Phosphatidylglycerol Molecules Research Articles

The F-pilus biomechanical adaptability accelerates conjugative dissemination of antimicrobial resistance and biofilm formation

Conjugation is used by bacteria to propagate antimicrobial resistance (AMR) in the environment. Central to this process are widespread conjugative F-pili that establish the connection between donor and recipient cells, thereby facilitating the spread of IncF plasmids among enteropathogenic bacteria. Here, we show that the F-pilus is highly flexible but robust at the same time, properties that increase its resistance to thermochemical and mechanical stresses. By a combination of biophysical and molecular dynamics methods, we establish that the presence of phosphatidylglycerol molecules in the F-pilus contributes to the structural stability of the polymer. Moreover, this structural stability is important for successful delivery of DNA during conjugation and facilitates rapid formation of biofilms in harsh environmental conditions. Thus, our work highlights the importance of F-pilus structural adaptations for the efficient spread of AMR genes in a bacterial population and for the formation of biofilms that protect against the action of antibiotics.

Crucial importance of length of fatty-acyl chains bound to the sn-2 position of phosphatidylglycerol for growth and photosynthesis of Synechocystis sp. PCC 6803

Phosphatidylglycerol (PG) in thylakoid membrane is essential for growth and photosynthesis of photosynthetic organisms. Although the sn-2 position of PG in thylakoid membrane is exclusively esterified with C16 fatty acids, the functional importance of the C16 fatty-acyl chains at the sn-2 position has not been clarified. In this study, we chemically synthesized non-metabolizable PG molecules: we introduced linoleic acid (18:2, fatty acid containing 18 carbons with 2 double bonds) and one of the saturated fatty acids with different chain length (12:0, 14:0, 16:0, 18:0 and 20:0) by ether linkage to the sn-1 and sn-2 positions, respectively. With the synthesized ether-linked PG molecules, we checked whether they could complement the growth and photosynthesis of pgsA mutant cells of Synechocystis sp. PCC 6803 to understand the importance of length of fatty chains at the sn-2 position of PG. The pgsA mutant is incapable of synthesizing PG, so it requires exogenous PG added to medium for growth. The growth rate and photosynthetic activity of mutant cells depended on the length of fatty chains: the PG molecular species binding 16:0 most effectively complemented the growth and photosynthesis of mutant cells, and other PG molecular species with fatty chains shorter or longer than 16:0 were less effective; especially, those binding 12:0 inhibited the growth and photosynthetic activity of the mutant cells. These data demonstrate that length of fatty chains bound to the sn-2 position of PG is critical for PG performance in growth and photosynthesis.

Specific Incorporation of Polyunsaturated Fatty Acids into the sn-2 Position of Phosphatidylglycerol Accelerates Photodamage to Photosystem II under Strong Light.

Free fatty acids (FFAs) are generated by the reaction of lipases with membrane lipids. Generated polyunsaturated fatty acids (PUFAs) containing more than two double bonds have toxic effects in photosynthetic organisms. In the present study, we examined the effect of exogenous FFAs in the growth medium on the activity of photosystem II (PSII) under strong light in the cyanobacterium Synechocystis sp. PCC 6803 (Synechocystis). PUFAs but not monounsaturated fatty acids accelerated the rate of photodamage to PSII by inactivating electron transfer at the oxygen-evolving complex. Moreover, supplemented PUFAs were specifically incorporated into the sn-2 position of phosphatidylglycerol (PG), which usually contains C16 fatty acids at the sn-2 position in Synechocystis cells. The disruption of the gene for an acyl-ACP synthetase reduced the effect of PUFAs on the photoinhibition of PSII. Thus, the specific incorporation of PUFAs into PG molecules requires acyl-ACP synthetase and leads to an unstable PSII, thereby accelerating photodamage to PSII. Our results are a breakthrough into elucidating the molecular mechanism of the toxicity of PUFAs to photosynthetic organisms.

