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Disturbance of cell-size determination by forced overproduction of sulfoquinovosyl diacylglycerol in the cyanobacterium Synechococcus elongatus PCC 7942
Biochemical and Biophysical Research Communications xxx (2017) 1e6
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Disturbance of cell-size determination by forced overproduction of sulfoquinovosyl diacylglycerol in the cyanobacterium Synechococcus elongatus PCC 7942 Norihiro Sato a, *, Yuki Ebiya a, Ryutaro Kobayashi a, Yoshitaka Nishiyama b, Mikio Tsuzuki a a
School of Life Sciences, Tokyo University of Pharmacy and Life Sciences, Horinouchi, Hachioji, Tokyo 192-0392, Japan Department of Biochemistry and Molecular Biology, Graduate School of Science and Engineering, Saitama University, 255 Shimo-Okubo, Sakura-ku, Saitama 338-8570, Japan
b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 17 April 2017 Accepted 23 April 2017 Available online xxx
Sulfoquinovosyl diacylglycerol (SQDG) is present in the membranes of cyanobacteria or their descendants, plastids at species-dependent levels. We investigated the physiological significance of the intrinsic SQDG content in the cyanobacterium Synechococcus elongatus PCC 7942, with the use of its mutant, in which the genes for SQDG synthesis, sqdB and sqdX, were overexpressed. The mutant showed a 1.3-fold higher content of SQDG (23.6 mol% relative to total cellular lipids, cf., 17.1 mol% in the control strain) with much less remarkable effects on the other lipid classes. Simultaneously observed were 1.6- to 1.9-fold enhanced mRNA levels for the genes responsible for the synthesis of the lipids other than SQDG, as if to compensate for the SQDG overproduction. Meanwhile, the mutant showed no injury to cell growth, however, cell length was increased (6.1 ± 2.3, cf., 3.8 ± 0.8 mm in the control strain). Accordingly with this, a wide range of genes responsible for cell division were 1.6e2.4-fold more highly expressed in the mutant. These results suggested that a regulatory mechanism for lipid homeostasis functions in the mutant, and that SQDG has to be kept from surpassing the intrinsic content in S. elongatus for repression of the abnormal expression of cell division-related genes and, inevitably, for normal cell division. © 2017 Elsevier Inc. All rights reserved.
1. Introduction Sulfoquinovosyl diacylglycerol (SQDG) is present in oxygenic photosynthetic organisms from cyanobacteria to their postulated descendants, plastids of plants, and contributes to construction of their membrane systems including thylakoid membranes, together with two galactolipids, monogalactosyl diacylglycerol (MGDG) and digalactosyl diacylglycerol (DGDG), and a sole phospholipid, phosphatidylglycerol (PG) [1e3]. Exceptionally, SQDG, distinct from other lipid classes, is absent in Gloeobacter violaceus PCC 7421, a cyanobacterium, that is regarded as the most primitive of extant cyanobacteria based on the molecular phylogenetic analysis of the
Abbreviations: Chl a, chlorophyll a; DG, diacylglycerol; DGDG, digalactosyl diacylglycerol; MGDG, monogalactosyl diacylglycerol; PG, phosphatidylglycerol; PBS, phycobilisome; SQDG, sulfoquinovosyl diacylglycerol. * Corresponding author. School of Life Sciences, Tokyo University of Pharmacy and Life Sciences, Horinouchi 1432-1, Hachioji, Tokyo 192-0392, Japan. E-mail address: [email protected] (N. Sato).
16s rRNA gene sequence, and correspondingly possesses no thylakoid membrane with a photosynthetic electron transport system functioning in cell membranes [4e6]. SQDG thus seems to have appeared later during the evolution of cyanobacteria than the other three lipids did [7]. As regards the electrochemical properties of polar head groups, SQDG is similar to PG in the possession of a negatively charged head group, but is distinct from MGDG and DGDG that contain noncharged ones [1]. The content of SQDG relative to total lipids in cyanobacterial cells or to those in plant plastids depends on the species and environmental conditions even in the same species (Fig. 1). SQDG amounts to ca. 10e20 mol% in the cells of freshwater and/or coastal cyanobacteria such as Synechococcus elongatus PCC 7942 and Synechococcus sp. PCC 7002 [7e10], and to ca. 2 to >20 mol% in plastids of red and green algae, including C. reinhartdii [11e14], and in those of seed plants such as A. thaliana [15,16]. However, ambient stress conditions such as phosphorus (P)- or sulfur (S)-depletion cause changes in the SQDG content (Fig. 1): Pdepletion in S. elongatus PCC 7942, e.g., leads to an increase of the
http://dx.doi.org/10.1016/j.bbrc.2017.04.129 0006-291X/© 2017 Elsevier Inc. All rights reserved.
Please cite this article in press as: N. Sato, et al., Disturbance of cell-size determination by forced overproduction of sulfoquinovosyl diacylglycerol in the cyanobacterium Synechococcus elongatus PCC 7942, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/j.bbrc.2017.04.129
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C. reinhardtii, but not in S. elongatus, Synechococcus sp. PCC 7002, or A. thaliana [reviewed in 21]. An extreme observation was the essentiality of SQDG for cell growth in Synechocystis sp. PCC 6803, but not in other organisms including S. elongatus and Synechococcus sp. PCC 7002, which could be explained by the specific requirement of SQDG in Synechocystis among cyanobacteria for progression of the cell cycle, in particular, for DNA synthesis [8,22]. In each case above, however, the defective phenotype caused by the loss of SQDG synthesis only indicate whether or not the presence of SQDG is physiologically important, but does not lead to a comprehensive understanding of the physiological significance of the intrinsic SQDG content that depends on the species. In particular, no information has so far been obtained as to whether or not overproduction of SQDG has an impact on oxygenic photosynthetic organisms. In cyanobacteria, SQDG is synthesized through two successive reactions catalyzed by UDP-sulfoquinovose and SQDG synthases, which are coded by the sqdB and sqdX genes, respectively [1]. S. elongatus contains these two genes as a probable sqdBX operon [23]. In this study, we overexpressed sqdBX in S. elongatus for investigation of the effects of over-produced SQDG on the physiological processes in the cells.
Fig. 1. SQDG contents in cellular lipids of cyanobacteria and in plant plastid lipids. Species 1e7 are cyanobacteria (circles), whereas species 8 and 9, 10 and 11, 12 and 13, and 14 and 15 belong to red algae (squares), green algae (squares), diatoms (triangles), and seed plants (diamonds), respectively, as described below. 1, Synechocystis sp. PCC 6803 [8]. 2, S. elongatus [9]. 3, Trichodesmium erythraem [10]. 4, Synechococcus sp. PCC 7002 [7]. 5, Prochlorococcus MED4 [10]. 6, Synechococcus WH8113 [10]. 7, Synechococcus WH8101 [10]. 8, Polysiphonia lanosa [11]. 9, Cyanidioschyzon merolae [12]. 10, C. reinhardtii [13,17]. 11, Parietochloris incisa [14]. 12, Skeletonema costatum [19]. 13, Thalassiosira pseudonana [18]. 14, Arabidopsis thaliana [15]. 15, Spinacia oleracea [16]. Shown are the SQDG contents relative to total cellular lipids in cyanobacteria and those relative to total plastid lipids, i.e., the summed contents of MGDG, DGDG, SQDG, and PG. The SQDG contents relative to total plastid lipids were estimated on the basis of data concerning cellular lipid compositions in plants including algae, and on that of data concerning thylakoid membranes in S. oleracea. Open symbols, non-stressed conditions. Closed symbols, phosphate-limited conditions. Grey square, sulfurlimited conditions.
