Salinigranum marinum sp. nov. and Halohasta salina sp. nov., halophilic archaea isolated from sediment of a marine saltern and inland saline soil.

Two halophilic archaeal strains, ZS-10T and GSL13T, were isolated from the Zhoushan marine saltern in Zhejiang, and an inland saline soil from the Tarim Basin, Xinjiang, PR China, respectively. The cells of strain ZS-10T were pleomorphic while those of strain GSL13T were rod-shaped. Both of them stained Gram-negative and formed red-pigmented colonies on agar plates and their cells lysed in distilled water. The optimum growth of strain ZS-10T was observed at 40 °C, 3.4 M NaCl, 0.03 M MgCl2 and pH 7.5, while that of strain GSL13T was at 37 °C, 3.1 M NaCl, 0.5 M MgCl2 and pH 7.5. Phylogenetic and phylogenomic analyses indicated that these two strains were related to Salinigranum and Halohasta, respectively. Strains ZS-10T and GSL13T could be differentiated from the current members of Salinigranum and Halohasta based on the comparison of diverse phenotypic characteristics. The average amino acid identity, average nucleotide identity and digital DNA-DNA hybridization values among strain ZS-10T and current species of Salinigranum were 75.8-78.6 %, 80.6-81.9 % and 24.3-26.1 %, respectively. These values between strain GSL13T and current species of Halohasta were 78.4-80.8 %, 79.8-82.8% and 22.7-25.7 %, respectively, clearly below the threshold values for species demarcation. The polar lipids of strain ZS-10T were phosphatidylglycerol (PG), phosphatidylglycerol phosphate methyl ester (PGP-Me) and sulphated mannosyl glucosyl diether (S-DGD-1), while those of strain GSL13T were phosphatidic acid, PG, PGP-Me, phosphatidylglycerol sulphate and S-DGD-1. The polar lipid profile of strain GSL13T was identical to those of Halohasta, whereas strain ZS-10T did not contain the minor glycolipids detected in the current Salinigranum species. The phenotypic, phylogenetic and genome-based results suggested that strains ZS-10T (=CGMCC 1.12868T=JCM 30241T) and GSL13T (=CGMCC 1.15214T=JCM 30841T) represent two novel species, for which the names Salinigranum marinum sp. nov. and Halohasta salina sp. nov. are proposed.

Natrinema salsiterrestre sp. nov., an extremely halophilic archaeon isolated from a hypersaline soil.

An extremely halophilic archaeal strain, designated S1CR25-10T, was isolated from hypersaline soil sampled in the Odiel Saltmarshes Natural Area in Southwestern Spain (Huelva) and subjected to a polyphasic taxonomic characterization. The cells were Gram-stain-negative, motile and their colonies were pink-pigmented. It was a strictly aerobic haloarchaeon that could grow at 25-55 °C (optimum, 37 °C), at pH 6.0-9.0 (optimum, pH 7.0-8.0) and in the presence of 12-30 % (w/v) total salts (optimum, 20-25 %, w/v). The phylogenetic analysis based on the comparison of the 16S rRNA gene sequences revealed that strain S1CR25-10T belongs to the genus Natrinema, with 98.9 % similarity to Natrinema salinisoli SLN56T. In addition, the values of orthologous average nucleotide identity, digital DNA-DNA hybridization and average amino acid identity were below the threshold limits accepted for prokaryotic species delineation, with N. salinisoli SLN56T showing the highest relatedness values (92.6 % and 48.4 %, respectively). The major polar lipids were phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, phosphatidylglycerol sulfate and a glycolipid chromatographically identical to sulfated diglycosyl diether. The DNA G+C content of the isolate was 63.8 mol%. Based on the phylogenetic, phenotypic and chemotaxonomic characterization and the whole genome results, strain S1CR25-10T represents a new species within the genus Natrinema, for which the name Natrinema salsiterrestre sp. nov., with type strain S1CR25-10T (=CECT 30623T=CCM 9251T), is proposed.

Haladaptatus halobius sp. nov. and Haladaptatus salinisoli sp. nov., two extremely halophilic archaea isolated from Gobi saline soil.

