Biosynthesis Of Phosphatidylglycerol Research Articles

Anionic lipids facilitate membrane development and protochlorophyllide biosynthesis in etioplasts.

Dark-germinated angiosperm seedlings develop chloroplast precursors called etioplasts in cotyledon cells. Etioplasts develop lattice membrane structures called prolamellar bodies (PLBs), where the chlorophyll intermediate protochlorophyllide (Pchlide) forms a ternary complex with NADPH and light-dependent NADPH:Pchlide oxidoreductase (LPOR). The lipid bilayers of etioplast membranes are mainly composed of galactolipids, which play important roles in membrane-associated processes in etioplasts. Although etioplast membranes also contain 2 anionic lipids, phosphatidylglycerol (PG) and sulfoquinovosyldiacylglycerol (SQDG), their roles are unknown. To determine the roles of PG and SQDG in etioplast development, we characterized etiolated Arabidopsis (Arabidopsis thaliana) mutants deficient in PG and SQDG biosynthesis. A partial deficiency in PG biosynthesis loosened the lattice structure of PLBs and impaired the insertion of Mg2+ into protoporphyrin IX, leading to a substantial decrease in Pchlide content. Although a complete lack of SQDG biosynthesis did not notably affect PLB formation and Pchlide biosynthesis, lack of SQDG in addition to partial PG deficiency strongly impaired these processes. These results suggested that PG is required for PLB formation and Pchlide biosynthesis, whereas SQDG plays an auxiliary role in these processes. Notably, PG deficiency and lack of SQDG oppositely affected the dynamics of LPOR complexes after photoconversion, suggesting different involvements of PG and SQDG in LPOR complex organization. Our data demonstrate pleiotropic roles of anionic lipids in etioplast development.

Cytidine diphosphate diacylglycerol synthase is essential for mitochondrial structure and energy production in Arabidopsis thaliana.

Cytidine diphosphate diacylglycerol (CDP-DAG), an important intermediate for glycerolipid biosynthesis, is synthesized under the catalytic activity of CDP-DAG synthase (CDS) to produce anionic phosphoglycerolipids such as phosphatidylglycerol (PG) and cardiolipin (CL). Previous studies showed that Arabidopsis CDSs are encoded by a small gene family, termed CDS1-CDS5, the members of which are integral membrane proteins in endoplasmic reticulum (ER) and in plastids. However, the details on how CDP-DAG is provided for mitochondrial membrane-specific phosphoglycerolipids are missing. Here we present the identification of a mitochondrion-specific CDS, designated CDS6. Enzymatic activity of CDS6 was demonstrated by the complementation of CL synthesis in the yeast CDS-deficient tam41Δ mutant. The Arabidopsis cds6 mutant lacking CDS6 activity showed decreased mitochondrial PG and CL biosynthesis capacity, a severe growth deficiency finally leading to plant death. These defects were rescued partly by complementation with CDS6 or supplementation with PG and CL. The ultrastructure of mitochondria in cds6 was abnormal, missing the structures of cristae. The degradation of triacylglycerol (TAG) in lipid droplets and starch in chloroplasts in the cds6 mutant was impaired. The expression of most differentially expressed genes involved in the mitochondrial electron transport chain was upregulated, suggesting an energy-demanding stage in cds6. Furthermore, the contents of polar glycerolipids in cds6 were dramatically altered. In addition, cds6 seedlings lost the capacity for cell proliferation and showed a higher oxidase activity. Thus, CDS6 is indispensable for the biosynthesis of PG and CL in mitochondria, which is critical for establishing mitochondrial structure, TAG degradation, energy production and seedling development.

Two Different Phospholipases C, Isc1 and Pgc1, Cooperate To Regulate Mitochondrial Function.

