Ablation of glucosinolate accumulation in the oil crop Camelina sativa by targeted mutagenesis of genes encoding the transporters GTR1 and GTR2 and regulators of biosynthesis MYB28 and MYB29.

Camelina sativa is an oil crop with low input costs and resistance to abiotic and biotic stresses. The presence of glucosinolates, plant metabolites with adverse health effects, restricts the use of camelina for human and animal nutrition. Cas9 endonuclease-based targeted mutagenesis of the three homeologs of each of the glucosinolate transporters CsGTR1 and CsGTR2 caused a strong decrease in glucosinolate amounts, highlighting the power of this approach for inactivating multiple genes in a hexaploid crop. Mutagenesis of the three homeologs of each of the transcription factors CsMYB28 and CsMYB29 resulted in the complete loss of glucosinolates, representing the first glucosinolate-free Brassicaceae crop. The oil and protein contents and the fatty acid composition of the csgtr1csgtr2 and csmyb28csmyb29 mutant seeds were not affected. The decrease and elimination of glucosinolates improves the quality of the oil and press cake of camelina, which thus complies with international standards regulating glucosinolate levels for human consumption and animal feeding.

Triacylglycerol and phytyl ester synthesis in Synechocystis sp. PCC6803.

Cyanobacteria are unicellular prokaryotic algae that perform oxygenic photosynthesis, similar to plants. The cells harbor thylakoid membranes composed of lipids related to those of chloroplasts in plants to accommodate the complexes of photosynthesis. The occurrence of storage lipids, including triacylglycerol or wax esters, which are found in plants, animals, and some bacteria, nevertheless remained unclear in cyanobacteria. We show here that the cyanobacterium Synechocystis sp. PCC6803 accumulates both triacylglycerol and wax esters (fatty acid phytyl esters). Phytyl esters accumulate in higher levels under abiotic stress conditions. The analysis of an insertional mutant revealed that the acyltransferase slr2103, with sequence similarity to plant esterase/lipase/thioesterase (ELT) proteins, is essential for triacylglycerol and phytyl ester synthesis in Synechocystis The recombinant slr2103 enzyme showed acyltransferase activity with phytol and diacylglycerol, thus producing phytyl esters and triacylglycerol. Acyl-CoA thioesters were the preferred acyl donors, while acyl-ACP (acyl carrier protein), free fatty acids, or galactolipid-bound fatty acids were poor substrates. The slr2103 protein sequence is unrelated to acyltransferases from bacteria (AtfA) or plants (DGAT1, DGAT2, PDAT), and therefore establishes an independent group of bacterial acyltransferases involved in triacylglycerol and wax ester synthesis. The identification of the gene slr2103 responsible for triacylglycerol synthesis in cyanobacteria opens the possibility of using prokaryotic photosynthetic cells in biotechnological applications.

The Glycine-Glucolipid of Alcanivorax borkumensis Is Resident to the Bacterial Cell Wall.

