DesC1 and DesC2, Δ9 Fatty Acid Desaturases of Filamentous Cyanobacteria: Essentiality and Complementarity.

DesC1 and DesC2, which are fatty acid desaturases found in cyanobacteria, are responsible for introducing a double bond at the Δ9 position of fatty-acyl chains, which are subsequently esterified to the sn-1 and sn-2 positions of the glycerol moiety, respectively. However, since the discovery of these two desaturases in the Antarctic cyanobacterium Nostoc sp. SO-36, no further research has been reported. This study presents a comprehensive characterization of DesC1 and DesC2 through targeted mutagenesis and transformation using two cyanobacteria strains: Anabaena sp. PCC 7120, comprising both desaturases, and Synechocystis sp. PCC 6803, containing a single Δ9 desaturase (hereafter referred to as DesCs) sharing similarity with DesC1 in amino acid sequence. The results suggested that both DesC1 and DesC2 were essential in Anabaena sp. PCC 7120 and that DesC1, but not DesC2, complemented DesCs in Synechocystis sp. PCC 6803. In addition, DesC2 from Anabaena sp. PCC 7120 desaturated fatty acids esterified to the sn-2 position of the glycerol moiety in Synechocystis sp. PCC 6803.

Possible involvement of extracellular polymeric substrates of Antarctic cyanobacterium Nostoc sp. strain SO-36 in adaptation to harsh environments.

Cyanobacteria are some of the primary producers in extremely cold biospheres such as the Arctic, Antarctic, and vast ice sheets. Many genera of cyanobacteria are identified from these harsh environments, but their specific mechanisms for cold adaptation are not fully understood. Nostoc sp. strain SO-36 is a cyanobacterium isolated in Antarctica more than 30 years ago and regarded as a psychrotolelant species. To determine whether the strain is psychrotolelant or psychrophilic, it was first grown at 30 °C and 10 °C. The cells grew exponentially at 30 °C, but their growth stopped at 10 °C, indicating that the strain is only psychrotolerant. Microscopic analysis revealed that the morphology of the cells grown at 30 °C was filamentous and differentiated heterocysts, which are specialized cells for gaseous nitrogen fixation under nitrogen-deprived conditions, indicating that the strain can grow diazotrophically. The cells grown at 10 °C have a smaller size, shortened filament length and decreased chlorophyll content per cell. At 10 °C, the cells are aggregated with extracellular polymeric substrates (EPSs), which is a common mechanism to protect cells from ultraviolet light. These results imply that segmentation into short filaments was induced by photodamage at low temperatures. To fully understand the adaptation mechanisms of Nostoc sp. strain SO-36 for low-temperature conditions, next-generation sequencing analyses were conducted. Complete genome sequence of the strain revealed that it has one main chromosome of approximately 6.8 Mbp with 4 plasmids, including 6855 coding sequences, 48 tRNA genes, 4 copies of rRNA operons, and 5 CRISPR regions. Putative genes for EPS biosynthesis were found to be conserved in Nostocaceae regardless of their habitat. These results provide basic information to understand the adaptation mechanisms at low temperatures, and the strain can be a model organism to analyze adaptation to extreme environments.

Complete Replacement of the Galactolipid Biosynthesis Pathway with a Plant-Type Pathway in the Cyanobacterium Synechococcus elongatus PCC 7942.

Monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) are the major components of thylakoid membranes and well-conserved from cyanobacteria to chloroplasts. However, cyanobacteria and chloroplasts synthesize these galactolipids using different pathways and enzymes, but they are believed to share a common ancestor. This fact implies that there was a replacement of the cyanobacterial galactolipid biosynthesis pathway during the evolution of a chloroplast. In this study, we first replaced the cyanobacterial MGDG biosynthesis pathway in a model cyanobacterium, Synechococcus elongatus PCC 7942, with the corresponding plant-type pathway. No obvious phenotype was observed under the optimum growth condition, and the content of membrane lipids was not largely altered in the transformants. We next replaced the cyanobacterial DGDG biosynthesis pathway with the corresponding plant-type pathway using the strain described above and isolated the strain harboring the replaced plant-type pathway instead of the whole galactolipid biosynthesis pathway. This transformant, SeGPT, can grow photoautotrophically, indicating that cyanobacterial galactolipid biosynthesis pathways can be functionally complemented by the corresponding plant-type pathways and that the lipid products MGDG and DGDG, and not biosynthesis pathways, are important. While SeGPT does not show strong growth retardation, the strain has low cellular chlorophyll content but it retained a similar oxygen evolution rate per chlorophyll content compared with the wild type. An increase in total membrane lipid content was observed in SeGPT, which was caused by a significant increase in DGDG content. SeGPT accumulated carotenoids from the xanthophyll groups. These results suggest that cyanobacteria have the capacity to accept other pathways to synthesize essential components of thylakoid membranes.

Adaptations in chloroplast membrane lipid synthesis from synthesis in ancestral cyanobacterial endosymbionts.

Cyanobacteria and chloroplasts are believed to share a common ancestor, but synthetic pathways for membrane lipids are different. Lyso-phosphatidic acid (lyso-PA) is the precursor for the synthesis of all membrane lipids and synthesized by an acyl-ACP dependent glycerol-3-phosphate acyltransferase (GPAT) in chloroplasts. In cyanobacteria, GPAT genes are not found and, instead, genes coding for enzymes in the acyl-phosphate dependent lyso-PA synthetic pathway (plsX and plsY) are conserved. We report that the PlsX/Y dependent lyso-PA synthetic pathway is essential in cyanobacteria, but can be replaced by acyl-ACP dependent GPAT from Escherichia coli (plsB) and Arabidopsis thaliana (ATS1). Cyanobacteria thus display the capacity to accept enzymes from other organisms to synthesize essential components. This ability may have enabled them to evolve into current chloroplasts from their ancestral origins.

CyanoBase: a large-scale update on its 20th anniversary.

The first ever cyanobacterial genome sequence was determined two decades ago and CyanoBase (http://genome.microbedb.jp/cyanobase), the first database for cyanobacteria was simultaneously developed to allow this genomic information to be used more efficiently. Since then, CyanoBase has constantly been extended and has received several updates. Here, we describe a new large-scale update of the database, which coincides with its 20th anniversary. We have expanded the number of cyanobacterial genomic sequences from 39 to 376 species, which consists of 86 complete and 290 draft genomes. We have also optimized the user interface for large genomic data to include the use of semantic web technologies and JBrowse and have extended community-based reannotation resources through the re-annotation of Synechocystis sp. PCC 6803 by the cyanobacterial research community. These updates have markedly improved CyanoBase, providing cyanobacterial genome annotations as references for cyanobacterial research.

Sulfur-Containing Carotenoids from A Marine Coral Symbiont Erythrobacter flavus Strain KJ5.

Erythrobacter flavus strain KJ5 (formerly called Erythrobacter sp. strain KJ5) is a yellowish marine bacterium that was isolated from a hard coral Acropora nasuta in the Karimunjawa Islands, Indonesia. The complete genome sequence of the bacterium has been reported recently. In this study, we examined the carotenoid composition of this bacterium using high-performance liquid chromatography coupled with ESI-MS/MS. We found that the bacterium produced sulfur-containing carotenoids, i.e., caloxanthin sulfate and nostoxanthin sulfate, as the most abundant carotenoids. A new carotenoid zeaxanthin sulfate was detected based on its ESI-MS/MS spectrum. The unique presence of sulfated carotenoids found among the currently known species of the Erythrobacter genus were discussed.

Digalactosyldiacylglycerol is essential in Synechococcus elongatus PCC 7942, but its function does not depend on its biosynthetic pathway.

