C 12 t 30 26 1 14 27 2 22³C 9 1 34 23 t 10 19 13 SQDG 34³C 17 t 65 7 1 21 5 t 22³C 11 t 60 6 t 18 10 5 PG 34³C 16 t 54 3 1 27 15 t 22³C 23 t 50 3 1 11 27 8 The values represent the averages of two experiments. The deviation of the values was within þ 2%. a Wild-type cells of Synechococcus sp. PCC 7002 (strain NIBB) were grown under continuous illumination (70 WE m32 s31 ) at 34³C or 22³C. b t: trace amount, less than 0.5%
FEMSLE 7645 27-8-97
T. Sakamoto et al. / FEMS Microbiology Letters 152 (1997) 313^320
315
described [5]. In the experiments presented in Table 3, the lyophilized cell pellets were directly methanolyzed with 5% (w/w) HCl in methanol, and the resultant methylesters extracted with n-hexane were analyzed by a gas-liquid chromatograph. Total RNA of 5 Wg was separated on formaldehyde-agarose gels, and transferred onto nylon membrane ¢lters. The amount of RNA on the blot was normalized to the intensity of the ribosomal RNA bands for the lane, which was obtained from the ethidium bromide stained gel. RNA blots was hybridized with radiolabeled probes speci¢c for the desA (v12 desaturase), desB (g3 or v15 desaturase) and desC (v9 desaturase) genes [11]. 3. Results
3.1. Changes in lipids in response to growth temperature in Synechococcus sp. PCC7002 (strain NIBB)
Fig. 1. Changes in fatty acid composition of the total lipids after a temperature shift-down from 34³C to 22³C. Wild-type cells of Synechococcus sp. PCC 7002 (strain NIBB) grown at 34³C in medium A under continuous illumination of 70 WE m32 s31 were transferred at 22³C with equivalent illumination. The values are the means obtained in two experiments. The deviation of values was within þ 2%.
autotrophically in medium A supplemented with 1 mg ml31 NaNO3 [12] with aeration of 1% (v/v) CO2 in air under constant illumination from incandescent lamps (strain NIBB) or from cool-white £uorescent lamps (strain PR6000). The strain, light intensity and growth temperature for each experiment are described in the legends for the ¢gures and tables. For lipid analyses, cells were collected by centrifugation, and lipids were extracted from the cell pellet as described [13]. The total lipids were fractionated into lipid classes by thin-layer chromatography [14]. Total lipids and the fractionated lipid classes were subjected to methanolysis, and the resultant methylesters were analyzed with a gas-liquid chromatograph equipped with a capillary column and a hydrogen £ame-ionization detector as previously
Table 1 shows the class composition of the total lipids, as well as their associated fatty acid compositions, for the NIBB strain of Synechococcus sp. PCC 7002. MGDG represented a little more than 50% of the total glycerolipids, while the relative levels of DGDG, SQDG, and PG ranged from 9 to 23%. The PG level increased signi¢cantly in cells grown at 22³C, while the levels of SQDG and DGDG decreased slightly. All lipid classes contained 18:1(9), 18:2(9, 12) and 18:3(9, 12, 15), and the unsaturation levels of the C18 fatty acids changed in response to growth temperature. Although 18:3 fatty acids were only present in trace amounts in the lipids of cells grown at 34³C, g3- (or v15-) desaturated C18 fatty acids were subTable 2 Doubling time of wild-type cells of Synechococcus sp. PCC7002 (strain PR6000)a Light intensity Temperature 38³C 22³C 3 2 31 4 þ 0.3 h 10 þ 1 h 250 WE m s 50 WE m32 s31 20 þ 1 h 20 þ 1 h The values represent the averages of at least three experiments. a Growth was monitored by the optical density at 550 nm.