The marine bacterium Alcanivorax borkumensis produces a surface-active glycine-glucolipid during growth with long-chain alkanes. A high-performance liquid chromatography (HPLC) method was developed for absolute quantification. This method is based on the conversion of the glycine-glucolipid to phenacyl esters with subsequent measurement by HPLC with diode array detection (HPLC-DAD). Different molecular species were separated by HPLC and identified as glucosyl-tetra(3-hydroxy-acyl)-glycine with varying numbers of 3-hydroxy-decanoic acid or 3-hydroxy-octanoic acid groups via mass spectrometry. The growth rate of A. borkumensis cells with pyruvate as the sole carbon source was elevated compared to hexadecane as recorded by the increase in cell density as well as oxygen/carbon dioxide transfer rates. The amount of the glycine-glucolipid produced per cell during growth on hexadecane was higher compared with growth on pyruvate. The glycine-glucolipid from pyruvate-grown cells contained considerable amounts of 3-hydroxy-octanoic acid, in contrast to hexadecane-grown cells, which almost exclusively incorporated 3-hydroxy-decanoic acid into the glycine-glucolipid. The predominant proportion of the glycine-glucolipid was found in the cell pellet, while only minute amounts were present in the cell-free supernatant. The glycine-glucolipid isolated from the bacterial cell broth, cell pellet, or cell-free supernatant showed the same structure containing a glycine residue, in contrast to previous reports, which suggested that a glycine-free form of the glucolipid exists which is secreted into the supernatant. In conclusion, the glycine-glucolipid of A. borkumensis is resident to the cell wall and enables the bacterium to bind and solubilize alkanes at the lipid-water interface. IMPORTANCE Alcanivorax borkumensis is one of the most abundant marine bacteria found in areas of oil spills, where it degrades alkanes. The production of a glycine-glucolipid is considered an essential element for alkane degradation. We developed a quantitative method and determined the structure of the A. borkumensis glycine-glucolipid in different fractions of the cultures after growth in various media. Our results show that the amount of the glycine-glucolipid in the cells by far exceeds the amount measured in the supernatant, confirming the proposed cell wall localization. These results support the scenario that the surface hydrophobicity of A. borkumensis cells increases by producing the glycine-glucolipid, allowing the cells to attach to the alkane-water interface and form a biofilm. We found no evidence for a glycine-free form of the glucolipid.

Polar lipid characterization and description of Chryseobacterium capnotolerans sp. nov., isolated from high CO2-containing atmosphere and emended descriptions of the genus Chryseobacterium, and the species C. balustinum, C. daecheongense, C. formosense, C. gleum, C. indologenes, C. joostei, C. scophthalmum and C. ureilyticum.

Modified atmosphere (MA) packaging plays an important role in improving food quality and safety. By using different gas mixtures and packaging materials the shelf life of fresh produce can significantly be increased. A Gram-negative-staining, rod-shaped, orange-pigmented strain DH-B6T, has been isolated from MA packed raw pork sausage (20% CO2, 80% O2). The strain produced biofilms and showed growth at high CO2 levels of up to 40%. Complete 16S rRNA gene and whole-genome sequences revealed that strain DH-B6T belongs to the genus Chryseobacterium, being closely related to strain Chryseobacterium indologenes DSM 16777T (98.4%), followed by Chryseobacterium gleum NCTC11432T (98.3%) and Chryseobacterium lactis KC1864T (98.2%). Average nucleotide identity value between DH-B6T and C. indologenes DSM 16777T was 81.1% and digital DNA-DNA hybridisation was 24.9%, respectively. The DNA G+C content was 35.51 mol%. Chemotaxonomical analysis revealed the presence of the rare glycine lipid cytolipin, the serine-glycine lipid flavolipin and the sulfonolipid sulfobacin A, as well as phosphatidylethanolamine, monohexosyldiacylglycerol and ornithine lipid, including the hydroxylated forms. Major fatty acids were iC15 : 0 (50.7%) and iC17 : 1 cis 9 (28.7%), followed by iC15 : 0 2-OH (7.0%) and iC17 : 0 3-OH (6.2%). The isolated strain contained MK-6 as the only respiratory quinone and flexirubin-like pigments were detected as the major pigments. Based on the phenotypic, chemotaxonomic and phylogenetic characteristics, the strain DH-B6T (=DSM 110542T=LMG 31915T) represents a novel species of the genus Chryseobacterium, for which the name Chryseobacterium capnotolerans sp. nov. is proposed. Emended descriptions of the genus Chryseobacterium and eight species of this genus based on polar lipid characterisation are also proposed.

Synthesis of the unusual lipid bis(monoacylglycero)phosphate in environmental bacteria.

