Presence Of Phosphatidylethanolamine Research Articles

In-Depth Genome Characterization and Pan-Genome Analysis of Strain KMM 296, a Producer of Highly Active Alkaline Phosphatase; Proposal for the Reclassification of Cobetia litoralis and Cobetia pacifica as the Later Heterotypic Synonyms of Cobetia amphilecti and Cobetia marina, and Emended Description of the Species Cobetia amphilecti and Cobetia marina.

A strictly aerobic, Gram-stain-negative, rod-shaped, and motile bacterium, designated strain KMM 296, isolated from the coelomic fluid of the mussel Crenomytilus grayanus, was investigated in detail due to its ability to produce a highly active alkaline phosphatase CmAP of the structural family PhoA. A previous taxonomic study allocated the strain to the species Cobetia marina, a member of the family Halomonadaceae of the class Gammaproteobacteria. However, 16S rRNA gene sequencing showed KMM 296's relatedness to Cobetia amphilecti NRIC 0815T. The isolate grew with 0.5-19% NaCl at 4-42 °C and hydrolyzed Tweens 20 and 40 and L-tyrosine. The DNA G+C content was 62.5 mol%. The prevalent fatty acids were C18:1 ω7c, C12:0 3-OH, C18:1 ω7c, C12:0, and C17:0 cyclo. The polar lipid profile was characterized by the presence of phosphatidylethanolamine, phosphatidylglycerol, phosphatidic acid, and also an unidentified aminolipid, phospholipid, and a few unidentified lipids. The major respiratory quinone was Q-8. According to phylogenomic and chemotaxonomic evidence, and the nearest neighbors, the strain KMM 296 represents a member of the species C. amphilecti. The genome-based analysis of C. amphilecti NRIC 0815T and C. litoralis NRIC 0814T showed their belonging to a single species. In addition, the high similarity between the C. pacifica NRIC 0813T and C. marina LMG 2217T genomes suggests their affiliation to one species. Based on the rules of priority, C. litoralis should be reclassified as a later heterotypic synonym of C. amphilecti, and C. pacifica is a later heterotypic synonym of C. marina. The emended descriptions of the species C. amphilecti and C. marina are also proposed.

Genome-Based Classification of Strain 16-SW-7, a Marine Bacterium Capable of Converting B Red Blood Cells, as Pseudoalteromonas distincta and Proposal to Reclassify Pseudoalteromonas paragorgicola as a Later Heterotypic Synonym of Pseudoalteromonas distincta.

A strictly aerobic, Gram-stain-negative, rod-shaped, and motile bacterium, designated strain 16-SW-7, isolated from a seawater sample, was investigated in detail due to its ability to produce a unique α-galactosidase converting B red blood cells into the universal type blood cells. The phylogenetic analysis based on 16S rRNA gene sequences revealed that the strain 16-SW-7 is a member of the Gammaproteobacteria genus Pseudoalteromonas. The closest relatives of the environmental isolate were Pseudoalteromonas distincta KMM 638T and Pseudoalteromonas paragorgicola KMM 3548T, with the plural paralogous 16S rRNA genes of 99.87–100% similarity. The strain 16-SW-7 grew with 1–10% NaCl and at 4–34°C, and hydrolyzed casein, gelatin, tyrosine, and DNA. The genomic DNA G+C content was 39.3 mol%. The prevalent fatty acids were C16:1 ω7c, C16:0, C17:1 ω8c, C18:1 ω7c, C17:0, and C12:0 3-OH. The polar lipid profile was characterized by the presence of phosphatidylethanolamine, phosphatidylglycerol, two unidentified amino lipids, and three unidentified lipids. The major respiratory quinone was Q-8. The finished genome of the strain 16-SW-7 (GenBank assembly accession number: GCA_005877035.1) has a size of 4,531,445 bp and comprises two circular chromosomes L1 and S1, deposited in the GenBank under the accession numbers CP040558 and CP040559, respectively. The strain 16-SW-7 has the ANI values of 98.2% with KMM 638T and KMM 3548T and the DDH values of 84.4 and 83.5%, respectively, indicating clearly that the three strains belonged to a single species. According to phylogenetic evidence and similarity for the chemotaxonomic and genotypic properties, the strain 16-SW-7 (= KCTC 52772 = KMM 701) represents a novel member of the species Pseudoalteromonas distincta. Also, we have proposed to reclassify Pseudoalteromonas paragorgicola as a later heterotypic synonym of P. distincta based on the rules of priority with the emendation of the species.

