Low-temperature-induced desaturation of fatty acids and expression of desaturase genes in the cyanobacterium Synechococcus sp. PCC 7002

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 ω3 desaturation occurred specifically in cells grown at low temperature. While the level of 18:1(9) fatty acids declined, desaturation at the ω3 position of C₁₈ fatty acids increased gradually during a 12-h period after a temperature shift-down to 22°C. However, the mRNA levels of the desA (Δ12 desaturase), desB (ω3 desaturase) and desC (Δ9 desaturase) genes increased within 15 min after a temperature shift-down to 22°C; the desaturase gene mRNA levels also rapidly declined within 15 min after a temperature shift-up to 38°C. 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 22°C and 38°C 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.

Cyanobacterium, Gene regulation, Fatty acid desaturase, Homeoviscous adaptation, Low temperature acclimatization

1 Introduction

Cyanobacteria are classified 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], 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 classified 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, Δ12 desaturase; desB, ω3 or Δ15 desaturase; and desC, Δ9 desaturase), encoding the enzymes required for the conversion of the C₁₈ fatty acid stearate to α-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 effects dependent upon growth rate to be distinguished from effects directly dependent upon the growth temperature. To establish the direct influence 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 μE m⁻² s⁻¹), under which conditions the cells grew at an identical rate at 22°C and 38°C. 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 photoautotrophically in medium A supplemented with 1 mg ml⁻¹ NaNO₃[12] with aeration of 1% (v/v) CO₂ in air under constant illumination from incandescent lamps (strain NIBB) or from cool-white fluorescent lamps (strain PR6000). The strain, light intensity and growth temperature for each experiment are described in the legends for the figures 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 flame-ionization detector as previously 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.

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)
250 μE m−2 s−1
38°C	55±2	6±1	1	17±3	18±2	1
22°C	51±2	7±1	1	7±1	15±1	19±2
22°C (12 h)b	53±2	7±1	2±1	7±1	12±1	19±1
50 μE m−2 s−1
38°C	57±1	9±1	2±1	20±1	11±1	tc
22°C	46±1	13±1	1	15±1	21±1	5±1

The values represent the averages of at least two experiments. ^aWild-type cells of Synechococcus sp. PCC 7002 (strain PR6000) were grown under continuous illumination (250 μE m⁻² s⁻¹ or 50 μE m⁻² s⁻¹) at 38°C, 22°C or 15°C. ^bCells grown at 38°C were shifted to 22°C for 12 h. ^ct: trace amount, less than 0.5%.

Total RNA of 5 μg was separated on formaldehyde-agarose gels, and transferred onto nylon membrane filters. 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 specific for the desA (Δ12 desaturase), desB (ω3 or Δ15 desaturase) and desC (Δ9 desaturase) genes[11].

3 Results

3.1 Changes in lipids in response to growth temperature in Synechococcus sp. PCC7002 (strain NIBB)

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 significantly in cells grown at 22°C, while the levels of SQDG and DGDG decreased slightly.

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
34°C		1	40	16	1	23	18	tb
22°C		1	35	19	t	10	25	10
MGDG
34°C	55	t	35	20	t	24	20	1
22°C	57	t	23	29	t	7	29	12
DGDG
34°C	12	t	30	26	1	14	27	2
22°C	9	1	34	23	t	10	19	13
SQDG
34°C	17	t	65	7	1	21	5	t
22°C	11	t	60	6	t	18	10	5
PG
34°C	16	t	54	3	1	27	15	t
22°C	23	t	50	3	1	11	27	8

The values represent the averages of two experiments. The deviation of the values was within ±2%. ^aWild-type cells of Synechococcus sp. PCC 7002 (strain NIBB) were grown under continuous illumination (70 μE m⁻² s⁻¹) at 34°C or 22°C. ^bt: trace amount, less than 0.5%

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 34°C, ω3- (or Δ15-) desaturated C₁₈ fatty acids were substantial constituents of all lipid classes in cells grown at 22°C; the levels of 18:1(9) fatty acids decreased in all classes at 22°C. 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 significant 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 22°C.