The bacterial membrane is constantly remodelled in response to environmental conditions and the external supply of precursor molecules. Some bacteria are able to acquire exogenous lyso-phospholipids and convert them to the corresponding phospholipids. Here, we report that some soil-dwelling bacteria have alternative options to metabolize lyso-phosphatidylglycerol (L-PG). We find that the plant-pathogen Agrobacterium tumefaciens takes up this mono-acylated phospholipid and converts it to two distinct isoforms of the non-canonical lipid bis(monoacylglycero)phosphate (BMP). Chromatographic separation and quadrupole-time-of-flight MS/MS analysis revealed the presence of two possible BMP stereo configurations acylated at either of the free hydroxyl groups of the glycerol head group. BMP accumulated in the inner membrane and did not visibly alter cell morphology and growth behaviour. The plant-associated bacterium Sinorhizobium meliloti was also able to convert externally provided L-PG to BMP. Other bacteria like Pseudomonas fluorescens and Escherichia coli metabolized L-PG after cell disruption, suggesting that BMP production in the natural habitat relies both on dedicated uptake systems and on head-group acylation enzymes. Overall, our study adds two previously overlooked phospholipids to the repertoire of bacterial membrane lipids and provides evidence for the remarkable condition-responsive adaptation of bacterial membranes.

Sphingomonas aliaeris sp. nov., a new species isolated from pork steak packed under modified atmosphere.

Species belonging to the genus Sphingomonas have been isolated from environments such as soil, water and plant tissues. Many strains are known for their capability of degrading aromatic molecules and producing extracellular polymers. A Gram-stain-negative, strictly aerobic, motile, red-pigmented, oxidase-negative, catalase-positive, rod-shaped strain, designated DH-S5T, has been isolated from pork steak packed under CO2-enriched modified atmosphere. Cell diameters were 1.5×0.9 µm. Growth optima were at 30 °C and at pH 6.0. Phylogenetic analyses based on both complete 16S rRNA gene sequence and whole-genome sequence data revealed that strain DH-S5T belongs to the genus Sphingomonas, being closely related to Sphingomonas alpina DSM 22537T (97.4 % gene sequence similarity), followed by Sphingomonas qilianensis X1T (97.4 %) and Sphingomonas hylomeconis GZJT-2T (97.3 %). The DNA G+C content was 64.4 mol%. The digital DNA-DNA hybridization value between the isolate strain and S. alpina DSM 22537T was 21.0 % with an average nucleotide identity value of 77.03 %. Strain DH-S5T contained Q-10 as the ubiquinone and major fatty acids were C18 : 1 cis 11 (39.3 %) and C16 : 1 cis 9 (12.5 %), as well as C16 : 0 (12.1 %) and C14 : 0 2-OH (11.4 %). As for polar lipids, phosphatidylcholine, phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine, dimethylphosphatidylethanolamine and sphingoglycolipid could be detected, alongside traces of monomethylphosphatidylethanolamine. Based on its phenotypic, chemotaxonomic and phylogenetic characteristics, strain DH-S5T (=DSM 110829T=LMG 31606T) is classified as a representative of the genus Sphingomonas, for which the name Sphingomonas aliaeris sp. nov. is proposed.

Arthrobacter bussei sp. nov., a pink-coloured organism isolated from cheese made of cow's milk.