SQDG content from 10 to >20 mol% with a concomitant decrease of PG, which would reflect a decrease of the P-quota due to substitution of a non-phosphorus lipid, SQDG, for PG [9]. On the contrary, sulfur-starvation induces almost complete degradation of SQDG in C. reinhardtii with a concomitant increase of PG [17]. Meanwhile, absolute marine cyanobacteria including Synechococcus and Prochlorococcus species, or a marine diatom, Thalassiosira pseudonana, which inhabit oceans with severely limited P, generally accumulate SQDG to as high as >50 mol% in total lipids of cyanobacterial cells or in those of plastid ones (Fig. 1) [10,18]. These observations imply that the extent of the contribution of SQDG synthesis to the construction of membrane systems has become diversified depending on the species, e.g., on their respective prerequisites for ecophysiological adaptation. To answer the question of why SQDG has been conserved throughout the evolution of oxygenic photosynthetic organisms, the physiological roles of SQDG have been investigated in several species through the generation and characterization of mutants deficient in SQDG synthesis of cyanobacteria, S. elongatus [9], Synechocystis sp. PCC 6803 [8], and Synechococcus sp. PCC 7002 [7], a green alga, Chlamydomonas reinhardtii [20], and a seed plant, Arabidopsis thaliana [15]. Despite the evolutionary conservation of SQDG, it has been clarified that SQDG plays not common, but, if any, species-dependent roles in these organisms under normal growth conditions: as regards photosynthesis, SQDG is necessary for full functioning of the PSII complex in Synechocystis sp. PCC 6803 and
2. Materials and methods 2.1. Cyanobacterial strains and growth conditions The wild type (WT) cells of S. elongatus were cultured at 30 C in a tube containing BG11 medium, with illumination (10 W m2) and aeration, until the OD730 value of the culture became ca. 1.0 for transformation [24]. Meanwhile, BXOE and EMP cells (see below) were cultured under the same conditions, but with the exception of spectinomycine addition (20 mg ml1). The OD730 value, and the chlorophyll a (Chl a) and phycobilisome (PBS) contents in the cultures were measured with a spectrophotometer (DU 640, Beckman) [24]. The BXOE and EMP cells that were cultured until the OD730 value became ca. 1.0 were collected by centrifugation at 3000g for 15 min for storage at 80 C until use for lipid or mRNA analysis.
2.2. Transformation of S. elongatus A DNA fragment covering the coding regions of sqdB and sqdX, which are aligned in tandem in the genome of S. elongatus, was amplified by PCR, with KOD-Plus, primer set 1 (Table S1), and genomic DNA of S. elongatus as a template. The amplified DNA sequence of sqdB-sqdX was ligated to the SmaI site of the pAM1044 vector. The resultant plasmid (pBXOE) was introduced into cells of S. elongatus for integration of the sqdB-sqdX sequence into a neutral site in the genome through homologous recombination and its expression under the control of the strong constitutive promoter conII [25]. The transformant (BXOE) that gained the spectinomycine-resistance phenotype was selected. Otherwise, the empty vector was introduced into cells of S. elongatus, a transformant (EMP) that showed spectinomycine-resistance being generated.
2.3. Lipid analysis Lipid analysis was performed as previously described [24]. Total lipids were extracted from cells, and thereafter separated into individual lipid classes by TLC. The spots of individual lipid classes were used for the preparation of fatty acid methyl esters for analysis by capillary GLC.
Please cite this article in press as: N. Sato, et al., Disturbance of cell-size determination by forced overproduction of sulfoquinovosyl diacylglycerol in the cyanobacterium Synechococcus elongatus PCC 7942, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/j.bbrc.2017.04.129
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2.4. PCR analyses of transcripts Semi-quantitative reverse-transcriptase PCR was performed with RNA prepared from BXOE or EMP [24]. Primer sets 2e4 in Table S1 were used for amplification of the sqdB-, sqdX-, and sqdBXrelated sequences, respectively. The expression level of rnpB, the gene for a subunit of ribonuclease P, was examined with the use of primer set 5, as a control [24]. Meanwhile, RNA was also subjected to quantitative real-time (RT) PCR involving a Rotor-Gene Q PCR System (Qiagen) with primer sets 6e12 for the genes responsible for lipid synthesis, and with sets 13e25 for the genes responsible for cell division (Table S1). The expression was also investigated for the gene for the small subunit of ribulose 1,5- bisphosphate carboxylase/oxygenase (rbcS) with the use of primer set 26. The expression levels of the respective genes were normalized as to that of rnpB, with the use of primer set 27. 2.5. Microscopic analysis of cell morphology Cells were observed under a Nomarski microscope (BX53; Olympus, Tokyo, Japan) equipped with an image-capture camera (DP73; Olympus, Tokyo, Japan). Analysis of cell size was performed on the images with the use of an FV1000 viewer (Olympus, Tokyo, Japan). 3. Results and discussion 3.1. Effects of sqdBX overexpression in S. elongatus on lipid contents and lipid-synthesis gene expression A transformant (BXOE) was generated in S. elongatus through
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introduction of the sqdB-sqdX sequence under the control of conII promoter with the use of pAM1044 vector [25]. Otherwise, S. elongatus cells were transformed with the empty vector for the generation of a control strain (EMP). Semi-quantitative PCR analysis demonstrated that both sqdB and sqdX were more highly expressed in BXOE than in EMP (Fig. 2a). Similar results were obtained with the primer set for sqdBX, demonstrating that sqdB and sqdX definitely constitute an operon. More than 16-fold higher expression levels in BXOE were confirmed as to sqdB or sqdX through quantitative RT-PCR analysis (Fig. 2b). Accordingly with the stronger expression of sqdB and sqdX, i.e., that of sqdBX, the SQDG content became 1.3-fold higher in BXOE (23.6 mol% relative to total lipids on the basis of fatty acids) than in EMP (17.1 mol%, Fig. 2c and d). This higher SQDG content was compensated for by a 0.9-fold lower MGDG content in BXOE (Fig. 2d; 47.6 mol%, cf., 53.0 mol% in EMP). Meanwhile, neither DGDG nor PG was significantly affected (Fig. 2d). Collectively, we could genetically manipulate the SQDG content for its increase with little or less remarkable alteration in the other three lipid classes. The expression levels of two genes responsible for MGDG synthesis, i.e., mgdA for MGlcDG synthase that transfers the Glc moiety of UDP-Glc to diacylglycerol (DG) and mgdE for MGlcDG epimerase that converts MGlcDG into MGDG, were respectively 1.7-fold upregulated in BXOE, relative to in EMP (Fig. 2b). In line, the expression level of dgdA for DGDG synthase, which converts MGDG into DGDG through galactosylation, was 1.9-fold higher in BXOE (Fig. 2b). Meanwhile, pgsA for phosphatidylglycerophosphate synthase, which is responsible for PG synthesis, was 1.6-fold more highly expressed in BXOE (Fig. 2b). In view of probable competition of MgdA with SqdX for DG, the enhanced expression level of mgdA in BXOE would have been balanced with the overexpression of
Fig. 2. Effects of sqdBX overexpression on the expression levels of lipid synthesis genes and lipid composition in S. elongatus. (a) Semi-quantitative analysis of expression of sqdB, sqdX, and sqdBX at the transcript level in BXOE or EMP. Expression of rnpB mRNA is shown as an internal control. (b) The ratios of transcript levels of BXOE relative to EMP, as to the respective lipid synthesis genes, were estimated through quantitative RT-PCR analysis. The expression of the respective genes was normalized as to that of rnpB. The values are averages ± SE for three biological replicates. Note that BXOE was little affected in the transcript level of rbcS, one of the key proteins for photosynthesis, compatible with the little effect on cell growth. The genes of which the expression levels were evaluated to be significantly higher than that of rbcS with Student's t tests, are indicated by * (P < 0.1) or ** (P < 0.05). (c) Lipid profile revealed on TLC for BXOE or EMP. (d) Lipid composition of BXOE or EMP. White and grey bars indicate BXOE and EMP, respectively. The values are averages ± SD for three biological replicates.