Elucidating Duramycin's Bacterial Selectivity and Mode of Action on the Bacterial Cell Envelope.

The use of naturally occurring antimicrobial peptides provides a promising route to selectively target pathogenic agents and to shape microbiome structure. Lantibiotics, such as duramycin, are one class of bacterially produced peptidic natural products that can selectively inhibit the growth of other bacteria. However, despite longstanding characterization efforts, the microbial selectivity and mode of action of duramycin are still obscure. We describe here a suite of biological, chemical, and physical characterizations that shed new light on the selective and mechanistic aspects of duramycin activity. Bacterial screening assays have been performed using duramycin and Populus-derived bacterial isolates to determine species selectivity. Lipidomic profiles of selected resistant and sensitive strains show that the sensitivity of Gram-positive bacteria depends on the presence of phosphatidylethanolamine (PE) in the cell membrane. Further the surface and interface morphology were studied by high resolution atomic force microscopy and showed a progression of cellular changes in the cell envelope after treatment with duramycin for the susceptible bacterial strains. Together, these molecular and cellular level analyses provide insight into duramycin’s mode of action and a better understanding of its selectivity.

Flavisolibacter metallilatus sp. nov., isolated from an automotive air conditioning system and emended description of the genus Flavisolibacter.

A Gram-stain-negative, aerobic, non-motile, rod-shaped and pale yellow-pigmented bacterium, designated strain TX0661T, was isolated from an automotive air conditioning system collected in the Republic of Korea. 16S rRNA gene sequence analysis showed that the strain TX0661T was grouped with members of the genus Flavisolibacter and the strain had 98.2-95.3 % 16S rRNA gene sequence similarities to the species of the genus Flavisolibacter. DNA-DNA relatedness between TX0661T and Flavisolibacter ginsenosidimutans KCTC 22818T and Flavisolibacter ginsengisoli KCTC 12657T was less than 30 %. The low levels of DNA-DNA relatedness identified strain TX0661T as a novel species in the genus Flavisolibacter. The strain grew at 28-37 °C (optimum, 37 °C), at pH 6.0-7.0 (optimum, pH 6.5) and in the presence of 0-0.5 % (w/v, optimum, 0.5 %) NaCl. It contained summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c), iso-C15 : 0 and iso-C17 : 0 3-OH as major fatty acids and MK-7 as the predominant menaquinone. The polar lipid profile revealed that the presence of phosphatidylethanolamine, aminoglycophospholipid, two unidentified aminolipids and two unidentified lipids. The DNA G+C content of the strain was 49.1 mol%. Based on phenotypic, genotypic and chemotaxonomic data, strain TX0661T represents a novel species in the genus Flavisolibacter, for which the name Flavisolibactermetallilatus sp. nov. (=KACC 19145T=KCTC 52779T=NBRC 111784T) is proposed.

The folding, stability and function of lactose permease differ in their dependence on bilayer lipid composition

Lipids play key roles in Biology. Mechanical properties of the lipid bilayer influence their neighbouring membrane proteins, however it is unknown whether different membrane protein properties have the same dependence on membrane mechanics, or whether mechanics are tuned to specific protein processes of the protein. We study the influence of lipid lateral pressure and electrostatic effects on the in vitro reconstitution, folding, stability and function of a representative of the ubiquitous major facilitator transporter superfamily, lactose permease. Increasing the outward chain lateral pressure in the bilayer, through addition of lamellar phosphatidylethanolamine lipids, lowers lactose permease folding and reconstitution yields but stabilises the folded state. The presence of phosphatidylethanolamine is however required for correct folding and function. An increase in headgroup negative charge through the addition of phosphatidylglycerol lipids favours protein reconstitution but is detrimental to topology and function. Overall the in vitro folding, reconstitution, topology, stability and function of lactose permease are found to have different dependences on bilayer composition. A regime of lipid composition is found where all properties are favoured, even if suboptimal. This lays ground rules for rational control of membrane proteins in nanotechnology and synthetic biology by manipulating global bilayer properties to tune membrane protein behaviour.