A pink-coloured bacterium (strain KR32T) was isolated from cheese and assigned to the 'Arthrobacter agilis group'. Members of the 'pink Arthrobacter agilis group' form a stable clade (100 % bootstrap value) and contain the species Arthrobacter agilis, Arthrobacter ruber and Arthrobacter echini, which share ≥99.0 % 16S rRNA gene sequence similarity. Isolate KR32T showed highest 16S rRNA gene sequence similarity (99.9 %) to A. agilis DSM 20550T. Additional multilocus sequence comparison confirmed the assignment of strain KR32T to the clade 'pink A. agilis group'. Average nucleotide identity and digital DNA-DNA hybridization values between isolate KR32T and A. agilis DSM 20550T were 82.85 and 26.30 %, respectively. The G+C content of the genomic DNA of isolate KR32T was 69.14 mol%. Chemotaxonomic analysis determined anteiso-C15 : 0 as the predominant fatty acid and MK-9(H2) as the predominant menaquinone. Polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol and monoacyldimannosyl-monoacylglycerol. The peptidoglycan type of the isolate was A3α. The carotenoid bacterioruberin was detected as the major pigment. At 10 °C, strain KR32T grew with increased concentrations of bacterioruberin and production of unsaturated fatty acids. Strain KR32T was a Gram-stain-positive, catalase-positive, oxidase-positive and coccus-shaped bacterium with optimal growth at 27-30 °C and pH 8. The results of phylogenetic and phenotypic analyses enabled the differentiation of the isolate from other closely related species of the 'pink A. agilis group'. Therefore, strain KR32T represents a novel species for which the name Arthrobacter bussei sp. nov. is proposed. The type strain is KR32T (=DSM 109896T=LMG 31480T=NCCB 100733T).

Synthesis and transfer of galactolipids in the chloroplast envelope membranes of Arabidopsis thaliana.

Galactolipids [monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG)] are the hallmark lipids of photosynthetic membranes. The galactolipid synthases MGD1 and DGD1 catalyze consecutive galactosyltransfer reactions but localize to the inner and outer chloroplast envelopes, respectively, necessitating intermembrane lipid transfer. Here we show that the N-terminal sequence of DGD1 (NDGD1) is required for galactolipid transfer between the envelopes. Different diglycosyllipid synthases (DGD1, DGD2, and Chloroflexus glucosyltransferase) were introduced into the dgd1-1 mutant of Arabidopsis in fusion with N-terminal extensions (NDGD1 and NDGD2) targeting to the outer envelope. Reconstruction of DGDG synthesis in the outer envelope membrane was observed only with diglycosyllipid synthase fusion proteins carrying NDGD1, indicating that NDGD1 enables galactolipid translocation between envelopes. NDGD1 binds to phosphatidic acid (PA) in membranes and mediates PA-dependent membrane fusion in vitro. These findings provide a mechanism for the sorting and selective channeling of lipid precursors between the galactolipid pools of the two envelope membranes.

Identification and characterization of a plastidial phosphatidylglycerophosphate phosphatase in Arabidopsis thaliana.

Phosphatidylglycerol (PG) is the only phospholipid in the thylakoid membranes of chloroplasts of plants, and it is also found in extraplastidial membranes including mitochondria and the endoplasmic reticulum. Previous studies showed that lack of PG in the pgp1-2 mutant of Arabidopsis deficient in phosphatidylglycerophosphate (PGP) synthase strongly affects thylakoid biogenesis and photosynthetic activity. In the present study, the gene encoding the enzyme for the second step of PG synthesis, PGP phosphatase, was isolated based on sequence similarity to the yeast GEP4 and Chlamydomonas PGPP1 genes. The Arabidopsis AtPGPP1 protein localizes to chloroplasts and harbors PGP phosphatase activity with alkaline pH optimum and divalent cation requirement. Arabidopsis pgpp1-1 mutant plants contain reduced amounts of chlorophyll, but photosynthetic quantum yield remains unchanged. The absolute content of plastidial PG (34:4; total number of acyl carbons:number of double bonds) is reduced by about 1/3, demonstrating that AtPGPP1 is involved in the synthesis of plastidial PG. PGP 34:3, PGP 34:2 and PGP 34:1 lacking 16:1 accumulate in pgpp1-1, indicating that the desaturation of 16:0 to 16:1 by the FAD4 desaturase in the chloroplasts only occurs after PGP dephosphorylation.

Remobilization of Phytol from Chlorophyll Degradation Is Essential for Tocopherol Synthesis and Growth of Arabidopsis.

