Methods for Analyzing Archaeal Lipids

Methods for Analyzing Archaeal Lipids Detection of Regioisomeric Macrocyclic Tetraethers in the Lipids of Methanobacterium thermoautotrophicum and other Archaeal Organisms Otto Gräther and Duilio Arigoni. J. Chem. Soc., Chem. Commun. 405-406 (1995) Chemical degradation of GDGTs from cultured microorganisms revealed a near-racemic mixture of caldarchaeol to isocaldarchaeol (45:55). Special methods for the analysis of ether lipid structure and metabolism in archaea. Yosuke Koga & Hiroyuki Morii. Analytical Biochemistry 348, (2006): 1-14. An overview of chemical methods used to analyze archaeal lipids, focusing on diether lipids. Extraction using an acidified Bligh-Dyer method. Extraction and Composition of Polar Lipids from the Archaebacterium, Methanobacterium thermoautotrophicum: Effective Extraction of Tetraether Lipids by an Acidified Solvent Nishihara, Masateru, Yosuke Koga., and J. Biochem. 101, (1987): 997-1005. Instead of using a regular Bligh-Dyer solvent mixture for the extraction (MeOH/CHCl3/H2O, 2:1:0.8), the water is replaced with a 5% TCA (trichloroacetic acid) aqueous solution. TCA is a strong protein denaturing agent and commonly used to precipitate DNA and RNA in biochemistry. Lipids comprised 0.98% of the cell dry weight with the regular solvent mixture and 5.58% with the TCA mixture. Nishihara and Koga identified polar archaeal lipids using TLC (Thin Layer Chromatography). Solvent B: chloroform, methanol, acetic acid, water (85:30:15:5) Spray reagents used to identify IPLs (Intact Polar Membrane Lipids): Ninhydrin Acid molybdate 0.5% α-naphthol reagent Periodate-Schiff reagent 30% H2SO4 Solvent A Solvent A: chloroform, methanol, 7M aqueous ammonia (60:35:8) amino groups phospholipids glycolipids vicinal hydroxyl groups all lipids Solvent B Wet chemistry techniques for the preparation of core lipids. HCL Methanolysis: 5% HCl in MeOH at 100°C for 3h Cleaves glycosidic bonds, but will not cleave phosphodiester bonds in diether lipids. Acetolysis with acid methanolysis: HF cleavage with mild HCl methanolysis: Acetic acid, acetic anhydride (3:2) at 160°C for 16 h 46% HF at 0°C for 24 h then 0.18% HCl at 50°C for 24 h Cleaves phosphodiester bonds, forming an acetylated core lipid which is removed by subsequent methanolysis. Cleaves phosphodiester bonds while preserving hydroxyarachaeols. Ether cleavage methods. HI cleavage: 47% HI under reflux for 24 h Preferred by Koga and Morii because it reportedly gives a sharper peak in GC analysis. BBr3/BCl3 + Lithium aluminium hydride (LAH): Cleaves ether bonds while preserving hydroxyl groups. Kates, et al. Biochim. Biophys. Acta, 98 (1965): 252-268. LAH cleavage: 100°C for 90 min in the presence of LAH Cleaves allyl ether bonds, yielding unsaturated Acid methanolysis: 5% HCl in methanol 80°C for 1 h Allyl ether bonds cleaved, while non-allyl ether bonds remain intact. Gas Chromatography Mass Spectroscopy of Archaeal Lipids Archaeal diether lipids are GC-amenable after derivatization. Archaeal tetraether lipids are not GC-amenable, and ether cleavage is required to resolve the alkyl chains bound between the glycerol moieties. HPLC (High Performance Liquid Chromatography) Overview Types of Columns: Normal Phase: Uses a polar stationary phase and a non-polar mobile phase. Hydrophobic compounds elute first. Reverse Phase: Uses a non-polar stationary phase and a polar mobile phase. Hydrophilic compounds elute first Size Exclusion: Uses a stationary phase composed of porous beads. Ion Exchange: Operates on the basis of selective exchange of ions in the sample with counterions in the stationary phase. Liquid-Liquid: Components in the sample capable of hydrogen bonding with the stationary phase are retained longer. Analysis of intact tetraether lipids in archaeal cell material and sediments by high performance liquid chromatography/atmospheric pressure chemical ionization mass spectrometry. Hopmans, et al. Rapid Commun. Mass Spectrom 14, (2000): 585-589. Uses a normal phase column to separate GDGTs of different unsaturations, allowing for rapid determination of the relative distribution of intact GDGTs. Intact polar membrane lipids in prokaryotes and sediments deciphered by high-performance liquid chromatography/electrospray ionization multistage mass spectrometry—new biomarkers for biogeochemistry and microbial ecology. Sturt, et al. Rapid Commun. Mass Spectrom 18, (2004): 617-628. Uses a normal phase column to separate and identify IPLs of prokaryotic communities. A combination of positive and negative ion modes can distinguish between diacyl, diether, and acyl-ether glycerol backbones as well as identify the polar head groups of IPLs. Review and re-analysis of domain-specific 16S primers Baker, et al. Journal of Microbiological Methods 55, (2003): 541-555. PCR Overview: 1) DNA denaturing: DNA and primers heated to 94-98°C for 1-5 min. 2) Annealing: Temperature lowers so primers can attach themselves to single strands of DNA. Usually 5°C below the melting temp. of the primers for 1-2 min. 3) Polymerization: DNA polymerase is added, binds to the primer, and replicates the DNA. Taq polymerase replicates optimally at 72°C. This step typically lasts 10-15 min. Baker et al. note that current “universal” primers for prokaryotes do not amplify Archaea well, introducing bias when constructing libraries of environmental samples. Using as many available genomes as possible, they propose new primers designed to amplify prokaryotes from the Archaeal domain: A571F/UA1204R and A751F/UA1406R. These new primers were tested, and observed to amplify more sequences from environmental samples than previously reported, but did not amplify sequences from E. coli. Inosine residues and degenerate bases were used in primer design when mismatches occurred in documented genomes in order to maximize complementarity.