Spirosoma luteolum sp. nov. isolated from water.

A novel Gram-negative and rod-shaped bacterial strain, designated as 16F6ET, was isolated from a water sample. Cells were yellowish in color and catalase- and oxidase-positive. The strain grew at 10-37°C (optimum at 25°C) but not at 4 and 42°C, and pH 5-7 (optimum at pH 7). It showed moderate resistance to gamma-ray irradiation. Comparative phylogenetic analysis showed that strain 16F6ET belonged to the family Cytophagaceae of the class Cytophagia. Furthermore, this isolate showed relatively low 16S rRNA gene sequence similarities (90.7-93.1%) to the members of the genus Spirosoma. The major fatty acids were summed feature 3 (C16:1 ω7c/C16:1 ω6c), C16:1 ω5c, C16:0 N alcohol, and C16:0. The polar lipid profile indicated presence of phosphatidylethanolamine, unknown aminophospholipids, an unknown amino lipid, unknown phospholipids, and unknown polar lipids. The predominant isoprenoid quinone was MK-7. The genomic DNA G+C content of strain 16F6ET was 56.5 mol%. Phenotypic, phylogenetic, and chemotaxonomic properties indicated that isolate 16F6ET represents a novel species within the genus Spirosoma, for which the name Spirosoma luteolum sp. nov. is proposed. The type strain is 16F6ET (=KCTC 52199T =JCM 31411T).

Lacinutrix cladophorae sp. nov., a flavobacterium isolated from the green alga Cladophora stimpsonii, transfer of Flavirhabdus iliipiscaria Shakeela et al. 2015 to the genus Lacinutrix as Lacinutrix iliipiscaria comb. nov. and emended description of the genus Lacinutrix.

A strictly aerobic, Gram-stain-negative, rod-shaped, motile by gliding and yellow-pigmented bacterium, designated strain 7Alg 4T, was isolated from the green alga Cladophora stimpsonii. Phylogenetic analysis based on 16S rRNA gene sequences revealed that the novel strain was affiliated to the family Flavobacteriaceae of the phylum Bacteroidetes, and was most closely related to the recognized species of the genera Lacinutrixand Flavirhabdus, with 16S rRNA gene sequence similarities of 95.1-98.1 and 97.0 %, respectively. Strain 7Alg 4T grew in the presence of 1-5 % NaCl and at 4-32 °C, and hydrolysed aesculin, gelatin, starch and Tween 80. The prevalent fatty acids were iso-C15 : 1 G, iso-C15 : 0, iso-C17 : 0 3-OH, iso-C15 : 0 3-OH and C15 : 0. The polar lipid profile was characterized by the presence of phosphatidylethanolamine, three unidentified aminolipids and four unidentified lipids. The major respiratory quinone was MK-6. The DNA G+C content was 31.9 mol%. On the basis of the differences in 16S rRNA gene sequences, chemotaxonomic and phenotypic characteristics, it is suggested that strain 7Alg 4T represents a novel species of the genus Lacinutrix, for which the name Lacinutrixcladophorae sp. nov. is proposed. The type strain is 7Alg 4T (=KCTC 23036T=KMM 6381T). Reclassification of Flavirhabdus iliipiscaria as Lacinutrix iliipiscaria comb. nov. and an emend of the genus Lacinutrix are also proposed.

Pseudoalteromonas fenneropenaei sp. nov., a marine bacterium isolated from sediment of a Fenneropenaeus chinensis pond.