Phytol from chlorophyll degradation can be phosphorylated to phytyl-phosphate and phytyl-diphosphate, the substrate for tocopherol (vitamin E) synthesis. A candidate for the phytyl-phosphate kinase from Arabidopsis thaliana (At1g78620) was identified via a phylogeny-based approach. This gene was designated VITAMIN E DEFICIENT6 (VTE6) because the leaves of the Arabidopsis vte6 mutants are tocopherol deficient. The vte6 mutant plants are incapable of photoautotrophic growth. Phytol and phytyl-phosphate accumulate, and the phytyl-diphosphate content is strongly decreased in vte6 leaves. Phytol feeding and enzyme assays with Arabidopsis and recombinant Escherichia coli cells demonstrated that VTE6 has phytyl-P kinase activity. Overexpression of VTE6 resulted in increased phytyl-diphosphate and tocopherol contents in seeds, indicating that VTE6 encodes phytyl-phosphate kinase. The severe growth retardation of vte6 mutants was partially rescued by introducing the phytol kinase mutation vte5. Double mutant plants (vte5 vte6) are tocopherol deficient and contain more chlorophyll, but reduced amounts of phytol and phytyl-phosphate compared with vte6 mutants, suggesting that phytol or phytyl-phosphate are detrimental to plant growth. Therefore, VTE6 represents the missing phytyl-phosphate kinase, linking phytol release from chlorophyll with tocopherol synthesis. Moreover, tocopherol synthesis in leaves depends on phytol derived from chlorophyll, not on de novo synthesis of phytyl-diphosphate from geranylgeranyl-diphosphate.

Lipids in plant-microbe interactions.

Bacteria and fungi can undergo symbiotic or pathogenic interactions with plants. Membrane lipids and lipid-derived molecules from the plant or the microbial organism play important roles during the infection process. For example, lipids (phospholipids, glycolipids, sphingolipids, sterol lipids) are involved in establishing the membrane interface between the two organisms. Furthermore, lipid-derived molecules are crucial for intracellular signaling in the plant cell, and lipids serve as signals during plant-microbial communication. These signal lipids include phosphatidic acid, diacylglycerol, lysophospholipids, and free fatty acids derived from phospholipase activity, apocarotenoids, and sphingolipid breakdown products such as ceramide, ceramide-phosphate, long chain base, and long chain base-phosphate. Fatty acids are the precursors for oxylipins, including jasmonic acid, and for azelaic acid, which together with glycerol-3-phosphate are crucial for the regulation of systemic acquired resistance. This article is part of a Special Issue titled "Plant Lipid Biology," guest editors Kent Chapman and Ivo Feussner.

DGDG and Glycolipids in Plants and Algae.

Photosynthetic organelles in plants and algae are characterized by the high abundance of glycolipids, including the galactolipids mono- and digalactosyldiacylglycerol (MGDG, DGDG) and the sulfolipid sulfoquinovosyldiacylglycerol (SQDG). Glycolipids are crucial to maintain an optimal efficiency of photosynthesis. During phosphate limitation, the amounts of DGDG and SQDG increase in the plastids of plants, and DGDG is exported to extraplastidial membranes to replace phospholipids. Algae often use betaine lipids as surrogate for phospholipids. Glucuronosyldiacylglycerol (GlcADG) is a further glycolipid that accumulates under phosphate deprived conditions. In contrast to plants, a number of eukaryotic algae contain very long chain polyunsaturated fatty acids of 20 or more carbon atoms in their glycolipids. The pathways and genes for galactolipid and sulfolipid synthesis are largely conserved between plants, Chlorophyta, Rhodophyta and algae with complex plastids derived from secondary or tertiary endosymbiosis. However, the relative contribution of the endoplasmic reticulum- and plastid-derived lipid pathways for glycolipid synthesis varies between plants and algae. The genes for glycolipid synthesis encode precursor proteins imported into the photosynthetic organelles. While most eukaryotic algae contain the plant-like galactolipid (MGD1, DGD1) and sulfolipid (SQD1, SQD2) synthases, the red alga Cyanidioschyzon harbors a cyanobacterium-type DGDG synthase (DgdA), and the amoeba Paulinella, derived from a more recent endosymbiosis event, contains cyanobacterium-type enzymes for MGDG and DGDG synthesis (MgdA, MgdE, DgdA).

