The Acyl-Acyl Carrier Protein Synthetase from Synechocystis sp. PCC6803 mediates Fatty Acid Import

Transfer of fatty acids across biological membranes is a largely uncharacterized process although essential at membranes of several higher plant organelles like chloroplasts, peroxisomes or the endoplasmic reticulum. Here, we analyzed loss of function mutants of the unicellular cyanobacteria Synechocystis sp. PCC 6803 as a model system to circumvent redundancy problems encountered in eukaryotic organisms. Cells deficient in the only cytoplasmic Synechocystis acyl-acyl carrier protein synthetase (SynAas) were highly resistant to externally provided α -linolenic acid while wild-type cells bleached upon this treatment. Bleaching of wild-type cells was accompanied by a continuous increase of α -linolenic acid in total lipids while no such accumulation could be observed in SynAas deficient cells ( Δ synaas ). When SynAas was disrupted in the tocopherol-deficient, α -linolenic acid-hypersensitive Synechocystis mutant Δ slr1736 , double mutant cells displayed the same resistance phenotype as Δ synaas . Moreover, heterologous expression of SynAas in yeast mutants lacking the major yeast fatty acid import protein Fat1p ( Δ fat1 ) led to restoration of wild-type sensitivity against exogenous α -linolenic acid of the otherwise resistant Δ fat1 mutant indicating that SynAas is functionally equivalent to Fat1p. In addition, liposome assays provided direct evidence for the ability of purified SynAas protein to mediate α -[ 14 C]linolenic acid retrieval from preloaded liposome membranes via synthesis of [ 14 C]linolenoyl-ACP. Taken together our data show that an acyl-activating enzyme like SynAas is necessary and sufficient to mediate transfer of fatty acids across a biological membrane. enzyme can process free fatty acids from artificial membranes, we established an acyl-ACP synthetase assay where the fatty acid substrate is embedded in an artificial liposome membrane. Using this assay we could show that the recombinant acyl-activating enzyme from Synechocystis is able to highly C ] -linolenic acid (Hartmann Analytic) for 10 min at 30°C followed by three wash steps with Tris-HCl pH 8. The [ 1- 14 C ] -linolenic acid loaded liposomes were incubated with 400 µl of acyl-ACP-synthetase assay according to Kaczmarzyk et al. (2010) for 15 min at 30°C. Liposomes and supernatant were separated by centrifugation at 16,000 g counted separately in a Beckmann scintillation counter.


Introduction
Fatty acids have many functions in plants for example as structural components of phospho-and galactolipids, as storage reserves in triacyl glycerols or as precursors for signaling molecules and plant hormones. As essential components of biological membranes they enable the subcellular compartmentalization of plant cells and ensure the vital separation of biochemical reactions and pathways that require different chemical environments.
Transmembrane transport of fatty acids has been characterized in detail in unicellular organisms (Black and DiRusso, 2003). In gram-negative bacteria like Escherichia coli two components contribute to the uptake of fatty acids from the external media. One is the integral membrane protein FadL that is localized in the outer membrane of the E. coli envelope and exhibits typical transport protein characteristics. Consequently, FadL defective mutants do not show any uptake of exogenous long-chain fatty acids (Black et al., 1985). The other component, FadD, displays acyl-CoA synthetase activity and is associated with the inner membrane of the cell envelope. Both FadL and FadD are necessary and act in concert in a process termed vectorial acylation since mutant cells lacking either activity are unable to grow on media containing long-chain fatty acids as the sole carbon source (Nunn and Simons, 1978).
In Saccharomyces cerevisiae a similar vectorial acylation system consisting of a transmembrane protein and acyl-CoA synthetases exists. Here both, the membrane protein Fat1p and the acyl-activating enzymes Faa1p and Faa4p are associated with the plasma membrane. Mutants lacking either Fat1p or any of Faa1 or Faa4 activity are 6 (Gardiner et al., 1982). However, while knowledge about the mechanisms involved in transfer of lipids from the chloroplast to the endoplasmic reticulum and vice versa has greatly advanced in recent years (Benning, 2008), understanding of fatty acid transport across the chloroplast membrane is still limited. Tracer experiments applying [ 18 O] or [ 14 C]acetate to spinach or pea leaves (Pollard and Ohlrogge, 1999;Koo et al., 2004) provided initial experimental verification for a model first outlined by Shine et al. (1976).
This model involves thioesterase mediated cleavage of acyl-ACP (acyl carrier protein) at the inner envelope membrane, transfer of the free fatty acids to the outer envelope membrane and acyl-activating enzyme activity at the outer envelope membrane.
Moreover, incubation of isolated chloroplasts with radiolabeled fatty acids revealed that chloroplasts are also able to take up exogenously supplied fatty acids and elongate and incorporate them into lipids (Thompson et al., 1986;Koo et al., 2005). Koo et al. (2005) provided evidence that the acyl-activating enymes AAE15 and AAE16 are essential in that process. Their study showed that isolated Arabidopsis thaliana chloroplasts activated exogenously applied laureate very rapidly through esterification to acyl-carrier protein and that this reaction is dependent mostly on AAE15.
In both E. coli and yeast, acyl-activating enzymes are involved in transmembrane fatty acid transport. Since the large superfamily of 63 acyl activating enzymes in Arabidopsis suggested possible obstruction of mutant analyses through functional redundancy (Schnurr et al., 2002;Shockey et al., 2003), we employed the more simple unicellular cyanobacteria Synechocystis sp. PCC 6803 as a model. Previous studies had revealed the presence of only a single protein in Synechocystis as homologous to higher plant acylactivating enzymes (Kaczmarzyk and Fulda, 2010). These studies also established that the Synechocystis enzyme (Aas) specifically uses acyl carrier protein (ACP) as cosubstrate to recycle free fatty acids occurring naturally in Synechocystis cells (Kaczmarzyk and Fulda, 2010). Here we provide evidence that Aas from Synechocystis (SynAas), is also involved in transfer of free fatty acids across membranes by vectorial acylation and discuss an analogous mechanism for fatty acid transfer across the chloroplast envelope.