A novel Gram-stain-negative, motile, oxidase- and catalase-positive, non-spore-forming, non-pigmented, aerobic bacterium, designated rzy34T, was isolated from the sediment of a pond containing farmed Fenneropenaeus chinensis Rizhao, China. The strain was able to grow at pH 6-10 (optimum pH 7), 20-40 °C (optimum 30 °C) and in the presence of 1.0-6.0 % NaCl (optimum 1.0-2.0 %). Phylogenetic analysis based on 16S rRNA gene sequences revealed that the nearest relative of strain rzy34T was Pseudoalteromonas xiamenensis Y2T, with the highest sequence similarity of 96.09 %. Within the genus Pseudoalteromonas, it showed the lowest similarity of 92.7 % to Pseudoalteromonas donghaensis. The G+C content of the chromosomal DNA of strain rzy34T was 45.3 mol%. The predominant cellular fatty acids were summed feature 3 (C16 : 1ω6c and/or C16 : 1ω7c), C16 : 0, C17 : 1ω8c and summed feature 8 (C18 : 1ω6c and/or C18 : 1ω7c). The major respiratory quinone was Q-8. Polar lipid analysis indicated the presence of phosphatidylethanolamine and phosphatidylcholine. On the basis of the phenotypic and phylogenetic characteristics, strain rzy34T represents a novel species of the genus Pseudoalteromonas, for which the name Pseudoalteromonas fenneropenaei sp. nov. is proposed. The type strain is rzy34T (=CGMCC 1.15325T=KCTC 42730T).

Amylibacter ulvae sp. nov., a new alphaproteobacterium isolated from the Pacific green alga Ulva fenestrata.

A strictly aerobic, Gram-stain-negative, rod-shaped and non-motile bacterium, designated strain 6Alg 255(T), was isolated from the green alga Ulva fenestrata. Phylogenetic analysis based on 16S rRNA gene sequencing revealed that the novel strain affiliated to the family Rhodobacteraceae of the class Alphaproteobacteria being most closely related to Amylibacter marinus LMG 28364(T) with 16S rRNA gene sequence similarity of 97.2 %. Strain 6Alg 255(T) grew with 0.5-6.0 % NaCl and at 4-33 °C, hydrolysed aesculin, casein, gelatin and urea. The DNA G + C content was 50.4 mol%. The prevalent fatty acids were C18:1 ω7c and C16:0. The polar lipid profile was characterized by the presence of phosphatidylethanolamine, phosphatidylglycerol, phosphatidylcholine and unidentified aminolipid. The major respiratory quinone was Q-10. The significant molecular distinctiveness between the novel isolate and its nearest neighbour was strongly supported by the differences in physiological and biochemical tests. Therefore, strain 6Alg 255(T) represents a novel species of the genus Amylibacter, for which the name Amylibacter ulvae sp. nov. is proposed. The type strain is 6Alg 255(T) (=KCTC 32465(T) = KMM 6515(T)).

Brassicibacter thermophilus sp. nov., a thermophilic bacterium isolated from coastal sediment.

A novel thermophilic, obligately anaerobic bacterium, strain Cel2f(T), was isolated from a cellulolytic community enriched from coastal marine sediment. Cells were Gram-stain-negative, non-motile, non-spore-forming and rod-shaped. Optimal growth temperature and pH of strain Cel2f(T) were 55 °C and pH 7.0, respectively. NaCl was essential for the growth of strain Cel2f(T) and the strain showed enhanced growth in the presence of sea salt; the optimum sea salt concentration for growth was 7% (w/v). Thiosulfate, sulfate and sulfite were potential electron acceptors. The major fatty acids of strain Cel2f(T) were iso-C15 : 0, C16 : 0, and C18 : 0. Polar lipid analysis indicated the presence of phosphatidylethanolamine and phosphatidylglycerol. Strain Cel2f(T) contained menaquinone MK-7 as the isoprenoid quinone, and the DNA G+C content was 31.3 mol%. Phylogenetic analysis revealed that the nearest relative of strain Cel2f(T) was Brassicibacter mesophilus BM(T) with 93.8% 16S rRNA gene sequence similarity. Based on phenotypic, chemotaxonomic and phylogenetic characteristics, strain Cel2f(T) represents a novel species of genus Brassicibacter, for which the name Brassicibacter thermophilus sp. nov. is proposed. The type strain is Cel2f(T) ( = JCM 30480(T) = CGMCC 1.5200(T)).