Promiscuous phospholipid biosynthesis enzymes in the plant pathogen Pseudomonas syringae

Bacterial membranes are primarily composed of phosphatidylethanolamine (PE), phosphatidylglycerol (PG) and cardiolipin (CL). In the canonical PE biosynthesis pathway, phosphatidylserine (PS) is decarboxylated by the Psd enzyme. CL formation typically depends on CL synthases (Cls) using two PG molecules as substrates. Only few bacteria produce phosphatidylcholine (PC), the hallmark of eukaryotic membranes. Most of these bacteria use phospholipid N-methyltransferases to successively methylate PE to PC and/or a PC synthase (Pcs) to catalyze the condensation of choline and CDP-diacylglycerol (CDP-DAG) to PC. In this study, we show that membranes of Pseudomonas species able to interact with eukaryotes contain PE, PG, CL and PC. More specifically, we report on PC formation and a poorly characterized CL biosynthetic pathway in the plant pathogen P. syringae pv. tomato. It encodes a Pcs enzyme responsible for choline-dependent PC biosynthesis. CL formation is catalyzed by a promiscuous phospholipase D (PLD)-type enzyme (PSPTO_0095) that we characterized in vivo and in vitro. Like typical bacterial CL biosynthesis enzymes, it uses PE and PG for CL production. This enzyme is also able to convert PE and glycerol to PG, which is then combined with another PE molecule to synthesize CL. In addition, the enzyme is capable of converting ethanolamine or methylated derivatives into the corresponding phospholipids such as PE both in P. syringae and in E. coli. It can also hydrolyze CDP-DAG to yield phosphatidic acid (PA). Our study adds an example of a promiscuous Cls enzyme able to synthesize a suite of products according to the available substrates.

A promiscuous archaeal cardiolipin synthase enables construction of diverse natural and unnatural phospholipids

Cardiolipins (CL) are a class of lipids involved in the structural organization of membranes, enzyme functioning, and osmoregulation. Biosynthesis of CLs has been studied in eukaryotes and bacteria, but has been barely explored in archaea. Unlike the common fatty acyl chain–based ester phospholipids, archaeal membranes are made up of the structurally different isoprenoid-based ether phospholipids, possibly involving a different cardiolipin biosynthesis mechanism. Here, we identified a phospholipase D motif–containing cardiolipin synthase (MhCls) from the methanogen Methanospirillum hungatei. The enzyme was overexpressed in Escherichia coli, purified, and its activity was characterized by LC-MS analysis of substrates/products. MhCls utilizes two archaetidylglycerol (AG) molecules in a transesterification reaction to synthesize glycerol-di-archaetidyl-cardiolipin (Gro-DACL) and glycerol. The enzyme is nonselective to the stereochemistry of the glycerol backbone and the nature of the lipid tail, as it also accepts phosphatidylglycerol (PG) to generate glycerol-di-phosphatidyl-cardiolipin (Gro-DPCL). Remarkably, in the presence of AG and PG, MhCls formed glycerol-archaetidyl-phosphatidyl-cardiolipin (Gro-APCL), an archaeal-bacterial hybrid cardiolipin species that so far has not been observed in nature. Due to the reversibility of the transesterification, in the presence of glycerol, Gro-DPCL can be converted back into two PG molecules. In the presence of other compounds that contain primary hydroxyl groups (e.g., alcohols, water, sugars), various natural and unique unnatural phospholipid species could be synthesized, including multiple di-phosphatidyl-cardiolipin species. Moreover, MhCls can utilize a glycolipid in the presence of phosphatidylglycerol to form a glycosyl-mono-phosphatidyl-cardiolipin species, emphasizing the promiscuity of this cardiolipin synthase that could be of interest for bio-catalytic purposes.

Quantitative molecular tissue atlas of Bis(monoacylglycero)phosphate and phosphatidylglycerol membrane lipids in rodent organs generated by methylation assisted high resolution mass spectrometry

Bis(monoacylglycero)phosphate (BMP) and phosphatidylglycerol (PG) are structural isomeric phospholipids with very different properties and biological functions. Due to their isomeric nature, it has thus far been challenging to simultaneously quantify BMP and PG lipids in tissue samples by mass spectrometry. Therefore, we have developed a sensitive LC-MS/MS based approach with prior methylation derivatization that is able to handle large batches of samples. Using this high throughput platform, a simulated MS/MS database was established for confident lipid assignment. In this work, we have simultaneously identified and quantified BMP and PG lipid molecules in different body tissues of rats and mice. We report for the first time a quantitative molecular atlas of BMP and PG lipids for 14 different tissues and organs in Wistar rats, NMRI and CD1 mice. Organ- and species-specificity was analyzed and compared for both lipid molecule classes. A total of 34 BMP and 10 PG molecules were quantified, with PG concentrations being generally much higher across tissues than BMP, but BMP lipids showing a much higher molecular diversity between animal organs. The large diversity of the BMP lipids with regard to their abundance and molecular composition suggests distinct biological function(s) of the individual BMP molecules in different tissues and organs of body. Particularly high tissue levels of BMP were seen in spleen, lung, liver, kidney and small intestines, i.e. tissues that are known for their high abundance and/or activity level of lysosomes late and endosomes. Elevated BMP levels in brain tissue of APP/PSEN transgenic compared to age matched wild-type mice were also observed using this platform. This analytical methodology presented a high throughput LC-based approach incorporating simulated MS/MS database to identify and quantify BMP lipids as well as PG molecules.