Please cite this article in press as: N. Sato, et al., Disturbance of cell-size determination by forced overproduction of sulfoquinovosyl diacylglycerol in the cyanobacterium Synechococcus elongatus PCC 7942, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/j.bbrc.2017.04.129
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sqdBX, which, together with the higher expression levels of mgdE and dgdA, would have contributed to the synthesis of MGDG or DGDG at a level as close as possible to the intrinsic one. The higher expression level of pgsA in BXOE might have been partially responsible for the ongoing PG synthesis at a normal level even when sqdBX was overexpressed (Fig. 2b, d). In C. reinhardtii cells exposed to P-starvation, the increase of the SQDG content with a decrease in the PG content was accompanied by up- and downregulation of the expression levels of the SQDG and PG synthesis genes, respectively, which seemed to reflect the functioning of the regulatory mechanism for anionic lipid homeostasis at a genetic level [26]. In S. elongatus cells under normal conditions, however, the forced overexpression of SQDG synthesis genes had no impact as to positive down-regulation of the PG content or expression levels of PG synthesis genes (Fig. 2b, d). In view of the essentiality of PG for cyanobacterial cell growth [21], the ongoing PG synthesis seems to precede the balancing of total anionic lipid contents under normal environmental conditions. Collectively, a mutant of S. elongatus that possesses excess SQDG could be generated through overexpression of sqdBX, and was determined to probably utilize a genetic system for the maintenance of lipid homeostasis.
pattern of the OD730 value, or on those of the Chl and phycobilisome contents (Fig. 3a and b), similar to in the case of the SQDG-deficient DsqdB mutant of S. elongatus [8,9]. These results indicated that the content of SQDG, whether it is lower or higher than the intrinsic level, at least within the levels we examined, has little deleterious impact on the construction of the photosynthetic machinery, and inevitably on cell growth under normal conditions. At the same time, it might be interpreted that the regulatory mechanism for lipid homeostasis, as suggested above, allowed the BXOE cells to undergo vigorous growth. Meanwhile, microscopic analysis revealed that BXOE cells were distributed preferentially at larger sizes than EMP ones in the histogram of cell size (Fig. 3c and d). Inevitably, BXOE showed a greater cell length on average (6.1 ± 2.3 mm) than EMP did (3.8 ± 0.8 mm). These results suggested that SQDG overproduction at a certain level beyond its intrinsic one could disturb some mechanism for the determination of cell size in S. elongatus, although SQDG-loss hardly interferes with the mechanism [22].
3.2. Effects of the elevated SQDG content on cellular physiological processes
The elongated cell morphology of BXOE implied malfunctioning of the cell division machinery, which might be accompanied by abnormal expression of the related genes. The respective transcript levels of 13 genes that are responsible for the progress of cell division were then measured by RT-PCR analysis. Bacterial cell
Comparison of BXOE with EMP as to cell growth showed no deleterious effect of the elevated SQDG content on the increasing
3.3. Elevated expression levels of the genes responsible for cell division progress in BXOE
Fig. 3. Effects of sqdBX overexpression on cell growth and length in S. elongatus. Cell growth (a), and the Chl or PBS content (b) on the basis of the OD730 value in BXOE or EMP. Open and closed circles indicate EMP and BXOE, respectively. (c) Photographs of BXOE and EMP cells under a microscope. The bars show 5 mm. (d) Distribution of cell lengths in BXOE or EMP (n ¼ 300 for each strain). Open and closed bars indicate EMP and BXOE, respectively. The values in (a) and (b) are the averages ± SE for three biological replicates.
Please cite this article in press as: N. Sato, et al., Disturbance of cell-size determination by forced overproduction of sulfoquinovosyl diacylglycerol in the cyanobacterium Synechococcus elongatus PCC 7942, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/j.bbrc.2017.04.129
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Fig. 4. Effects of sqdBX overexpression on the expression levels of the genes for progress of the cell division in S. elongatus. The ratios of transcript levels of BXOE relative to EMP, as to the respective genes responsible for cell division, were estimated through quantitative RT-PCR analysis, as explained in Fig. 2b. Two homologs of ftsW, Synpcc7942_0324 and Synpcc7942_1104, are denoted as 0324 and 1104, respectively. The values are averages ± SE for three biological replicates. The value of rbcS is the same as that in Fig. 2b. The genes of which the expression levels were evaluated to be significantly higher than that of rbcS with Student's t tests, were marked by * (P < 0.1) or ** (P < 0.05).
division is driven by the multi-protein machinery through the formation and constriction of the division ring at mid-cell [27,28]. The FtsZ, Q, and W proteins participate in the construction of a proto-ring, transduction of signals from the periplasm to the Z ring for the coordination of its constriction, and septal peptideglycan synthesis, respectively, through the progression of cell division. Meanwhile, MinC-E, and MinC/D and DivIVA comprise the system that regulates the positioning of Z ring formation in Escherichia coli and Bacillus subtilis, respectively. Eleven genes were found to be more highly expressed in BXOE (1.6e2.4-fold), relative to in EMP, comprising cdv3 (divIVA-like gene), cikA, ftn6, ftsQ/W/Z, and minC-E, the exception being cdv1/2 (Fig. 4). Meanwhile, dnaA is responsible for the initiation of DNA replication, which is one of the prerequisites for the progress of cell division, its expression level also having being 1.9-fold higher in BXOE than in EMP (Fig. 4). These observations suggested that SQDG overproduction disturbed the expression of a large member of the genes that are responsible for cell division. Anionic phospholipids or the membrane potential in cell membranes is crucial for some protein members of the cell division machinery such as MinD to bind to the membranes [29,30]. The membrane lipids of cyanobacteria participate predominantly in the construction of thylakoid membranes, and also in that of cell membranes, although to a much lesser extent. The observed difference in lipid composition between BXOE and EMP (Fig. 2d) should thus more directly reflect that in thylakoid membranes. However, it is highly probable that cell membranes of BXOE possess more abundant SQDG than those of EMP. Meanwhile, in S. elongatus, cell length became greater through overexpression of ftsZ, minC, minD or minE, besides through disruption of cdv1, cdv2, cdv3, cikA, ftn2 or ftn6 [31e33]. One possible scenario is as follows: in BXOE, this higher content of cell-membranous SQDG, which is anionic like the cyanobacterial sole phospholipid PG, might interfere with the localization pattern or functionality of MinD, and those of FtsW and FtsQ as well, which should also be localized in cell membranes [27]. Such perturbation of the protein components of the cell division machinery might cause the abnormal construction or functionality of the machinery that consequently elevates the expression levels of a wide range of cell-division related genes including ftsZ and minC-E, the cell length eventually being increased (Fig. 4). In any case, we succeeded in increasing the SQDG content in photosynthetic organisms, and clarified that the content has to be kept from surpassing the intrinsic level.
Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.bbrc.2017.04.129. Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.bbrc.2017.04.129. References [1] N. Sato, H. Wada, Lipid biosynthesis and its regulation in cyanobacteria, in: H. Wada, N. Murata (Eds.), Lipids in Photosynthesis, Essential and Regulatory Functions, Springer, Dordrecht, 2009, pp. 157e177. [2] M. Shimojima, H. Ohta, Y. Nakamura, Biosynthesis and function of chloroplast lipids, in: H. Wada, N. Murata (Eds.), Lipids in Photosynthesis, Essential and Regulatory Functions, Springer, Dordrecht, 2009, pp. 35e55. [3] R. Goss, C. Wilhelm, Lipids in algae, lichens and mosses, in: H. Wada, N. Murata (Eds.), Lipids in Photosynthesis, Essential and Regulatory Functions, Springer, Dordrecht, 2009, pp. 117e135. [4] E. Selstam, D. Campbell, Membrane lipid composition of the unusual cyanobacterium Gloeobacter violaceus sp. PCC 7421, which lacks sulfoquinovosyl diacylglycerol, Arch. Microbiol. 166 (1996) 132e135. [5] G. Guglielmi, G. Cohen-Bazire, D.A. Bryant, The structure of Gloeobacter violaceus and its phycobilisomes, Arch. Microbiol. 129 (1981) 181e189. [6] D. Honda, A. Yokota, J. Sugiyama, Detection of seven major evolutionary lineages in cyanobacteria based on the 16S rRNA gene sequence analysis with new sequences of five marine Synechococcus strains, J. Mol. Evol. 48 (1999) 723e739. [7] N. Sato, R. Kamimura, M. Tsuzuki, Dispensability of a sulfolipid for photoautotrophic cell growth and photosynthesis in a marine cyanobacterium, Synechococcus sp. PCC 7002, Biochem. Biophys. Res. Commun. 477 (2016) 854e860. [8] M. Aoki, N. Sato, A. Meguro, M. Tsuzuki, Differing involvement of sulfoquinovosyl diacylglycerol in photosystem II in two species of unicellular cyanobacteria, Eur. J. Biochem. 271 (2004) 685e693. €rtel, et al., A null mutant of Synechococcus sp. [9] S. Güler, A. Seeliger, H. Ha PCC7942 deficient in the sulfolipid sulfoquinovosyl diacylglycerol, J. Biol. Chem. 271 (1996) 7501e7507. [10] B.A. Van Mooy, G. Rocap, H.F. Fredricks, et al., Sulfolipids dramatically decrease phosphorus demand by picocyanobacteria in oligotrophic marine environments, Proc. Natl. Acad. Sci. U. S. A. 103 (2006) 8607e8612. [11] T.R. Pettitt, A.L. Jones, J.L. Harwood, Lipid metabolism in the red marine algae Chondrus crispus and Polysiphonia lanosa as modified by temperature, Phytochemistry 28 (1989) 2053e2058. [12] N. Sato, T. Moriyama, Genomic and biochemical analysis of lipid biosynthesis in the unicellular rhodophyte Cyanidioschyzon merolae: lack of a plastidic desaturation pathway results in the coupled pathway of galactolipid synthesis, Eukaryot. Cell 6 (2007) 1006e1017. [13] W.R. Riekhof, M.E. Ruckle, T.A. Lydic, et al., The sulfolipids 2'-O-acyl-sulfoquinovosyldiacylglycerol and sulfoquinovosyldiacylglycerol are absent from a Chlamydomonas reinhardtii mutant deleted in SQD1, Plant Physiol. 133 (2003)
Please cite this article in press as: N. Sato, et al., Disturbance of cell-size determination by forced overproduction of sulfoquinovosyl diacylglycerol in the cyanobacterium Synechococcus elongatus PCC 7942, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/j.bbrc.2017.04.129
6
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864e874. [14] C. Bigogno, I. Khozin-Goldberg, S. Boussiba, et al., Lipid and fatty acid composition of the green oleaginous alga Parietochloris incisa, the richest plant source of arachidonic acid, Phytochemistry 60 (2002) 497e503. [15] B. Yu, C. Xu, C. Benning, Arabidopsis disrupted in SQD2 encoding sulfolipid synthesase is impaired in phosphate-limited growth, Proc. Natl. Acad. Sci. U. S. A. 99 (2002) 5732e5737. [16] M.A. Block, A.-J. Dorne, J. Joyard, R. Douce, Preparation and characterization of membrane fractions enriched in outer and inner envelope membranes from spinach chloroplasts, J. Biol. Chem. 258 (1983) 13281e13286. [17] K. Sugimoto, N. Sato, M. Tsuzuki, Utilization of a chloroplast membrane sulfolipid as a major internal sulfur source for protein synthesis in the early phase of sulfur starvation in Chlamydomonas reinhardtii, FEBS Lett. 581 (2007) 4519e4522. [18] P. Martin, B.A. Van Mooy, A. Heithoff, S.T. Dyhrman, Phosphorus supply drives rapid turnover of membrane phospholipids in the diatom Thalassiosira pseudonana, ISME J. 5 (2011) 1057e1060. [19] J.-P. Berge, J.-P. Gouygou, J.-P. Dubacq, P. Durand, Reassesment of lipid composition of the diatom, Skeletonema costatum, Phytochemistry 39 (1995) 1017e1021. [20] N. Sato, K. Sonoike, M. Tsuzuki, A. Kawaguchi, Impaired photosystem II in a mutant of Chlamydomonas reinhardtii defective in sulfoquinovosyl diacylglycerol, Eur. J. Biochem. 234 (1995) 16e23. [21] N. Sato, Roles of the acidic lipids sulfoquinovosyl diacylglycerol and phosphatidylglycerol in photosynthesis: their specificity and evolution, J. Plant Res. 117 (2004) 495e505. [22] M. Aoki, M. Tsuzuki, N. Sato, Involvement of sulfoquinovosyl diacylglycerol in DNA synthesis in Synechocystis sp. PCC 6803, BMC Res. Notes 5 (2012) 98. [23] N. Sato, S. Kobayashi, M. Aoki, et al., Identification of genes for sulfolipid synthesis in primitive red alga Cyanidioschyzon merolae, Biochem. Biophys.
Res. Commun. 470 (2016) 123e129. [24] S. Kobayashi, M. Tsuzuki, N. Sato, Sulfite-stress induced functional and structural changes in the complexes of photosystems I and II in a cyanobacterium, Synechococcus elongatus PCC 7942, Plant Cell Physiol. 56 (2015) 1521e1532. [25] P. Sae-Tang, Y. Hihara, I. Yumoto, et al., Overexpressed superoxide dismutase and catalase act synergistically to protect the repair of PSII during photoinhibition in Synechococcus elongatus PCC 7942, Plant Cell Physiol. 57 (2016) 1899e1907. [26] C.-H. Hung, K. Endo, K. Kobayashi, et al., Characterization of Chlamydomonas reinhardtii phosphatidylglycerophosphate synthase in Synechocystis sp. PCC 6803, Front. Microbiol. 6 (2015) 842. nez, G. Rivas, P. Schwille, Towards a bottom-up reconsti[27] A. Martos, M. Jime tution of bacterial cell division, Trends Cell Biol. 22 (2012) 634e643. [28] D.P. Haeusser, W. Margolin, Splitsville: structural and functional insights into the dynamic bacterial Z ring, Nat. Rev. Microbiol. 14 (2016) 305e319. [29] S. Mazor, T. Regev, E. Mileykovskaya, et al., Mutual effects of MinD-membrane interaction: I. Changes in the membrane properties induced by MinD binding, Biochim. Biophys. Acta 1778 (2008) 2496e2504. [30] H. Strahl, L.W. Hamoen, Membrane potential is important for bacterial cell division, Proc. Natl. Acad. Sci. U. S. A. 107 (2010) 12281e12286. [31] S. Miyagishima, C.P. Wolk, K.W. Osteryoung, Identification of cyanobacterial cell division genes by comparative and mutational analyses, Mol. Microbiol. 56 (2005) 126e143. [32] T. Mori, C.H. Johnson, Independence of circadian timing from cell division in cyanobacteria, J. Bacteriol. 183 (2001) 2439e2444. [33] J.S. MacCready, J. Schossau, K.W. Osteryoung, D.C. Ducat, Robust. Min-system oscillation in the presence of internal photosynthetic membranes in cyanobacteria, Mol. Microbiol. 103 (2017) 483e503.