The absence of Isc1, the yeast homologue of mammalian neutral sphingomyelinase type 2, leads to severe mitochondrial dysfunction. We show that the deletion of another type C phospholipase, the phosphatidylglycerol (PG)-specific phospholipase Pgc1, rescues this defect. Phosphatidylethanolamine (PE) levels and cytochrome c oxidase activity, which were reduced in isc1Δ cells, were restored to wild-type levels in the pgc1Δ isc1Δ mutant. The Pgc1 substrate PG inhibited the in vitro activities of Isc1 and the phosphatidylserine decarboxylase Psd1, an enzyme crucial for PE biosynthesis. We also identify a mechanism by which the balance between the current demand for PG and its consumption is controlled. We document that the product of PG hydrolysis, diacylglycerol, competes with the substrate of PG-phosphate synthase, Pgs1, and thereby inhibits the biosynthesis of excess PG. This feedback loop does not work in the absence of Pgc1, which catalyzes PG degradation. Finally, Pgc1 activity is partially inhibited by products of Isc1-mediated hydrolysis. The described functional interconnection of the two phospholipases contributes significantly to lipid homeostasis throughout the cellular architecture. IMPORTANCE In eukaryotic cells, mitochondria are constantly adapting to changes in the biological activity of the cell, i.e., changes in nutrient availability and environmental stresses. We propose a model in which this adaptation is mediated by lipids. Specifically, we show that mitochondrial phospholipids regulate the biosynthesis of cellular sphingolipids and vice versa. To do this, lipids move by free diffusion, which does not require energy and works under any condition. This model represents a simple way for the cell to coordinate mitochondrial structure and performance with the actual needs of overall cellular metabolism. Its simplicity makes it a universally applicable principle of cellular regulation.

Leaf Bacteriome in Sugar Beet Shows Differential Response against Beet curly top virus during Resistant and Susceptible Interactions

Beet curly top virus (BCTV) significantly reduces sugar beet yield in semi-arid production areas. Genetic resistance to BCTV is limited; therefore, identification of additional resistance-associated factors is highly desired. Using 16S rRNA sequencing and BCTV resistant (R) genotypes (KDH13, KDH4-9) along with a susceptible (S) genotype (KDH19-17), we investigated leaf bacteriome changes during BCTV post inoculation (pi). At day 6 (~6-week-old plants), Cyanobacteria were predominant (~90%); whereas, at week 4 (~10-week-old plants) Firmicutes (11–66%), Bacteroidetes (17–26%), and Verrucomicrobia (12–29%) were predominant phyla and genotype dependent. Both Bacteroidetes and Verrucomicrobia, increased post infection only in the R lines. The bacterial genera Brevibacillus increased at 6 dpi, and Akkermansia and Bacteroides at 4 wkpi in the R lines. Linear discriminant analysis effect size (LEfSe) identified potential biomarkers in the R vs. S lines. Functional profiling revealed bacterial enrichment associated with the TCA cycle, polyisoprenoid, and L-methionine biosynthesis pathways only in KDH4-9 at 6 dpi. At 4 wkpi, bacteria associated with tryptophan and palmitate biosynthesis in the R lines, and uridine monophosphate, phosphatidyl glycerol, and phospholipid biosynthesis in the S line, were enriched. Future characterization of bacterial genera with antiviral properties will help establish their use as biocontrol agents/biomarkers against BCTV.

Genome-wide CRISPR screens identify GATA6 as a proviral host factor for SARS-CoV-2 via modulation of ACE2