Accumulation of novel glycolipids and ornithine lipids in Mesorhizobium loti under phosphate deprivation.

Glycolipids are found mainly in photosynthetic organisms (plants, algae, and cyanobacteria), Gram-positive bacteria, and a few other bacterial phyla. They serve as membrane lipids and play a role under phosphate deprivation as surrogates for phospholipids. Mesorhizobium loti accumulates different di- and triglycosyl diacylglycerols, synthesized by the processive glycosyltransferase Pgt-Ml, and two so far unknown glycolipids, which were identified in this study by mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy as O-methyl-digalactosyl diacylglycerol (Me-DGD) and glucuronosyl diacylglycerol (GlcAD). Me-DGD is a novel glycolipid, whose synthesis depends on Pgt-Ml activity and the involvement of an unknown methyltransferase, while GlcAD is formed by a novel glycosyltransferase encoded by the open reading frame (ORF) mlr2668, using UDP-glucuronic acid as a sugar donor. Deletion mutants lacking GlcAD are not impaired in growth. Our data suggest that the different glycolipids in Mesorhizobium can mutually replace each other. This may be an adaptation mechanism to enhance the competitiveness in natural environments. A further nonphospholipid in Mesorhizobium was identified as a hydroxylated form of an ornithine lipid with the additional hydroxy group linked to the amide-bound fatty acid, introduced by the hydroxylase OlsD. The presence of this lipid has not been reported for rhizobia yet. The hydroxy group is placed on the C-2 position of the acyl chain as determined by NMR spectroscopy. Furthermore, the isolated ornithine lipids contained up to 80 to 90% d-configured ornithine, a stereoform so far undescribed in bacteria.

A bifunctional glycosyltransferase from Agrobacterium tumefaciens synthesizes monoglucosyl and glucuronosyl diacylglycerol under phosphate deprivation.

Glycolipids are mainly found in phototrophic organisms (like plants and cyanobacteria), in Gram-positive bacteria, and a few other bacterial phyla. Besides the function as bulk membrane lipids, they often play a role under phosphate deprivation as surrogates for phospholipids. The Gram-negative Agrobacterium tumefaciens accumulates four different glycolipids under phosphate deficiency, including digalactosyl diacylglycerol and glucosylgalactosyl diacylglycerol synthesized by a processive glycosyltransferase. The other two glycolipids have now been identified by mass spectrometry and nuclear magnetic resonance spectroscopy as monoglucosyl diacylglycerol and glucuronosyl diacylglycerol. These two lipids are synthesized by a single promiscuous glycosyltransferase encoded by the ORF atu2297, with UDP-glucose or UDP-glucuronic acid as sugar donors. The transfer of sugars differing in their chemistry is a novel feature not observed before for lipid glycosyltransferases. Furthermore, this enzyme is the first glucuronosyl diacylglycerol synthase isolated. Deletion mutants of Agrobacterium lacking monoglucosyl diacylglycerol and glucuronosyl diacylglycerol or all glycolipids are not impaired in growth or virulence during infection of tobacco leaf discs. Our data suggest that the four glycolipids and the nonphospholipid diacylglyceryl trimethylhomoserine can mutually replace each other during phosphate deprivation. This redundancy of different nonphospholipids may represent an adaptation mechanism to enhance the competitiveness in nature.

Fatty acid phytyl ester synthesis in chloroplasts of Arabidopsis.

