C40,C40 lipid by TLC. The core lipids of examples from the genera Methanobacerritcm,. Meghambrevhcter, Methanogenium and Methanoplm#cr did not contain ...
Jounal of General Microbiology (1988), 134, 3159-3163. Printed m Great Britain
3159
A Simpie Chrcnnatographic procednre for the Detection of Cydzed Archaebacterid Glycerol-Bb&phytanyl-Glyceml Tetraetbr Core Lipids By A. TRINCONE,' M.DE ROSA,'s2 A. GAMBACORTA', V. LANZOTTI,' B. NICOLAUS,' J. E. HARRIS3 A N D W. D. GRANT4* 'Istituto di Chimica di Molecole di Interesse Bwlogico, del Consiglio Nazionale delle Ricerche, 8W72-Arco Felice, via Toiano 6, Italy 2Istituto di Biochimica dene hlaeromolede, Wdwsita' di Napdi, Napli, Italy 3Food Research Institute, cohtey Lme,Norwich NR4 7UA, UK 4Deprtment of Mkrobwlogy, University of hieester, Leicester LEI 7RH, UK
(&embed 26 May 1988) Archaebacterial glycerol-bisdiphytanyl-glyceroltetraether core lipids containing from one to eight cyclopentane rings could be resolved from each other and from the parent uncyclized C40,C40 lipid by TLC. The core lipids of examples from the genera Methanobacerritcm, Meghambrevhcter, Methanogenium and Methanoplm#cr did not contain cyclized forms of glycerol-bisdiphytanyl-glyceroltetraethers, whereas the core lipids of Methumsarcina barkeri contained glycerol-bisdiphytanyl-glyceroltetraethers with from one to three cyclopentane rings in each C40 isopranoid chain. INTRODUCTION
All membrane lipids of archaebacteria characterized to date are based on saturated isopranoid ethers formed by condensationof glycerol, or more complex poiyois, with isopranoid alcohols containing 20, 25 or 40 carbon atoms @e Rosa et d., 198th). Most halophilic
archaebacteria have membrane lipids based exclusively on 2,3 di-O-phytanyl-sn-8lycerol (C20,C20),although a few isolates, notably the alkaliphiles,also have significantamounts of C25 (sesterterpanyl)chains as Czo,C25glycerol diethers (Re.Rosaet al., 1982,1983~). The situation is more complex for methanogenic and thermophilic archaebacteria, since, in most cases, the These membranes have a significant component of glycerol-bisdiphytanyl-glycerolte-thers. are formed by dirnerization of diphytanyl diethers, where head-to-head linkage between the terminal methyls occurs. Methanogencore lipid structuresare particularlydiverse, in that in some isolates, glycerol may be replaced by a tetritol, both in the core lipid structures @e Rosa et al., 1986b)and in complex lipids(Ferranteet al., 1987).Furthermore,one isolate hasamacrocyclicdietherwith a 36-member ring, which originates from the condensationof a glycerol, in the 2,3-sn-coafiguration,with the 3,7,11,15,18,22,26,30-octamethyldotriacontane-1-32-diol(Comita & Gagmian, 1983). Thermophilic archaebacteria of the genera DesUklfsrtococnrs,Thenrtoproteus, Z%em$ilwnand Pyrodictiumhave ether lipids based on Czoand CWchains (Stetter et al., 1983;Zillig et al., 1981, 1982,1983)whereas 2'7wmwccu.s celer contains only C20,C20lipids @e Rosa et al., 1987). In Suifolobus spp. the lipids are based on c40,c40tetraethers, but there are two classes; one class consists of glycerol dialkyl glycerol tetraethers (GDGT),while the second M e r s from the first in that, nonitol, a nine-carbon polyol, replaces one of' the glycerols (GDNT).These C40,C40 lipids have the additional feature of containing up to four cyclopentane rings in each Ca chain. The degree of cyclization depends on the growth temperature of the organism (DeRosa et al., 1986~). ~
~
Abbreviations: GDGT, glycerol dialkyl glycerol tetraethers; GDNT, glycerol dialkyl nonitof tetraethers.