The global spread of SARS-CoV-2 led to major economic and health challenges worldwide. Revealing host genes essential for infection by multiple variants of SARS-CoV-2 can provide insights into the virus pathogenesis, and facilitate the development of novel therapeutics. Here, employing a genome-scale CRISPR screen, we provide a comprehensive data-set of cellular factors that are exploited by wild type SARS-CoV-2 as well as two additional recently emerged variants of concerns (VOCs), Alpha and Beta. We identified several host factors critical for SARS-CoV-2 infection, including various components belonging to the Clathrin-dependent transport pathway, ubiquitination, Heparan sulfate biogenesis and host phosphatidylglycerol biosynthesis. Comparative analysis of the different VOCs revealed the host factors KREMEN2 and SETDB1 as potential unique candidates required only to the Alpha variant. Furthermore, the analysis identified GATA6, a zinc finger transcription factor, as an essential proviral gene for all variants inspected. We show that GATA6 directly regulates ACE2 transcription and accordingly, is critical for SARS-CoV-2 cell entry. Analysis of clinical samples collected from SARS-CoV-2 infected individuals shows elevated levels of GATA6, suggesting a role in COVID-19 pathogenesis. Finally, pharmacological inhibition of GATA6 resulted in down-modulation of ACE2 and inhibition of viral infectivity. Overall, we show GATA6 may represent a target for the development of anti-SARS-CoV-2 therapeutic strategies and reaffirm the value of the CRISPR loss-of-function screens in providing a list of potential new targets for therapeutic interventions.

Biallelic variants in TAMM41 are associated with low muscle cardiolipin levels, leading to neonatal mitochondrial disease.

SummaryMitochondrial disorders are clinically and genetically heterogeneous, with variants in mitochondrial or nuclear genes leading to varied clinical phenotypes. TAMM41 encodes a mitochondrial protein with cytidine diphosphate-diacylglycerol synthase activity: an essential early step in the biosynthesis of phosphatidylglycerol and cardiolipin. Cardiolipin is a mitochondria-specific phospholipid that is important for many mitochondrial processes. We report three unrelated individuals with mitochondrial disease that share clinical features, including lethargy at birth, hypotonia, developmental delay, myopathy, and ptosis. Whole exome and genome sequencing identified compound heterozygous variants in TAMM41 in each proband. Western blot analysis in fibroblasts showed a mild oxidative phosphorylation (OXPHOS) defect in only one of the three affected individuals. In skeletal muscle samples, however, there was severe loss of subunits of complexes I–IV and a decrease in fully assembled OXPHOS complexes I–V in two subjects as well as decreased TAMM41 protein levels. Similar to the tissue-specific observations on OXPHOS, cardiolipin levels were unchanged in subject fibroblasts but significantly decreased in the skeletal muscle of affected individuals. To assess the functional impact of the TAMM41 missense variants, the equivalent mutations were modeled in yeast. All three mutants failed to rescue the growth defect of the Δtam41 strains on non-fermentable (respiratory) medium compared with wild-type TAM41, confirming the pathogenicity of the variants. We establish that TAMM41 is an additional gene involved in mitochondrial phospholipid biosynthesis and modification and that its deficiency results in a mitochondrial disorder, though unlike families with pathogenic AGK (Sengers syndrome) and TAFAZZIN (Barth syndrome) variants, there was no evidence of cardiomyopathy.

Impacts of phosphatidylglycerol on plastid gene expression and light induction of nuclear photosynthetic genes.

Phosphatidylglycerol (PG) is the only major phospholipid in the thylakoid membrane of chloroplasts. PG is essential for photosynthesis, and loss of PG in Arabidopsis thaliana results in severe defects of growth and chloroplast development, with decreased chlorophyll accumulation, impaired thylakoid formation, and down-regulation of photosynthesis-associated genes encoded in nuclear and plastid genomes. However, how the absence of PG affects gene expression and plant growth remains unclear. To elucidate this mechanism, we investigated transcriptional profiles of a PG-deficient Arabidopsis mutant pgp1-2 under various light conditions. Microarray analysis demonstrated that reactive oxygen species (ROS)-responsive genes were up-regulated in pgp1-2. However, ROS production was not enhanced in the mutant even under strong light, indicating limited impacts of photooxidative stress on the defects of pgp1-2. Illumination to dark-adapted pgp1-2 triggered down-regulation of photosynthesis-associated nuclear-encoded genes (PhANGs), while plastid-encoded genes were constantly suppressed. Overexpression of GOLDEN2-LIKE1 (GLK1), a transcription factor gene regulating chloroplast development, in pgp1-2 up-regulated PhANGs but not plastid-encoded genes along with chlorophyll accumulation. Our data suggest a broad impact of PG biosynthesis on nuclear-encoded genes partially via GLK1 and a specific involvement of this lipid in plastid gene expression and plant development.