Site-directed mutagenesis of two amino acid residues in cytochrome b559 α subunit that interact with a phosphatidylglycerol molecule (PG772) induces quinone-dependent inhibition of photosystem II activity

X-ray crystallographic analysis (1.9-Å resolution) of the cyanobacterial photosystem II (PSII) dimer showed the presence of five phosphatidylglycerol (PG) molecules per reaction center. One of the PG molecules, PG772, is located in the vicinity of the QB-binding site. To investigate the role of PG772 in PSII, we performed site-directed mutagenesis in the cytochrome (Cyt) b559 α subunit of Synechocystis sp. PCC 6803 to change two amino acids, Thr-5 and Ser-11, which interact with PG772. The photosynthetic activity of intact cells was slightly lower in all mutants than that of cells in the control strain; however, the oxygen-evolving PSII activity was decreased markedly in cells of mutants, as measured using artificial quinones (such as p-benzoquinone). Furthermore, electron transport from QA to QB was inhibited in mutants incubated with quinones, particularly under high-intensity light conditions. Lipid analysis of purified PSII showed approximately one PG molecule per reaction center, presumably PG772, was lost in the PSII dimer from the T5A and S11A mutants compared with that in the PSII dimer from the control strain. In addition, protein analysis of monomer and dimer showed decreased levels of PsbV and PsbU extrinsic proteins in the PSII monomer purified from T5A and S11A mutants. These results suggest that site-directed mutagenesis of Thr-5 and Ser-11, which presumably causes the loss of PG772, induces quinone-dependent inhibition of PSII activity under high-intensity light conditions and destabilizes the binding of extrinsic proteins to PSII.

Lipid-Controlled Stabilization of Charge-Separated States (P+QB-) and Photocurrent Generation Activity of a Light-Harvesting-Reaction Center Core Complex (LH1-RC) from Rhodopseudomonas palustris.

The photosynthetic light-harvesting-reaction center core complex (LH1-RC) is a natural excitonic and photovoltaic device embedded in a lipid membrane. In order to apply LH1-RCs as a biohybrid energy-producing material, some important issues must be addressed, including how to make LH1-RCs function as efficiently as possible. In addition, they should be characterized to evaluate how many active LH1-RCs efficiently work in artificial systems. We report here that an anionic phospholipid, phosphatidylglycerol (PG), stabilizes the charge-separated state (a photooxidized electron donor and reduced quinone pair, P+QB-) of LH1-RC (from Rhodopseudomonas palustris) and enhances its activity in photocurrent generation. Steady-state fluorometric analysis demonstrated that PG enhances the formation of the P+QB- state at lower irradiances. The photocurrent generation activity was analyzed via Michaelis-Menten kinetics, revealing that 38% of LH1-RCs reconstituted into the PG membrane generated photocurrent at a turnover frequency of 46 s-1. PG molecules, which interact with LH1-RC in vivo, play the role of an active effector component for LH1-RC to enhance its function in the biohybrid system.

Structural and Functional Basis for Lipid Synergy on the Activity of the Antibacterial Peptide ABC Transporter McjD

The lipid bilayer is a dynamic environment that consists of a mixture of lipids with different properties that regulate the function of membrane proteins; these lipids are either annular, masking the protein hydrophobic surface, or specific lipids, essential for protein function. In this study, using tandem mass spectrometry, we have identified specific lipids associated with the Escherichia coli ABC transporter McjD, which translocates the antibacterial peptide MccJ25. Using non-denaturing mass spectrometry, we show that McjD in complex with MccJ25 survives the gas phase. Partial delipidation of McjD resulted in reduced ATPase activity and thermostability as shown by circular dichroism, both of which could be restored upon addition of defined E. coli lipids. We have resolved a phosphatidylglycerol lipid associated with McjD at 3.4 Å resolution, whereas molecular dynamic simulations carried out in different lipid environments assessed the binding of specific lipids to McjD. Combined, our data show a synergistic effect of zwitterionic and negatively charged lipids on the activity of McjD; the zwitterionic lipids provide structural stability to McjD, whereas the negatively charged lipids are essential for its function.