Two extremely halophilic archaeal strains, PSR5T and PSR8T, were isolated from a saline soil sample collected from the Tarim Basin, Xinjiang, PR China. Both strains had two copies of the 16S rRNA genes rrn1 and rrn2, showing 2.6 and 3.9% divergence, respectively. The rrn1 gene of PSR5T showed 98.4 and 95.3% similarity to the rrn1 and rrn2 genes of strain PSR8T; the rrn2 gene of PSR5T displayed 97.4 and 96.7% similarity to those of strain PSR8T, respectively. Phylogenetic analyses based on the 16S rRNA and rpoB' genes revealed that strains PSR5T and PSR8T formed a single cluster, and then tightly clustered with the current four Haladaptatus species (93.5-97.1% similarities for the 16S rRNA gene and 89.3-90.9% similarities for the rpoB' gene, respectively). Several phenotypic characteristics differentiate strains PSR5T and PSR8T from current Haladaptatus members. The polar lipids of the two strains are phosphatidic acid, phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester phosphatidylglycerol sulphate and three glycolipids. One of the glycolipids is sulphated mannosyl glucosyl diether, and the remaining two glycolipids are unidentified. The average nucleotide identity, in silico DNA-DNA hybridization, amino acid identity and percentage of conserved proteins values between the two strains were 88.5, 39.1, 89.3 and 72.8 %, respectively, much lower than the threshold values proposed as a species boundary. These values among the two strains and Haladaptatus members were 77.9-79.2, 22.0-23.5, 75.1-78.2 and 56.8-69.9 %, respectively, much lower than the recommended threshold values for species delimitation. These results suggested that strains PSR5T and PSR8T represent two novel species of Haladaptatus. Based on phenotypic, chemotaxonomic, genomic and phylogenetic properties, strains PSR5T (=CGMCC 1.16851T=JCM 34141T) and PSR8T (=CGMCC 1.17025T=JCM 34142T) represent two novel species of the genus Haladaptatus, for which the names Haladaptatus halobius sp. nov. and Haladaptatus salinisoli sp. nov. are proposed.

Neorhizobium xiangyangii sp. nov., isolated from a highland barley cultivation soil in Qamdo, Tibet.

A novel Gram-negative, aerobic, rod-shaped and non-nitrogen fixing bacterium named T786T was isolated from a highland barley cultivation soil in Qamdo, Tibet Autonomous Region, PR China. Strain T786T grew at 5-30 â„ƒ and pH 6.0-10.0 (optimum, 20-25 â„ƒ and pH 7.0-8.0) with 0-4% (w/v) NaCl (optimum, 0%). The 16S rRNA gene sequences of strain T786T showed the highest similarity to Neorhizobium vignae CCBAU 05176T (98.7%), followed by Neorhizobium alkalisoli CCBAU 01393T (98.5%), Neorhizobium tomejilense T17_20T (98.4%), Neorhizobium huautlense S02T (98.4%), and Neorhizobium galegae ATCC 43677T (98.0%). Phylogenetic analysis based on 16S rRNA genes indicated that strain T786T was a new member of the genus Neorhizobium. The digital DNA-DNA hybridization and average nucleotide identity values between strain T786T and related strains were estimated as 20.2-20.6% and 76.6-80.0%, respectively. The genomic DNA G + C content based on the draft genome sequence was 60.2%. The major cellular fatty acids were Summed feature 8 (C18:1 ω7c or C18:1 ω6c), C16:0 and Summed feature 3 (C16:1 ω7c or C16:1 ω6c). The polar lipids were diphosphatidyl glycerol, phosphatidyl glycerol, phosphatidyl ethanolamine, phosphatidyl methyl ethanolamine, unidentified phospholipid and unidentified lipids (1-4). The isoprenoid quinone was ubiquinone-10. The DAP and sugar components of cell wall were meso-DAP and ribose, glucose, respectively. Based on phenotypic, phylogenetic, and genotypic data, for which the name Neorhizobium xiangyangii sp. nov. is proposed. The type strain is T786T (= JCM 35100T = CICC 25102T).

Algiphilus acroporae sp. nov. and Coraliihabitans acroporae gen. nov. sp. nov., isolated from scleractinian coral Acropora digitifera.