Results
We used the deduced amino acid sequence of Arabidopsis LACS9 to search for homologous proteins in the cyanobacteria Synechoccystis sp. PCC 6803. Analogously to Kazmarzcyk and Fulda (2010) Δ synaas when exposed to increasing concentrations in the growth media. Interestingly, Synechocystis wild-type cells were impaired in growth both on plates and in liquid culture supplemented with α -linolenic acid ( Figure 1). Whereas plate drop assays showed that wild-type cells when diluted from a pre-grown culture on control media simply would not grow in the presence of 10 µM α -linolenic acid or above, the phenotype in liquid culture was much more dramatic. Here, an initially green culture diluted to an OD 750 of 0.25 (compare 0 µM α -linolenic acid in Figure 1B) would completely bleach within 24 h when challenged with 20 µM α -linolenic acid or above ( Figure 1B). Moreover, when monitoring growth of wild-type α -linolenic acid challenged cultures as optical density, cell growth rapidly ceased and cells died ( Figure 1C) In contrast Δ synaas mutant cells were unaffected on plates, showed resistance in liquid culture to very high concentrations of α -linolenic acid of up to 200 µM and were able to maintain growth rates comparable to control conditions at 40 µM α -linolenic acid ( Figure 1B, C). These data show that high concentrations of α -linolenic acid in the external media have a detrimental effect on Synechocystis cells and that this effect is dependent on the intracellular, SynAasdependent activation of fatty acids.

Δ synaas cells
Since α -linolenic acid-challenged wild-type cells bleached completely after 24 h of incubation, we investigated the impact of exogenously fed α -linolenic acid on photosynthesis in wild-type and Δ synaas cells after shorter periods of time using chlorophyll fluorescence. We monitored the photosynthetic electron transport rate (ETR), as a sensitive parameter for intactness of the photosynthetic apparatus. The ETR in wildtype cells started to steeply decrease after 5 h of incubation with 150 µM α-linolenic acid until virtually no photosynthetic electron transport was detectable after 9 -10 h of incubation ( Figure 2). However, the wild-type cell culture did not visibly bleach in these conditions as after 24 h of α -linolenic acid treatment ( Figure 1B). In contrast, photosynthesis in Δ synaas cells remained unchanged in response to α -linolenic acid incubation ( Figure 2). In addition control treatment with only the α -linolenic acid solvent (ethanol) had no effect on ETR in wild-type or Δ synaas cells (Figure 2).