Vaginal Dysbiotic Microbiome in Women With No Symptoms of Genital Infections.

The vaginal microbiome plays a critical role in determining the progression of female genital tract infections; however, little is known about the vaginal microbiota of Indian women. We aimed to investigate the vaginal microbial architecture of women with asymptomatic bacterial vaginosis (BV) (n=20) and normal microbiota (n=19). Microbial diversity was analyzed in vaginal swabs from regularly menstruating women (18-45yrs) by 16S rRNA V3-V4 amplicon (MiSeq Illumina) sequencing. Rarefaction analysis showed a higher number of species in normal flora compared to BV. Alpha diversity as measured by Pielou’s evenness revealed microbial diversity was significantly greater in BV samples than normal microbiota (p= 0.0165). Beta diversity comparison using UniFrac metrics indicated distinct microbial communities clustering between normal and BV flora. Firmicutes were the major phyla observed in vaginal specimens of normal microbiota whereas Actinobacteria, Fusobacteria, Bacteroidetes were significantly abundant in BV samples. Notably, the relative abundance of Lactobacillus was significantly high in normal microbiota. Conversely Gardnerella, Sneathia, Prevotella, Atopobium, Ureaplasma, Dialister significantly dominated dysbiotic microbiota. Relative frequency of Lactobacillus decreased significantly in BV (6%) as compared to normal microbiota (35.2%). L. fermentum, L. gasseri, L. iners, L. jensenii, L. mucosae, L. ruminis, L. salivarius, L. coleohominis was more exclusively present in normal microbiota. L. iners was detected from both the groups with a relative frequency of 50.4% and 17.2% in normal and BV microbiota respectively. Lefse analysis indicated Atopobium vaginae, Sneathia amnii, Mycoplasma hominis Prevotella disiens in the vaginal microbiota as a biomarker for dysbiosis and L. jensenii as a biomarker of a healthy microbiota. Firmicutes were negatively correlated to Tenericutes, Actinobacteria, Bacteroidetes, and Fusobacteria. Proteobacteria positively correlated to Tenericutes, and Bacteroidetes were shown to be positively correlated to Fusobacteria. Predicted functional analysis indicated differences in the functional profiles between BV and normal microbiota. Normal microbiota utilized pathways essential for phosphatidylglycerol biosynthesis I & II, peptidoglycan biosynthesis, geranylgeranyl diphosphate biosynthesis I, mevalonate pathway, CoA biosynthesis pathway I and pyrimidine nucleotide salvage; whereas BV bacteria had characteristic aromatic amino acid biosynthesis, pentose phosphate pathway, carbohydrate degradation. In conclusion, women with asymptomatic BV have vaginal microbiota significantly different than women with normal microbiota. Furthermore, the study provides insights into the vaginal microbial structure of Indian women that will enable us to explore the prospective candidates for restoring the vaginal microbiota.

Plastid Anionic Lipids Are Essential for the Development of Both Photosynthetic and Non-Photosynthetic Organs in Arabidopsis thaliana.