During stress or senescence, thylakoid membranes in chloroplasts are disintegrated, and chlorophyll and galactolipid are broken down, resulting in the accumulation of toxic intermediates, i.e., tetrapyrroles, free phytol, and free fatty acids. Chlorophyll degradation has been studied in detail, but the catabolic pathways for phytol and fatty acids remain unclear. A large proportion of phytol and fatty acids is converted into fatty acid phytyl esters and triacylglycerol during stress or senescence in chloroplasts. We isolated two genes (PHYTYL ESTER SYNTHASE1 [PES1] and PES2) of the esterase/lipase/thioesterase family of acyltransferases from Arabidopsis thaliana that are involved in fatty acid phytyl ester synthesis in chloroplasts. The two proteins are highly expressed during senescence and nitrogen deprivation. Heterologous expression in yeast revealed that PES1 and PES2 have phytyl ester synthesis and diacylglycerol acyltransferase activities. The enzymes show broad substrate specificities and can employ acyl-CoAs, acyl carrier proteins, and galactolipids as acyl donors. Double mutant plants (pes1 pes2) grow normally but show reduced phytyl ester and triacylglycerol accumulation. These results demonstrate that PES1 and PES2 are involved in the deposition of free phytol and free fatty acids in the form of phytyl esters in chloroplasts, a process involved in maintaining the integrity of the photosynthetic membrane during abiotic stress and senescence.

Accumulation of glycolipids and other non-phosphorous lipids in Agrobacterium tumefaciens grown under phosphate deprivation.

Phosphate deficiency is characteristic for many natural habitats, resulting in different physiological responses in plants and bacteria including the replacement of phospholipids by glycolipids and other phosphorous-free lipids. The plant pathogenic bacterium Agrobacterium tumefaciens, which is free of glycolipids under full nutrition, harbors an open reading frame (ORF) coding for a processive glycosyltransferase (named as Pgt). This glycosyltransferase was previously shown to synthesize glucosylgalactosyldiacylglycerol (GGD) and digalactosyldiacylglycerol (DGD) after heterologous expression. The native function of this enzyme and the conditions for its activation remained unknown. We show here that Pgt is active under phosphate deprivation synthesizing GGD and DGD in Agrobacterium. A corresponding deletion mutant (Δpgt) is free of these two glycolipids. Glycolipid accumulation is mainly regulated by substrate (diacylglycerol) availability. Diacylglycerol and the total fatty acid pool are characterized by an altered acyl composition in dependence of the phosphate status with a strong decrease of 18:1 and concomitant increase of 19:0 cyclo during phosphate deprivation. Furthermore, Agrobacterium accumulates two additional unknown glycolipids and diacylglycerol trimethylhomoserine (DGTS) during phosphate deprivation. Accumulation of all these lipids is accompanied by a reduction in phospholipids from 75 to 45% in the wild type. A further non-phosphorous lipid, ornithine lipid, was not increased but its degree of hydroxylation was elevated under phosphate deprivation. The lack of GGD and DGD in the Δpgt mutant has no effect on growth and virulence of Agrobacterium, suggesting that these two lipids are functionally replaced by DGTS and the two unknown glycolipids under phosphate deprivation.

Implications of Below-Ground Allelopathic Interactions of Camelina sativa and Microorganisms for Phosphate Availability and Habitat Maintenance.

Toxic breakdown products of young Camelina sativa (L.) Crantz, glucosinolates can eliminate microorganisms in the soil. Since microorganisms are essential for phosphate cycling, only insensitive microorganisms with phosphate-solubilizing activity can improve C. sativa's phosphate supply. In this study, 33P-labeled phosphate, inductively coupled plasma mass spectrometry and pot experiments unveiled that not only Trichoderma viride and Pseudomonas laurentiana used as phosphate-solubilizing inoculants, but also intrinsic soil microorganisms, including Penicillium aurantiogriseum, and the assemblies of root-colonizing microorganisms solubilized as well phosphate from apatite, trigger off competitive behavior between the organisms. Driving factors in the competitiveness are plant and microbial secondary metabolites, while glucosinolates of Camelina and their breakdown products are regarded as key compounds that inhibit the pathogen P. aurantiogriseum, but also seem to impede root colonization of T. viride. On the other hand, fungal diketopiperazine combined with glucosinolates is fatal to Camelina. The results may contribute to explain the contradictory effects of phosphate-solubilizing microorganisms when used as biofertilizers. Further studies will elucidate impacts of released secondary metabolites on coexisting microorganisms and plants under different environmental conditions.