α -linolenic acid treatment
We analyzed the impact of α -linolenic acid treatment on cell structure and integrity in wild-type and Δ synaas cells using electron microscopy. Samples taken after 0, 6 and 10 h of incubation with 150 µM α -linolenic acid showed a gradual change in cell structure with duration of α -linolenic acid treatment in wild-type cells. Here, an accumulation of electron-dense particles could be observed after 6 h ( Figure 3). In electron microscopy, lipophilic structures often appear as dark regions (Neiss, 1983). Hence, the detected electron-dense particles could be assigned either to collapsed membrane material or accumulation of free fatty acids and lipids. These particles increased massively in size and number after 10 h of incubation and the subcellular compartmentation into peripheral thylakoid membranes and central carboxysome and DNA was almost completely lost ( Figure 3), congruent with the observed lack of photosynthetic electron transport in these cells ( Figure 2). In contrast

Wild-type but not
Δ synaas cells accumulate

α -linolenic acid in short and long-term incubation experiments
In order to test whether exogenously fed α -linolenic acid accumulates in Synechocystis cells, we exposed wild-type and The increase in α -linolenic acid in wild-type cells over the course of 10 h was specific for this fatty acid, since the other fatty acids did not display any change in levels ( Figure 4C).

α -linolenic acid accumulates in lipids and as free fatty acid
In order to assess the distribution of α-linolenic acid absorbed from the external media among cellular lipid species we applied thin layer chromatography. Lipids were extracted from cell samples after 0, 6 and 10 h of α-linolenic acid incubation and separated by thin layer chromatography. A substantial increase of free α-linolenic acid is visible in the wild-type strain after 6 and 10 hours of incubation ( Figure 5) indicating that free fatty acids constitute a major component of the electron dense accumulations apparent in the electron micrographs ( Figure 5). Additionally there is an increase in monogalactosyl diacylglycerol (MGDG), sulfoquinovosyl diacylglycerol (SQDG) and phospatidyl glycerol (PG) over time. When applying α-[ 14 C]linolenic acid for incubation times of up to 60 minutes radio label could clearly be detected in major lipid classes after 20 minutes (Supplemental Figure 2) showing that α-linolenic acid is incorporated into most of the predominant lipid species of Synechocystis (Wada and Murata, 1998). We suspect that the additional band, appearing right above MGDG ( Figure 5), represents an MGDG with two 18:3 fatty acid residues, which would lead to a more lipophilic molecule than the common MGDG with 16:0 fatty acids at the sn-2 position (Wada and Murata, 1990). An unknown very polar molecule, running underneath phosphatidyl glycerol (PG) is also increasing over time. However, we could not clarify its identity with our standards. In contrast to the findings in wild type, there is no increase in any of the lipid species in the Δsynaas mutant ( Figure 5). to grow on plates containing as little as 5 µM α -linolenic acid, a concentration that did not affect growth of Synechocystis wild-type cells ( Figure 1A, Figure 6, Supplemental Figure 4). In addition Δ slr1736 growth in liquid culture was as severely impaired in the presence of α -linolenic acid as wild-type cells ( Figure 1C). In contrast, the Δ slr1736/Δsynaas double mutant grew not only at concentrations that were still tolerated by wild-type cells but also at much higher concentrations that are tolerated by unable to grow in the absence of any additional carbon source (data not shown), however, reduced growth of wild-type cells compared to control plates could be observed when glucose was added at a low concentration (0.55 mM, Figure 7B). In contrast Δfat1 single mutant cells showed α -linolenic acid-resistant growth on the same plates ( Figure 7B).
Since yeast cell growth in liquid culture measured as OD 600 could more reliably be characterized at a glucose concentration of 5.5 mM in the media we determined the effect of α-linolenic acid on wild-type and Δfat1 cells at moderate glucose concentrations ( Figure 7A). Under these conditions wild-type cell growth in the presence of 3.6 mM α-linolenic acid is only weakly inhibited compared to control conditions and does not differ from Δfat1 growth (Figure 7 A). However, overexpression of a SynAas-GFP fusion protein in the Δfat1 mutant and wild-type background lead to a significant depression of cell growth ( Figure 7A) which was even more evident in the presence of low glucose in the media ( Figure 7B). Here, the residual growth of SynAas-GFP expressing wild-type cells observed in liquid culture ( Figure 7A) was completely abolished in SynAas-GFP over-expressing cells ( Figure 7B). These data support the conclusion that the GFP-tagged SynAas protein can functionally complement Fat1p-mediated α -linolenic acid uptake and that an acyl-activating enzyme alone is sufficient to mediate import of fatty acids from the external media into the cell.