Effect of Reactive Aldehydes on Ionophore-Mediated Transmembrane Translocations of H+ and K+

Reactive aldehydes (RA) are involved in the onset and progression of many pathologies such as cardiovascular and neurodegenerative diseases. The precise mechanisms by which aldehydes contribute to these diseases remain unclear. Previously, we and other groups suggested that RAs modify the membrane protein function by either binding to the proteins directly(1) or to aminophosphosholipids, with a subsequent alteration in protein conformation. Here, we investigated the influence of biologically important RAs such as 4-hydroxy-2-nonenal (HNE), 4-oxo-2-nonenal (ONE) and 4-hydroxy-2-hexenal (HHE) on the transport kinetics of the ionophores carbonyl-cyanide-m-chlorophenylhydrazone (CCCP) and valinomycin (Val). We found that RA increases the membrane conductance, G, in the presence of Val in the following order ONE>HNE>HHE. In contrast G decreases in the presence of CCCP and ONE. The presence of phosphatidylethanolamine in the membrane was crucial for the effect to occur. The results are consistent with the hypothesis that RA adducts alter membrane boundary potential.1. E. A. Malingriaux, et al., “Fatty Acids are Key in 4-Hydroxy-2-Nonenal-Mediated Activation of Uncoupling Proteins 1 and 2” PLoS. ONE. 8(10), e77786 (2013).

Interaction of the Spo20 membrane-sensor motif with phosphatidic acid and other anionic lipids, and influence of the membrane environment.

The yeast protein Spo20 contains a regulatory amphipathic motif that has been suggested to recognize phosphatidic acid, a lipid involved in signal transduction, lipid metabolism and membrane fusion. We have investigated the interaction of the Spo20 amphipathic motif with lipid membranes using a bioprobe strategy that consists in appending this motif to the end of a long coiled-coil, which can be coupled to a GFP reporter for visualization in cells. The resulting construct is amenable to in vitro and in vivo experiments and allows unbiased comparison between amphipathic helices of different chemistry. In vitro, the Spo20 bioprobe responded to small variations in the amount of phosphatidic acid. However, this response was not specific. The membrane binding of the probe depended on the presence of phosphatidylethanolamine and also integrated the contribution of other anionic lipids, including phosphatidylserine and phosphatidyl-inositol-(4,5)bisphosphate. Inverting the sequence of the Spo20 motif neither affected the ability of the probe to interact with anionic liposomes nor did it modify its cellular localization, making a stereo-specific mode of phosphatidic acid recognition unlikely. Nevertheless, the lipid binding properties and the cellular localization of the Spo20 alpha-helix differed markedly from that of another amphipathic motif, Amphipathic Lipid Packing Sensor (ALPS), suggesting that even in the absence of stereo specific interactions, amphipathic helices can act as subcellular membrane targeting determinants in a cellular context.

Shewanella aestuarii sp. nov., a marine bacterium isolated from a tidal flat

A Gram-stain-negative, non-spore-forming, facultatively anaerobic bacterium, designated strain SC18(T), was isolated from a tidal flat of Suncheon bay in South Korea. Cells were rod-shaped and motile by means of a polar flagellum. Cells were catalase-, oxidase- and β-haemolysis-positive. Growth was observed at 4-37 °C (optimum, 25-30 °C), at pH 5.0-9.0 (optimum, pH 7.0) and in the presence of 0-5.0 % (w/v) NaCl (optimum, 0-2 %). The major cellular fatty acids were summed feature 3 (comprising C16 : 1ω7c and/or C16 : 1ω6c), iso-C15 : 0 and C16 : 0. The polar lipid pattern indicated the presence of phosphatidylethanolamine, phosphatidylglycerol, phosphatidylmethylethanolamine, an unidentified aminolipid and three unidentified lipids. Strain SC18(T) contained Q-7, Q-8, MK-7 and MMK-7 as the dominant respiratory quinones and the G+C content of the genomic DNA was 41.3 mol%. Phylogenetic analysis based on 16S rRNA and gyrase B (gyrB) gene sequences showed that strain SC18(T) formed a tight phyletic lineage with members of the genus Shewanella. Strain SC18(T) was related most closely to Shewanella denitrificans OS217(T) (97.3 % 16S rRNA gene sequence similarity) and Shewanella gaetbuli TF-27(T) (97.1 %), but the DNA-DNA relatedness levels between strain SC18(T) and the type strains of S. denitrificans and S. gaetbuli were 18.3±2.8 and 22.5±1.6 % (mean±sd), respectively. On the basis of phenotypic, chemotaxonomic and molecular features, strain SC18(T) represents a novel species of the genus Shewanella, for which the name Shewanella aestuarii sp. nov. is proposed. The type strain is SC18(T) ( = KACC 16187(T) = JCM 17801(T)).

