The regulator gene phoB mediates phosphate stress-controlled synthesis of the membrane lipid diacylglyceryl-N,N,N-trimethylhomoserine in Rhizobium (Sinorhizobium) meliloti

Bacteria react to phosphate starvation by activating genes involved in the transport and assimilation of phosphate as well as other phosphorous compounds. Some soil bacteria have evolved an additional mechanism for saving phosphorous. Under phosphate-limiting conditions, they replace their membrane phospholipids by lipids not containing phosphorus. Here, we show that the membrane lipid pattern of the free-living microsymbiotic bacterium Rhizobium (Sinorhizobium) meliloti is altered at low phosphate concentrations. When phosphate is growth limiting, an increase in sulpholipids, ornithine lipids and the de novo synthesis of diacylglyceryl trimethylhomoserine (DGTS) lipids is observed. Rhizobium meliloti phoCDET mutants, deficient in phosphate uptake, synthesize DGTS constitutively at low or high medium phosphate concentrations, suggesting that reduced transport of phosphorus sources to the cytoplasm causes induction of DGTS biosynthesis. Rhizobium meliloti phoU or phoB mutants are unable to form DGTS at low or high phosphate concentrations. However, the functional complementation of phoU or phoB mutants with the phoB gene demonstrates that, of the two genes, only intact phoB is required for the biosynthesis of the membrane lipid DGTS.     

Annotation of genes involved in glycerolipid biosynthesis in Chlamydomonas reinhardtii: discovery of the betaine lipid synthase BTA1Cr

Lipid metabolism in flowering plants has been intensely studied, and knowledge regarding the identities of genes encoding components of the major fatty acid and membrane lipid biosynthetic pathways is very extensive. We now present an in silico analysis of fatty acid and glycerolipid metabolism in an algal model, enabled by the recent availability of expressed sequence tag and genomic sequences of Chlamydomonas reinhardtii. Genes encoding proteins involved in membrane biogenesis were predicted on the basis of similarity to proteins with confirmed functions and were organized so as to reconstruct the major pathways of glycerolipid synthesis in Chlamydomonas. This analysis accounts for the majority of genes predicted to encode enzymes involved in anabolic reactions of membrane lipid biosynthesis and compares and contrasts these pathways in Chlamydomonas and flowering plants. As an important result of the bioinformatics analysis, we identified and isolated the C. reinhardtii BTA1 (BTA1Cr) gene and analyzed the bifunctional protein that it encodes; we predicted this protein to be sufficient for the synthesis of the betaine lipid diacylglyceryl-N,N,N-trimethylhomoserine (DGTS), a major membrane component in Chlamydomonas. Heterologous expression of BTA1Cr led to DGTS accumulation in Escherichia coli, which normally lacks this lipid, and allowed in vitro analysis of the enzymatic properties of BTA1Cr. In contrast, in the bacterium Rhodobacter sphaeroides, two separate proteins, BtaARs and BtaBRs, are required for the biosynthesis of DGTS. Site-directed mutagenesis of the active sites of the two domains of BTA1Cr allowed us to study their activities separately, demonstrating directly their functional homology to the bacterial orthologs BtaARs and BtaBRs.     