The lipid bilayer matrix of the thylakoid membrane of cyanobacteria and chloroplasts of plants and algae is mainly composed of uncharged galactolipids, but also contains anionic lipids sulfoquinovosyldiacylglycerol (SQDG) and phosphatidylglycerol (PG) as major constituents. The necessity of PG for photosynthesis is evident in all photosynthetic organisms examined to date, whereas the requirement of SQDG varies with species. In plants, although PG and SQDG are also found in non-photosynthetic plastids, their importance for the growth and functions of non-photosynthetic organs remains unclear. In addition, plants synthesize another anionic lipid glucuronosyldiacylglycerol (GlcADG) during phosphorus starvation, but its role in plant cells is not elucidated yet. To understand the functional relationships among PG, SQDG, and GlcADG, we characterized several Arabidopsis thaliana mutants defective in biosynthesis of these lipids. The mutants completely lacking both PG and SQDG biosynthesis in plastids showed developmental defects of roots, hypocotyls, and embryos in addition to leaves, which suggests that these lipids are pleiotropically required for the development of both photosynthetic and non-photosynthetic organs. Furthermore, our analysis revealed that SQDG, but not GlcADG, is essential for complementing the role of PG, particularly in photosynthesis under PG-deficient conditions such as phosphorus starvation.

The phosphatidylglycerol phosphate synthase PgsA utilizes a trifurcated amphipathic cavity for catalysis at the membrane-cytosol interface

Phosphatidylglycerol is a crucial phospholipid found ubiquitously in biological membranes of prokaryotic and eukaryotic cells. The phosphatidylglycerol phosphate (PGP) synthase (PgsA), a membrane-embedded enzyme, catalyzes the primary reaction of phosphatidylglycerol biosynthesis. Mutations in pgsA frequently correlate with daptomycin resistance in Staphylococcus aureus and other prevalent infectious pathogens. Here we report the crystal structures of S. aureus PgsA (SaPgsA) captured at two distinct states of the catalytic process, with lipid substrate (cytidine diphosphate-diacylglycerol, CDP-DAG) or product (PGP) bound to the active site within a trifurcated amphipathic cavity. The hydrophilic head groups of CDP-DAG and PGP occupy two different pockets in the cavity, inducing local conformational changes. An elongated membrane-exposed surface groove accommodates the fatty acyl chains of CDP-DAG/PGP and opens a lateral portal for lipid entry/release. Remarkably, the daptomycin resistance-related mutations mostly cluster around the active site, causing reduction of enzymatic activity. Our results provide detailed mechanistic insights into the dynamic catalytic process of PgsA and structural frameworks beneficial for development of antimicrobial agents targeting PgsA from pathogenic bacteria.

A Pair of Arabidopsis Diacylglycerol Kinases Essential for Gametogenesis and Endoplasmic Reticulum Phospholipid Metabolism in Leaves and Flowers.

Phosphatidic acid (PA) is a key phospholipid in glycerolipid metabolism and signaling. Diacylglycerol kinase (DGK) produces PA by phosphorylating diacylglycerol, a crucial step in PA metabolism. Although DGK activity is known to be involved in plant development and stress response, how specific DGK isoforms function in development and phospholipid metabolism remains elusive. Here, we showed that Arabidopsis (Arabidopsis thaliana) DGK2 and DGK4 are crucial for gametogenesis and biosynthesis of phosphatidylglycerol and phosphatidylinositol in the endoplasmic reticulum (ER). With comprehensive transcriptomic data of seven DGKs and genetic crossing, we found that dgk2-1/- dgk4-1/- plants were gametophyte lethal, although parental single homozygous plants were viable. The dgk2-1/+ dgk4-1/+ double heterozygote showed defective pollen tube growth and seed development because of nonviable mutant gametes. DGK2 and DGK4 were localized to the ER and were involved in PA production for pollen tube growth. Transgenic knockdown lines of DGK2 and DGK4 confirmed the gametophyte defect and also revealed defective leaf and root growth. Glycerolipid analysis in the knockdown lines showed that phosphatidylglycerol and phosphatidylinositol metabolism was affected differently in floral buds and leaves. These results suggest that DGK2 and DGK4 are essential during gametogenesis and are required for ER-localized phospholipid metabolism in vegetative and reproductive growth.

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.