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Disturbance of cell-size determination by forced overproduction of sulfoquinovosyl diacylglycerol in the cyanobacterium Synechococcus elongatus PCC 7942 Norihiro Sato a, *, Yuki Ebiya a, Ryutaro Kobayashi a, Yoshitaka Nishiyama b, Mikio Tsuzuki a a
School of Life Sciences, Tokyo University of Pharmacy and Life Sciences, Horinouchi, Hachioji, Tokyo 192-0392, Japan Department of Biochemistry and Molecular Biology, Graduate School of Science and Engineering, Saitama University, 255 Shimo-Okubo, Sakura-ku, Saitama 338-8570, Japan
b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 17 April 2017 Accepted 23 April 2017 Available online xxx
Sulfoquinovosyl diacylglycerol (SQDG) is present in the membranes of cyanobacteria or their descendants, plastids at species-dependent levels. We investigated the physiological significance of the intrinsic SQDG content in the cyanobacterium Synechococcus elongatus PCC 7942, with the use of its mutant, in which the genes for SQDG synthesis, sqdB and sqdX, were overexpressed. The mutant showed a 1.3-fold higher content of SQDG (23.6 mol% relative to total cellular lipids, cf., 17.1 mol% in the control strain) with much less remarkable effects on the other lipid classes. Simultaneously observed were 1.6- to 1.9-fold enhanced mRNA levels for the genes responsible for the synthesis of the lipids other than SQDG, as if to compensate for the SQDG overproduction. Meanwhile, the mutant showed no injury to cell growth, however, cell length was increased (6.1 ± 2.3, cf., 3.8 ± 0.8 mm in the control strain). Accordingly with this, a wide range of genes responsible for cell division were 1.6e2.4-fold more highly expressed in the mutant. These results suggested that a regulatory mechanism for lipid homeostasis functions in the mutant, and that SQDG has to be kept from surpassing the intrinsic content in S. elongatus for repression of the abnormal expression of cell division-related genes and, inevitably, for normal cell division. © 2017 Elsevier Inc. All rights reserved.
1. Introduction Sulfoquinovosyl diacylglycerol (SQDG) is present in oxygenic photosynthetic organisms from cyanobacteria to their postulated descendants, plastids of plants, and contributes to construction of their membrane systems including thylakoid membranes, together with two galactolipids, monogalactosyl diacylglycerol (MGDG) and digalactosyl diacylglycerol (DGDG), and a sole phospholipid, phosphatidylglycerol (PG) [1e3]. Exceptionally, SQDG, distinct from other lipid classes, is absent in Gloeobacter violaceus PCC 7421, a cyanobacterium, that is regarded as the most primitive of extant cyanobacteria based on the molecular phylogenetic analysis of the
Abbreviations: Chl a, chlorophyll a; DG, diacylglycerol; DGDG, digalactosyl diacylglycerol; MGDG, monogalactosyl diacylglycerol; PG, phosphatidylglycerol; PBS, phycobilisome; SQDG, sulfoquinovosyl diacylglycerol. * Corresponding author. School of Life Sciences, Tokyo University of Pharmacy and Life Sciences, Horinouchi 1432-1, Hachioji, Tokyo 192-0392, Japan. E-mail address: [email protected] (N. Sato).
16s rRNA gene sequence, and correspondingly possesses no thylakoid membrane with a photosynthetic electron transport system functioning in cell membranes [4e6]. SQDG thus seems to have appeared later during the evolution of cyanobacteria than the other three lipids did [7]. As regards the electrochemical properties of polar head groups, SQDG is similar to PG in the possession of a negatively charged head group, but is distinct from MGDG and DGDG that contain noncharged ones [1]. The content of SQDG relative to total lipids in cyanobacterial cells or to those in plant plastids depends on the species and environmental conditions even in the same species (Fig. 1). SQDG amounts to ca. 10e20 mol% in the cells of freshwater and/or coastal cyanobacteria such as Synechococcus elongatus PCC 7942 and Synechococcus sp. PCC 7002 [7e10], and to ca. 2 to >20 mol% in plastids of red and green algae, including C. reinhartdii [11e14], and in those of seed plants such as A. thaliana [15,16]. However, ambient stress conditions such as phosphorus (P)- or sulfur (S)-depletion cause changes in the SQDG content (Fig. 1): Pdepletion in S. elongatus PCC 7942, e.g., leads to an increase of the
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Please cite this article in press as: N. Sato, et al., Disturbance of cell-size determination by forced overproduction of sulfoquinovosyl diacylglycerol in the cyanobacterium Synechococcus elongatus PCC 7942, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/j.bbrc.2017.04.129
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C. reinhardtii, but not in S. elongatus, Synechococcus sp. PCC 7002, or A. thaliana [reviewed in 21]. An extreme observation was the essentiality of SQDG for cell growth in Synechocystis sp. PCC 6803, but not in other organisms including S. elongatus and Synechococcus sp. PCC 7002, which could be explained by the specific requirement of SQDG in Synechocystis among cyanobacteria for progression of the cell cycle, in particular, for DNA synthesis [8,22]. In each case above, however, the defective phenotype caused by the loss of SQDG synthesis only indicate whether or not the presence of SQDG is physiologically important, but does not lead to a comprehensive understanding of the physiological significance of the intrinsic SQDG content that depends on the species. In particular, no information has so far been obtained as to whether or not overproduction of SQDG has an impact on oxygenic photosynthetic organisms. In cyanobacteria, SQDG is synthesized through two successive reactions catalyzed by UDP-sulfoquinovose and SQDG synthases, which are coded by the sqdB and sqdX genes, respectively [1]. S. elongatus contains these two genes as a probable sqdBX operon [23]. In this study, we overexpressed sqdBX in S. elongatus for investigation of the effects of over-produced SQDG on the physiological processes in the cells.
Fig. 1. SQDG contents in cellular lipids of cyanobacteria and in plant plastid lipids. Species 1e7 are cyanobacteria (circles), whereas species 8 and 9, 10 and 11, 12 and 13, and 14 and 15 belong to red algae (squares), green algae (squares), diatoms (triangles), and seed plants (diamonds), respectively, as described below. 1, Synechocystis sp. PCC 6803 [8]. 2, S. elongatus [9]. 3, Trichodesmium erythraem [10]. 4, Synechococcus sp. PCC 7002 [7]. 5, Prochlorococcus MED4 [10]. 6, Synechococcus WH8113 [10]. 7, Synechococcus WH8101 [10]. 8, Polysiphonia lanosa [11]. 9, Cyanidioschyzon merolae [12]. 10, C. reinhardtii [13,17]. 11, Parietochloris incisa [14]. 12, Skeletonema costatum [19]. 13, Thalassiosira pseudonana [18]. 14, Arabidopsis thaliana [15]. 15, Spinacia oleracea [16]. Shown are the SQDG contents relative to total cellular lipids in cyanobacteria and those relative to total plastid lipids, i.e., the summed contents of MGDG, DGDG, SQDG, and PG. The SQDG contents relative to total plastid lipids were estimated on the basis of data concerning cellular lipid compositions in plants including algae, and on that of data concerning thylakoid membranes in S. oleracea. Open symbols, non-stressed conditions. Closed symbols, phosphate-limited conditions. Grey square, sulfurlimited conditions.