Two Gram-staining-negative, aerobic, rod-shaped bacteria NNCM1T and NNCM2T were isolated from the scleractinian coral Acropora digitifera. NNCM1T grew with 0.5-12 % (w/v) NaCl (optimum, 3-6 %), at 18-37 °C (optimum, 28 °C) and at pH 6.0-10.0 (optimum, 7.0-8.0). NNCM2T grew with 0.5-10 % (w/v) NaCl (optimum, 2 %), at 18-37 °C (optimum, 28 °C) and at pH 6.5-9.0 (optimum, 7.0). The results of phylogenetic analysis based on 16S rRNA gene sequences indicated that NNCM1T formed a lineage within the genus Algiphilus of the family Algiphilaceae, and it was distinct from the most closely related species Algiphilus aromaticivorans DG1253T, with a 16S rRNA gene sequences similarity of 97.05 %. NNCM2T formed a lineage within the family Rhodobacteraceae, and it was distinct from the closely related genera Limibaculum halophilum CAU 1123T, Paroceanicella profunda D4M1T and Pseudoruegeria aestuarii MME-001T with 93.41, 92.78 and 91.09% identities, respectively. The major respiratory quinone was Q-8 and Q-10 for NNCM1T and NNCM2T, respectively. The predominant fatty acids (more than 10 %) were summed feature 8 (39.4 %) and C16 : 0 (19.4 %) for NNCM1T and summed feature 8 (62.8 %) and C16 : 0 (12.4 %) for NNCM2T. The DNA G+C contents of NNCM1T and NNCM2T were 63.3 and 63.4 mol% respectively. The polar lipids of NNCM1T comprised one diphosphatidylglycerol, one phosphatidylethanolamine, one phosphatidylglycerol and one unknown polar lipid, while those of NNCM2T comprised one phosphatidylethanolamine, one phosphatidylglycerol, one aminolipid and four unknown polar lipids. Phenotypic characteristics (physiological, biochemical and chemotaxonomic) also supported the taxonomic novelty of the two isolates. Thus, NNCM1T is considered to represent a novel species within genus Algiphilus, for which the name Algiphilus acroporae sp. nov. is proposed. The type strain is NNCM1T (=KCTC 82966T=MCCC 1K06445T). NNCM2T represents a novel genus and species within the family Rhodobacteraceae, for which the name Coraliihabitans acroporae gen. nov. sp. nov. is proposed. The type strain is NNCM2T (=KCTC 82967T=MCCC 1K06408T).

Quantitative Analysis of Polyphosphoinositide, Bis(monoacylglycero)phosphate, and Phosphatidylglycerol Species by Shotgun Lipidomics After Methylation.

Phospholipids play important roles in biological process even at a very low level. For example, bis(monoacylglycerol)phosphate (BMP) is involved in the pathogenesis of lysosomal storage diseases, and polyphosphoinositides (PPI) play critical roles in cellular signaling and functions. Phosphatidylglycerol (PG), a structural isomer of BMP, mediates lipid-protein and lipid-lipid interactions, and inhibits platelet activating factor and phosphatidylcholine transferring. However, due to their low abundance, the analysis of these phospholipids from biological samples is technically challenging. Therefore, the cellular function and metabolism of these phospholipids are still elusive. This chapter overviews a novel method of shotgun lipidomics after methylation with trimethylsilyl-diazomethane (TMS-D) for accurate and comprehensive analysis of these phospholipid species in biological samples. Firstly, a modified Bligh and Dyer procedure is performed to extract tissue lipids for PPI analysis, whereas modified methyl-tert-butylether (MTBE) extraction and modified Folch extraction methods are described to extract tissue lipids for PPI analysis. Secondly, TMS-D methylation is performed to derivatize PG/BMP and PPI, respectively. Then, we described the shotgun lipidomics strategies that can be used as cost-effective and relatively high-throughput methods to determine BMP, PG, and PPI species and isomers with different phosphate position(s) and fatty acyl chains. The described method of shotgun lipidomics after methylation achieves feasible and reliable quantitative analysis of low-abundance lipid classes. The application of this novel method should enable us to reveal the metabolism and functions of these phospholipids in healthy and disease states.

Delta or Omega? Δ12 (ω6) fatty acid desaturases count 3C after the pre-existing double bond.