The lipid droplet protein Pgc1 controls the subcellular distribution of phosphatidylglycerol.

The biosynthesis of yeast phosphatidylglycerol (PG) takes place in the inner mitochondrial membrane. Outside mitochondria, the abundance of PG is low. Here, we present evidence that the subcellular distribution of PG is maintained by the locally controlled enzymatic activity of the PG-specific phospholipase, Pgc1. A fluorescently labeled Pgc1 protein accumulates on the surface of lipid droplets (LD). We show, however, that LD are not only dispensable for Pgc1-mediated PG degradation, but do not even host any phospholipase activity of Pgc1. Our in vitro assays document the capability of LD-accumulated Pgc1 to degrade PG upon entry to the membranes of the endoplasmic reticulum, mitochondria and even of artificial phospholipid vesicles. Fluorescence recovery after photobleaching analysis confirms the continuous exchange of GFP-Pgc1 within the individual LD in situ, suggesting that a steady-state equilibrium exists between LD and membranes to regulate the immediate phospholipase activity of Pgc1. In this model, LD serve as a storage place and shelter Pgc1, preventing its untimely degradation, while both phospholipase activity and degradation of the enzyme occur in the membranes.

Phosphatidylglycerophosphate phosphatase is required for root growth in Arabidopsis

Phosphatidylglycerol (PG) is an indispensable lipid class in photosynthetic activity. However, the importance of PG biosynthesis in non-photosynthetic organs remains elusive. We previously identified phosphatidylglycerophosphate phosphatase 1 (PGPP1), which catalyzes the last step of PG biosynthesis in Arabidopsis thaliana. In the present report, we noted considerably shorter roots of the pgpp1-1 mutant compared to the wild type. We observed defective order of columella cells in the root apices, which was complemented by introducing the wild-type PGPP1 gene. Although PGPP1 is chloroplast-localized in leaf mesophyll cells, we observed mitochondrial localization of PGPP1 in root cells, suggesting possible dual targeting of PGPP1. Moreover, we identified previously uncharacterized 2 protein tyrosine phosphatase-like proteins as functional PGPPs. These proteins, designated PTPMT1 and PTPMT2, complemented growth and lipid phenotypes of Δgep4, a Saccharomyces cerevisiae mutant of PGPP. The ptpmt1-1 ptpmt2-1 exhibited no visible phenotype; however, the pgpp1-1 ptpmt1-1 ptpmt2-1 significantly enhanced the root phenotype of pgpp1-1 without further affecting the photosynthesis, suggesting that these newly found PGPPs are involved in the root phenotype. Radiolabeling experiment of mutant roots showed that decreased PG biosynthesis is associated with the mutation of PGPP1. These results suggest that PG biosynthesis is required for the root growth.

Arabidopsis phosphatidylglycerophosphate phosphatase 1 involved in phosphatidylglycerol biosynthesis and photosynthetic function.

Phosphatidylglycerol (PG) is an indispensable lipid constituent of photosynthetic membranes, whose function is essential in photosynthetic activity. In higher plants, the biological function of the last step of PG biosynthesis remains elusive because an enzyme catalyzing this reaction step, namely phosphatidylglycerophosphate phosphatase (PGPP), has been a missing piece in the entire glycerolipid metabolic map. Here, we report the identification and characterization of AtPGPP1 encoding a PGPP in Arabidopsis thaliana. Heterologous expression of AtPGPP1 in yeast Δgep4 complemented growth phenotype and PG-producing activity, suggesting that AtPGPP1 encodes a functional PGPP. The GUS reporter assay showed that AtPGPP1 was preferentially expressed in hypocotyl, vasculatures, trichomes, guard cells, and stigmas. A subcellular localization study with GFP reporter indicated that AtPGPP1 is mainly localized at chloroplasts. A T-DNA-tagged knockout mutant of AtPGPP1, designated pgpp1-1, showed pale green phenotype with reduced PG and chlorophyll contents but no defect in embryo development. In the pgpp1-1 mutant, ultrastructure of plastids indicated defective development of chloroplasts and measurement of photosynthetic parameters showed impaired photosynthetic activity. These results suggest that AtPGPP1 is a primary plastidic PGPP required for PG biosynthesis and photosynthetic function in Arabidopsis.