Digalactosyldiacylglycerol (DGDG) is a major component of thylakoid membranes, occupying approximately 20% of the membrane system. This lipid composition is conserved from cyanobacteria to the chloroplasts of terrestrial plants, suggesting that DGDG is important for the function of photosynthetic membranes. Here we isolated the gene for DGDG synthase in the cyanobacterium Synechococcus elongatus PCC 7942 (7942dgdA) and found that this gene is essential for this species. 7942dgdA could be knocked out only when genes for cyanobacterial or plant DGDG synthases were expressed, indicating that the important factor was not the specific synthetic pathway but the lipid product. Lack of DGDG could not be compensated by the other membrane lipids in S. elongatus PCC 7942 or by glucosylgalactosyldiacylglycerol synthesized by the ß-GlcT gene of Chloroflexus aurantiacus. These results reveal that DGDG has an indispensable role in S. elongatus PCC 7942 and that the second galactose molecule is key. Conservation and distribution of the galactolipid synthetic pathway among oxygenic phototrophs is discussed. This article is part of a Special Issue entitled: Plant Lipid Biology edited by Kent D. Chapman and Ivo Feussner.

Thylakoid Development and Galactolipid Synthesis in Cyanobacteria.

Cyanobacteria carry out oxygenic photosynthesis and share many features with chloroplasts, including thylakoid membranes, which are mainly composed of membrane lipids and protein complexes that mediate photosynthetic electron transport. Although the functions of the various thylakoid protein complexes have been well characterized, the details underlying the biogenesis of thylakoid membranes remain unclear. Galactolipids are the major constituents of the thylakoid membrane system, and all the genes involved in galactolipid biosynthesis were recently identified. In this chapter, I summarize recent advances in our understanding of the factors involved in thylakoid development, including regulatory proteins and enzymes that mediate lipid biosynthesis.

Oxygenic photosynthesis without galactolipids.

The thylakoid membranes of oxygenic photosynthetic organisms are dominated by the galactolipids monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG). In cyanobacteria, MGDG is synthesized via monoglucosyldiacylglycerol (GlcDG). However, the putative epimerase involved in the conversion of GlcDG to MGDG has not been identified. Here we report the identification of the gene for the glucolipid epimerase (mgdE) by comparative genomic analysis. Knockout mutants of mgdE in Synechocystis sp. PCC 6803 lacked both MGDG and DGDG and accumulated GlcDG. The mutants did possess thylakoid membranes and showed normal maximal photosynthetic activity, albeit with reduced utilization of light energy. These results cast doubt on the long-standing belief that oxygenic photosynthesis is absolutely dependent on galactolipids.

Fatty alcohols can complement functions of heterocyst specific glycolipids in Anabaena sp. PCC 7120.

Heterocyst glycolipid synthase (HglT) catalyzes the final step of heterocyst glycolipid (Hgl) biosynthesis, in which a glucose is transferred to the aglycone (fatty alcohol). Here we describe the isolation of hglT null mutants. These mutants lacked Hgls under nitrogen-starved conditions and instead accumulated fatty alcohols. Differentiated heterocyst cells in the mutants were morphologically indistinguishable from those of the wild-type cells. Interestingly, the mutants grew under nitrogen starvation but fixed nitrogen with lower nitrogenase activity than did the wild-type. The mutants had a pale green phenotype with a decreased chlorophyll content, especially under nitrogen-starved conditions. These results suggest that the glucose moiety of the Hgls may be necessary for optimal protection against oxygen influx but is not essential and that aglycones can function as barriers against oxygen influx in the heterocyst cells.

Changes in intracellular energetic and metabolite states due to increased galactolipid levels in Synechococcus elongatus PCC 7942.

The lipid composition of thylakoid membranes is conserved from cyanobacteria to green plants. However, the biosynthetic pathways of galactolipids, the major components of thylakoid membranes, are known to differ substantially between cyanobacteria and green plants. We previously reported on a transformant of the unicellular rod-shaped cyanobacterium Synechococcus elongatus PCC 7942, namely SeGPT, in which the synthesis pathways of the galactolipids monogalactosyldiacylglycerol and digalactosyldiacylglycerol are completely replaced by those of green plants. SeGPT exhibited increased galactolipid content and could grow photoautotrophically, but its growth rate was slower than that of wild-type S. elongatus PCC 7942. In the present study, we investigated pleiotropic effects that occur in SeGPT and determined how its increased lipid content affects cell proliferation. Microscopic observations revealed that cell division and thylakoid membrane development are impaired in SeGPT. Furthermore, physiological analyses indicated that the bioenergetic state of SeGPT is altered toward energy storage, as indicated by increased levels of intracellular ATP and glycogen. We hereby report that we have identified a new promising candidate as a platform for material production by modifying the lipid synthesis system in this way.