Arenibacter hampyeongensis sp. nov., a marine bacterium isolated from a tidal flat

A Gram-staining-negative, dark orange, strictly aerobic bacterium, designated strain HP12(T), was isolated from a tidal flat at Hampyeong in South Korea. Cells were moderately halotolerant, catalase- and oxidase-positive, non-motile rods. Growth was observed at 5-35 °C (optimum, 25 °C), at pH 6.0-8.5 (optimum, pH 7.0-7.5), and in the presence of 1-6 % (w/v) NaCl (optimum, 1-2 %). The major cellular fatty acids were summed feature 3 (C(16 : 1)ω7c and/or iso-C(15 : 0) 2-OH), iso-C(17 : 0) 3-OH, C(15 : 0), iso-C(15 : 1) G and iso-C(15 : 0). The polar lipid pattern indicated the presence of phosphatidylethanolamine and two unidentified lipids. The G+C content of the genomic DNA was 37.1 mol% and the predominant respiratory quinone was menaquinone-6. Phylogenetic analysis based on 16S rRNA gene sequences showed that the novel strain formed a tight phyletic lineage with members of the genus Arenibacter and was most closely related to Arenibacter palladensis KMM 3961(T), Arenibacter troitsensis KMM 3674(T) and Arenibacter echinorum KMM 6032(T), with 16S rRNA gene sequence similarities of 98.1 %, 98.0 % and 97.8 %, respectively. However, the DNA-DNA relatedness values between strain HP12(T) and A. palladensis JCM 13509(T), A. troitsensis KCTC 12362(T) and A. echinorum KCTC 22013(T) were only 20.2±0.3 %, 22.6±0.6 % and 9.1±2.6 %, respectively. On the basis of phenotypic and molecular features, strain HP12(T) represents a novel species of the genus Arenibacter, for which the name Arenibacter hampyeongensis sp. nov. is proposed. The type strain is HP12(T) ( = KACC 16193(T) = JCM 17788(T)).

Analysis of AcrB and AcrB/DARPin ligand complexes by LILBID MS

The AcrA/AcrB/TolC complex is responsible for intrinsic multidrug resistance (MDR) in Escherichia coli. Together with the periplasmic adaptor protein AcrA and the outer membrane channel TolC, the inner membrane component AcrB forms an efflux complex that spans both the inner and outer membrane and bridges the periplasm of the Gram-negative cell. Within the entire tripartite complex, homotrimeric AcrB plays a central role in energy transduction and substrate selection. In vitro selected designed ankyrin repeat proteins (DARPin) that specifically bind to the periplasmic domain of AcrB were shown to ameliorate diffraction resolution of AcrB/DARPin protein co-crystals (G. Sennhauser, P. Amstutz, C. Briand, O. Storchenegger, M.G. Grutter, Drug export pathway of multidrug exporter AcrB revealed by DARPin inhibitors, PLoS Biol 5 (2007) e7). Structural analysis by X-ray crystallography revealed that 2 DARPin molecules were bound to the trimeric AcrB wildtype protein in the crystal, whereas the V612F and G616N AcrB variant crystal structures show 3 DARPin molecules bound to the trimer. These specific stoichiometric differences were analyzed in solution via densitometry after microchannel electrophoresis, analytical ultracentrifugation and via laser-induced liquid bead ion desorption mass spectrometry (LILBID-MS). Using the latter technology, we investigated the gradual disassembly of the AcrB trimer and bound DARPin ligands in dependence on laser intensity in solution. At low laser intensity, the release of the detergent molecule micelle from the AcrB/DARPin complex was observed. By increasing laser intensity, dimeric and monomeric AcrB species with bound DARPin molecules were detected showing the high affinity binding of DARPin to monomeric AcrB species. High laser intensity LILBID MS experiments indicated a spectral shift of the monomeric AcrB peak of 3.1kDa, representing a low molecular weight ligand in all detergent-solubilized AcrB samples and in the AcrB crystal. The identity of this ligand was further investigated using phospholipid analysis of purified AcrB and AcrB variant samples, and indicated the presence of phosphatidylethanolamine and possibly cardiolipin, both constituents of the Escherichia coli membrane.