Fatty acid desaturases (FADs) represent a class of oxygen-dependent enzymes that dehydrogenate C-C bonds in the fatty acids (FAs) producing unsaturated CC double bonds that markedly change the properties of biological membranes. FADs are highly specific towards their acyl substrates, the position and configuration of the introduced double bonds. The double bond positioning of soluble acyl-carrier-protein Δ9-FADs was determined relative to the carboxyl end of a FA. Similar mode was suggested for the acyl-lipid Δ12-FADs (also known as ω6-FADs), however, their exact counting order remain unknown. Here we used monounsaturated odd- (17:1Δ10) and even-chain (18:1Δ11) FAs to show that acyl-lipid Δ12-FADs of, at least, two cyanobacterial species, Gloeobacter violaceus and Synechocystis sp. strain PCC 6803, use neither end of the fatty acid (Δ or ω) as a counting reference point; but count three carbons toward the methyl end from an existing double bond in the monoene precursors irrespective of a FA chain length.

The composition of phospholipid model bacterial membranes determines their endurance to secretory phospholipase A2 attack - The role of cardiolipin.

Soil bacteria are decomposer organisms crucial for the biodegradation of organic pollutants, mineralization of dead organic matter and the turnover of biogenic elements. In their environment they are constantly exposed to membrane-lytic enzymes emitted to the soil by other microorganisms competing for the same niche. Therefore, the composition and structure of their membranes is of utmost importance for survival in the harsh environment. Although soil bacteria species can be Gram-negative or Gram-positive and their membranes differ significantly, they are formed by phospholipids belonging mainly to three classes: phosphatidylethanolamines (PE), phosphatidylglycerols (PG) and cardiolipins (CL). The correlation of the membrane phospholipid composition and its susceptibility to secretory membrane-lytic enzymes is widely unknown; thus, to shed light on these phenomena we applied the Langmuir monolayer technique to construct models of soil bacteria membranes differing in the mutual proportion of the main phospholipids. To characterize the systems we studied their elasticity, mesoscopic texture, 2D crystalline structure and discussed the thermodynamics of the interactions between their components. The model membranes were exposed to secretory phospholipase A2. It turned out that in spite of the structural similarities the model membranes differed significantly in their susceptibility to s-PLA2 attack. The membranes devoid of cardiolipin were completely degraded, whereas, these containing cardiolipin were much more resistant to the enzymatic hydrolysis. It also turned out that the sole presence of cardiolipin in the model membrane did not guarantee the membrane durability and that the interplay between cardiolipin and the zwitterionic phosphatidylethanolamine was here of crucial importance.

Simultaneous Quantification of DPPG, DEPC and Cholesterol in Propofol Liposome by HPLC-ELSD Using Alkaline Hydrolysis.

A high-performance liquid chromatography method with evaporative light-scattering detection (ELSD) was performed for simultaneous determination of dipalmitoyl phosphatidylglycerol (DPPG), dierucoyl phosphatidylcholine (DEPC) and cholesterol in propofol liposome by the pretreatment of alkaline hydrolysis (temperature, concentration of KOH anhydrous ethanol solution and reaction time were 90°C, 1 mol · L-1 and 10 min, respectively). The analysis was carried out on an Agilent TC-C18 column (4.6 mm × 250 mm, 5 µm) with isocratic elution of methanol and 0.1% acetic acid aqueous solution (95:5, v/v) at a flow rate of 1.0 mL · min-1. The column temperature was 30°C. The drift tube temperature of the ELSD system was set at 30°C, and the pressure of carrier gas was 350 KPa. The regression equation revealed a good linear relationship (r = 0.9990-0.9993) during the test ranges. The RSD of stability and repeatability (n = 6) was found to be less than 1.96 and 1.46%, respectively. The average recoveries ranged from 97.90 to 101.00%. The proposed method was validated and showed good precision, stability, repeatability and recovery, which indicated that the method could be readily utilized as a quality evaluation method for the determination of DPPG, DEPC and cholesterol in propofol liposome.

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.

Neptunomonas marina sp. nov., isolated from seawater.