苜蓿根瘤菌17560细胞膜膜脂组成分析及DGTS合成基因克隆与表达

【目的】分析一株分离自黑龙江省的苜蓿根瘤菌在低磷胁迫及正常磷含量条件下细胞膜脂的组成,并从该菌中克隆和鉴定细胞膜无磷脂二酰基甘油三甲基高丝氨酸(DGTS)合成基因。【方法】分别在不同磷含量的Sherwood基本培养基中进行根瘤菌培养,采用Bligh-Dyer方法提取细胞膜脂,以文献报道Sinorhizobium meliloti(苜蓿中华根瘤菌)菌株1021的脂类图谱和磷脂PE、PG、PC标准品作为参照,利用薄层层析方法分析不同磷含量条件下培养菌株的细胞膜脂组成。根据GenBank中已发表的DGTS合成基因btaA和btaB序列设计引物,以产DGTS菌株基因组DNA为模板,扩增btaA和btaB同源基因,并在E.coil BL21(DE3)表达。同时检测表达菌株是否合成细胞膜无磷脂DGTS以验证基因功能。对菌株17560进行16S rRNA基因序列分析。【结果】分离自黑龙江省的苜蓿根瘤菌17560与Sinorhizobium meliloti的16S rRNA基因序列相似性高达99.8%,但其细胞膜脂组成明显不同于参比菌株Sinorhizobium meliloti 1021的膜脂组成。在低磷胁迫条件下,该菌株的细胞膜脂主要由OL和DGTS等无磷脂组成,但OL的组成明显不同,该菌株含有3种不同类型的鸟氨酸脂(OLs),而参比菌株Sinorhizobium meliloti 1021只含有一种类型的鸟氨酸脂(OL)。在正常磷含量条件下,该菌株的细胞膜脂主要由PE和一种未知的含氨基磷脂组成,PG与PC的含量均较少,而参比菌株Sinorhizobium meliloti 1021的细胞膜脂主要由PE、PG与PC组成。通过PCR扩增从产DGTS菌株17560中获得1 913 bpDNA片段,经序列分析发现其中有两个ORF与菌株Sinorhizobium meliloti 1021的btaA和btaB基因序列相似性均为99%。将该DNA片段克隆于pET-30a(+)得到重组质粒pLH01,转化宿主菌获得表达菌株E.coli BL21(DE3).pLH01,经IPTG诱导后产生相对分子量约为45 kD和25 kD的蛋白。薄层层析验证重组菌细胞膜脂组成,结果表明,表达菌株E.coliBL21(DE3).pLH01可以在IPTG诱导后合成无磷脂DGTS,而转入空载体pET-30a(+)的阴性对照菌株E.coli BL21(DE3).pET-30a(+)则不能合成。【结论】系统发育地位相同的苜蓿根瘤菌株的细胞膜脂组成明显不同;苜蓿根瘤菌的细胞膜组成随培养基中的磷含量不同而变化,低磷胁迫条件下其细胞膜脂主要由OL和DGTS等无磷脂组成;在Sinorhizobium膜脂中首次发现一种未知的氨基磷脂及3种不同类型的鸟氨酸脂(OLs);从菌株17560中克隆获得2个DGTS合成基因btaA和btaB,在大肠杆菌中成功表达,并证实了所表达基因的功能。     

Biosynthesis of diacylglyceryl-N,N,N-trimethylhomoserine in Rhodobacter sphaeroides and evidence for lipid-linked N methylation.

Rhodobacter sphaeroides, which produces diacylglyceryl-N,N,N-trimethylhomoserine (DGTS) under phosphate-limiting conditions, was incubated with L-[1-14C]- and L-[methyl-14C]methionine in pulse and pulse-chase experiments. The label was incorporated specifically into the polar part of DGTS and of three other compounds. One of them (compound 3) could be identified as diacylglyceryl-N,N-dimethylhomoserine by cochromatography with a reference obtained semisynthetically from DGTS. It was labelled when using L-[1-14C]- as well as L-[methyl-14C]methionine as a precursor and was converted to DGTS when incubated with the DGTS-forming eukaryotic alga Ochromonas danica (Chrysophyceae). Of the other two compounds labelled with L-[1-14C]methionine, compound 2 was also labelled with L-[methyl-14C]methionine whereas compound 1 was not, suggesting that these two intermediates are the corresponding N-methyl and nonmethylated lipids, respectively. The methyltransferase inhibitor 3'-deazaadenosine enhanced the amounts of compounds 1 to 3 but decreased the amount of DGTS. It is concluded that in R. sphaeroides, DGTS is synthesized by the same pathway as in eukaryotic organisms and that the N methylation is the terminal step in this process and occurs on the preformed lipid. Since the phosphatidylcholine-deficient mutant CHB20, lacking the phosphatidylcholine-forming N-methyltransferase was able to synthesize DGTS, one or several separate N-methyltransferases are suggested to be responsible for the synthesis of DGTS.     