Site-directed mutagenesis of amino acid residues of D1 protein interacting with phosphatidylglycerol affects the function of plastoquinone QB in photosystem II.

Recent X-ray crystallographic analysis of photosystem (PS) II at 1.9-Å resolution identified 20 lipid molecules in the complex, five of which are phosphatidylglycerol (PG). In this study, we mutagenized amino acid residues S232 and N234 of D1, which interact with two of the PG molecules (PG664 and PG694), by site-directed mutagenesis in Synechocystis sp. PCC 6803 to investigate the role of the interaction in PSII. The serine and asparagine residues at positions 232 and 234 from the N-terminus were mutagenized to alanine and aspartic acid, respectively, and a mutant carrying both amino acid substitutions was also produced. Although the obtained mutants, S232A, N234D, and S232AN234D, exhibited normal growth, they showed decreased photosynthetic activities and slower electron transport from QA to QB than the control strain. Thermoluminescence analysis suggested that this slower electron transfer in the mutants was caused by more negative redox potential of QB, but not in those of QA and S2. In addition, the levels of extrinsic proteins, PsbV and PsbU, were decreased in PSII monomer purified from the S232AN234D mutant, while that of Psb28 was increased. In the S232AN234D mutant, the content of PG in PSII was slightly decreased, whereas that of monogalactosyldiacylglycerol was increased compared with the control strain. These results suggest that the interactions of S232 and N234 with PG664 and PG694 are important to maintain the function of QB and to stabilize the binding of extrinsic proteins to PSII.

The Sensing Sites for the Membrane Inner Lipids Regulate the Activation Gating of the KcsA Potassium Channel

The activity of the ion channels are influenced by membrane lipid compositions and specific lipid molecules are indispensable for maintaining channel activities. For the KcsA potassium channel, the presence of anionic phospholipids such as phosphatidylglycerol (PG) is prerequisite for the channel activity. Previously we demonstrated by means of single-channel current recordings in the asymmetric lipid bilayer that the PG molecule on the inner leaflet, rather than the outer leaflet, renders the KcsA channel highly active. The fluorescent method revealed that the helix-bundle gate is kept open in the PG liposome but not much in the liposomes made of neutral or cationic phospholipids. To elucidate the underlying mechanism of the interaction between anionic lipids on the inner leaflet and the activation gate, charge-neutralizing mutations to positively charged residues were introduced. Several amino acid residues lying at the inner boundary of the membrane were found to be sensitive to the PG effect on the gating. Mechanism underlying lipid-mediated regulation of the activation gating of the KcsA channel will be discussed.

Discovery of a cardiolipin synthase utilizing phosphatidylethanolamine and phosphatidylglycerol as substrates

Depending on growth phase and culture conditions, cardiolipin (CL) makes up 5-15% of the phospholipids in Escherichia coli with the remainder being primarily phosphatidylethanolamine (PE) and phosphatidylglycerol (PG). In E. coli, the cls and ybhO genes (renamed clsA and clsB, respectively) each encode a CL synthase (Cls) that catalyzes the condensation of two PG molecules to form CL and glycerol. However, a ΔclsAB mutant still makes CL in the stationary phase, indicating the existence of additional Cls. We identified a third Cls encoded by ymdC (renamed clsC). ClsC has sequence homology with ClsA and ClsB, which all belong to the phospholipase D superfamily. The ΔclsABC mutant lacks detectible CL regardless of growth phase or growth conditions. CL can be restored to near wild-type levels in stationary phase in the triple mutant by expressing either clsA or clsB. Expression of clsC alone resulted in a low level of CL in the stationary phase, which increased to near wild-type levels by coexpression of its neighboring gene, ymdB. CL synthesis by all Cls is increased with increasing medium osmolarity during logarithmic growth and in stationary phase. However, only ClsA contributes detectible levels of CL at low osmolarity during logarithmic growth. Mutation of the putative catalytic motif of ClsC prevents CL formation. Unlike eukaryotic Cls (that use PG and CDP-diacylglycerol as substrates) or ClsA, the combined YmdB-ClsC used PE as the phosphatidyl donor to PG to form CL, which demonstrates a third and unique mode for CL synthesis.