SynAas can retrieve and activate α -linolenic acid from artificial liposome membranes
In order to analyze the ability of SynAas to retrieve and activate fatty acids from artificial membranes, we labeled large unilamellar vesicles (0.2 µm) with α -[ 14 C]linolenic acid and incubated these liposomes with purified SynAas. When α-linolenoyl-ACP was determined as water-soluble radioactivity in the supernatant of [ 14 C]-labeled liposomes, substantial synthesis of α-linolenoyl-ACP was detected in assay conditions containing SynAas, ACP, ATP and Mg 2+ ( Figure 8A). A low, about four times reduced level of radioactivity was observed in the absence of either ATP (with SynAas and ACP present, 'no ATP' in Figure 8A) or SynAas and ACP (with ATP present, 'buffer only' in Figure   8A) or when adding boiling-inactivated SynAas to otherwise complete assay media ('boiled SynAas in Figure 8A). Complementary, the [ 14 C]-label was lowest in assay conditions providing all components and approximately equally high when carried out without either ATP or SynAas and ACP or with boiling-inactivated SynAas when liposome-associated radioactivity was determined from the liposome pellet after assay performance ( Figure 8B). This indicates that α-[ 14 C]linolenic acid could be retrieved and activated from liposome membranes through action of SynAas.

Discussion
Although polyunsaturated fatty acids are part of all plant membranes, they are toxic to the cell in higher concentrations. In plants, accumulation of α-linolenic acid within the cell is detrimental to the integrity of chloroplasts by unfolding of grana stacks and subsequent changes in the thylakoid membrane ultra structure (Siegenthaler, 1972;Okamoto et al., 1977;Kunz et al., 2009). Here we used the cytotoxic effect of intracellularly accumulating α-linolenic acid as a tool to demonstrate the involvement of an acyl activating enzyme in fatty acid translocation across biological membranes. Since a negative impact of α-linolenic acid on ultrastructure and photosynthetic electron transport has also been described for isolated chloroplasts (Siegenthaler, 1972;Okamoto et al., 1977;Golbeck et al., 1980;Kunz et al., 2009) it appears reasonable that the accumulation of excess amounts of α-linolenic acid within Synechocystis cells is the major cause for the observed toxicity. No accumulation of α-linolenic acid could be observed in Δsynaas mutant cells, neither in short-nor in long-term experiments ( Figure   4, 5). Consequently, α-linolenic acid resistant growth could be observed in the nonaccumulating Δsynaas mutant cells (Figure 1, 4). The exact mechanism of α-linolenic acid toxicity to cells remains unclear. Although our study did not aim at exploring the physiological basis for the observed toxicity in Synechocystis and yeast wild-type cells, we adopt its occurrence as an argument for strongly reduced or absent α-linolenic acid uptake into mutant cells.

Loss of function of the acyl-activating enzyme protects the cell from the toxic effect of
In fact, our analyses of Synechocystis wild-type cells showed strongly elevated levels of α-linolenic acid in lipids and as free fatty acid ( Figure 5). Since it had been shown previously that hydrogen atoms adjacent to olefinic bonds such as those found in polyunsaturated fatty acids are susceptible to oxidative attack (Singh et al., 2002), we speculated that the observed detrimental effects of α-linolenic acid might partly be caused by lipid peroxidation. Intracellular lipid peroxidation results in secondary effects like protein modification and degradation and DNA damage (McIntyre et al., 1999) which would fit well with the observed general reduction in cell viability of α-linolenic acid accumulating wild-type cells. In order to protect polyunsaturated fatty acid acyl chains from oxidative damage, cyanobacteria, algae and higher plants have evolved a scavenger mechanism employing α-tocopherol as antioxidant (Maeda and DellaPenna, 2007;Hunter and Cahoon, 2007 unique ability to prevent oxidative damage to polyunsaturated fatty acid acyl chains through interception of fatty acid peroxyl radicals and hence interruption of the chain reaction process inherent to lipid peroxidation (Schneider, 2005). Moreover, recent studies on Synechocystis mutants suggest that α-tocopherol is also important for repair of photodamaged photosystem II (PSII) by protecting de novo biosynthesis of high-turnover