基于液质联用的莱茵衣藻极性甘油酯组定性定量分析

以模式藻株莱茵衣藻(Chlamydomonas reinhardtii)为材料, 基于液质联用技术对其极性甘油酯组进行定性定量分析。通过综合利用UPLC-ESI-Q-Trap/MS的一级质谱扫描(中性丢失或母离子扫描)及UPLC-ESI-Orbitrap/MS²的二级碎片信息扫描, 共鉴定出109种极性甘油酯分子; 再通过外标法利用UPLC-ESI-Q-Trap/MS在多级反应监测模式下对各分子进行靶向定量分析。结果表明, 莱茵衣藻的极性脂以糖脂MGDG、DGDG及甜菜碱脂DGTS为主, 所有极性脂的分子组成表明, DGDG、SQDG、DGTS及PI是C18脂肪酸的去饱和载体。该研究利用液质联用技术建立了莱茵衣藻极性甘油酯组的结构图谱及定量分析技术平台, 为微藻极性脂生物学功能及脂质代谢研究奠定了基础。     

Seasonal changes of lipid content in the leaves of some ferns

Seasonal distribution of total lipids, of total and individual phospholipids as well as of one betaine lipid, diacylglyceryl-N,N,N-trimethylhomoserine (DGTS), found in the leaves of Dryopteris carthusiana, Dryopteris filix-mas, Matteuccia struthiopteris and Pteridium aquilinum was studied. Using thin-layer chromatography it was shown that lipid composition was different in these species and changed substantially throughout the development of scrolls (unrolled young leaves) to vegetatively mature leaves. The maximum concentration of DGTS for all the examined species was found in scrolls. DGTS then decreased to its minimum level or entirely disappeared to reappear at later stages of leaf development. This may indicate a specific role of DGTS in the plant membrane.     

Primary structure of the hypertrehalosaemic factor II from the corpus cardiacum of the Indian stick insect, Carausius morosus, determined by fast atom bombardment mass spectrometry

The primary structure of hypertrehalosaemic factor II, isolated from the corpus cardiacum of the Indian Stick Insect Carausius morosus, has been assigned as Glu-Leu-Thr-Phe-Thr-Pro-Asn-Trp-Gly-Thr-NH2 from its fast atom bombardment (FAB) mass spectrum and metastable scans of its FAB spectrum. The structure assigned shows close homology to other insect neuropeptides. A synthetic sample of this peptide gave the same FAB spectra, reversed-phase high-performance liquid chromatographic behavior, and biological behavior as the natural material. Mass spectrometric fragmentation of the synthetic peptide was examined by B/E linked scan and MIKES techniques in a two-sector mass spectrometer and by the MS/MS technique in a four-sector (tandem) spectrometer.     

Enhancement of structural information in FAB ionized carbohydrate samples by neutral gas collision

Fast atom bombardment (FAB) ionization and two coupled analyzers (BE-EB) have been combined with neutral gas collision (C) to enhance structural information in the mass spectra of oligosaccharides. (B and E are abbreviations for magnetic and electric sectors respectively.) FAB ionization and the first analysers (BE) have provided parent ions free from biological and liquid matrix contaminants. Structural detail of these products were observed after collision and daughter ion analysis in a second coupled analyser (EB). Starting from complex mixtures, this instrumental approach, BE-C-EB, has provided specific oligomeric sequence information which was not observed in the normal FAB mass spectra. Collision spectra obtained from isomeric linear and branched oligosaccharides show unique fragments that can be directly related to structure.     

Identification of diacylglycerol-O-(N,N,N-trimethyl)-homoserine in the halotolerant alga, Dunaliella parva

An unusual zwitterionic polar lipid component isolated from the halotolerant alga, Dunaliella parva Lerche, has been identified as 1(3),2-diacylglyceryl-3(1)-O-4′-(N,N,N-trimethyl) homoserine by means of infrared spectrometry, ¹H- and ¹³C-NMR spectrometry and field desorption mass spectrometry of the intact lipid, as well as by its TLC mobilities and staining behaviour. Mass spectrometry of this lipid indicated the presence of the following major molecular species: 16:0–18:0 (24%); 18:3–18:3 (19%); 16:0–18:2 (16%); 16:0–18:1 (8%).     