Methods of Lipid Analyses for Microalgae: Charophytes, Eustigmatophytes, and Euglenophytes.

Algae are ecologically important organisms and are widely used for basic research, with a focus on for example photosynthesis, evolution, and lipid metabolism. Many biosynthetic pathways of algal lipids have been deciphered using available genomic information. Here we describe methods for lipid analyses from three representative algae, including Archaeplastida, the SAR lineage (Stramenopiles, Alveolata, Rhizaria), and Excavata. Archaeplastida acquired their plastids by primary endosymbiosis, and the others by secondary endosymbiosis with a Rhodophyceae-type plastid in SAR and a Chlorophyceae-type plastid in Excavata (Euglenozoa). Analytical methods for these algae are described for membrane lipids and neutral lipids including triacylglycerol and wax esters.

Type-B monogalactosyldiacylglycerol synthases are involved in phosphate starvation-induced lipid remodeling, and are crucial for low-phosphate adaptation.

Mono- and digalactosyldiacylglycerol (MGDG and DGDG, respectively) constitute the bulk of membrane lipids in plant chloroplasts. Mutant analyses in Arabidopsis have shown that these galactolipids are essential for chloroplast biogenesis and photoautotrophic growth. Moreover, these non-phosphorous lipids are proposed to participate in low-phosphate (Pi) adaptations. Under Pi-limited conditions, a drastic accumulation of DGDG occurs concomitantly with a large reduction in membrane phospholipids, suggesting that plants substitute DGDG for phospholipids during Pi starvation. Previously, we reported that among the three MGDG synthase genes (MGD1, MGD2 and MGD3), the type-B MGD2 and MGD3 are upregulated in parallel with DGDG synthase genes during Pi starvation. Here, we describe the identification and characterization of T-DNA insertional mutants of Arabidopsis type-B MGD genes. Under Pi-starved conditions, the mgd3-1 mutant showed a drastic reduction in DGDG accumulation, particularly in the root, indicating that MGD3 is the main isoform responsible for DGDG biosynthesis in Pi-starved roots. Moreover, in the roots of mgd2 mgd3 plants, Pi stress-induced accumulation of DGDG was almost fully abolished, showing that type-B MGD enzymes are essential for membrane lipid remodeling in Pi-starved roots. Reductions in fresh weight, root growth and photosynthetic performance were also observed in these mutants under Pi-starved conditions. These results demonstrate that Pi stress-induced membrane lipid remodeling is important in plant growth during Pi starvation. The widespread distribution of type-B MGD genes in land plants suggests that membrane lipid remodeling mediated by type-B MGD enzymes is a potent adaptation to Pi deficiency for land plants.

A polyketide synthase HglEA, but not HglE2, synthesizes heterocyst specific glycolipids in Anabaena sp. PCC 7120.

Heterocysts are the specialized cells for nitrogen fixation in some filamentous cyanobacteria. To protect the oxygen labile nitrogen fixing enzyme, nitrogenase, heterocysts keep their inner environment microoxic by developing layers of barrier on the outside of their outer membranes. Heterocyst specific glycolipids (Hgls) are constituents of the layer of barrier and amphipathic compounds, synthesized from a very long chain fatty alcohol as a hydrophobic tail and a sugar as a polar head. In the model heterocystous cyanobacterium Anabaena sp. PCC 7120, Hgls are made of fatty alcohol with 26 carbons and a glucose, linked by an ether bond in alpha configuration. The fatty alcohol is synthesized via reactions of a polyketide synthase, HglEA. In Anabaena sp. PCC 7120, another polyketide synthase HglE2 shared more than 50% identity in an amino acid sequence with HglEA and is expected to be involved in Hgls synthesis. However, no direct evidence has been reported. Here, we experimentally show that HglEA is the contributor of Hgls synthesis, and that HglE2 is not involved in the development of the heterocyst specific glycolipid layer.

hetN and patS Mutations Enhance Accumulation of Fatty Alcohols in the hglT Mutants of Anabaena sp. PCC 7120.