Exploring the role of the phospholipid ligand in endothelial protein C receptor: A molecular dynamics study

Endothelial protein C receptor (EPCR) is a CD1-like transmembrane glycoprotein with important regulatory roles in protein C (PC) pathway, enhancing PC's anticoagulant, anti-inflammatory, and antiapoptotic activities. Similarly to homologous CD1d, EPCR binds a phospholipid [phosphatidylethanolamine (PTY)] in a groove corresponding to the antigen-presenting site, although it is not clear if lipid exchange can occur in EPCR as in CD1d. The presence of PTY seems essential for PC gamma-carboxyglutamic acid (Gla) domain binding. However, the lipid-free form of the EPCR has not been characterized. We have investigated the structural role of PTY on EPCR, by multiple molecular dynamics (MD) simulations of ligand bound and unbound forms of the protein. Structural changes, subsequent to ligand removal, led to identification of two stable and folded ligand-free conformations. Compared with the bound form, unbound structures showed a narrowing of the A' pocket and a high flexibility of the helices around it, in agreement with CD1d simulation. Thus, a lipid exchange with a mechanism similar to CD1d is proposed. In addition, unbound conformations presented a reduced interaction surface for Gla domain, confirming the role of PTY in establishing the proper EPCR conformation for the interaction with its partner protein. Single MD simulations were also obtained for 29 mutant models with predicted structural stability and impaired binding ability. Ligand affinity calculations, based on linear interaction energy method, showed that substitution-induced conformational changes affecting helices around the A' pocket were associated to a reduced binding affinity. Mutants responsible for this effect may represent useful reagents for experimental tests.

Ekhidna lutea gen. nov., sp. nov., a member of the phylum Bacteroidetes isolated from the South East Pacific Ocean

A novel aerobic, heterotrophic bacterium, designated BiosLi39(T), was isolated from the South East Pacific Ocean. Cells were Gram-negative gliding rods forming yellow colonies on marine agar. The isolate was oxidase-, catalase- and alkaline phosphatase-positive and β-galactosidase-negative. Strain BiosLi39(T) grew at 20-37°C (optimum 30°C), at pH7.0-9.0 (optimum pH8.0) and with 20-60 g NaCl l(-1) (optimum 30-50 g NaCl l(-1)). The fatty acids (>1 %) comprised iso-C(14 : 0), iso-C(15 : 1) G, iso-C(15 : 0), anteiso-C(15 : 0), C(15 : 1) G, C(15 : 0), iso-C(15 : 0) 2-OH, iso-C(16 : 1) G, iso-C(16 : 0), iso-C(16 : 0) 3-OH, iso-C(16 : 0) 2-OH, iso-C(17 : 0) 3-OH, C(17 : 0) 2-OH and three unidentified components with equivalent chain lengths of 17.87, 18.10 and 18.71. A significant proportion of the hydroxylated fatty acids are amide-linked. The lipid pattern indicated the presence of phosphatidylethanolamine, two unidentified aminolipids and three unidentified polar lipids. The strain contained menaquinone 7 as the sole respiratory lipoquinone and did not produce flexirubin-type pigments. The G+C content of the genomic DNA was 37.2 mol%. Comparative 16S rRNA gene sequence analysis indicated that strain BiosLi39(T) was distantly related to all of the representatives of the phylum Bacteroidetes. Its closest relative was Marinoscillum furvescens IFO 15994(T), with which it shared 92.5 % 16S rRNA gene sequence similarity. On the basis of genotypic, phenotypic and chemotaxonomic characteristics, we propose a novel genus and species, Ekhidna gen. nov., sp. nov., with type strain BiosLi39(T) (=DSM 19307(T) =CIP 109600(T) =OOB 398(T)).