Strain HPM-16T, isolated from seawater, was characterized using a polyphasic taxonomy approach. Phylogenetic analyses based on 16S rRNA gene sequences and coding sequences of an up-to-date bacterial core gene set (92 protein clusters) indicated that strain HPM-16T formed a phylogenetic lineage in the genus Neptunomonas. Strain HPM-16T was most closely related to Neptunomonas concharum LHW37T with 16S rRNA gene sequence similarity of 96.7%. Cells were Gram-stain negative, facultatively anaerobic, motile by means of a single polar flagellum, rod-shaped and formed white colonies. Optimal growth occurred at 30-35 °C, pH 6.5-8, and in the presence of 2-5% NaCl. C18:1ω7c and summed feature 3 (C16:1ω7c and/or C16:1ω6c) were the predominant fatty acids. The only isoprenoid quinone was Q-8. The polar lipid profile revealed the presence of phosphatidylethanolamine, phosphatidylglycerol and several uncharacterized lipids. The major polyamines were putrescine and spermidine. The draft genome was approximately 3.68 Mb in size with a G + C content of 50.5 mol%. Differential phenotypic properties, together with the phylogenetic inference, demonstrate that strain HPM-16T should be classified as a novel species of the genus Neptunomonas, for which the name Neptunomonas marina sp. nov. is presented. The type strain is HPM-16T (= BCRC 80980T = LMG 29560T = KCTC 52235T).

Simultaneous Relative Quantification of Various Polyglycerophospholipids with Isotope-Labeled Methylation by Nanoflow Ultrahigh Performance Liquid Chromatography-Tandem Mass Spectrometry.

Herein, we introduce a comprehensive analytical method for the separation and relative quantification of polyglycerophospholipids (PGPLs), including phosphatidylglycerol (PG), bis(monoacylglycero)phosphate (BMP), bis(diacylglycero)phosphate (BDP), Hemi BDP, cardiolipin (CL), monolysocardiolipin (MLCL), and dilysocardiolipin (DLCL), using isotope-labeled methylation (ILM) with nanoflow ultrahigh performance liquid chromatography-electrospray ionization-tandem mass spectrometry (nUHPLC-ESI-MS/MS). Abnormal levels of BMP and CL have been associated with the pathology of lysosomal storage and neurodegenerative diseases. Thus, simultaneous analysis of all PGPLs is important to understand the mechanisms and pathologies of such diseases. In this study, improved separation and MS detection of PGPLs, including their regioisomers, was achieved by the methylation of PGPL. ILM-based relative quantification was applied to lipid extracts from a dopaminergic cell line (SH-SY5Y) treated with drugs commonly used for Parkinson's disease (PD), resulting in the identification of 229 unique PGPLs, including 121 CLs, 71 MLCLs, and 16 Hemi BDP species. The drug treatment induced significant increases in the amount of CLs containing polyunsaturated fatty acyl chains, including 20:4 and 22:6, as well as decreased levels of BMP, Hemi BDP, and BDP species, demonstrating the feasibility of using ILM for the comprehensive and high-speed relative quantification of PGPLs.

Expanding lipidomics coverage: effective ultra performance liquid chromatography-high resolution mass spectrometer methods for detection and quantitation of cardiolipin, phosphatidylglycerol, and lysyl-phosphatidylglycerol.

INTRODUCTION: Lipidomics can reveal global alterations in a broad class of molecules whose functions are innately linked to physiology. Monitoring changes in the phospholipid composition of biological membranes in response to stressors can aid the development of targeted therapies. However, exact quantitation of cardiolipins is not a straightforward task due to low ionization efficiencies and poor chromatographic separation of these compounds. OBJECTIVE: The aim of this study was to develop a quantitative method for the detection of cardiolipins and other phospholipids using both a targeted and untargeted analyses with a Q-Exactive. METHODS: HILIC chromatography and high-resolution mass spectrometry with parallel reaction monitoring was used to measure changes in lipid concentration. Internal standards and fragmentation techniques allowed for the reliable quantitation of lipid species including: lysyl-phosphatidylglycerol, phosphatidylglycerol, and cardiolipin. RESULTS: The untargeted analysis was capable to detecting 6 different phospholipid classes as well as free fatty acids. The targeted analysis quantified up to 23 cardiolipins, 10 phosphatidylglycerols and 10 lysyl-phosphatidylglycerols with detection limits as low as 50 nM. Biological validation with Enterococcus faecalis demonstrates sensitivity in monitoring the incorporation of exogenously supplied free fats into membrane phospholipids. When supplemented with oleic acid, the amount of free oleic acid in the membrane was 100 times greater and the concentration of polyunsaturated cardiolipin increased to over 3.5 µM compared to controls. CONCLUSIONS: This lipidomics method is capable of targeted quantitation for challenging biologically relevant cardiolipins as well as broad, untargeted lipid profiling.