The heterocysts present in filamentous cyanobacteria such as Anabaena sp. PCC 7120 are known to be regulated by HetN and PatS, the repressors of heterocyst differentiation; therefore, the inactivation of these proteins will result in the formation of multiple heterocysts. To enhance the accumulation of fatty alcohols synthesized in the heterocyst, we introduced mutations of these repressors to increase heterocyst frequency. First, we isolated double mutants of hetN and patS and confirmed that the null mutation of these genes promoted higher frequencies of heterocyst formation and higher accumulation of heterocyst-specific glycolipids (Hgls) compared with its wild type. Next, we combined hetN and patS mutations with an hglT (encoding glycosyltransferase, an enzyme involved in Hgl synthesis) mutation to increase the accumulation of fatty alcohols since knockout mutation of hglT results in accumulation of very long chain fatty alcohol, the precursor of Hgl. We also observed retarded growth, lower chlorophyll content and up to a five-fold decrease in photosynthetic activity of the hetN/patS/hglT triple mutants. In contrast, the triple mutants showed three times higher heterocyst formation frequencies than the hglT single mutant and wild type. The production rate of fatty alcohol in the triple mutants attained a value 1.41 nmol/mL OD730, whereas accumulation of Hgls in the wild type was 0.90 nmol/mL OD730. Aeration of culture improved the accumulation of fatty alcohols in hetN/patS/hglT mutant cells up to 2.97 nmol/mL OD730 compared with cells cultured by rotation. Our study outlines an alternative strategy for fatty alcohol production supported by photosynthesis and nitrogen fixation.

Relationship Between Glycerolipids and Photosynthetic Components During Recovery of Thylakoid Membranes From Nitrogen Starvation-Induced Attenuation in Synechocystis sp. PCC 6803.

Thylakoid membranes, the site of photochemical and electron transport reactions of oxygenic photosynthesis, are composed of a myriad of proteins, cofactors including pigments, and glycerolipids. In the non-diazotrophic cyanobacterium Synechocystis sp. PCC 6803, the size and function of thylakoid membranes are reduced under nitrogen (N) starvation but are quickly recovered after N addition to the starved cells. To understand how the functionality of thylakoid membranes is adjusted in response to N status in Synechocystis sp. PCC 6803, we examined changes in thylakoid components and the photosynthetic activity during the N starvation and recovery processes. In N-starved cells, phycobilisome content, photosystem II protein levels and the photosynthetic activity substantially decreased as compared with those in N-sufficient cells. Although the content of chlorophyll (Chl) a, total protein and total glycerolipid also decreased under the N-starved condition based on OD730 reflecting cell density, when based on culture volume, the Chl a and total protein content remained almost constant and total glycerolipid content even increased during N starvation, suggesting that cellular levels of these components decrease under the N-starved condition mainly through dilution due to cell growth. With N addition, the photosynthetic activity quickly recovered, followed by full restoration of photosynthetic pigment and protein levels. The content of phosphatidylglycerol (PG), an essential lipid constituent of both photosystems, increased faster than that of Chl a, whereas the content of glycolipids, the main constituents of the thylakoid lipid bilayer, gradually recovered after N addition. The data indicate differential regulation of PG and glycolipids during the construction of the photosynthetic machinery and regeneration of thylakoid membranes. Of note, addition of PG to the growth medium slightly accelerated the Chl a accumulation in wild-type cells during the recovery process. Because PG is required for the biosynthesis of Chl a and the formation of functional photosystem complexes, rapid PG biosynthesis in response to N acquisition may be required for the rapid formation of the photosynthetic machinery during thylakoid regeneration.

Isolation and characterization of a tandem-repeated cysteine protease from the symbiotic dinoflagellate Symbiodinium sp. KB8.