SynAas mediates fatty acid import when heterologously expressed in Saccharomyces cerevisiae
In order to further examine the involvement of acyl activating enzymes in the import of fatty acids, we tested the Saccharomyces cerevisiae mutant Δfat1 upon treatment with α-linolenic acid. Fat1p, as the postulated fatty acid transporter, acts in concert with Faa1p and Faa4p in vectorial acylation in Saccharomyces cerevisiae (Obermeyer et al., 2007).
Analysis of the Δfat1 mutant had revealed that the import of oleate or the fatty acid analogon BODIPY-3823 is strongly reduced (Faergeman et al., 1997). However, earlier studies also revealed that the two acyl-activating enzymes Faa1p and Faa4p are essential for uptake of fatty acids (Knoll et al., 1995). Here, we describe a new phenotype of the Δfat1 mutant, which displays α-linolenic acid-resistant growth on plates containing high concentrations of α-linolenic acid, whereas Saccharomyces cerevisiae wild-type cells are highly sensitive under these conditions (Figure 7). We propose that, as in Synechocystis, the reduced uptake of toxic α-linolenic acid is the major cause for the resistance phenotype.
In our studies, we focused on Fat1p because it is the only acyl-activating enzyme with several membrane spans, characteristics of a classical transport protein, and has also been proven to have a major impact on fatty acid import in yeast (Faergeman et al., 1997).
Overexpression of the cyanobacterial SynAas protein fused to GFP in the Δfat1 mutant lead to an increased sensitivity when grown in media containing 3.6 mM α-linolenic acid and 5.5 mM glucose ( Figure 7A). More strikingly, α-linolenic acid hypersensitiy was imposed in wild-type cells over-expressing SynAas-GFP upon α-linolenic acid treatment ( Figure 7). We postulate that in addition to the functional, endogenous import mechanism of wild-type cells, SynAas mediates increased import of toxic fatty acids, which causes the observed growth retardation. A generally stronger α-linolenic acid sensitivity in SynAas-expressing wild-type and Δfat1 mutants could be observed by reducing the glucose content in the media to 0.55 mM ( Figure 7B). This effect probably reflects the fact that the uptake of fatty acids becomes less important the more glucose is provided as a carbon source and implies that the native yeast fatty acid import mechanism may be www.plantphysiol.org on August 20, 2017 -Published by Downloaded from Copyright © 2012 American Society of Plant Biologists. All rights reserved.
down-regulated at the transcriptional or post-transcriptional level in the presence of sufficient quantities of glucose.
The recovery of the Δfat1/SynAas α-linolenic acid-sensitive phenotype and the hypersensitive phenotype of the WT/SynAas cells suggests that the acyl-activating enzyme from Synechocystis is functionally equivalent to Fat1p and further strengthens our assumption that SynAas is able to mediate fatty acid import without the help of any additional transport protein.

Fatty acids embedded in membranes serve as substrate for the acyl-activating enzyme
It had been demonstrated previously that uncharged fatty acids can rapidly flip-flop between the exoplasmic face and the cytoplasmic face of membranes without the help of proteins (Kamp and Hamilton, 1992). In order to generate an influx of fatty acids across membranes, a mechanism needs to be present that removes fatty acids from the membrane at the cytosolic side. Flipping of free fatty acids from one leaflet to the other is reversible, however, when fatty acids are removed on one side, a continuous, transmembrane flux of fatty acids would be created. Such a mechanism can be driven by an intracellular acyl-activating enzyme, which retrieves the lipohilic free fatty acid from the membrane and converts it to the water-soluble acyl-ACP or acyl-CoA ester. This socalled vectorial acylation has been discussed and investigated in great detail for S.
cerevisiae and E. coli as the main mediator of cellular fatty acid uptake (Black and DiRusso, 2003;Black and DiRusso, 2007). For this mechanism to work properly it would be beneficial if the acyl-activating enzyme was membrane associated. Although SynAas does not contain any apparent membrane domains according to various prediction programs, it has been purified from the membrane fraction of heterologous expression systems in this study and previous reports (Kaczmarzyk and Fulda, 2010). Moreover, SynAas has been identified in a proteomics study of Synechocystis plasma membrane proteins (Pisareva et al., 2007) indicating that it is probably membrane associated in vivo.
In order to analyze whether an acyl-activating enzyme can process free fatty acids from artificial membranes, we established an acyl-ACP synthetase assay where the fatty acid substrate is embedded in an artificial liposome membrane. Using this assay we could show that the recombinant acyl-activating enzyme from Synechocystis is able to highly increase the concentration of water-soluble α-linolenoyl-ACP to 79.8±13.3 nmol/l SynAas, whereas without ATP in the assay the concentration remains at 24.7±3.8 nmol/l SynAas in the supernatant (Figure 8). Concomitantly the amount of membrane embedded free fatty acids is decreasing from 417.8±33.0 nmol/l to 387.0±21.7 nmol/l in an ATPdependent manner (Figure 8). From these results it is evident that fatty acids embedded in artificial liposome membranes can serve as substrate for an acyl-activating enzyme like SynAas and hence be transferred across a biological membrane solely by the action of such an enzyme.