Seasonal variations of diacylglyceryl-N,N,N-trimethylhomoserine content in Polypodiophyta

Diacylglyceryl-N,N,N-trimethylhomoserine (DGTS) belongs to a family of three known betaine lipids. It is synthesized by a number of bacteria, fungi, brown, red and green algae, in addition to lichens, and is also found in higher plants Bryophyta, Equisetophyta and Polydiophyta. It has been determined that DGTS plants can be DGTS-positive or DGTS-negative. The purpose of the present study was to examine the leaves of the ferns Drytopteris filix-mas (L.) Tod. and Pteridium aquilinum (L.) Kuhn, collected at different time periods during a single vegetation season for their DGTS contents. The results obtained show that amounts of DGTS in all of the species examined varies relative to the time periods in which they were collected.     

Differences in betaine lipids and fatty acids between Pseudoisochrysis paradoxa VLP and Diacronema vlkianum VLP isolates (Haptophyta)

Two Haptophytes were isolated from extensive aquaculture ponds at Veta La Palma state (Guadalquivir estuary, SW Spain). They were identified as Pseudoisochrysis paradoxa VLP and Diacronema vlkianum VLP based on their SSU rDNA homology to other Haptophytes and positioned in the Isochrysidaceae and Pavlovaceae families, respectively. Both Haptophytes had phosphatidilglycerol (PG) as the only phospholipid (PL), representing a low proportion of the total lipid content (0.8% in P. paradoxa VLP and 3.3% in D. vlkianum VLP). Instead, they were found to have different types of betaine lipids (BL) that were identified and characterized by HPLC/ESI-TOF-MS operating in multiple reacting monitoring (MRM) modes. P. paradoxa VLP had 2.2% of total lipids as diacylgyceryl-N-trimethylhomoserine (DGTS): it is the first Haptophyte reported to have this BL. Its total lipid fraction also contained 12.0% of diacylglyceryl-carboxyhydroxymethylcholine (DGCC) as the main BL and no diacylglyceryl-hydroxymethyl-N,N,N-trimethyl-β-alanine (DGTA) was detected. DGTA was only present (4.6% of total lipids) in D. vlkianum VLP: this was the main difference in BL content relative to P. paradoxa. D. vlkianum VLP also had DGTS (4.1%) and DGCC (7.6%): it is the first microalgae in which the simultaneous presence of these three BL has been demonstrated.The fatty acid profiles of P. paradoxa VLP and D. vlkianum VLP were close to those described for the major part of known members of the Isochrisidaceae and Pavlovaceae families, respectively, with the main differences due to the higher percentages of 18:1n9 (18.5%), 18:4n3 (12.6%) and 22:6n3 (9.3%) in the former. The corresponding fatty acid percentages for D. vlkianum VLP were 3.9%, 3.5% and 3.9%, respectively. D. vlkianum VLP showed higher 16:1n7 (16.1%) and 20:5n3 (9.4%) contents, whereas P. paradoxa VLP had significantly lower percentages of 16:1n7 (1.7%) and 20:5n3 (0.6%). Fatty acids of BL differed between both haptophytes. In DGTS from P. paradoxa VLP, 90.9% of total molecular species consisted of the 14:0–18:1 fatty acid combination, whereas DGTS from D. vlkianum showed a more diverse range of fatty acids. The unsaturation index (UI) of DGTS was lower (55.8) than that of total lipid UI (178.3) in P. paradoxa VLP. In D. vlkianum VLP the UI of DGTS was higher (146.9) and similar to that for total cell lipids (145.9). DGTA from D. vlkianum VLP had the highest UI (321.8) of all BL studied and it contained maximum levels (27.7%) of 22:6n3, representing 7.1 times the proportion of this fatty acid in the whole lipid extract. DGCC was enriched in 20:5n3 by a factor of around four in both microalgae. Due to different levels of this fatty acid in the two microalgae their respective 20:5n3 content in DGCC varied from 2.2% (P. paradoxa VLP) to 41.0% (D. vlkianum VLP) and these concentrations were also associated with UI values of 92.2 and 271.0, respectively. The specific differences in BL and fatty acids described in the present work for two phylogenetic distant Hatophytes is a contribution to a better understanding on the complex relationship between lipid composition and taxonomy of this important Division of microalgae. Present results can also be useful for a more accurate identification of primary producers in food web studies using fatty acids and intact polar lipids as trophic markers.     