FEMSLE 7645 27-8-97
316
T. Sakamoto et al. / FEMS Microbiology Letters 152 (1997) 313^320
stantial constituents of all lipid classes in cells grown at 22³C; the levels of 18:1(9) fatty acids decreased in all classes at 22³C. C16 fatty acids accounted for 52^ 57% of the total fatty acids in MGDG, DGDG and PG; the total C16 fatty acids were highest in SQDG (approximately 70%). The highest levels (20^29%) of 16:1(9) were found in MGDG and DGDG, while much lower levels (3^7%) were found in SQDG and PG. Growth temperature had no signi¢cant effect on the total amount of C16 fatty acids in any lipid class; however, an increase in the content of 16:1(9) was observed in MGDG at 22³C. Fig. 1 shows the time course for changes of fatty acid composition of total lipids that occur in Synechococcus sp. PCC 7002 (strain NIBB) after a temperature shift-down from 34³C to 22³C. Detectable changes in the levels of C18 fatty acids occurred about 4 h after the temperature shift-down to 22³C. The level of 18:1(9) fatty acids decreased to approximately half the initial amount, while 18:3(9, 12, 15) fatty acids increased signi¢cantly, and 18:2(9, 12) increased slightly, in the period 4^12 h after the temperature shift-down. The relative level of 16:0 fatty acids began to decrease slightly, and the level of 16:1 fatty acids began to increase correspondingly, about 10^12 h after a temperature shift-down to 22³C. However, the total level of the C16 fatty acids remained nearly constant after the temperature shift-down (Table 1). When the time of treatment at 22³C was expanded to 33 h, the levels of all fatty acids reached the steady-state levels found in cells grown continuously at 22³C.
3.2. E¡ect of light intensity and temperature on growth rate and fatty acid composition in Synechococcus sp. PCC 7002 (strain PR6000)
Table 2 shows the doubling times calculated from exponentially growing cultures of strain PR6000 of Synechococcus sp. PCC 7002. For this strain, cells growing at high light intensity (250 WE m32 s31 ) at 38³C have the maximum growth rate: a doubling time of approximately 4 h. As expected for a phototroph, cells grown under lower light intensity (50 WE m32 s31 ) have a much longer doubling time of 20 h. Importantly, however, at this light intensity the doubling time for Synechococcus sp. PCC 7002 is identical at both growth temperatures (38³C and 22³C). When cells growing at 38³C under 50 WE m32 s31 were transferred to 22³C at the same light intensity, no lag phase occurred and cells continued to grow at 22³C with the same growth rate (data not shown). These results strongly suggest that cells are light-energy limited when grown at this light intensity (50 WE m32 s31 ). Table 3 shows the fatty acid composition of total lipids in strain PR6000 of Synechococcus sp. PCC 7002 when grown under di¡erent conditions of light intensity and temperature. Desaturation at the g3 (or v15) position increased at 22³C under both light intensities tested (250 WE m32 s31 and 50 WE m32 s31 ), although the increase at low light intensity was smaller than that observed at high light intensity. Some di¡erences in fatty acid composition were noted between the NIBB strain and the PR6000
Table 3 Total fatty acid composition of wild-type cells of Synechococcus sp. PCC 7002 (strain PR6000)a Growth conditions Fatty acid (Mol%) 16:0 16:1(9) 18:0 18:1(9) 18:2(9, 12) 18:3(9, 12, 15) 3 2 31 250 WE m s 38³C 55 þ2 6þ1 1 17 þ 3 18 þ 2 1 22³C 51 þ2 7þ1 1 7þ1 15 þ 1 19þ 2 22³C (12 h)b 53 þ2 7þ1 2þ1 7þ1 12 þ 1 19þ 1 50 WE m32 s31 38³C 57 þ1 9þ1 2þ1 20 þ 1 11 þ 1 tc 22³C 46 þ1 13 þ 1 1 15 þ 1 21 þ 1 5þ1 The values represent the averages of at least two experiments. a Wild-type cells of Synechococcus sp. PCC 7002 (strain PR6000) were grown under continuous illumination (250 WE m32 s31 or 50 WE m32 s31 ) at 38³C, 22³C or 15³C. b Cells grown at 38³C were shifted to 22³C for 12 h. c t: trace amount, less than 0.5%.
FEMSLE 7645 27-8-97
T. Sakamoto et al. / FEMS Microbiology Letters 152 (1997) 313^320
317
Fig. 2. Temperature-dependent expression of the acyl-lipid desaturase genes of Synechococcus sp. PCC 7002 (strain PR6000). RNA was isolated from cells grown at 38³C (lane 1) or at 22³C (lane 2) under low light intensity (50 WE m32 s31 ). To investigate temperature-induced changes in the mRNA levels for the acyl-lipid desaturase genes, RNA was isolated from cells grown at 38³C under 50 WE m32 s31 (lane 3; untreated control), from cells grown at 38³C and treated at 22³C for 15 min (lane 4), or from cells returned to 38³C for 15 min after a 15-min treatment at 22³C (lane 5). The temperature treatments were performed under a constant light intensity of 50 WE m32 s31 . RNA blots was hybridized with radiolabeled probes speci¢c for the desA (v12 desaturase), desB (g3 or v15 desaturase) and desC (v9 desaturase) genes.