Paraburkholderia dokdonella sp. nov., isolated from a plant from the genus Campanula.

The novel Gram-stain-negative, rod-shaped, aerobic bacterial strain DCR-13T was isolated from a native plant belonging to the genus Campanula on Dokdo, an island in the Republic of Korea. Comparative analysis of the 16S rRNA gene sequence indicated that this strain is closely related to Paraburkholderia peleae PP52-1T (98.43% 16S rRNA gene sequence similarity), Paraburkholderia oxyphila NBRC 105797T (98.42%), Paraburkholderia sacchari IPT 101T (98.28%), Paraburkholderia mimosarum NBRC 106338T (97.80%), Paraburkholderia denitrificans KIS30-44T (97.46%), and Paraburkholderia paradise WAT (97.45%). This analysis of the 16S rRNA gene sequence also suggested that DCR-13T and the six closely related strains formed a clade within the genus Paraburkholderia, but that DCR-13T was clearly separated from the established species. DCR-13T had ubiquinone 8 as its predominant respiratory quinone, and its genomic DNA G + C content was 63.9 mol%. The isolated strain grew at a pH of 6.0-8.0 (with an optimal pH of 6.5), 0-4% w/v NaCl (with an optimal level of 0%), and a temperature of 18-42°C (with an optimal temperature of 30°C). The predominant fatty acids were C16:0, summed feature 8 (C18:1ω7c/C18:1ω6c), C17:0 cyclo, C19:0 cyclo ω8c, summed feature 3 (C16:1ω6c/C16:1ω7c) and summed feature 2 (C12:0 aldehyde), and the major polar lipids were phosphatidylglycerol and phosphatidylethanolamine. On the basis of polyphasic evidence, it is proposed that strain DCR-13T (= KCTC 62811T = LMG 30889T) represents the type strain of a novel species, Paraburkholderia dokdonella sp. nov.

Follicular dynamics of glycerophospholipid and sphingolipid metabolisms in polycystic ovary syndrome patients.

Polycystic ovary syndrome (PCOS) is a common heterogeneous disease, affecting up to 5-10% women at reproductive age. Although PCOS patients could produce morphologically normal metaphase II oocytes undergoing assisted reproductive techniques (ART), oocyte developmental competence and embryo development have been impaired in following in-vitro fertilization (IVF) steps. Follicular fluid (FF) provides a variety of information in oocyte environment when oocytes grow. In the present work, based on ultra-performance liquid chromatography coupled with mass spectrometry (UPLC-MS), the metabolic signatures of PCOS FF have been compared with healthy women using untargeted metabolomics approach. Significant abundance differences of a series of glycerolipid, glycerophospholipids, sphingolipids, and carboxylic acids have been discovered. Among them, reduced levels of phosphatidylglycerolphosphate (PGP) and a triglyceride (TG) were highly related to the lower fertilization rate in PCOS; increased abundance of lysoPE and decreased amount of PC were significantly correlated with LH/FSH (ratio of luteinizing hormone to follicle stimulating hormone). Some metabolites, including decreased sphingolipids, glycerophospholipids, and fluctuated fatty acyls, also performed close relationship with other ART and clinical results. We concluded that dysfunctions in the metabolism of glycerolipid, glycerophospholipid, sphingolipid, and glycosphingolipid biosynthesis in PCOS patients' follicles play a non-ignorable role in declining the 2 pronuclei (PN) fertilization rate during IVF procedure.

High-resolution plasma metabolomics analysis to detect Mycobacterium tuberculosis-associated metabolites that distinguish active pulmonary tuberculosis in humans.