Betaine Lipids in Lower Plants. Biosynthesis of DGTS and DGTA in Ochromonas danica (Chrysophyceae) and the Possible Role of DGTS in Lipid Metabolism

Membrane lipids and fatty acids of Ochromonas danica were analyzed.Of the two betaine lipids, the homoserine lipid DGTS mainlycontains 14:0 and 18:2 fatty acids, while the alanine lipidDGTA is enriched in 18:0, 18:2 and 22:5 fatty acids. Of thepolar moiety of DGTA, improved NMR data are presented. On incubationof cells with [3,4-¹⁴C]methionine, DGTS as well as DGTA werelabelled. With [1-¹⁴C]methionine as a substrate, the label appearedin DGTS only. If double labelled [³H](glycerol)/[¹⁴C](polarpart)DGTS was used as a precursor, radioactivity was incorporatedspecifically into DGTA in which the isotope ratio was unchangedcompared to the precursor. Thus, the glyceryltrimethylhomoserinepart of DGTS acts as the precursor of the polar group of DGTA.Labelling of cells with [1-¹⁴C]oleate in a pulse-chase mannerand subsequent analysis of the label in the fatty acids andmolecular species of different lipids including DGTS and DGTA,suggested a clearly different role of the two betaine lipids:DGTS acts as a i) primary acceptor for exogenous C₁₈ monoeneacid, ii) substrate for the desaturation of 18:1 to 18:2 acid,and iii) donor of mainly 18:2 fatty acid to be distributed amongPE and other membrane lipids. Into DGTA, in contrast, fattyacids are introduced only after elongation and desaturation.As a result, the biosynthesis of DGTA from DGTS involves a decarboxylationand recarboxylation of the polar part and a simultaneous deacylationand reacylation of the glycerol moiety. (Received January 28, 1992; Accepted March 11, 1992)     

The characterisation and cyclic production of a highly unsaturated homoserine lipid in Chlorella minutissima

The marine alga Chlorella minutissima contains DGTS (diacylglyceryl-N,N,N-trimethylhomoserine) as a major component (up to 44% of total lipids). This lipid is absent from other members of the Chlorococcales, except for C. fusca, which contains DGTS as 1.3% of total lipids. Contrary to expectation, the DGTS is accompanied by PC (phosphatidylcholine) as the major phospholipid. DGTS is normally highly saturated in the C-1 position of glycerol, but in C. minutissima, both C-1 and C-2 are acylated with EPA (eicosapentaenoic acid, 20:5) in the major molecular species (over 90% of total). The DGTS level shows a marked rhythmic fluctuation with time which is inversely correlated with the level of MGDG (monogalactosyldiacylglycerol), the other major lipid. Improved NMR data and the first electrospray MS data on this lipid are presented.     

The electron impact, chemical ionization and fast atom bombardment positive ion mass spectra of 1,2-bis(sulfonyl)methylhydrazines.

A series of bis(sulfonyl)-1-methylhydrazines were analyzed by positive ion electron impact (EI), chemical ionization (CI) and fast atom bombardment (FAB) mass spectrometry. Since these compounds showed activity against the L1210 leukemia, an understanding of their mass spectral behavior is important should the structural characterization of metabolites be required. FAB proved to be the most useful technique, generally providing abundant protonated molecule ion peaks, in contrast to the weak peaks observed with CI (ammonia or isobutane) and the total absence of molecular ion peaks in the EI mass spectra. In addition, utilizing FAB eliminated the problem of thermal decomposition, which was very difficult to control under EI and CI experimental conditions. Fragments observed in FAB and CI mass spectra were consistent with protonation at the methyl-bearing nitrogen. One can locate the R1 and R2 moieties relative to the methyl-bearing nitrogen in FAB and CI by assigning that nitrogen as the site of protonation, with subsequent elimination of R2SO2H.     

Application of fast atom bombardment mass spectrometry to biological samples: analysis of urinary metabolites of acetaminophen

Fast atom bombardment (FAB) is useful for the characterization of all major metabolites of the analgesic acetaminophen (APAP). It is particularly useful for providing mass spectra of the polar glucuronide and sulfate conjugates which eluded identification by field desorption and other more conventional methods of ionization. A protocol is described for the use of FAB in the identification of urinary APAP metabolites isolated by reversed phase high-performance liquid chromatography (HPLC) following therapeutic dosages of the drug. A tentative set of recommendations for the off-line use of HPLC and FAB is directed towards solving problems encountered when using these two analytical techniques in concert. In addition, a method for calculating the signal to background ratio (S/B) for analyte peaks in FAB spectra from selected relative ion intensities is proposed. Examples are presented that show the potential of S/B as an empirical parameter for judging the quality of FAB spectra.     