A cysteine protease belonging to peptidase C1A superfamily from the eukaryotic, symbiotic dinoflagellate, Symbiodinium sp. strain KB8, was characterized. The protease was purified to near homogeneity (566-fold) by (NH4)2SO4 fractionation, ultrafiltration, and column chromatography using a fluorescent peptide, butyloxycarbonyl-Val-Leu-Lys-4-methylcoumaryl-7-amide (Boc-VLK-MCA), as a substrate for assay purposes. The enzyme was termed VLKP (VLK protease), and its activity was strongly inhibited by cysteine protease inhibitors and activated by reducing agents. Based on the results for the amino acid sequence determined by liquid chromatography-coupled tandem mass spectrometry, a cDNA encoding VLKP was synthesized. VLKP was classified into the peptidase C1A superfamily of cysteine proteases (C1AP). The predicted amino acid sequence of VLKP indicated a tandem array of highly conserved precursors of C1AP with a molecular mass of approximately 71 kDa. The results of gel-filtration chromatography and SDS-PAGE suggested that VLKP exists as a monomer of 31-32 kDa, indicating that the tandem array is likely divided into two mass-equivalent halves that undergo equivalent posttranslational modifications. The VLKP precursor contains an inhibitor prodomain that might become activated after acidic autoprocessing at approximately pH 4. Both purified and recombinant VLKPs had a similar substrate specificity and kinetic parameters for common C1AP substrates. Most C1APs reside in acidic organelles such as the vacuole and lysosomes, and indeed VLKP was most active at pH 4.5. Since VLKP exhibited maximum activity during the late logarithmic growth phase, these attributes suggest that, VLKP is involved in the metabolism of proteins in acidic organelles.

Complete Genome Sequence of the Marine Bacterium Erythrobacter flavus Strain KJ5.

Erythrobacter flavus strain KJ5 (formerly called Erythrobacter sp. strain KJ5) is a yellowish marine bacterium that was isolated from a hard coral in the Karimunjawa Islands of Indonesia. Here, we report the complete genome sequence of the bacterium and provide a useful resource for studies of the biosynthetic pathways of its unique carotenoids.

Non-vesicular and vesicular lipid trafficking involving plastids.

In plants, newly synthesized fatty acids are either directly incorporated into glycerolipids in the plastid or exported and assembled into lipids at the endoplasmic reticulum (ER). ER-derived glycerolipids serve as building blocks for extraplastidic membranes. Alternatively, they can return to the plastid where their diacylglycerol backbone is incorporated into the glycerolipids of the photosynthetic membranes, the thylakoids. Thylakoid lipids are assembled at the plastid envelope membranes and are transferred to the thylakoids. Under phosphate-limited growth conditions, galactolipids are exported from the outer plastid envelope membranes to extraplastidic membranes. Proteins, such as TRIGALACTOSYLDIACYLGLYCEROL1 (TGD1) or VESICLE-INDUCING PROTEIN IN PLASTIDS1 (VIPP1), which are involved in different aspects of plastid lipid trafficking phenomena have recently been identified and mechanistic models that are based on the analysis of these components have begun to emerge.

Configuration of the sugar head of glycolipids in thylakoid membranes.

Glycolipids constitute the majority of membrane components in oxygenic photosynthetic organisms, whereas they are minor lipids in other organisms. In cyanobacteria, three glycolipids comprise ~90 mol% of the total lipids in thylakoid membranes, where photosynthetic electron transport occurs. Among these glycolipids, 80 mol% are galactolipids (monogalactosyldiacylglycerol and digalactosyldiacylglycerol). Galactolipids are well conserved in oxygenic photosynthetic organisms and are believed to be essential for the integrity of the membrane system. It remains unclear, however, which part(s) of the galactolipid structure is the key factor for their function, e.g., the sugar moiety and/or the anomeric configuration. To address this issue, several bacterial membrane glycolipid synthase genes have been introduced into cyanobacteria to test for complementation of knocked-out genes involved in galactolipid biosynthesis. In this review, we summarize recent advances in the analyses of sugar species and configurations of glycolipids heterologously synthesized in the thylakoid membrane and discuss their functional importance.