In Vivo and in Vitro Synthesis of Phosphatidylglycerol by an Escherichia coli Cardiolipin Synthase

Phosphatidylglycerol (PG) makes up 5-20% of the phospholipids of Escherichia coli and is essential for growth in wild-type cells. PG is synthesized from the dephosphorylation of its immediate precursor, phosphatidylglycerol phosphate (PGP) whose synthase in E. coli is PgsA. Using genetic, biochemical, and highly sensitive mass spectrometric approaches, we identified an alternative mechanism for PG synthesis in E. coli that is PgsA independent. The reaction of synthesis involves the conversion of phosphatidylethanolamine and glycerol into PG and is catalyzed by ClsB, a phospholipase D-type cardiolipin synthase. This enzymatic reaction is demonstrated herein both in vivo and in vitro as well as by using the purified ClsB protein. When the growth medium was supplemented with glycerol, the expression of E. coli ClsB significantly increased PG and cardiolipin levels, with the growth deficiency of pgsA null strain also being complemented under such conditions. Identification of this alternative mechanism for PG synthesis not only expands our knowledge of bacterial anionic phospholipid biosynthesis, but also sheds light on the biochemical functions of the cls gene redundancy in E. coli and other bacteria. Finally, the PGP-independent PG synthesis in E. coli may also have important implications for the understanding of PG biosynthesis in eukaryotes that remains incomplete.

Sulfoquinovosyldiacylglycerol has an Essential Role in Thermosynechococcus elongatus BP-1 Under Phosphate-Deficient Conditions.

Anionic lipids, sulfoquinovosyldiacylglycerol (SQDG) and phosphatidylglycerol (PG), are major classes of the thylakoid membrane lipids in cyanobacteria and plant chloroplasts. PG is essential for growth and photosynthesis of cyanobacteria, algae and plants, but the requirement for SQDG differs even among cyanobacterial species. Although SQDG and PG can compensate each other in part presumably to maintain proper balance of anionic charge in lipid bilayers, the functional relationship of these lipids is largely unknown. In this study, we inactivated the sqdB gene, encoding a UDP-sulfoquinovose synthase and involved in SQDG biosynthesis, in Thermosynechococcus elongatus BP-1. In wild-type cells, PG accounted for only approximately 3.5 mol% of total membrane lipids, but its content was substantially increased along with complete loss of SQDG in the sqdB mutant. Under phosphate (Pi)-sufficient conditions, the growth rate and PSII activity were slightly lower in sqdB than in wild-type cells. In addition, the formation of PSI trimers and PSII dimers and energy transfer in phycobilisomes were perturbed in the mutant. Under Pi-deficient conditions, the growth of sqdB cells was severely impaired, with a decrease in PSII activity. PG supplementation could partially rescue the defective growth and PSII activity of Pi-deficient sqdB cells but fully recovered the impaired growth of the pgsA mutant of T. elongatus, which is deficient in PG biosynthesis. These data suggest that SQDG has a specific role in the growth and photosynthesis of T. elongatus, which cannot be complemented by PG, particularly under Pi-deficient conditions.

Multiple Impacts of Loss of Plastidic Phosphatidylglycerol Biosynthesis on Photosynthesis during Seedling Growth of Arabidopsis.