A model for fatty acid translocation across membranes
Through analyses of Synechocystis wild-type and mutant cells, complementation of a yeast fatty acid transporter mutant and liposome experiments, we provide direct evidence for a model in which fatty acids can cross a membrane bilayer by first integrating into the membrane according to their physicochemical properties and subsequent retrieval through the action of SynAas at the cytosolic side (Figure 9). Although this model has been derived from studying cellular fatty acid uptake in a prokaryotic organism, it might be speculated that an analogous mechanism is operating at endomembranes of eukaryotic cells. Transferring this model to fatty acid export from chloroplasts of higher plants would implicate that fatty acids synthesized as acyl-ACP esters inside chloroplasts, would have to be released as free fatty acids into the inner envelope membrane by the action of a stroma localized thioesterase such as FatA1 or FatB1 (Jones et al., 1995). In the intermembrane space, an acyl-activating enzyme would activate the free fatty acid through esterification to coenzyme-A, providing fast export rates by vectorial acylation.
The acyl moiety might then either be directly incorporated into phosphatidylcholine via acyl editing as recently suggested (Tjellström et al., 2012) or transferred by a second vectorial-acylation step to the cytosol for incorporation into ER derived lipids. The identity of many of the molecular components involved in this process in higher plants is still elusive. However, the Synechocystis and yeast mutant phenotypes reported here may prove to be useful in future research aiming to demonstrate the putative involvement of candidate proteins in fatty acid export from chloroplasts.

Strains and growth conditions
Synechocystis sp. PCC6803 wildtype and mutant strains were grown under constant light conditions at 45 µE m -2 s -1 and 30°C. Liquid cultures were grown in BG11 media (Rippka et al., 1979), mutant strains were cultivated in the presence of antibiotics for selection (25 µg ml -1 kanamycin or 25 µg ml -1 spectinomycin). Sacharomyces cerevisiae wild type and mutant strains were cultivated in liquid SD media (0.67% yeast nitrogen base, 2% glucose and amino acids with uracil for wildtype and without uracil for transformation selection) at 29°C. SLR1736 knock-out cells were created as described before (Savidge et al., 2002): the SLR1736 open reading frame was amplified from genomic DNA using primers with added NdeI and BamHI (underlined) restriction sites: SLR1736_s tattcatatggcaactatccaagctttttg and SLR1736_as ggatcctaattgaagaagatactaaatagttc.

Generation of Sacharomyces cerevisiae complementation strains
The coding sequence of SynAas was amplified using the Primer SynAas_CDS_s:   Figure 5).

Liposome assay
Liposomes were prepared as described previously (Takei et al., 1998)      Circles and asterisks indicate significant (p ≤ 0.01) differences compared to wild type at the same time point or compared to time point zero, respectively.
Error bars: SEM, n =3. C: Fatty acid profile of total lipids in wild type and Δsynaas upon long-term incubation with α-linolenic acid (18:3(n-3)). Only the 18:3(n-3) concentration in wild-type cells is substantially increasing over time and other fatty acids remain unaltered.  A : wild type, wild type/SynAas, Δfat1, Δfat1/SynAas cultures were diluted to OD 600 0.05 and grown in liquid YNB media supplemented with 5.5 mM glucose, 1% tergitol and 3.6 mM α-linolenic acid. EtOH was used as control. Asterisks indicate significantly (p ≤ 0.01) different values compared to wild type. Error bars: SEM, n = 4-5 B: wild-type, wild-type/SynAas, Δfat1, Δfat1/SynAas cells were diluted to OD 600 0.05 and spotted on YNB plates supplemented with 0.55 mM glucose, 1% tergitol and 3.6 mM α-linolenic acid. EtOH was used as control.  Free fatty acids like α-linolenic acid integrate into the plasma membrane and are retrieved from the membrane phase by SynAas action through simultaneous activation to acyl-ACP (large inset). Synechocystis wild-type cells accumulate an excess of α-linolenic acid in cell lipids when exposed to elevated concentrations in the external media that leads to lipid peroxidation.
In regular growth conditions lipid peroxidation can largely be prevented through the presence of α-tocopherol. LD: lipophilic droplets (as depicted in