ORIGINAL PAPER Characterization of the precursor of tetraether lipid biosynthesis in the thermoacidophilic archaeon Thermoplasma acidophilum Received: 27 August 2002 / Accepted: 7 January 2003 / Published online: 27 February 2003 Ó Springer-Verlag 2003 Abstract Polar lipid biosynthesis in the thermoacido- philic archaeon Thermoplasma acidophilum was ana- lyzed using terbinafine, an inhibitor of tetraether lipid biosynthesis. Cells of T. acidophilum were labeled with [ 14 C]mevalonic acid, and their lipids were extracted and analyzed by two-dimensional thin-layer chroma- tography. Lipids labeled with [ 14 C]mevalonic acid, [ 14 C]glycerol, and [ 32 P]orthophosphoric acid were extracted and hydrolyzed under different conditions to determine the structure of polar lipids. The polar lipids were estimated to be archaetidylglycerol, glycerophos- phatidylcaldarchaetidylglycerol, caldarchaetidylglycer- ol, and b-L-gulopyranosylcaldarchaetidylglycerol, the main polar lipid of T. acidophilum. Pulse and chase experiments with terbinafine revealed that one tetrae- ther 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 pre- cursor accumulated in the presence of terbinafine with a fast-atom-bombardment mass spectrometry peak m/z 806 was compatible with archaetidylglycerol. The rel- ative height of the peak m/z 806 decreased after re- moval of the inhibitor. The results suggest that most of the precursor, archaetidylglycerol, is in fully saturated form. Keywords Archaea Æ Ether lipid Æ Lipid biosynthesis Æ Thermoacidophile Æ Thermoplasma acidophilum Introduction Core structures of membrane lipids in Archaea are dif- ferent from those in Bacteria and Eukarya. Membrane lipids in Archaea are composed of ether lipids contain- ing saturated isopranyl chains, archaeol, and caldar- chaeol. Archaeol is composed of two isopranoid groups with 20 or 25 carbon atoms attached to one glycerol molecule. Caldarchaeol is composed of two isopranyl groups with 40 carbon atoms attached to two glycerol molecules (Koga et al. 1993). A lipid containing archaeol or caldarchaeol as its core lipid is a diether lipid or tet- raether lipid, respectively (see Fig. 1). Thermoplasma acidophilum is a thermoacidophilic archaeon that grows optimally at pH 1–2 and at 55– 59 °C. About 90% of the membrane lipids of this or- ganism are tetraether lipid and the remainder is diether lipid (Langworthy et al. 1972). Approximately one-half of the polar ether lipids are b-L-gulopyranosylcaldar- chaetidylglycerol, known as the main polar lipid (MPL) in T. acidophilum (Swain et al. 1997). The other half of the polar lipids have been extensively analyzed, and their structures were previously reported as two phospholi- pids (diether and tetraether lipid), seven glycolipids (tetraether lipid having mono- or oligosaccharides with gulose [Gul] or glucose and zero to three mannose groups as the sugar moiety), and eight phosphoglycoli- pids (tetraether lipids having glycerophosphate as the phosphoester moiety and gulose or glucose and zero to three mannose groups as the sugar moiety) (Shimada et al. 2002; Uda et al. 1999, 2000b). The biosynthetic pathway of polar ether lipids has been analyzed by pulse and chase experiments using radiola- beled compounds in Halobacterium cutirubrum (Mold- oveanu and Kates 1988) and Methanobacterium Extremophiles (2003) 7:235–243 DOI 10.1007/s00792-003-0315-x Naoki Nemoto Æ Yasuo Shida Æ Haruo Shimada Tairo Oshima Æ Akihiko Yamagishi Communicated by G. Antranikian N. Nemoto Æ H. Shimada Æ T. Oshima Æ A. Yamagishi (&) Department of Molecular Biology, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan E-mail: Yamagish@LS.toyaku.ac.jp Tel.: +81-426-767139 Fax: +81-426-767145 Y. Shida Department of Chemical Analysis, Tokyo University of Pharmacy and Life Science, Tokyo, Japan