Naoki Nemoto

Complete Polar Lipid Composition of Thermoplasma acidophilum HO-62 Determined by High-Performance Liquid Chromatography with Evaporative Light-Scattering Detection

Polar ether lipids of Thermoplasma acidophilum HO-62 were purified by high-performance liquid chromatography with an evaporative light-scattering detector. Structures of purified lipids were investigated by capillary gas chromatography, mass spectrometry, and nuclear magnetic resonance. Three types of ether lipids were found: phospholipids, glycolipids, and phosphoglycolipids. The two phospholipids had glycerophosphate as the phosphoester moiety. The seven glycolipids had different combinations of gulose, mannose, and glucose, which formed mono- or oligosaccharides. The eight phosphoglycolipids with two polar head groups contained glycerophosphate as the phosphoester moiety and gulose alone or gulose and mannose, which formed mono- or oligosaccharides, as the sugar moiety. Although gulose is an unusual sugar in nature, several glyco- and phosphoglycolipids contained gulose as one of the sugar moieties in Thermoplasma acidophilum. All the ether lipids had isopranoid chains of C(40) or C(20) with zero to three cyclopentane rings. The structures of these lipids including four new glycolipids and three new phosphoglycolipids were determined, and a glycosylation process for biosynthesis of these glycolipids was suggested.

Effects of pH and Temperature on the Composition of Polar Lipids in Thermoplasma acidophilum HO-62

Thermoplasma acidophilum HO-62 was grown at different pHs and temperatures, and its polar lipid compositions were determined. Although the number of cyclopentane rings in the caldarchaeol moiety increased when T. acidophilum was cultured at high temperature, the number decreased at low pHs. Glycolipids, phosphoglycolipids, and phospholipids were analyzed by high-performance liquid chromatography with an evaporative light-scattering detector. The amount of caldarchaeol with more than two sugar units on one side increased under low-pH and high-temperature conditions. The amounts of glycolipids increased and those of phosphoglycolipids decreased under these conditions. The proton permeability of the liposomes obtained from the phosphoglycolipids that contained two or more sugar units was lower than that of the liposomes obtained from the phosphoglycolipids that contained one sugar unit. From these results, we propose the hypothesis that T. acidophilum adapts to low pHs and high temperatures by extending sugar chains on their cell surfaces, as well as by varying the number of cyclopentane rings.

Characterization of the precursor of tetraether lipid biosynthesis in the thermoacidophilic archaeon Thermoplasma acidophilum

Polar lipid biosynthesis in the thermoacidophilic archaeon Thermoplasma acidophilum was analyzed using terbinafine, an inhibitor of tetraether lipid biosynthesis. Cells of T. acidophilum were labeled with [14C]mevalonic acid, and their lipids were extracted and analyzed by two-dimensional thin-layer chromatography. Lipids labeled with [14C]mevalonic acid, [14C]glycerol, and [32P]orthophosphoric acid were extracted and hydrolyzed under different conditions to determine the structure of polar lipids. The polar lipids were estimated to be archaetidylglycerol, glycerophosphatidylcaldarchaetidylglycerol, caldarchaetidylglycerol, and β-l-gulopyranosylcaldarchaetidylglycerol, the main polar lipid of T. acidophilum. Pulse and chase experiments with terbinafine revealed that one tetraether lipid molecule is synthesized by head-to-head condensation of two molecules of archaetidylglycerol and that a sugar group of tetraether phosphoglycolipid is expected to attach to the tetraether lipid core after head-to-head condensation in T. acidophilum. A precursor accumulated in the presence of terbinafine with a fast-atom-bombardment mass spectrometry peak m/z 806 was compatible with archaetidylglycerol. The relative height of the peak m/z 806 decreased after removal of the inhibitor. The results suggest that most of the precursor, archaetidylglycerol, is in fully saturated form.

An Actin Homolog of the Archaeon Thermoplasma acidophilum That Retains the Ancient Characteristics of Eukaryotic Actin

Actin, a central component of the eukaryotic cytoskeleton, plays a crucial role in determining cell shape in addition to several other functions. Recently, the structure of the archaeal actin homolog Ta0583, isolated from the archaeon Thermoplasma acidophilum, which lacks a cell wall, was reported by Roeben et al. (J. Mol. Biol. 358:145-156, 2006). Here we show that Ta0583 assembles into bundles of filaments similar to those formed by eukaryotic actin. Specifically, Ta0583 forms a helix with a filament width of 5.5 nm and an axial repeating unit of 5.5 nm, both of which are comparable to those of eukaryotic actin. Eukaryotic actin shows a greater resemblance to Ta0583 than to bacterial MreB and ParM in terms of polymerization characteristics, such as the requirement for Mg(2+), critical concentration, and repeating unit size. Furthermore, phylogenetic analysis also showed a closer relationship between Ta0583 and eukaryotic actin than between MreB or ParM and actin. However, the low specificity of Ta0583 for nucleotide triphosphates indicates that Ta0583 is more primitive than eukaryotic actin. Taken together, our results suggest that Ta0583 retains the ancient characteristics of eukaryotic actin.

Quinone Profiles of Thermoplasma acidophilum HO-62

Quinones of Thermoplasma acidophilum HO-62 were analyzed by high-performance liquid chromatography, mass spectrometry, and nuclear magnetic resonance. Menaquinone, methionaquinone, and 2-trans and 2-cis forms of thermoplasmaquinone were identified. The relative amount of thermoplasmaquinone increased under anaerobic conditions, and those of menaquinone and methionaquinone increased under aerobic conditions.

Effects of a squalene epoxidase inhibitor, terbinafine, on ether lipid biosyntheses in a thermoacidophilic archaeon, Thermoplasma acidophilum.

The archaeal plasma membrane consists mainly of diether lipids and tetraether lipids instead of the usual ester lipids found in other organisms. Although a molecule of tetraether lipid is thought to be synthesized from two molecules of diether lipids, there is no direct information about the biosynthetic pathway(s) or intermediates of tetraether lipid biosynthesis. In this study, we examined the effects of the fungal squalene epoxidase inhibitor terbinafine on the growth and ether lipid biosyntheses in the thermoacidophilic archaeon Thermoplasma acidophilum. Terbinafine was found to inhibit the growth of T. acidophilum in a concentration-dependent manner. When growing T. acidophilum cells were pulse-labeled with [2-(14)C]mevalonic acid in the presence of terbinafine, incorporation of radioactivity into the tetraether lipid fraction was strongly suppressed, while accumulation of radioactivity was noted at the position corresponding to diether lipids, depending on the concentration of terbinafine. After the cells were washed with fresh medium and incubated further without the radiolabeled substrate and the inhibitor, the accumulated radioactivity in the diether lipid fraction decreased quickly while that in the tetraether lipids increased simultaneously, without significant changes in the total radioactivity of ether lipids. These results strongly suggest that terbinafine inhibits the biosynthesis of tetraether lipids from a diether-type precursor lipid(s). The terbinafine treatment will be a tool for dissecting tetraether lipid biosynthesis in T. acidophilum.

Processing of the Tail Lysozyme (gp5) of Bacteriophage T4

The processing site of gp5 has been determined to be between residues Val-390 and His-391, instead of Ser-351 and Ala-352 as previously reported (H. Kanamaru, N. C. Gassner, N. Ye, S. Takeda, and F. Arisaka, J. Bacteriol. 181:2739-2744). Moreover, the maturation of gp5 is abolished by null mutations in other hub genes, indicating that cleavage requires the interactions of several baseplate proteins.