strain (compare Tables 1 and 3). Notably, the total content of C16 fatty acids was somewhat higher in the PR6000 strain (V60%) compared to the NIBB strain (V55%), but the content of 16:1(9) fatty acids was signi¢cantly greater in the NIBB strain. Nevertheless, these results indicate that temperature, but not growth rate, regulates the g3 desaturation in membrane lipids in this cyanobacterium. 3.3. Responses to shifts in temperature for mRNAs of three desaturase genes in Synechococcus sp. PCC 7002 (strain PR6000)
Fig. 2 shows the steady-state mRNA levels for the ( 12 desaturase), desB (g3 or v15 desaturase) and ( 9 desaturase) genes, as well as transient changes in mRNA levels of the desA, desB and desC genes induced by a temperature-shift treatment, in cells of Synechococcus sp. PCC 7002 (strain PR6000) grown under 50 WE m32 s31 . When the desA hybridization probe was used, 1.2-kb transcripts were detected in RNA extracted from cells grown at both temperatures (38³C and 22³C). The signal intensity of the hybridization showed that the desA mRNA level was about two-fold higher in cells grown at 22³C than in those grown at 38³C (Fig. 2, lanes 1 and 2, desA panel). When a desB hybridization probe was used to probe the RNA blot, 1.2-kb desA v desC v
transcripts were detected in the total RNA of cells grown at 22³C but not in the RNA of cells grown at 38³C (Fig. 2, lanes 1 and 2, desB panel), indicating that desB transcripts only accumulate in cells grown at 22³C. When the desC hybridization probe was used, transcripts of two size classes, 0.95 and 1.2 kb, were detected in total RNA isolated from cells grown at both temperatures (Fig. 2, lanes 1 and 2, desC panel). In order to investigate transient changes in mRNA levels of the desA, desB, and desC genes induced by a temperature-shift treatment at low light intensity, strain PR6000 cells grown at 38³C under 50 WE m32 s31 were transferred to 22³C at the same light intensity for 15 min. The relative abundance of the transcripts for all three desaturase genes increased signi¢cantly within 15 min after a temperature shift-down to 22³C under 50 WE m32 s31 (Fig. 2, lanes 4). These transcript levels quickly returned to the initial levels found in 38³C-grown cells when cells were subsequently shifted back to 38³C for 15 min (Fig. 2, lanes 5). These results indicate that mRNA abundances for the desaturase genes change very rapidly in response to ambient temperature, even under conditions where the growth rate is not altered. In contrast, the fatty acid composition of glycerolipids changes very slowly over a period of hours when cells are shifted to 22³C (see Fig. 1).
FEMSLE 7645 27-8-97
T. Sakamoto et al. / FEMS Microbiology Letters 152 (1997) 313^320
318
strain M3 [3,4], and
4. Discussion
[5]. In
A. nidulans,
Synechocystis
sp. PCC 6803
a unicellular group 1 strain that
In this study the fatty acid compositions of two
cannot synthesize polyunsaturated fatty acids [9],
laboratory wild-type strains (NIBB and PR6000) of
conversion of 16 :0 to 16 :1(9) fatty acids occurs dur-
Synechococ-
ing a 10^40-h period after a temperature shift-down ;
sp. PCC 7002 were compared. Both strains con-
subsequently, the proportion of shorter chain-length
the unicellular marine cyanobacterium
cus
A. variabilis
tained 16 :0, 16 :1(9), 18 :1(9), 18 :2(9, 12) and 18 :3(9,
fatty acids also increases [6]. In
12, 15) fatty acids (Tables 1 and 3). These character-
M3, a ¢lamentous strain of group 2 [9], a rapid but
istics, along with the presence of 16 :1(9) fatty acids,
transient exchange of 16 :0 to 16 :1(9) fatty acids at
demonstrate that this organism is a member of group
the
2 [11]. However, unlike some other group 2 members
after a temperature shift-down, and polyunsaturated
Anabaena
sn-2
strain
position of MGDG took place within 10 h
sp.), no doubly unsaturated 16 :2(9,
C16 fatty acids (16 :2(9, 12)) were also produced in
12) fatty acids were detected. Although the types of
cells at 22³C [3]. The low-temperature-induced con-
fatty acids synthesized by the two strains were iden-
version of 16 :0 to 16 :1(9) fatty acids at the
tical, the proportions of speci¢c fatty acids di¡ered
position of MGDG occurred in the dark as well as
(e.g.