Importance of Direct Interactions with Lipids for the Function of the Mechanosensitive Channel MscL

We have studied the effects of lipid structure on the function of the mechanosensitive channel of large conductance (MscL) from Escherichia coli to determine whether effects follow from direct interaction between the lipids and protein or whether they follow indirectly from changes in the curvature stress in the membrane. The G22C mutant of MscL was reconstituted into sealed vesicles containing the fluorescent molecule calcein, and the release of calcein from the vesicles was measured following opening of the channel by reaction with [2-(triethylammonium)ethyl] methanethiosulfonate (MTSET), which introduces five positive charges into the region of the pore constriction. The presence of anionic lipids in the vesicle membrane changed the rates and amplitudes of calcein release, the effects not correlating with calculated changes in lipid spontaneous curvature. Mutation of charged residues in the Arg-104, Lys-105, Lys-106 cluster removed high-affinity binding of anionic lipids to MscL, and the presence of anionic lipid no longer affected calcein flux through MscL. Changing the zwitterionic lipid from phosphatidylcholine to phosphatidylethanolamine resulted in a large decrease in the rate of calcein release, the change in rate varying linearly with lipid composition, as expected if spontaneous curvature affected the rate of release. However, rates of release of calcein measured in the presence of phosphatidylethanolamine- N-methyl and phosphatidylethanolamine- N, N-dimethyl did not fit the correlation between rate and curvature established for the phosphatidylcholine/phosphatidylethanolamine mixtures. Rather, the effects of zwitterionic lipid headgroup on calcein flux suggested that what was important was the presence of a proton in the headgroup, able to take part in hydrogen bonding to MscL. We conclude that the function of MscL is likely to be modulated by direct interaction with the surrounding, annular phospholipids that contact the protein in the membrane.

Pseudochrobactrum glaciei sp. nov., isolated from sea ice collected from Peter the Great Bay of the Sea of Japan

An aerobic, Gram-negative, non-pigmented, non-motile bacterium, KMM 3858(T), was isolated from a sea-ice sample collected from Peter the Great Bay of the Sea of Japan, Russia, and subjected to a phenotypic and phylogenetic study. Comparative analyses based on the 16S rRNA and recA gene sequences placed strain KMM 3858(T) within the genus Pseudochrobactrum. The major chemotaxonomic characteristics were found to be the presence of phosphatidylethanolamine, phosphatidylmonomethylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, an unknown aminolipid and phosphatidylcholine, major fatty acids C(18 : 1)omega7c and C(19 : 0) cyclo, and ubiquinone Q-10, confirming the affiliation of strain KMM 3858(T) to the genus Pseudochrobactrum. On the basis of the phylogenetic analysis and the physiological and biochemical characterization, strain KMM 3858(T) should be classified as representing a novel species of the genus Pseudochrobactrum, for which the name Pseudochrobactrum glaciei sp. nov. is proposed. The type strain is strain Pi26(T) (=KMM 3858(T)=NRIC 0733(T)=JCM 15115(T)).

Function of Beta2-GPI in Inhibiting the Oxidized Phospholipids Dependent APC Activity by Antiphospholipid IgG from APS Patients.

Beta2-GPI (ß2GPI), a plasma glycoprotein with phospholipid-binding properties, is the actual target antigen for autoimmune type antiphospholipid antibodies. Certain groups of these antibodies exert lupus anticoagulant (LA) activity by directly inhibiting the Protein C anticoagulant pathway and associated with an increased risk of thrombotic disease. Our previous studies have shown that the activated protein C (APC) activity and the ability to be inhibited by antiphospholipid antibodies associated with thrombosis are strongly augmented by the presence of Phosphatidyl-ethanolamine (PE) and phospholipid oxidation. In this study, we investigated the relationship between ß2GPI and anti-APC activity by IgG derived from APS patients.IgG from patients with APS syndrome were tested using a modified oxidized PE containing phospholipids dependent dRVVT test, in the presence of APC at concentration of 0.2ug/ml, the clotting times was prolonged to 3 fold of the baseline level, when the anti-phospholipids IgG antibodies added into the system, its showed a significant impact on the APC prolongation clotting times in the presence of ß2GPI. However, no affect was observed on the APC prolongation clotting times in the absence of ß2GPI. To eliminate the possible confounding effect from other plasma component, we investigated the effect on factor Va inactivation by APC in a purified system. As expected, the inhibition was observed only when the B2GPI was present. We also found that the presence of beta2GPI-IgG complex had its strongest effect when Protein S was included.We conclude that ß2GPI is necessary to inhibit the oxidized phospholipids dependent APC activity by IgG antibodies from APS patients. On oxidized lipids surface, ß2GPI -APS IgG complex had its strongest effect on APC activity. This is a potentially important result indicating the functional assay may be more sensitive for pathological antibodies.