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 34°C to 22°C. Detectable changes in the levels of C₁₈ fatty acids occurred about 4 h after the temperature shift-down to 22°C. The level of 18:1(9) fatty acids decreased to approximately half the initial amount, while 18:3(9, 12, 15) fatty acids increased significantly, 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 22°C. 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 22°C was expanded to 33 h, the levels of all fatty acids reached the steady-state levels found in cells grown continuously at 22°C.

1

Changes in fatty acid composition of the total lipids after a temperature shift-down from 34°C to 22°C. Wild-type cells of Synechococcus sp. PCC 7002 (strain NIBB) grown at 34°C in medium A under continuous illumination of 70 μE m⁻² s⁻¹ were transferred at 22°C with equivalent illumination. The values are the means obtained in two experiments. The deviation of values was within ±2%.

3.2 Effect 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 μE m⁻² s⁻¹) at 38°C have the maximum growth rate: a doubling time of approximately 4 h. As expected for a phototroph, cells grown under lower light intensity (50 μE m⁻² s⁻¹) 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 (38°C and 22°C). When cells growing at 38°C under 50 μE m⁻² s⁻¹ were transferred to 22°C at the same light intensity, no lag phase occurred and cells continued to grow at 22°C 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 μE m⁻² s⁻¹).

2

Doubling time of wild-type cells of Synechococcus sp. PCC7002 (strain PR6000)^a

Light intensity	Temperature
	38°C	22°C
250 μE m−2 s−1	4±0.3 h	10±1 h
50 μE m−2 s−1	20±1 h	20±1 h

The values represent the averages of at least three experiments. ^aGrowth was monitored by the optical density at 550 nm.

Table 3 shows the fatty acid composition of total lipids in strain PR6000 of Synechococcus sp. PCC 7002 when grown under different conditions of light intensity and temperature. Desaturation at the ω3 (or Δ15) position increased at 22°C under both light intensities tested (250 μE m⁻² s⁻¹ and 50 μE m⁻² s⁻¹), although the increase at low light intensity was smaller than that observed at high light intensity. Some differences in fatty acid composition were noted between the NIBB strain and the PR6000 strain (compare Tables 1 and 3). Notably, the total content of C₁₆ fatty acids was somewhat higher in the PR6000 strain (∼60%) compared to the NIBB strain (∼55%), but the content of 16:1(9) fatty acids was significantly greater in the NIBB strain. Nevertheless, these results indicate that temperature, but not growth rate, regulates the ω3 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 desA (Δ12 desaturase), desB (ω3 or Δ15 desaturase) and desC (Δ9 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 μE m⁻² s⁻¹. When the desA hybridization probe was used, 1.2-kb transcripts were detected in RNA extracted from cells grown at both temperatures (38°C and 22°C). The signal intensity of the hybridization showed that the desA mRNA level was about two-fold higher in cells grown at 22°C than in those grown at 38°C (Fig. 2, lanes 1 and 2, desA panel). When a desB hybridization probe was used to probe the RNA blot, 1.2-kb transcripts were detected in the total RNA of cells grown at 22°C but not in the RNA of cells grown at 38°C (Fig. 2, lanes 1 and 2, desB panel), indicating that desB transcripts only accumulate in cells grown at 22°C. 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).

2

Temperature-dependent expression of the acyl-lipid desaturase genes of Synechococcus sp. PCC 7002 (strain PR6000). RNA was isolated from cells grown at 38°C (lane 1) or at 22°C (lane 2) under low light intensity (50 μE m⁻² s⁻¹). To investigate temperature-induced changes in the mRNA levels for the acyl-lipid desaturase genes, RNA was isolated from cells grown at 38°C under 50 μE m⁻² s⁻¹ (lane 3; untreated control), from cells grown at 38°C and treated at 22°C for 15 min (lane 4), or from cells returned to 38°C for 15 min after a 15-min treatment at 22°C (lane 5). The temperature treatments were performed under a constant light intensity of 50 μE m⁻² s⁻¹. RNA blots was hybridized with radiolabeled probes specific for the desA (Δ12 desaturase), desB (ω3 or Δ15 desaturase) and desC (Δ9 desaturase) genes.