SQDG content from 10 to >20 mol% with a concomitant decrease of PG, which would reflect a decrease of the P-quota due to substitution of a non-phosphorus lipid, SQDG, for PG [9]. On the contrary, sulfur-starvation induces almost complete degradation of SQDG in C. reinhardtii with a concomitant increase of PG [17]. Meanwhile, absolute marine cyanobacteria including Synechococcus and Prochlorococcus species, or a marine diatom, Thalassiosira pseudonana, which inhabit oceans with severely limited P, generally accumulate SQDG to as high as >50 mol% in total lipids of cyanobacterial cells or in those of plastid ones (Fig. 1) [10,18]. These observations imply that the extent of the contribution of SQDG synthesis to the construction of membrane systems has become diversified depending on the species, e.g., on their respective prerequisites for ecophysiological adaptation. To answer the question of why SQDG has been conserved throughout the evolution of oxygenic photosynthetic organisms, the physiological roles of SQDG have been investigated in several species through the generation and characterization of mutants deficient in SQDG synthesis of cyanobacteria, S. elongatus [9], Synechocystis sp. PCC 6803 [8], and Synechococcus sp. PCC 7002 [7], a green alga, Chlamydomonas reinhardtii [20], and a seed plant, Arabidopsis thaliana [15]. Despite the evolutionary conservation of SQDG, it has been clarified that SQDG plays not common, but, if any, species-dependent roles in these organisms under normal growth conditions: as regards photosynthesis, SQDG is necessary for full functioning of the PSII complex in Synechocystis sp. PCC 6803 and
2. Materials and methods 2.1. Cyanobacterial strains and growth conditions The wild type (WT) cells of S. elongatus were cultured at 30 C in a tube containing BG11 medium, with illumination (10 W m2) and aeration, until the OD730 value of the culture became ca. 1.0 for transformation [24]. Meanwhile, BXOE and EMP cells (see below) were cultured under the same conditions, but with the exception of spectinomycine addition (20 mg ml1). The OD730 value, and the chlorophyll a (Chl a) and phycobilisome (PBS) contents in the cultures were measured with a spectrophotometer (DU 640, Beckman) [24]. The BXOE and EMP cells that were cultured until the OD730 value became ca. 1.0 were collected by centrifugation at 3000g for 15 min for storage at 80 C until use for lipid or mRNA analysis.
2.2. Transformation of S. elongatus A DNA fragment covering the coding regions of sqdB and sqdX, which are aligned in tandem in the genome of S. elongatus, was amplified by PCR, with KOD-Plus, primer set 1 (Table S1), and genomic DNA of S. elongatus as a template. The amplified DNA sequence of sqdB-sqdX was ligated to the SmaI site of the pAM1044 vector. The resultant plasmid (pBXOE) was introduced into cells of S. elongatus for integration of the sqdB-sqdX sequence into a neutral site in the genome through homologous recombination and its expression under the control of the strong constitutive promoter conII [25]. The transformant (BXOE) that gained the spectinomycine-resistance phenotype was selected. Otherwise, the empty vector was introduced into cells of S. elongatus, a transformant (EMP) that showed spectinomycine-resistance being generated.
2.3. Lipid analysis Lipid analysis was performed as previously described [24]. Total lipids were extracted from cells, and thereafter separated into individual lipid classes by TLC. The spots of individual lipid classes were used for the preparation of fatty acid methyl esters for analysis by capillary GLC.
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2.4. PCR analyses of transcripts Semi-quantitative reverse-transcriptase PCR was performed with RNA prepared from BXOE or EMP [24]. Primer sets 2e4 in Table S1 were used for amplification of the sqdB-, sqdX-, and sqdBXrelated sequences, respectively. The expression level of rnpB, the gene for a subunit of ribonuclease P, was examined with the use of primer set 5, as a control [24]. Meanwhile, RNA was also subjected to quantitative real-time (RT) PCR involving a Rotor-Gene Q PCR System (Qiagen) with primer sets 6e12 for the genes responsible for lipid synthesis, and with sets 13e25 for the genes responsible for cell division (Table S1). The expression was also investigated for the gene for the small subunit of ribulose 1,5- bisphosphate carboxylase/oxygenase (rbcS) with the use of primer set 26. The expression levels of the respective genes were normalized as to that of rnpB, with the use of primer set 27. 2.5. Microscopic analysis of cell morphology Cells were observed under a Nomarski microscope (BX53; Olympus, Tokyo, Japan) equipped with an image-capture camera (DP73; Olympus, Tokyo, Japan). Analysis of cell size was performed on the images with the use of an FV1000 viewer (Olympus, Tokyo, Japan). 3. Results and discussion 3.1. Effects of sqdBX overexpression in S. elongatus on lipid contents and lipid-synthesis gene expression A transformant (BXOE) was generated in S. elongatus through
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introduction of the sqdB-sqdX sequence under the control of conII promoter with the use of pAM1044 vector [25]. Otherwise, S. elongatus cells were transformed with the empty vector for the generation of a control strain (EMP). Semi-quantitative PCR analysis demonstrated that both sqdB and sqdX were more highly expressed in BXOE than in EMP (Fig. 2a). Similar results were obtained with the primer set for sqdBX, demonstrating that sqdB and sqdX definitely constitute an operon. More than 16-fold higher expression levels in BXOE were confirmed as to sqdB or sqdX through quantitative RT-PCR analysis (Fig. 2b). Accordingly with the stronger expression of sqdB and sqdX, i.e., that of sqdBX, the SQDG content became 1.3-fold higher in BXOE (23.6 mol% relative to total lipids on the basis of fatty acids) than in EMP (17.1 mol%, Fig. 2c and d). This higher SQDG content was compensated for by a 0.9-fold lower MGDG content in BXOE (Fig. 2d; 47.6 mol%, cf., 53.0 mol% in EMP). Meanwhile, neither DGDG nor PG was significantly affected (Fig. 2d). Collectively, we could genetically manipulate the SQDG content for its increase with little or less remarkable alteration in the other three lipid classes. The expression levels of two genes responsible for MGDG synthesis, i.e., mgdA for MGlcDG synthase that transfers the Glc moiety of UDP-Glc to diacylglycerol (DG) and mgdE for MGlcDG epimerase that converts MGlcDG into MGDG, were respectively 1.7-fold upregulated in BXOE, relative to in EMP (Fig. 2b). In line, the expression level of dgdA for DGDG synthase, which converts MGDG into DGDG through galactosylation, was 1.9-fold higher in BXOE (Fig. 2b). Meanwhile, pgsA for phosphatidylglycerophosphate synthase, which is responsible for PG synthesis, was 1.6-fold more highly expressed in BXOE (Fig. 2b). In view of probable competition of MgdA with SqdX for DG, the enhanced expression level of mgdA in BXOE would have been balanced with the overexpression of
Fig. 2. Effects of sqdBX overexpression on the expression levels of lipid synthesis genes and lipid composition in S. elongatus. (a) Semi-quantitative analysis of expression of sqdB, sqdX, and sqdBX at the transcript level in BXOE or EMP. Expression of rnpB mRNA is shown as an internal control. (b) The ratios of transcript levels of BXOE relative to EMP, as to the respective lipid synthesis genes, were estimated through quantitative RT-PCR analysis. The expression of the respective genes was normalized as to that of rnpB. The values are averages ± SE for three biological replicates. Note that BXOE was little affected in the transcript level of rbcS, one of the key proteins for photosynthesis, compatible with the little effect on cell growth. The genes of which the expression levels were evaluated to be significantly higher than that of rbcS with Student's t tests, are indicated by * (P < 0.1) or ** (P < 0.05). (c) Lipid profile revealed on TLC for BXOE or EMP. (d) Lipid composition of BXOE or EMP. White and grey bars indicate BXOE and EMP, respectively. The values are averages ± SD for three biological replicates.