sn-2
signi¢cantly in the two strains. The content of C16
in the light [4]. The amount of 16 :1(9) fatty acids
fatty acids was greater in strain PR6000, but the
recovered to the steady-state level as an increase in
proportion of 16 :1(9) fatty acids was generally lower
the proportion of 18 :3(9, 12, 15) fatty acids occurred
in this strain than in the NIBB strain (compare Ta-
at the
bles 1 and 3). Two possible explanations for this
sn-1 position of all lipid chocystis sp. strain PCC6803, a
di¡erence can be proposed : (1) a spontaneous muta-
group 4 [9], desaturation at the
tion(s) (e.g. a mutation in a gene for the C16 fatty
duced after a temperature shift-down, and 18 :3(9,
acyl
desaturase) has occurred in strain PR6000
12, 15) and 18 :4(6, 9, 12, 15) fatty acids were syn-
that causes a reduced level of 16 :1(9) fatty acids ;
thesized in cells grown at 22³C [5]. No signi¢cant
v9
or (2) uncontrolled, minor di¡erences in the growth conditions a¡ect the expression of the C16
v9
desat-
sn-2
position in
compositions were detected in the two strains, it is
Synechocystis sp. PCC echococcus sp. PCC 7002
desC
position was in-
alter their fatty acid com-
desA,
positions in response to growth temperature (Tables
genes are identical for both strains
1 and 3) and synthesize 18 :3(9, 12, 15) fatty acids
species. The local restriction maps for the and
g3
Synechocystis sp. PCC 6803 A. variabilis [3,4] and 6803 [5], both strains of Syn-
[5]. As is the case for both in
desB
Syne-
unicellular strain of
change was observed in the C16 fatty acids bound at the
urase. Although signi¢cant di¡erences in fatty acid
highly unlikely that these two strains are di¡erent
classes [3]. In
(data not shown). It is perhaps noteworthy that the
after a temperature shift-down. However,
percentage of 16 :1(9) fatty acids in strain PR6000
coccus
Synecho-
sp. PCC 7002 does not synthesize 16 :2(9, 12)
A. variabilis,
roughly corresponds to the molar excess of C16 fatty
fatty acids, which are characteristic of
acids over C18 fatty acids. This may indicate that the
and transient desaturation of 16 :0 fatty acids after
low level of unsaturated C16 fatty acids is produced
temperature shift-down was likewise not observed in
by the activity of the DesC desaturase acting on C16 fatty acids attached at the erolipids. The
desE
sn-1
position of the glyc-
gene encoding the putative C16
v9/sn-2 acyl lipid desaturase has recently been cloned and partially characterized [15]. The
desE
Synechococcus
sp. PCC 7002 (Fig. 1).
The expression of three desaturase genes (desA,
v12
desaturase ;
desC, v9
desB, g3
or
v15
desaturase ; and
desaturase) and fatty acid composition
gene is
were studied as a function of growth temperature
expressed at much lower levels in strain PR6000
(38³C and 22³C) in cells maintained at identical
than the other three desaturase genes, and it will be interesting to determine whether this gene is ex-
growth rates (doubling time=20 h) under light-limit2 1 s ) in strain PR6000. ing conditions (50 E m
pressed at higher levels in the NIBB strain.
The overall fatty acid compositions of cells grown
Desaturation of fatty acids following a temperature shift-down has also been studied in the cyanobacteria
Anacystis nidulans
[6],
Anabaena variabilis
W
3
3
at high or low light intensity at 38³C were similar, and
g3
desaturation increased signi¢cantly in cells
grown at 22³C under both light intensity conditions
FEMSLE 7645 27-8-97
T. Sakamoto et al. / FEMS Microbiology Letters 152 (1997) 313^320 (Table 3). Since transcripts from the
desB
gene are
predominantly by ambient growth temperature with
only observed in 22³C-grown cells (Fig. 2), it can be
relatively
concluded that fatty acid desaturation at the
growth rate.
g3 po-
sition is regulated by the temperature-dependent expression of the
desB
gene.