INTRODUCTION: Pulmonary tuberculosis (TB) is a major worldwide health problem that lacks robust blood-based biomarkers for detection of active disease. High-resolution metabolomics (HRM) is an innovative method to discover low-abundance metabolites as putative blood biomarkers to detect TB disease, including those known to be produced by the causative organism, Mycobacterium tuberculosis (Mtb). METHODS: We used HRM profiling to measure the plasma metabolome for 17 adults with active pulmonary TB disease and 16 of their household contacts without active TB. We used a suspect screening approach to identify metabolites previously described in cell culture studies of Mtb based on retention time and accurate mass matches. RESULTS: The association of relative metabolite abundance in active TB disease subjects compared to their household contacts predicted three Mtb-associated metabolites that were significantly increased in the active TB patients based on accurate mass matches: phosphatidylglycerol (PG) (16:0_18:1), lysophosphatidylinositol (Lyso-PI) (18:0) and acylphosphatidylinositol mannoside (Ac1PIM1) (56:1) (p<0.001 for all). These three metabolites provided excellent classification accuracy for active TB disease (AUC = 0.97). Ion dissociation spectra (tandem MS/MS) supported the identification of PG (16:0_18:1) and Lyso-PI (18:0) in the plasma of patients with active TB disease, though the identity of Ac1PIM1 could not be definitively confirmed. CONCLUSIONS: Presence of the Mtb-associated lipid metabolites PG (16:0_18:1) and Lyso-PI (18:0) in plasma accurately identified patients with active TB disease. Consistency of in vitro and in vivo data suggests suitability for exploring these in future studies for possible development as TB disease biomarkers.

Mechanism of High-Level Daptomycin Resistance in Corynebacterium striatum.

Daptomycin, a last-line-of-defense antibiotic for treating Gram-positive infections, is experiencing clinical failure against important infectious agents, including Corynebacterium striatum The recent transition of daptomycin to generic status is projected to dramatically increase availability, use, and clinical failure. Here we confirm the genetic mechanism of high-level daptomycin resistance (HLDR; MIC = >256 µg/ml) in C. striatum, which evolved within a patient during daptomycin therapy, a phenotype recapitulated in vitro In all 8 independent cases tested, loss-of-function mutations in phosphatidylglycerol synthase (pgsA2) were necessary and sufficient for high-level daptomycin resistance. Through lipidomic and biochemical analysis, we demonstrate that daptomycin's activity is dependent on the membrane phosphatidylglycerol (PG) concentration. Until now, the verification of PG as the in vivo target of daptomycin has proven difficult since tested cell model systems were not viable without membrane PG. C. striatum becomes daptomycin resistant at a high level by removing PG from the membrane and changing the membrane composition to maintain viability. This work demonstrates that loss-of-function mutation in pgsA2 and the loss of membrane PG are necessary and sufficient to produce high-level resistance to daptomycin in C. striatumIMPORTANCE Antimicrobial resistance threatens the efficacy of antimicrobial treatment options, including last-line-of-defense drugs. Understanding how this resistance develops can help direct antimicrobial stewardship efforts and is critical to designing the next generation of antimicrobial therapies. Here we determine how Corynebacterium striatum, a skin commensal and opportunistic pathogen, evolved high-level resistance to a drug of last resort, daptomycin. Through a single mutation, this pathogen was able to remove the daptomycin's target, phosphatidylglycerol (PG), from the membrane and evade daptomycin's bactericidal activity. We found that additional compensatory changes were not necessary to support the removal of PG and replacement with phosphatidylinositol (PI). The ease with which C. striatum evolved high-level resistance is cause for alarm and highlights the importance of screening new antimicrobials against a wide range of clinical pathogens which may harbor unique capacities for resistance evolution.

Brevibacterium anseongense sp. nov., isolated from soil of ginseng field.