Cardiolipin biosynthesis in Streptococcus mutans is regulated in response to external pH

Streptococcus mutans, a causative agent of dental caries in humans, adapts to changing environmental conditions, such as pH, in order to survive and cause disease in the oral cavity. Previously, we have shown that S. mutans increases the proportion of monounsaturated membrane fatty acids as part of its acid-adaptive strategy. Membrane lipids function as carriers of membrane fatty acids and therefore it was hypothesized that lipid backbones themselves could participate in the acid adaptation process. Lipids have been shown to protect other bacterial species from rapid changes in their environment, such as shifts in osmolality and the need for long-term survival. In the present study, we have determined the contribution of cardiolipin (CL) to acid resistance in S. mutans. Two ORFs have been identified in the S. mutans genome that encode presumptive synthetic enzymes for the acidic phospholipids: phosphatidylglycerol (PG) synthase (pgsA, SMU.2151c) and CL synthase (cls, SMU.988), which is responsible for condensing two molecules of PG to create CL. A deletion mutant of the presumptive cls gene was created using PCR-mediated cloning; however, attempts to delete pgsA were unsuccessful, indicating that pgsA may be essential. Loss of the presumptive cls gene resulted in the inability of the mutant strain to produce CL, indicating that SMU.988 encodes CL synthase. The defect in cls rendered the mutant acid sensitive, indicating that CL is required for acid adaptation in S. mutans. Addition of exogenous CL to the mutant strain alleviated acid sensitivity. MS indicated that S. mutans could assimilate exogenous CL into the membrane, halting endogenous CL incorporation. This phenomenon was not due to repression, as a cls gene transcriptional reporter fusion exhibited elevated activity when cells were supplemented with exogenous CL. Lipid analysis, via MS, indicated that CL is a reservoir for monounsaturated fatty acids in S. mutans. We demonstrated that the cls mutant exhibits elevated F-ATPase activity but it is nevertheless unable to maintain the normal membrane proton gradient, indicating cytoplasmic acidification. We conclude that the control of lipid backbone synthesis is part of the acid-adaptive repertoire of S. mutans.

The Asymmetric Lipid Bilayer Revealed Sidedness of the Effective Phospholipids on the Single-Channel Properties of the KcsA Potassium Channel

Function of ion channels is modulated by the composition of lipid bilayer. Some ion channels lose their activity when they are reconstituted into alien membrane environment. Thus, membrane lipids act as a co-factor for maintaining the channel activity as in the case for other membrane proteins. For the KcsA potassium channel, an anionic phospholipid has been thought to play an essential role in its function because KcsA exhibited reduced ion conducting activity in the artificial membrane without phosphatidylglycerol (PG). Relevance of PG to the KcsA function has been further supported by crystallographic studies that revealed specific binding of PG molecules to the transmembrane region of the channel. However, little is known about the mechanism on how lipid molecules, including PG, modulate the function of KcsA channel. In this study we analyzed effects of lipids on the single-channel current properties of KcsA using artificial lipid bilayer having asymmetric lipid composition. We revealed that presence of PG in the membrane significantly increased the open probability and unitary conductance of KcsA channel. We also found that these effects were substitutable with other anionic phospholipids such as phosphatidylserine and phosphatidic acid but not by neutral or cationic phospholipids such as phosphatidylcholine or ethyl phosphatidylcholine. Furthermore, the sidedness of these anionic lipid effects was analyzed. We concluded that the binding of anionic lipids to the specific lipid-binding sites, which had been found in the KcsA crystal structure, would not be an origin of these anionic lipid effects.