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 38°C under 50 μE m⁻² s⁻¹ were transferred to 22°C at the same light intensity for 15 min. The relative abundance of the transcripts for all three desaturase genes increased significantly within 15 min after a temperature shift-down to 22°C under 50 μE m⁻² s⁻¹ (Fig. 2, lanes 4). These transcript levels quickly returned to the initial levels found in 38°C-grown cells when cells were subsequently shifted back to 38°C 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 22°C (see Fig. 1).

4 Discussion

In this study the fatty acid compositions of two laboratory wild-type strains (NIBB and PR6000) of the unicellular marine cyanobacterium Synechococcus sp. PCC 7002 were compared. Both strains contained 16:0, 16:1(9), 18:1(9), 18:2(9, 12) and 18:3(9, 12, 15) fatty acids (Tables 1 and 3). These characteristics, along with the presence of 16:1(9) fatty acids, demonstrate that this organism is a member of group 2[11]. However, unlike some other group 2 members (e.g. Anabaena sp.), no doubly unsaturated 16:2(9, 12) fatty acids were detected. Although the types of fatty acids synthesized by the two strains were identical, the proportions of specific fatty acids differed significantly in the two strains. The content of C₁₆ fatty acids was greater in strain PR6000, but the proportion of 16:1(9) fatty acids was generally lower in this strain than in the NIBB strain (compare Tables 1 and 3). Two possible explanations for this difference can be proposed: (1) a spontaneous mutation(s) (e.g. a mutation in a gene for the C₁₆ fatty acyl Δ9 desaturase) has occurred in strain PR6000 that causes a reduced level of 16:1(9) fatty acids; or (2) uncontrolled, minor differences in the growth conditions affect the expression of the C₁₆Δ9 desaturase. Although significant differences in fatty acid compositions were detected in the two strains, it is highly unlikely that these two strains are different species. The local restriction maps for the desA, desB and desC genes are identical for both strains (data not shown). It is perhaps noteworthy that the percentage of 16:1(9) fatty acids in strain PR6000 roughly corresponds to the molar excess of C₁₆ fatty acids over C₁₈ fatty acids. This may indicate that the low level of unsaturated C₁₆ fatty acids is produced by the activity of the DesC desaturase acting on C₁₆ fatty acids attached at the sn-1 position of the glycerolipids. The desE gene encoding the putative C₁₆Δ9/sn-2 acyl lipid desaturase has recently been cloned and partially characterized[15]. The desE gene is expressed at much lower levels in strain PR6000 than the other three desaturase genes, and it will be interesting to determine whether this gene is expressed at higher levels in the NIBB strain.

Desaturation of fatty acids following a temperature shift-down has also been studied in the cyanobacteria Anacystis nidulans[6], Anabaena variabilis strain M3 [3, 4], and Synechocystis sp. PCC 6803[5]. In A. nidulans, a unicellular group 1 strain that cannot synthesize polyunsaturated fatty acids[9], conversion of 16:0 to 16:1(9) fatty acids occurs during a 10–40-h period after a temperature shift-down; subsequently, the proportion of shorter chain-length fatty acids also increases[6]. In A. variabilis strain M3, a filamentous strain of group 2[9], a rapid but transient exchange of 16:0 to 16:1(9) fatty acids at the sn-2 position of MGDG took place within 10 h after a temperature shift-down, and polyunsaturated C₁₆ fatty acids (16:2(9, 12)) were also produced in cells at 22°C[3]. The low-temperature-induced conversion of 16:0 to 16:1(9) fatty acids at the sn-2 position of MGDG occurred in the dark as well as in the light[4]. The amount of 16:1(9) fatty acids recovered to the steady-state level as an increase in the proportion of 18:3(9, 12, 15) fatty acids occurred at the sn-1 position of all lipid classes[3]. In Synechocystis sp. strain PCC6803, a unicellular strain of group 4[9], desaturation at the ω3 position was induced after a temperature shift-down, and 18:3(9, 12, 15) and 18:4(6, 9, 12, 15) fatty acids were synthesized in cells grown at 22°C[5]. No significant change was observed in the C₁₆ fatty acids bound at the sn-2 position in Synechocystis sp. PCC 6803[5]. As is the case for both in A. variabilis[3, 4] and Synechocystis sp. PCC 6803[5], both strains of Synechococcus sp. PCC 7002 alter their fatty acid compositions in response to growth temperature (Tables 1 and 3) and synthesize 18:3(9, 12, 15) fatty acids after a temperature shift-down. However, Synechococcus sp. PCC 7002 does not synthesize 16:2(9, 12) fatty acids, which are characteristic of A. variabilis, and transient desaturation of 16:0 fatty acids after temperature shift-down was likewise not observed in Synechococcus sp. PCC 7002 (Fig. 1).