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sqdBX, which, together with the higher expression levels of mgdE and dgdA, would have contributed to the synthesis of MGDG or DGDG at a level as close as possible to the intrinsic one. The higher expression level of pgsA in BXOE might have been partially responsible for the ongoing PG synthesis at a normal level even when sqdBX was overexpressed (Fig. 2b, d). In C. reinhardtii cells exposed to P-starvation, the increase of the SQDG content with a decrease in the PG content was accompanied by up- and downregulation of the expression levels of the SQDG and PG synthesis genes, respectively, which seemed to reflect the functioning of the regulatory mechanism for anionic lipid homeostasis at a genetic level [26]. In S. elongatus cells under normal conditions, however, the forced overexpression of SQDG synthesis genes had no impact as to positive down-regulation of the PG content or expression levels of PG synthesis genes (Fig. 2b, d). In view of the essentiality of PG for cyanobacterial cell growth [21], the ongoing PG synthesis seems to precede the balancing of total anionic lipid contents under normal environmental conditions. Collectively, a mutant of S. elongatus that possesses excess SQDG could be generated through overexpression of sqdBX, and was determined to probably utilize a genetic system for the maintenance of lipid homeostasis.
pattern of the OD730 value, or on those of the Chl and phycobilisome contents (Fig. 3a and b), similar to in the case of the SQDG-deficient DsqdB mutant of S. elongatus [8,9]. These results indicated that the content of SQDG, whether it is lower or higher than the intrinsic level, at least within the levels we examined, has little deleterious impact on the construction of the photosynthetic machinery, and inevitably on cell growth under normal conditions. At the same time, it might be interpreted that the regulatory mechanism for lipid homeostasis, as suggested above, allowed the BXOE cells to undergo vigorous growth. Meanwhile, microscopic analysis revealed that BXOE cells were distributed preferentially at larger sizes than EMP ones in the histogram of cell size (Fig. 3c and d). Inevitably, BXOE showed a greater cell length on average (6.1 ± 2.3 mm) than EMP did (3.8 ± 0.8 mm). These results suggested that SQDG overproduction at a certain level beyond its intrinsic one could disturb some mechanism for the determination of cell size in S. elongatus, although SQDG-loss hardly interferes with the mechanism [22].
3.2. Effects of the elevated SQDG content on cellular physiological processes
The elongated cell morphology of BXOE implied malfunctioning of the cell division machinery, which might be accompanied by abnormal expression of the related genes. The respective transcript levels of 13 genes that are responsible for the progress of cell division were then measured by RT-PCR analysis. Bacterial cell
Comparison of BXOE with EMP as to cell growth showed no deleterious effect of the elevated SQDG content on the increasing
3.3. Elevated expression levels of the genes responsible for cell division progress in BXOE
Fig. 3. Effects of sqdBX overexpression on cell growth and length in S. elongatus. Cell growth (a), and the Chl or PBS content (b) on the basis of the OD730 value in BXOE or EMP. Open and closed circles indicate EMP and BXOE, respectively. (c) Photographs of BXOE and EMP cells under a microscope. The bars show 5 mm. (d) Distribution of cell lengths in BXOE or EMP (n ¼ 300 for each strain). Open and closed bars indicate EMP and BXOE, respectively. The values in (a) and (b) are the averages ± SE for three biological replicates.
Please cite this article in press as: N. Sato, et al., Disturbance of cell-size determination by forced overproduction of sulfoquinovosyl diacylglycerol in the cyanobacterium Synechococcus elongatus PCC 7942, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/j.bbrc.2017.04.129
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Fig. 4. Effects of sqdBX overexpression on the expression levels of the genes for progress of the cell division in S. elongatus. The ratios of transcript levels of BXOE relative to EMP, as to the respective genes responsible for cell division, were estimated through quantitative RT-PCR analysis, as explained in Fig. 2b. Two homologs of ftsW, Synpcc7942_0324 and Synpcc7942_1104, are denoted as 0324 and 1104, respectively. The values are averages ± SE for three biological replicates. The value of rbcS is the same as that in Fig. 2b. The genes of which the expression levels were evaluated to be significantly higher than that of rbcS with Student's t tests, were marked by * (P < 0.1) or ** (P < 0.05).
division is driven by the multi-protein machinery through the formation and constriction of the division ring at mid-cell [27,28]. The FtsZ, Q, and W proteins participate in the construction of a proto-ring, transduction of signals from the periplasm to the Z ring for the coordination of its constriction, and septal peptideglycan synthesis, respectively, through the progression of cell division. Meanwhile, MinC-E, and MinC/D and DivIVA comprise the system that regulates the positioning of Z ring formation in Escherichia coli and Bacillus subtilis, respectively. Eleven genes were found to be more highly expressed in BXOE (1.6e2.4-fold), relative to in EMP, comprising cdv3 (divIVA-like gene), cikA, ftn6, ftsQ/W/Z, and minC-E, the exception being cdv1/2 (Fig. 4). Meanwhile, dnaA is responsible for the initiation of DNA replication, which is one of the prerequisites for the progress of cell division, its expression level also having being 1.9-fold higher in BXOE than in EMP (Fig. 4). These observations suggested that SQDG overproduction disturbed the expression of a large member of the genes that are responsible for cell division. Anionic phospholipids or the membrane potential in cell membranes is crucial for some protein members of the cell division machinery such as MinD to bind to the membranes [29,30]. The membrane lipids of cyanobacteria participate predominantly in the construction of thylakoid membranes, and also in that of cell membranes, although to a much lesser extent. The observed difference in lipid composition between BXOE and EMP (Fig. 2d) should thus more directly reflect that in thylakoid membranes. However, it is highly probable that cell membranes of BXOE possess more abundant SQDG than those of EMP. Meanwhile, in S. elongatus, cell length became greater through overexpression of ftsZ, minC, minD or minE, besides through disruption of cdv1, cdv2, cdv3, cikA, ftn2 or ftn6 [31e33]. One possible scenario is as follows: in BXOE, this higher content of cell-membranous SQDG, which is anionic like the cyanobacterial sole phospholipid PG, might interfere with the localization pattern or functionality of MinD, and those of FtsW and FtsQ as well, which should also be localized in cell membranes [27]. Such perturbation of the protein components of the cell division machinery might cause the abnormal construction or functionality of the machinery that consequently elevates the expression levels of a wide range of cell-division related genes including ftsZ and minC-E, the cell length eventually being increased (Fig. 4). In any case, we succeeded in increasing the SQDG content in photosynthetic organisms, and clarified that the content has to be kept from surpassing the intrinsic level.
Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.bbrc.2017.04.129. Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.bbrc.2017.04.129. References [1] N. Sato, H. Wada, Lipid biosynthesis and its regulation in cyanobacteria, in: H. Wada, N. Murata (Eds.), Lipids in Photosynthesis, Essential and Regulatory Functions, Springer, Dordrecht, 2009, pp. 157e177. [2] M. Shimojima, H. Ohta, Y. Nakamura, Biosynthesis and function of chloroplast lipids, in: H. Wada, N. Murata (Eds.), Lipids in Photosynthesis, Essential and Regulatory Functions, Springer, Dordrecht, 2009, pp. 35e55. [3] R. Goss, C. Wilhelm, Lipids in algae, lichens and mosses, in: H. Wada, N. Murata (Eds.), Lipids in Photosynthesis, Essential and Regulatory Functions, Springer, Dordrecht, 2009, pp. 117e135. [4] E. Selstam, D. Campbell, Membrane lipid composition of the unusual cyanobacterium Gloeobacter violaceus sp. PCC 7421, which lacks sulfoquinovosyl diacylglycerol, Arch. Microbiol. 166 (1996) 132e135. [5] G. Guglielmi, G. Cohen-Bazire, D.A. Bryant, The structure of Gloeobacter violaceus and its phycobilisomes, Arch. Microbiol. 129 (1981) 181e189. [6] D. Honda, A. Yokota, J. Sugiyama, Detection of seven major evolutionary lineages in cyanobacteria based on the 16S rRNA gene sequence analysis with new sequences of five marine Synechococcus strains, J. Mol. Evol. 48 (1999) 723e739. [7] N. Sato, R. Kamimura, M. Tsuzuki, Dispensability of a sulfolipid for photoautotrophic cell growth and photosynthesis in a marine cyanobacterium, Synechococcus sp. PCC 7002, Biochem. Biophys. Res. Commun. 477 (2016) 854e860. [8] M. Aoki, N. Sato, A. Meguro, M. Tsuzuki, Differing involvement of sulfoquinovosyl diacylglycerol in photosystem II in two species of unicellular cyanobacteria, Eur. J. Biochem. 271 (2004) 685e693. €rtel, et al., A null mutant of Synechococcus sp. [9] S. Güler, A. Seeliger, H. Ha PCC7942 deficient in the sulfolipid sulfoquinovosyl diacylglycerol, J. Biol. Chem. 271 (1996) 7501e7507. [10] B.A. Van Mooy, G. Rocap, H.F. Fredricks, et al., Sulfolipids dramatically decrease phosphorus demand by picocyanobacteria in oligotrophic marine environments, Proc. Natl. Acad. Sci. U. S. A. 103 (2006) 8607e8612. [11] T.R. Pettitt, A.L. Jones, J.L. Harwood, Lipid metabolism in the red marine algae Chondrus crispus and Polysiphonia lanosa as modified by temperature, Phytochemistry 28 (1989) 2053e2058. [12] N. Sato, T. Moriyama, Genomic and biochemical analysis of lipid biosynthesis in the unicellular rhodophyte Cyanidioschyzon merolae: lack of a plastidic desaturation pathway results in the coupled pathway of galactolipid synthesis, Eukaryot. Cell 6 (2007) 1006e1017. [13] W.R. Riekhof, M.E. Ruckle, T.A. Lydic, et al., The sulfolipids 2'-O-acyl-sulfoquinovosyldiacylglycerol and sulfoquinovosyldiacylglycerol are absent from a Chlamydomonas reinhardtii mutant deleted in SQD1, Plant Physiol. 133 (2003)
Please cite this article in press as: N. Sato, et al., Disturbance of cell-size determination by forced overproduction of sulfoquinovosyl diacylglycerol in the cyanobacterium Synechococcus elongatus PCC 7942, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/j.bbrc.2017.04.129
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N. Sato et al. / Biochemical and Biophysical Research Communications xxx (2017) 1e6
864e874. [14] C. Bigogno, I. Khozin-Goldberg, S. Boussiba, et al., Lipid and fatty acid composition of the green oleaginous alga Parietochloris incisa, the richest plant source of arachidonic acid, Phytochemistry 60 (2002) 497e503. [15] B. Yu, C. Xu, C. Benning, Arabidopsis disrupted in SQD2 encoding sulfolipid synthesase is impaired in phosphate-limited growth, Proc. Natl. Acad. Sci. U. S. A. 99 (2002) 5732e5737. [16] M.A. Block, A.-J. Dorne, J. Joyard, R. Douce, Preparation and characterization of membrane fractions enriched in outer and inner envelope membranes from spinach chloroplasts, J. Biol. Chem. 258 (1983) 13281e13286. [17] K. Sugimoto, N. Sato, M. Tsuzuki, Utilization of a chloroplast membrane sulfolipid as a major internal sulfur source for protein synthesis in the early phase of sulfur starvation in Chlamydomonas reinhardtii, FEBS Lett. 581 (2007) 4519e4522. [18] P. Martin, B.A. Van Mooy, A. Heithoff, S.T. Dyhrman, Phosphorus supply drives rapid turnover of membrane phospholipids in the diatom Thalassiosira pseudonana, ISME J. 5 (2011) 1057e1060. [19] J.-P. Berge, J.-P. Gouygou, J.-P. Dubacq, P. Durand, Reassesment of lipid composition of the diatom, Skeletonema costatum, Phytochemistry 39 (1995) 1017e1021. [20] N. Sato, K. Sonoike, M. Tsuzuki, A. Kawaguchi, Impaired photosystem II in a mutant of Chlamydomonas reinhardtii defective in sulfoquinovosyl diacylglycerol, Eur. J. Biochem. 234 (1995) 16e23. [21] N. Sato, Roles of the acidic lipids sulfoquinovosyl diacylglycerol and phosphatidylglycerol in photosynthesis: their specificity and evolution, J. Plant Res. 117 (2004) 495e505. [22] M. Aoki, M. Tsuzuki, N. Sato, Involvement of sulfoquinovosyl diacylglycerol in DNA synthesis in Synechocystis sp. PCC 6803, BMC Res. Notes 5 (2012) 98. [23] N. Sato, S. Kobayashi, M. Aoki, et al., Identification of genes for sulfolipid synthesis in primitive red alga Cyanidioschyzon merolae, Biochem. Biophys.
Res. Commun. 470 (2016) 123e129. [24] S. Kobayashi, M. Tsuzuki, N. Sato, Sulfite-stress induced functional and structural changes in the complexes of photosystems I and II in a cyanobacterium, Synechococcus elongatus PCC 7942, Plant Cell Physiol. 56 (2015) 1521e1532. [25] P. Sae-Tang, Y. Hihara, I. Yumoto, et al., Overexpressed superoxide dismutase and catalase act synergistically to protect the repair of PSII during photoinhibition in Synechococcus elongatus PCC 7942, Plant Cell Physiol. 57 (2016) 1899e1907. [26] C.-H. Hung, K. Endo, K. Kobayashi, et al., Characterization of Chlamydomonas reinhardtii phosphatidylglycerophosphate synthase in Synechocystis sp. PCC 6803, Front. Microbiol. 6 (2015) 842. nez, G. Rivas, P. Schwille, Towards a bottom-up reconsti[27] A. Martos, M. Jime tution of bacterial cell division, Trends Cell Biol. 22 (2012) 634e643. [28] D.P. Haeusser, W. Margolin, Splitsville: structural and functional insights into the dynamic bacterial Z ring, Nat. Rev. Microbiol. 14 (2016) 305e319. [29] S. Mazor, T. Regev, E. Mileykovskaya, et al., Mutual effects of MinD-membrane interaction: I. Changes in the membrane properties induced by MinD binding, Biochim. Biophys. Acta 1778 (2008) 2496e2504. [30] H. Strahl, L.W. Hamoen, Membrane potential is important for bacterial cell division, Proc. Natl. Acad. Sci. U. S. A. 107 (2010) 12281e12286. [31] S. Miyagishima, C.P. Wolk, K.W. Osteryoung, Identification of cyanobacterial cell division genes by comparative and mutational analyses, Mol. Microbiol. 56 (2005) 126e143. [32] T. Mori, C.H. Johnson, Independence of circadian timing from cell division in cyanobacteria, J. Bacteriol. 183 (2001) 2439e2444. [33] J.S. MacCready, J. Schossau, K.W. Osteryoung, D.C. Ducat, Robust. Min-system oscillation in the presence of internal photosynthetic membranes in cyanobacteria, Mol. Microbiol. 103 (2017) 483e503.
Please cite this article in press as: N. Sato, et al., Disturbance of cell-size determination by forced overproduction of sulfoquinovosyl diacylglycerol in the cyanobacterium Synechococcus elongatus PCC 7942, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/j.bbrc.2017.04.129