Characterization of daptomycin oligomerization with perylene excimer fluorescence: Stoichiometric binding of phosphatidylglycerol triggers oligomer formation

Daptomycin is a lipopeptide antibiotic that binds to and depolarizes bacterial cell membranes. Its antibacterial activity requires calcium and correlates with the content of phosphatidylglycerol in the target membrane. Daptomycin has been shown to form oligomers on liposome membranes. We here use perylene excimer fluorescence to further characterize the membrane-associated oligomer. To this end, the N-terminal fatty acyl chain was replaced with perylene-butanoic acid. The perylene derivative retains one third of the antibacterial activity of native daptomycin. On liposomes containing phosphatidylcholine and phosphatidylglycerol, as well as on Bacillus subtilis cells, the perylene-labeled daptomycin forms excimers, which shows that the N-terminal acyl chains of neighboring oligomer subunits are in immediate contact with one another. In a lipid bicelle system, oligomer formation can be titrated with stoichiometric amounts of phosphatidylglycerol. Therefore, the interaction of daptomycin with a single molecule of phosphatidylglycerol is sufficient to trigger daptomycin oligomerization.

Two functional sites of phosphatidylglycerol for regulation of reaction of plastoquinone QB in photosystem II

Functional roles of an anionic lipid phosphatidylglycerol (PG) were studied in pgsA-gene-inactivated and cdsA-gene-inactivated/phycobilisome-less mutant cells of a cyanobacterium Synechocystis sp. PCC 6803, which can grow only in PG-supplemented media. 1) A few days of PG depletion suppressed oxygen evolution of mutant cells supported by p-benzoquinone (BQ). The suppression was recovered slowly in a week after PG re-addition. Measurements of fluorescence yield indicated the enhanced sensitivity of QB to the inactivation by BQ. It is assumed that the loss of low-affinity PG (PGL) enhances the affinity for BQ that inactivates QB. 2) Oxygen evolution without BQ, supported by the endogenous electron acceptors, was slowly suppressed due to the direct inactivation of QB during 10days of PG depletion, and was recovered rapidly within 10h upon the PG re-addition. It is concluded that the loss of high-affinity PG (PGH) displaces QB directly. 3) Electron microscopy images of PG-depleted cells showed the specific suppression of division of mutant cells, which had developed thylakoid membranes attaching phycobilisomes (PBS). 4) Although the PG-depletion for 14days decreased the chlorophyll/PBS ratio to about 1/4, florescence spectra/lifetimes were not modified indicating the flexible energy transfer from PBS to different numbers of PSII. Longer PG-depletion enhanced allophycocyanin fluorescence at 683nm with a long 1.2ns lifetime indicating the suppression of energy transfer from PBS to PSII. 5) Action sites of PGH, PGL and other PG molecules on PSII structure are discussed.

Characterization of a 62-Kilodalton Acidic Phospholipid-Binding Protein Isolated from the Edible Mushroom Pleurotus ostreatus

Many lipid-binding proteins such as pleurotolysin and ostreolysin have been isolated from the edible mushroom Pleurotus ostreatus .I n this s tudy, we detected a novel lipid-binding protein with a molecular weight of 62kDa by measuring via centrifugation the association of aqueous extracts of the mushroom with lipid vesicles composed of various phospholipids. The 62-kDa protein (p62) was purified by sedimentation of the mixture of protein extracts and acidic phospholipid-containing lipid vesicles. The purified p62 bound to phosphatidylglycerol (PG)/phosphatidylcholine/cholesterol (5:45:50) vesicles but not to vesicles composed of other phospholipids including phosphatidylserine (PS), phosphatidylinositol, phosphatidic acid, lysoPS, and lysophosphatidylinositol. The p62 protein specifically associated with the PG-containing vesicles but not with other polyglycerophospholipid vesicles consisting of cardiolipin, bis(monoacylglycero)phosphate, monolysocardiolipin, or dilysocardiolipin, suggesting that p62 recognized a precise molecular structure of PG. Intrinsic tryptophan fluorescence of p62 was changed by incubation of p62 with PG-containing vesicles. Staining of giant unilamellar vesicles with fluorescence-labeled p62 showed that p62 bound to PG-containing vesicles but not PS-containing vesicles. These observations signify the potential usefulness of p62 as a tool for studying the functions of PG molecules in biological membranes.