319
little
in£uence
from
light
intensity
or
As an explanation for the chilling injury of cyanobacteria, it has been proposed that a phase transition
In the studies presented here (Fig. 2), the relative
in the plasma membrane causes some irreversible
mRNA level for each desaturase gene increased sig-
damage to the cells at low temperature [2,7]. The
ni¢cantly within 15 min after a temperature shift-
critical temperature at which irreversible damage to
down under low light intensity conditions under
cells occurs shifts to lower temperature when cells
which
have
are grown at lower temperatures. This suggests that
changed signi¢cantly. Previous studies have shown
low-temperature-induced changes of membrane lipid
the
overall
should
not
composition might a¡ect the susceptibility of cells to
sp. PCC 7002 are signi¢cantly more stable in cells at
low temperature [7]. Through genetic manipulation
low temperature [11], and this property could con-
of the
tribute substantially to the accumulation of these
been shown that desaturation of the
transcripts to high levels after a temperature shift-
in membrane lipids is essential for low-temperature
and
desB
rate
Synechococcus
that the
desA
growth
transcripts of
down. Since the stability of
desC
transcripts is not
desA
v
( 12 desaturase) genes [18^20], it has
tolerance of cyanobacteria. In
v12
Synechocystis
position
sp. PCC
signi¢cantly di¡erent at 22³C and 38³C [11], the
6803, diunsaturated fatty acids facilitate the recovery
transient increase in the mRNA levels for this acyl-
of photosystem II from photodamage and thereby
lipid desaturase gene presumably re£ects enhanced
increase the tolerance of cells to photoinhibition of
transcription of this gene initially after a temperature
photosynthesis at low temperature [21,22]. Although
shift-down.
the
the signi¢cantly increased levels of triunsaturated, or
desaturase genes increased very rapidly, the fatty
even tetraunsaturated, fatty acids in low-tempera-
acid composition of membrane lipids changed only
ture-grown cyanobacterial cells is a striking phenom-
very slowly during a 10^12-h period following the
enon [3^5] (Fig. 1 in this work), the biochemical and
temperature shift-down (Fig. 1, also see Table 3).
physiological signi¢cance of
These results clearly demonstrate that the rate-limit-
temperature acclimatization remains to be discov-
ing step for increasing the desaturation of membrane
ered.
Although
the
mRNA
levels
for
g3 desaturation in low-
lipids at low temperature is post-transcriptional. In
Synechocystis
sp. PCC 6803 the steady-state
mRNA level for the
desA
gene was shown to in-
Acknowledgments
crease when cells were grown at lower temperature [16]. However, in this cyanobacterium, no increase in the
desA
This work was supported by USPHS Grant GM-
mRNA was observed 15 min after temper-
31625 to D.A.B. T.S. is the recipient of a postdoc-
ature shift-down, and little or no decline of mRNA
toral fellowship from the Yamada Foundation (Osa-
level was observed 10 min after a temperature shift-
ka, Japan).
up [16]. Chemical hydrogenation of the cytoplasmic membrane stimulated accumulation of the
desA tran-
scripts without a temperature shift, implying that a change in membrane £uidity might be the primary signal for the onset of mRNA accumulation of the
desA gene [17], although the mechanism(s) regulating the mRNA levels of the desA gene is still not established. Although the pathway(s) for cyanobacterial regulation of gene expression in response to important environmental signals such as temperature and light remain to be determined, the expression of the acyl-lipid desaturase genes appears to be controlled
References [1] Gantt, E. (1994) Supramolecular membrane organization. In : The Molecular Biology of Cyanobacteria (Bryant, D.A., Ed.), pp. 119^138. Kluwer, Dordrecht. [2] Murata, N. and Nishida, I. (1987) Lipids of blue-green algae (cyanobacteria).
In :
The
Biochemistry
of
Plants
(Stumpf,
P.K., Ed.), Vol. 9, pp. 315^347. Academic Press, San Diego, CA. [3] Sato, N. and Murata, N. (1980) Temperature shift-induced responses in lipids in the blue-green alga,
FEMSLE 7645 27-8-97
Anabaena variabilis :
T. Sakamoto et al. / FEMS Microbiology Letters 152 (1997) 313^320
320
The central role of diacylmonogalactosylglycerol in thermo-
[4] Sato, N. and Murata, N. (1981) Studies on the temperature shift induced desaturation of fatty acids in monogalactosyl diacylglycerol in the blue-green alga (cyanobacterium),
baena variabilis. [5] Wada,
H.
and
Ana-
N.
(1990)
Temperature-induced
changes in the fatty acids composition of the cyanobacterium,
Synechocystis
[15] Sakamoto, T., Stirewalt, V.L. and Bryant, D.A. (1997) Two acyl-lipid
coccus
v9 desaturase genes of the cyanobacterium Synecho-
sp. PCC 7002. In : Physiology, Biochemistry, and Mo-
lecular Biology of Plant Lipids (Williams, J.P., Khan, M.U.