Exogenous fatty acids affect membrane properties and cold adaptation of Listeria monocytogenes.

Listeria monocytogenes is a food-borne pathogen that can grow at very low temperatures close to the freezing point of food and other matrices. Maintaining cytoplasmic membrane fluidity by changing its lipid composition is indispensable for growth at low temperatures. Its dominant adaptation is to shorten the fatty acid chain length and, in some strains, increase in addition the menaquinone content. To date, incorporation of exogenous fatty acid was not reported for Listeria monocytogenes. In this study, the membrane fluidity grown under low-temperature conditions was affected by exogenous fatty acids incorporated into the membrane phospholipids of the bacterium. Listeria monocytogenes incorporated exogenous fatty acids due to their availability irrespective of their melting points. Incorporation was demonstrated by supplementation of the growth medium with polysorbate 60, polysorbate 80, and food lipid extracts, resulting in a corresponding modification of the membrane fatty acid profile. Incorporated exogenous fatty acids had a clear impact on the fitness of the Listeria monocytogenes strains, which was demonstrated by analyses of the membrane fluidity, resistance to freeze-thaw stress, and growth rates. The fatty acid content of the growth medium or the food matrix affects the membrane fluidity and thus proliferation and persistence of Listeria monocytogenes in food under low-temperature conditions.

A processive glycosyltransferase involved in glycolipid synthesis during phosphate deprivation in Mesorhizobium loti.

Natural habitats are often characterized by a low availability of phosphate. In plants and many bacteria, phosphate deficiency causes different physiological responses, including the replacement of phosphoglycerolipids in the membranes with nonphosphorous lipids. We describe here a processive glycosyltransferase (Pgt) in Mesorhizobium loti (Rhizobiales) involved in the synthesis of di- and triglycosyldiacylglycerols (DGlycD and TGlycD) during phosphate deprivation. Cells of the corresponding Δpgt deletion mutant are deficient in DGlycD and TGlycD. Additional Pgt-independent lipids accumulate in Mesorhizobium after phosphate starvation, including diacylglyceryl trimethylhomoserine (DGTS) and ornithine lipid (OL). The accumulation of the nonphosphorous lipids during phosphate deprivation leads to the reduction of phosphoglycerolipids from 90 to 50%. Nodulation experiments of Mesorhizobium wild type and the Δpgt mutant with its host plant, Lotus japonicus, revealed that DGlycD and TGlycD are not essential for nodulation under phosphate-replete or -deficient conditions. Lipid measurements showed that the Pgt-independent lipids including OL and DGTS accumulate to higher proportions in the Δpgt mutant and therefore might functionally replace DGlycD and TGlycD during phosphate deprivation.

Thin-Layer Chromatography.

Lipid extracts from plants represent a mixture of polar membrane lipids and nonpolar lipids. The main constituents of the polar lipid fraction are glycerolipids, that is, galactolipids, sulfolipid, and phospholipids. In addition, betaine lipids are found in pteridophytes, bryophytes, and algae. Nonpolar lipids include the storage lipid triacylglycerol, wax esters, diacylglycerol and free fatty acids. The complex lipid mixtures from plant tissues can be separated by thin-layer chromatography (TLC) into different lipid classes. In most cases glass plates coated with a silica gel are used as stationary phase and an organic solvent as mobile phase. Different solvent systems are required to separate polar membrane lipids or nonpolar lipids by TLC. Depending on the complexity of the lipid mixture, lipids are separated using one- or two-dimensional TLC systems. Different dyes and reagents allow the visualization of all lipid classes, or the selective staining of glycolipids or phospholipids. Lipids can be isolated from the TLC plate for subsequent analysis, provided that nondestructive methods are used for visualization.