A Eukaryote-like Cardiolipin Synthase Is Present in Streptomyces coelicolor and in Most Actinobacteria

Cardiolipin (CL) is an anionic membrane lipid present in bacteria, plants, and animals, but absent from archaea. It is generally thought that bacteria use an enzyme belonging to the phospholipase D superfamily as cardiolipin synthase (Cls) catalyzing a reversible phosphatidyl group transfer from one phosphatidylglycerol (PG) molecule to another PG to form CL and glycerol. In contrast, in eukaryotes a Cls of the CDP-alcohol phosphatidyltransferase superfamily uses cytidine diphosphate-diacylglycerol (CDP-DAG) as the donor of the phosphatidyl group, which is transferred to a molecule of PG to form CL. Searching the genome of the actinomycete Streptomyces coelicolor A3(2) we identified a gene coding for a putative Cls of the CDP-alcohol phosphatidyltransferase superfamily (Sco1389). Here we show that expression of Sco1389 in a CL-deficient Rhizobium etli mutant restores CL formation. In an in vitro assay Sco1389 condenses CDP-DAG with PG to form CL and therefore catalyzes the same reaction as eukaryotic cardiolipin synthases. This is the first time that a CDP-alcohol phosphatidyltransferase from bacteria is shown to be responsible for CL formation. The broad occurrence of putative orthologues of Sco1389 among the actinobacteria suggests that CL synthesis involving a eukaryotic type Cls is common in actinobacteria.

The interactions between phosphatidylglycerol and phosphatidylethanolamines in model bacterial membranes: The effect of the acyl chain length and saturation

In this work the Langmuir monolayer technique was applied to study the interactions between phosphatidylglycerol (PG) and phosphatidylethanolamines (PEs). Since the technique of monomolecular layers is useful method of modeling of biomembranes and the investigated lipids are the major components of Gram-negative bacteria bilayers the results obtained were discussed in the context of the properties of bacterial membranes. To investigate the influence of the phosphatidylethanolamine acyl chain structure on its miscibility and interactions with dipalmitoylphosphatidylglycerol (DPPG) the chosen phosphatidylethanolamines differed both in the length (dipalmitoylphosphatidylethanolamine (DPPE), distearoylphosphatidylethanolamine (DSPE)), and saturation (dioleoylphosphatidylethanolamine (DOPE)) of the hydrocarbon chains. The obtained results prove non ideal behaviour of all of the mixed systems studied. It was found that in monolayers between DPPG and phosphatidylethanolamines with saturated acyl chains (DPPE, DSPE) strong attractions resulting from the formation of hydrogen bonds exist. These attractions are the strongest for equimolar mixtures indicating that PG and PE molecules form the “complexes” of 1:1 proportion. In DOPE/DPPG mixed system the distance between phospholipids is larger, due to the presence of the cis double bonds in the acyl chains of DOPE molecule, which makes the formation of the hydrogen bonds more difficult. The interactions between molecules, the molecular packing and the stability of the investigated mixed systems were thoroughly discussed from the point of view of the effect of polar head and PEs acyl chain structure. Basing on the obtained results it was suggested that PG molecules play in bacterial membranes similar role as cholesterol in mammalian bilayers.

Effects of phospholipase and lipase treatments on photosystem II core dimer from a thermophilic cyanobacterium

Lipids are important components of transmembrane protein complexes. In order to study the roles of lipids in photosystem II (PSII), we treated the PSII core dimer complex from a thermophilic cyanobacterium Thermosynechococcus vulcanus with phospholipase A(2) (PLA(2)) and lipase, and examined their effects on PSII structure and function. PLA(2)-treatment decreased the content of phospholipid, phosphatidylglycerol (PG) by 59%, leading to a decrease of oxygen evolution by 40%. On the other hand, although treatment with lipase specifically decreased the content of monogalactosyldiacylglycerol (MGDG) by 52%, it decreased oxygen evolution only by 16%. This indicates that PG plays a more important role in PSII than MGDG. Both PLA(2)- and lipase-treatments induced neither the dissociation of PSII dimer, nor any loss of polypeptides. The degradation of PG resulted in a damage to the Q(B)-binding site as demonstrated from photoreduction activity of 2,6-dichlorophenolindophenol and chlorophyll fluorescence yields in the absence or presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea, and the dependencies of oxygen evolution on various electron acceptors before and after PLA(2)- or lipase-treatments. However, there were approximately three and five molecules of PG and MGDG per PSII reaction center left in the PSII dimeric complex after the PLA(2)- and lipase-treatments. These lipids are therefore bound to the interior of the protein matrix and resistant to the lipase treatments. The resistance of these lipids against PLA(2)- and lipase-treatments may be a specific feature of PSII from the thermophilic cyanobacterium, suggesting a possible correlation between binding of lipids and thermostability of PSII.