The archaea are distinguished by their unique isoprenoid ether lipids, which typically consist of the sn-2,3-diphytanylglycerol diether or sn-2,3-dibiphytanyldiglycerol tetraether core modified with a variety of polar headgroups. However, many hyperthermophilic archaea also synthesize tetraether lipids with up to four pentacyclic rings per 40-carbon chain, presumably to improve membrane thermal stability at temperatures up to approximately 110 degrees C. This study aimed to correlate the ratio of tetraether to diether core lipid, as well as the presence of pentacyclic groups in tetraether lipids, with growth temperature for the hyperthermophilic archaeon, Archaeoglobus fulgidus. Analysis of the membrane core lipids of A. fulgidus using APCI-MS analysis revealed that the tetraether-to-diether lipid ratio increases from 0.3 +/- 0.1 for cultures grown at 70 degrees C to 0.9 +/- 0.1 for cultures grown at 89 degrees C. Thin-layer chromatography (TLC) followed by APCI-MS analysis provided evidence for no more than one pentacycle in the hydrocarbon chains of tetraether lipid from cultures grown at 70 degrees C and up to 2 pentacycles in the tetraether lipid from cultures grown at higher temperatures. Analysis of the polar lipid extract using TLC and negative-ion ESI-MS suggested the presence of diether and tetraether phospholipids with inositol, glycosyl, and ethanolamine headgroup chemistry.

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The membrane lipids of archaea are characterized by unique isoprenoid biochemistry, which typically is based on two core lipid structures, sn-2,3-diphytanylglycerol diether (archaeol) and sn-2,3-dibiphytanyldiglycerol tetraether (caldarchaeol). The biosynthetic pathway for the tetraether lipid entails unprecedented head-to-head coupling of isoprenoid intermediates by an unknown mechanism involving unidentified enzymes. To investigate the isoprenoid ether lipid biosynthesis pathway of the hyperthermophilic archaeon, Archaeoglobus fulgidus, its lipid synthesis machinery was reconstructed in an engineered E. coli strain in an effort to demonstrate, for the first time, efficient isoprenoid ether lipid biosynthesis for the production of the intermediate, digeranylgeranylglyceryl phosphate (DGGGP). The biosynthesis of DGGGP was verified using a LC/MS/MS technique and was accomplished by cloning and expressing the native E. coli gene for IPP isomerase (idi), along with the A. fulgidus genes for G1P dehydrogenase (egsA) and GGPP synthase (gps), under the control of the lac promoter. The A. fulgidus genes for GGGP synthase (GGGPS) and DGGGP synthase (DGGGPS), under the control of the araBAD promoter, were then introduced and expressed to enable DGGGP biosynthesis in vivo. This investigation established roles for four A. fulgidus genes in the isoprenoid ether lipid pathway for DGGGP biosynthesis and provides a platform useful for identification of subsequent, currently unknown, steps in tetraether lipid biosynthesis proceeding from DGGGP, which is the presumed substrate for the head-to-head coupling reaction yielding unsaturated caldarchaeol.

A practical approach for constructing enzyme-multiplied immunoassay technique (EMIT)-based protein/peptide assays is described. Normally used in small-molecule drug testing, EMIT is a homogeneous assay method that is attractive for its simplicity, sensitivity, and rapidity. The EMIT-based peptide/protein assay was developed by conjugating a cysteine-modified HA peptide (from influenza hemagglutinin A) to the reporter enzyme, glucose-6-phosphate dehydrogenase. The 13-min assay gave a free HA limit of detection of 10 nM and proved effective for detection of a high-molecular-weight model protein tagged with HA. Similar EMIT-based assay approaches may be developed for applications in biotoxin and infectious disease detection.