The expression of three desaturase genes (desA, Δ12 desaturase; desB, ω3 or Δ15 desaturase; and desC, Δ9 desaturase) and fatty acid composition were studied as a function of growth temperature (38°C and 22°C) in cells maintained at identical growth rates (doubling time = 20 h) under light-limiting conditions (50 μE m⁻² s⁻¹) in strain PR6000. The overall fatty acid compositions of cells grown at high or low light intensity at 38°C were similar, and ω3 desaturation increased significantly in cells grown at 22°C under both light intensity conditions (Table 3). Since transcripts from the desB gene are only observed in 22°C-grown cells (Fig. 2), it can be concluded that fatty acid desaturation at the ω3 position is regulated by the temperature-dependent expression of the desB gene.

In the studies presented here (Fig. 2), the relative mRNA level for each desaturase gene increased significantly within 15 min after a temperature shift-down under low light intensity conditions under which the overall growth rate should not have changed significantly. Previous studies have shown that the desA and desB transcripts of Synechococcus sp. PCC 7002 are significantly more stable in cells at low temperature[11], and this property could contribute substantially to the accumulation of these transcripts to high levels after a temperature shift-down. Since the stability of desC transcripts is not significantly different at 22°C and 38°C[11], the transient increase in the mRNA levels for this acyl-lipid desaturase gene presumably reflects enhanced transcription of this gene initially after a temperature shift-down. Although the mRNA levels for the desaturase genes increased very rapidly, the fatty acid composition of membrane lipids changed only very slowly during a 10–12-h period following the temperature shift-down (Fig. 1, also see Table 3). These results clearly demonstrate that the rate-limiting step for increasing the desaturation of membrane lipids at low temperature is post-transcriptional.

In Synechocystis sp. PCC 6803 the steady-state mRNA level for the desA gene was shown to increase when cells were grown at lower temperature[16]. However, in this cyanobacterium, no increase in the desA mRNA was observed 15 min after temperature shift-down, and little or no decline of mRNA level was observed 10 min after a temperature shift-up[16]. Chemical hydrogenation of the cytoplasmic membrane stimulated accumulation of the desA transcripts without a temperature shift, implying that a change in membrane fluidity 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 predominantly by ambient growth temperature with relatively little influence from light intensity or growth rate.

As an explanation for the chilling injury of cyanobacteria, it has been proposed that a phase transition in the plasma membrane causes some irreversible damage to the cells at low temperature [2, 7]. The critical temperature at which irreversible damage to cells occurs shifts to lower temperature when cells are grown at lower temperatures. This suggests that low-temperature-induced changes of membrane lipid composition might affect the susceptibility of cells to low temperature[7]. Through genetic manipulation of the desA (Δ12 desaturase) genes [18–20], it has been shown that desaturation of the Δ12 position in membrane lipids is essential for low-temperature tolerance of cyanobacteria. In Synechocystis sp. PCC 6803, diunsaturated fatty acids facilitate the recovery of photosystem II from photodamage and thereby increase the tolerance of cells to photoinhibition of photosynthesis at low temperature [21, 22]. Although the significantly increased levels of triunsaturated, or even tetraunsaturated, fatty acids in low-temperature-grown cyanobacterial cells is a striking phenomenon [3–5] (Fig. 1 in this work), the biochemical and physiological significance of ω3 desaturation in low-temperature acclimatization remains to be discovered.

Acknowledgements

This work was supported by USPHS Grant GM-31625 to D.A.B. T.S. is the recipient of a postdoctoral fellowship from the Yamada Foundation (Osaka, Japan).

Abbreviations

Abbreviations
• 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), α-linolenic acid