0001-4914 8 1988 SGM
3160
A. TRINCONE AND OTHERS
Cyclization of C40,C40core lipids has only been found in one methanogen to date (De Rosa et ul., 1986b), but detailed structural analyses of c4O,c40 lipids have only rarely been done (De Rosa et al., 1986b;Kramer et al., 1987;Kushwaha et al., 1981;Comita & Gagosian, 1983). The resolution and identification of cyclized forms currently involves a lengthy combination of column chromatography and HPLC, or GLC of the hydrocarbon chains after hydrolysis and derivatization(DeRosa et al.,19836; Tornabene & Langworthy, 1979; Makula & Singer, 1978). We report here a simple TLC procedure for the resolution of different cyclized forms of GDGT and the use of this procedure to survey the core lipids of a number of different methanogens. METHODS
Micro-organismsrmd d t w e conditions. Detailsof methanogens are shown in Table 1. Growth conditionswere as previously described(Grant et al., 1985).Sdfol&so~atmicur MT-4was grown in the standard medium at 87 "C as previously described (DeRosa et al., 1975). Cells were harvested at the stationary phase of growth and freezedried. Extraction and isolation of lipids. Polar lipids were extracted by the procedure of Bligh & Dyer (1959) as modified by Minnikin et al. (1979). Core lipids were extracted into chloroform or hexane, after hydrolysis of complex lipids by acid methanolysis (Rosset al., 1985). Purified samples of 2,3di-O-phytanyl-sn-glycerol(C20,C20diether), 3-0phytanyl-sn-glycerol (Cz0monoether), tetritol C20,C10diether, uncyclized c40,c40 tetraether and the glycerolbisdiphytanyl-glycerol tetraether (Ca,C4,, tetraether) fraction from Methmrosarcha borkeri(De Rosa et al., 1986b) were available for comparison by TLC. TLC.This was done on 0.25 mm layers of silica gel F 254 (Merck), activated by heating at 100 "Cfor 2 h. The d v e n t was n-hexane/ethyl acetate (7 :3, v/v), sinde development. Compounds were detected by spraying with 0.1% (w/v) ceS (o ,), in 11d-H2S04followed by heating at 150°C for 5 min. RESULTS AND DISCUSSION
In a previous study (Grant et al., 1985) the core lipids of a considerable number of different methanogens were analysed by TLC. Glycerol ethers with 20, 25 and 40 carbon atoms were detected, along with a number of new structures. In a later study, two of these new structures from Ms.burkeri were characterized as-a C20glycerol monoether and C20,C20tetritol diether (De Rosa et al., 19866). Furthermore, the c40,c40tetraether core lipids of this organism were shown to be of the GDGT class and to contain from one to three cyclopentanerings (DeRosa et a/., 19866). To date, Ms.batkerf is the only known methanogen known to possess cyclized core lipids; indeed this organism contains no uncyclized C4O,C40 lipids. The TLC system used by Table 1. Core lipids occurring in a range of methanogens Culture collection no.*
DSM 862 DSM 1535 DSM 1053 DSM 861 DSM 1093 DSM 744 DSM 1224 DSM 1537 DSM 2095 DSM 1497 DSM 1498 DSM 2373 DSM 2279 DSM 800
Core lipids
organism Methambacterium bryantii Methanobacterium formkicum Methanobacterirrm thennwutotrophicum Methanobrevibacter smithii Methonobrevibacter ruminantium M e t h u d r h t e r arboriphilicus Methanococcus &lii Methmrococcus voltae Methunocthennolithotmphicus Methamgenium cariaci Methanogenium marhigri Methanogenium themphilicum Methampllamcs lhicola Methonosarcina barkeri
Acyclic Acyclic Acyclic Acyclic Acyclic Acyclic -
-
Acyclic Acyclic Acyclic Acyclic cyclic
+ + + + + + + + + + + + +
+$ -, No CW,CWcomponents present. * DSM, Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, FRG. f Data from Grant et al. (1985). . , $ Also contains Cz0glycerol monoether, C25,C20glycerol diether and CZO,CtO tetritol diether (De Rosa et ai., 1986b).
TLC of cyclized archaebacterial lipids
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Fig. 1. TLC of core ether lipids using a simple development system (rehexane/ethylacetate,7 :3, v/v). Lanes:A, 2,3-di-O-phytat1yl-Jn-glycerol; B, uncyclizcd Ca,Ca derivative of GDGT;C, core lipids of S. so&ararimp (stnrchnes of compounds 1-9 are indicated in Fig. 2); D,Ca,Cm lipids of Ms.burkm'; E, 3-Gphytanyl-mglyced (lower band) and tetritol C20,Czodiether (upper band).
Grant et ul. (1985) does not resolve cyclized forms of C19,C40 core lipids from uncyclized forms and it is, therefore, not clear whether cyclized C4,,,C4, core lipids are present in the hydrolysed lipid extracts of those methanogens that have not been subject to detailed chemical characterization. Chromatographyof core lipid extracts on 0.25 mm layers of silica gel F 254 in n-hexane/ethyl acetate 7 :3 (v/v) resolved both uncyclized GDGT and the eight different cyclized variations of this C40,C40tetraether lipid found in the core lipids of S. solfatudcus (Fig. 1). The structuresof these compounds are shown in Fig. 2. Purified Bamples (De Rosa ef d.,1983b) were used to confinn the separation order shown in Fig. 1. A purified Coo,Coo core lipid fraction from Ms. barkeri was readily resolved into the three fractions corresponding to the cyclized forms of GDGT containing four, five or six cyclopentanerings, characterized in the previous study @e Rosa ef d.,1986b). Samplesof C20,C20 diether, tetritol C20,C20 diether and CzOmonoetherhave tetraethers (Fig. 1). GDNT ethers different RF values and were clearly distinct from C40,C40 remained at the origin using this solvent system. Table 1 shows the results of a survey of the core lipids of several different methanogensusing this TLC procedure. Of the strains tested, only Ms.barkeri possessed cycliied GDGT core
3 162
A . T R I N C O N E A N D OTHERS
core lipids (GDGTtype), the backbone of complex lipids of thermophilic The numbers 1-9 correspond to the bands in Fig. 1, lane C.
Fig. 2. Structuresof C&, arcb-ria.
lipids. Included in the survey were Methambactm’um thermoautotrophicum and Methanobrevibacter arboriphilicus, whose complex lipids have been extensively characterized as C20,C20 diethers and uncyclized c4&40 (GDGT) derivatives (Kramer et al., 1987; Morii et al., 1986). This preliminary survey suggests that cyclization of C40,C40tetraether lipids is likely to be uncommon amongst methanogenic archaebacteria that contain C4O,C40 tetraether lipids of the GDGT class. This supports and extends the results of the early surveys of Tornabene & Langworthy (1979) and Makula & Singer (1978). The pattern of complex lipids and core lipids of archaebacteria has some value in the taxonomy of the group (Rosset al., 1985; Langworthy &Pond, 1986;Grant et al., 1985).The use of this simple TLC procedure for the detection of cyclized GDGT core lipids extends the range of techniques that can readily be applied in non-specialist laboratories. We thank Dr M.R.Vaccaro and Mr G. Pinch for their technical assistance. REFERENCES
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