A spin label study of perturbation effects of N-(1-methyldodecyl)-N, N, N-trimethylammonium bromide and N-(1-methyldodecyl)-N, N-dimethylamine oxide on model membranes prepared from Escherichia coli-isolated lipids

Interaction of bactericidal surfactants N-(1-methyldodecyl)-N, N, N-trimethylammonium bromide (2-ATDBr) and N-(1-methyldodecyl)-N, N-dimethylamine oxide (2-ATDNO) with phospholipid membranes prepared from Escherichia coli -- isolated lipids was studied by ESR spectroscopy using m-doxyl stearic acid (m-DSA, m = 5, 12, 16) and N-cetyl-N, N-dimethyl-N-tempoylammonium bromide spin labels located in different membrane depths. 2-ATDBr was found to be a more potent membrane perturbant than 2-ATDNO both at equal membrane and sample concentrations; this is in compliance with the respective antimicrobial activities of these agents. Using the statistical model of hydrocarbon chains in lipid bilayers, the probabilities of the formation of gauche conformations and the effective energy differences between the trans and gauche conformations were calculated from m-DSA order parameters for two different bilayer regions. Based on these parameters, a molecular model of the location of surfactant molecules in bilayer has been formulated. It has been suggested that at low concentrations the surfactant molecules are located in structural defects between lipid clusters in the bilayer. After filling up these defects, the surfactant molecules penetrate into the clusters between lipid molecules, expand the bilayer laterally and increase the amount of gauche conformations in the hydrocarbon chains in the hydrophobic core of the bilayer.     

Fast atom bombardment mass spectrometry of bouvardin and selected analogs

The fast atom bombardment (FAB) mass spectra of bouvardin (1) 6-O-methylbouvardin (2), deoxybouvardin (3) and a synthetic analog (4) have been examined. The spectra of the bicyclic compounds 1-3 display site-directed fragmentations resulting from the presence of a phenolic bridged tyrosine moiety unique to this class of compounds. Comparison of the spectrum of 4, which lacks the rigid 14-membered ring, with the spectra of 1-3 shows some similarities, but clearly does not cleave in a site-directed manner as the other bouvardin analogs. Fragmentation pathways are postulated which account for most of the major ions observed in the FAB mass spectra of these potentially useful antitumor agents. Metastable ion analysis confirms the operation of the proposed pathways.     

A novel human erythrocyte glycosylphosphatidylinositol (GPI)-anchored glycoprotein ACA. Isolation,purification, primary structure determination,and molecular parameters of its lipid structure

A method has been elaborated to isolate and purify up to homogeneity a novel membrane glycoprotein containing a glycosyl-phosphatidylinositol (GPI) anchor by means of salting out with ammonium sulfate (40-80% saturation), followed by preparative SDS-PAGE, chromatography and acetone precipitation. The preparation obtained was homogeneous upon electrophoresis in the presence of 0.1% SDS after reduction with 2-mercaptoethanol. It is protein-soluble at its isoelectrical point (pH 5.5) with molecular mass of 65,000 daltons. The isolated protein is linked to the membrane via glycosyl-phosphatidylinositol susceptible to cleavage by purified phospholipase C. The hydrophobic portion of the glycolipid membrane anchor of the protein was radiolabeled with the photoactivated reagent 3-(trifluoromethyl)-3-(m-[(125)I]iodophenyl)diazirine and hydrolyzed with glycosyl-phosphatidylinositol-specific phospholipase C, followed by enzymatic deacetylation of the remaining lipid. Thin-layer chromatography showed that the generated radiolabeled fragment migrates with the same mobility as that of variant surface glycoprotein (VSG), obtained in the same manner. In this study we describe a novel erythrocyte membrane GPI-linked protein with the structural feature of an anchor that, in contrast to other GPI-linked erythrocyte proteins, has a non-acetylated inositol ring and diacylglycerol rather than alkyl-acyl glycerol as a lipid tail of the anchor.