Phosphatidylglycerol (PG) is the only major phospholipid in the thylakoid membrane in cyanobacteria and plant chloroplasts. Although PG accounts only for ~10% of total thylakoid lipids, it plays indispensable roles in oxygenic photosynthesis. In contrast to the comprehensive analyses of PG-deprived mutants in cyanobacteria, in vivo roles of PG in photosynthesis during plant growth remain elusive. In this study, we characterized the photosynthesis of an Arabidopsis thaliana T-DNA insertional mutant (pgp1-2), which lacks plastidic PG biosynthesis. In the pgp1-2 mutant, energy transfer from antenna pigments to the photosystem II (PSII) reaction center was severely impaired, which resulted in low photochemical efficiency of PSII. Unlike in the wild type, in pgp1-2, the PSII complexes were susceptible to photodamage by red light irradiation. Manganese ions were mostly dissociated from protein systems in pgp1-2, with oxygen-evolving activity of PSII absent in the mutant thylakoids. The oxygen-evolving complex may be disrupted in pgp1-2, which may accelerate the photodamage to PSII by red light. On the acceptor side of the mutant PSII, decreased electron-accepting capacity was observed along with impaired electron transfer. Although the reaction center of PSI was relatively active in pgp1-2 compared to the severe impairment in PSII, the cyclic electron transport was dysfunctional. Chlorophyll fluorescence analysis at 77K revealed that PG may not be needed for the self-organization of the macromolecular protein network in grana thylakoids but is essential for the assembly of antenna-reaction center complexes. Our data clearly show that thylakoid glycolipids cannot substitute for the role of PG in photosynthesis during plant growth.

Functional Specificity of Cardiolipin Synthase Revealed by the Identification of a Cardiolipin Synthase CrCLS1 in Chlamydomonas reinhardtii

Phosphatidylglycerol (PG) and cardiolipin (CL) are two essential classes of phospholipid in plants and algae. Phosphatidylglycerophosphate synthase (PGPS) and cardiolipin synthase (CLS) involved in the biosynthesis of PG and CL belong to CDP-alcohol phosphotransferase and share overall amino acid sequence homology. However, it remains elusive whether PGPS and CLS are functionally distinct in vivo. Here, we report identification of a gene encoding CLS in Chlamydomonas reinhardtii, CrCLS1, and its functional compatibility. Whereas CrCLS1 did not complement the growth phenotype of a PGPS mutant of Synechocystis sp. PCC 6803, it rescued the temperature-sensitive growth phenotype, growth profile with different carbon sources, phospholipid composition and enzyme activity of Δcrd1, a CLS mutant of Saccharomyces cerevisiae. These results suggest that CrCLS1 encodes a functional CLS of C. reinhardtii as the first identified algal CLS, whose enzyme function is distinct from that of PGPSs from C. reinhardtii. Comparison of CDP-alcohol phosphotransferase motif between PGPS and CLS among different species revealed a possible additional motif that might define the substrate specificity of these closely related enzymes.

Characterization of Chlamydomonas reinhardtii phosphatidylglycerophosphate synthase in Synechocystis sp. PCC 6803.

Phosphatidylglycerol (PG) is an indispensable phospholipid class with photosynthetic function in plants and cyanobacteria. However, its biosynthesis in eukaryotic green microalgae is poorly studied. Here, we report the isolation and characterization of two homologs (CrPGP1 and CrPGP2) of phosphatidylglycerophosphate synthase (PGPS), the rate-limiting enzyme in PG biosynthesis, in Chlamydomonas reinhardtii. Heterologous complementation of Synechocystis sp. PCC 6803 pgsA mutant by CrPGP1 and CrPGP2 rescued the PG-dependent growth phenotype, but the PG level and its fatty acid composition were not fully rescued in the complemented strains. As well, oxygen evolution activity was not fully recovered, although electron transport activity of photosystem II was restored to the wild-type level. Gene expression study of CrPGP1 and CrPGP2 in nutrient-starved C. reinhardtii showed differential response to phosphorus and nitrogen deficiency. Taken together, these results highlight the distinct and overlapping function of PGPS in cyanobacteria and eukaryotic algae.