Gram-positive, aerobic, non-motile, pale-yellow, and rodshaped bacterium, designated as Gsoil 188T, was isolated from the soil of a ginseng field in Pocheon, South Korea. A phylogenetic analysis based on 16S rRNA gene sequence comparison revealed that the strain formed a distinct lineage within the genus Brevibacterium and was most closely related to B. epidermidis NBRC 14811T (98.4%), B. sediminis FXJ8.269T (98.2%), B. avium NCFB 3055T (98.1%), and B. oceani BBH7T (98.1%), while it shared less than 98.1% identity with the other species of this genus. The DNA G + C content was 68.1 mol%. The predominant quinone was MK-8(H2). The major fatty acids were anteiso-C15:0 and anteiso-C17:0. The cell wall peptidoglycan of strain Gsoil 188T contained meso-diaminopimelic acid. The major polar lipids were phosphatidylglycerol, diphosphatidylglycerol, and an unidentified aminolipid. The physiological and biochemical characteristics, low DNA-DNA relatedness values, and taxonomic analysis allowed the differentiation of strain Gsoil 188T from the other recognized species of the genus Brevibacterium. Therefore, strain Gsoil 188T represents a novel species of the genus Brevibacterium, for which the name Brevibacterium anseongense sp. nov. is proposed, with the type strain Gsoil 188T (= KACC 19439T = LMG 30331T).

Thalassorhabdus alkalitolerans gen. nov., sp. nov., a novel Bacillaceae member isolated from marine sediment.

A novel Gram-stain-variable, rod-shaped, non-motile and non-endospore-forming bacterium (strain G27T) was isolated from near Dhuvaran, Gujarat, India. Based on 16S rRNA gene sequence analysis, strain G27T was identified as a member of the class Firmibacteria and was most closely related to Bacillus populi FJAT-45347T (94.9 % sequence similarity), Salipaludibacillus aurantiacus S9T (94.9 %), Salipaludibacillus neizhouensis KCTC 13187T (94.7 %), Alteribacillus iranensis DSM 23995T (94.6 %) and other Firmibacteria (<94.6 %). The DNA G+C content of strain G27T was 43.4±0.6 mol%. The cell-wall peptidoglycan contained meso-diaminopimelic acid. Polar lipids included diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, one unidentified phospholipid and five unidentified lipids. The predominant isoprenoid quinone was menaquinone MK-7. Major fatty acids (>5 %) included anteiso-C15:0, iso-C15 : 0, anteiso-C17:0, C16 : 0 and iso-C16 : 0. The results of phylogenetic, chemotaxonomic and biochemical tests allowed the clear differentiation of strain G27T from all other members of the family Bacillaceae.It is therefore considered to represent a novel species of a new genus, for which the name Thalassorhabdus alkalitolerans gen. nov., sp. nov., is proposed. The type strain of Thalassorhabdus alkalitolerans is G27T (=MCC 3411T=CGMCC 1.15772T=KCTC 33941T).

Zhongshania ponticola sp. nov., a novel lipolytic bacterium of the class Gammaproteobacteria isolated from seawater.

A bacterial strain, designated GM-8T, which was isolated from seawater around Pohang in Republic of Korea, was subjected to a polyphasic taxonomic study. It was lipolytic, Gram-stain-negative, aerobic, non-motile and coccoid, ovoid, or rod-shaped. Strain GM-8T grew optimally at 30 °C and in the presence of 2.0-3.0% (w/v) NaCl. The phylogenetic trees of 16S rRNA gene sequences based on three algorithms showed that strain GM-8T joined the clade comprising the type strains of Zhongshania species. The novel strain exhibited the highest 16S rRNA gene sequence similarity value (98.6%) to Zhongshania borealis CL-AS9T and sequence similarities of 97.7-98.3% to the type strains of three other Zhongshania species. Strain GM-8T contained Q-8 as the predominant ubiquinone and C17:1 ω8c, summed feature 3 (C16:1 ω7c and/or C16:1 ω6c), and C18:1 ω7c as the major fatty acids. Phosphatidylglycerol and phosphatidylethanolamine were major polar lipids found in strain GM-8T and the type strain of Zhongshania antarctica. The DNA G + C content of strain GM-8T was 50.9 mol%. Mean DNA-DNA relatedness values between strain GM-8T and the type strains of four other Zhongshania species were 13.3-20.3%. Its differential phenotypic traits, together with the phylogenetic and genetic evidences, revealed that strain GM-8T is distinct from recognized species of the genus Zhongshania. On the basis of the data presented, strain GM-8T represents a novel species of the genus Zhongshania, for which the name Zhongshania ponticola sp. nov. is proposed. The type strain is GM-8T (= KCTC 62425T = KACC 19616T = NBRC 113193T).