Plant Cell Physiol. 22, 1043^1050. Murata,
[14] Sato, N. and Murata, N. (1988) Membrane lipids. Methods Enzymol. 167, 251^259.
adaptation. Biochim. Biophys. Acta 619, 353^366.
and Lem, N.W., Eds.), pp. 380^381. Kluwer, Dordrecht. [16] Los, D., Horvath, I., Vigh, L. and Murata, N. (1993) The temperature-dependent
PCC6803. Plant Physiol. 92, 1062^1069.
[6] Murata, N., Ono, T.-A. and Sato, N. (1979) Lipid phase of
desA
in
Synechocystis
expression
of
the
desaturase
gene
PCC 6803. FEBS Lett. 318, 57^60.
Anacystis
[17] Vigh, L., Los, D.A., Horvath, I. and Murata, N. (1993) The
In : Low Temperature Stress in Crop Plants : The
primary signal in the biological perception of temperature :
Role of the Membrane (Lyons, J.M., Graham, D. and Radi-
Pd-catalyzed hydrogenation of membrane lipids stimulated
son, J.K., Eds.), pp. 337^345. Academic Press, New York.
the expression of the
membrane and chilling injury in the blue-green alga,
nidulans.
[7] Murata, N. (1989) Low-temperature e¡ects on cyanobacterial
desA
gene in
Synechocystis
PCC 6803.
Proc. Natl. Acad. Sci. USA 90, 9090^9094. [18] Wada, H., Gombos, Z. and Murata, N. (1990) Enhancement
membranes. J. Bioenerg. Biomembr. 21, 61^75. [8] Cossins, A.R. (1994) Homeoviscous adaptation of biological membranes and its functional signi¢cance. In : Temperature Adaptation of Biological Membranes (Cossins, A.R., Ed.),
of chilling tolerance of a cyanobacterium by genetic manipulation of fatty acid desaturation. Nature 347, 200^203. [19] Wada, H., Gombos, Z., Sakamoto, T. and Murata, N. (1992) Genetic manipulation of the extent of desaturation of fatty
pp. 63^76. Portland, London. [9] Murata, N., Wada, H. and Gombos, Z. (1992) Modes of fatty-acid desaturation in cyanobacteria. Plant Cell Physiol.
acids in membrane lipids in the cyanobacterium
Synechocystis
PCC6803. Plant Cell Physiol. 33, 535^540. [20] Wada, H., Gombos, Z. and Murata, N. (1994) Contribution
33, 933^941. [10] Sakamoto, T., Wada, H., Nishida, I., Ohmori, M. and Mu-
of membrane lipids to the ability of the photosynthetic machi-
rata, N. (1994) Identi¢cation of conserved domains in the
nery to tolerate temperature stress. Proc. Natl. Acad. Sci.USA
v12
desaturases of cyanobacteria. Plant Mol. Biol. 24, 643^650.
91, 4273^4277.
[11] Sakamoto, T. and Bryant, D.A. (1997) Temperature-regulated
[21] Gombos, Z., Wada, H. and Murata, N. (1994) The recovery
mRNA accumulation and stabilization for fatty acid desatur-
of photosynthesis from low-temperature photoinhibition is ac-
ase genes in the cyanobacterium
Synechococcus
sp. strain
celerated by the unsaturation of membrane lipids : A mechanism of chilling tolerance. Proc. Natl. Acad. Sci. USA 91,
PCC7002. Mol. Microbiol. 23, 1281^1292. [12] Stevens, Jr., S.E., Patterson, C.O.P. and Myers, J. (1973) The production of hydrogen peroxide by blue-green algae : A sur-
8787^8791. [22] Tasaka, Y., Gombos, Z., Nishiyama, Y., Mohanty, P., Ohba, T., Ohki, K. and Murata, N. (1996) Targeted mutagenesis of
vey. J. Phycol. 9, 427^430. [13] Bligh, E.G. and Dyer, W.J. (1959) A rapid method of total
acyl-lipid desaturases in
Synechocystis :
evidence for the im-
lipid extraction and puri¢cation. Can. J. Biochem. Physiol. 37,
portant roles of polyunsaturated membrane lipids in growth,
911^917.
respiration, and photosynthesis. EMBO J. 15, 6416^6425.
FEMSLE 7645 27-8-97