Incorporation of 13C-labelled glucose, acetate, pyruvate or erythrose allowed the determination of the origin of the carbon atoms of triterpenoids of the hopane series and/or of the ubiquinones from several bacteria (Zymomonas mobilis, Methylobacterium fujisawaense, Escherichia coli and Alicyclobacillus acidoterrestris) confirmed our earlier results obtained by incorporation of 13C-labelled acetate into the hopanoids of other bacteria and led to the identification of a novel biosynthetic route for the early steps of isoprenoid biosynthesis. The C5 framework of isoprenic units results most probably (i) from the condensation of a C2 unit derived from pyruvate decarboxylation (e.g. thiamine-activated acetaldehyde) on the C-2 carbonyl group of a triose phosphate derivative issued probably from dihydroxyacetone phosphate and not from pyruvate and (ii) from a transposition step. Although this hypothetical biosynthetic pathway resembles that of L-valine biosynthesis, this amino acid or its C5 precursors could be excluded as intermediates in the formation of isoprenic units.

-EJB 96 0709/5 35-0-/3-GaIacturonopyranosyl-, 35-0-~-3,S-anhydro-galacturonopyranosyl-and 35-0-a-altruronopyranosylbacteriohopanetetrol accompanied by their 2-methyl homologues have been isolated from Prochlorothrix hollandicu. We report here C,, triterpenoids of the hopane series in a prochlorophyte, a group of prokaryotic oxigenic phototrophs of the cyanobacterial lineage. Like many cyanobacteria, I? hollundicu contains a mixture of non-methylated as well as 2D-methylhopanoids. After side-chain cleavage by periodic acid oxidation followed by sodium borohydride reduction, these hopanoids could be localized in cell walls and thylakoids, in accordance with their role as membrane stabilizers.Keywords: hopanoid; prochlorophyte ; Prochlorothrix; triterpenoid.The prochlorophytes are a small polyphyletic group of prokaryotic oxigenic phototrophs. They belong to the cyanobacterial lineage, but contain chlorophyll u and b like plants and green algae and no phycobiliproteins like cyanobacteria (Urbach et al., 1992;Bullerjahn and Post, 1993). Lipid analysis (fatty acids and hydrocarbons) was in accordance with their classification into the prokaryotic kingdom. Diploptene (1) (Fig. l), a triterpene of the hopane series which is usually a minor compound in most of the hopanoid-producing eubacteria, has already been detected in Prochlorothrix hollundicu (Volkman et al., 1988). In all hopanoid-producing prokaryotes the major compounds are always the bacteriohopanepolyols resulting from a carbodcarbon linkage between the triterpenic hopane skeleton and a D-pentOSe derivative (Rohmer et al., 1984 Flesch and Rohmer, 1988). As hopanoids are essential metabolites for the bacteria producing them, acting mainly as membrane stabilizers (Flesch and Rohmer, 1987;Rohmer et al., 1979;Sahm et al., 1993; references cited in the latter), an axenic strain of Z? hollundica was examined for these compounds. In this paper we report the very peculiar hopanoid composition of this prokaryote containing only bacteriohopanetetrol glycosides with three different hexosuronic acid moieties and resembling the cyanobacterial hopanoids by the presence of a 2D-methyl group, as well as data on their intracellular localization. Bel, 4 rue Blaise Pascal, F-67070 Strasbourg Cedex, France trophically in BG 11 medium at pH 7.5 and 25 "C (Rippka et al., 1979). Mass cultures were prepared in a 10-1 Biostat E fermentor (Braun Diessel Biotech) without stirring. They were constantly gassed with a mixture of air and carbon dioxide at flow rates of 100 1 . h-' and 1 1 . h-' respectively and illuminated with white fluorescent light (500-1000 Ix). Cells were harvested by centrifugation (12000 g, 30 min) after 28 days growth, washed once with distilled water and subjected to lyophilization. Cell wall and thylakoid preparations were obtained and characterized as described previously (Jiirgens, 1990). MATERIALS AND METHODS StrainAnalytical methods and isolation of hopanoids. All analytical procedures and spectroscopic identifications were as previously described (Peiseler a...

Apart from a nuxture of bacteriohopanetetrols already found in other Acetobacter species, four new 3P-methylhopanoids have been isolated from Acetobacter europaeus. All of them present an ether linkage between a bacteriohopanetetrol or a bacteriohopanepentol and a carbapseudopentose moiety often found in bacterial hopanoids. Three of these ethers were shown by comparison with synthetic reference hopanoids to posess a supplementary methyl group at C31. This novel series of methylhopanoids may be the precursor of yet unidentified molecular fossils found in sediments.[rnethyl-2H,]Methionine was efficiently incorporated into the 3 1 -methylhopanoids with retention of all three deuterium atoms in the transferred methyl group. This labelling pattern might be consistent with a rather rarely found methylation reaction of an enol.Acetic acid bacteria are all good producers of hopanoids, a series of bacterial triterpenoids widely distributed among eubacteria [l] and acting as membrane stabilizers [2]. Acetobacter species present the most complicated pattern of structural variations on the pentacyclic skeleton : double-bond at C6 and/or C11, additional methyl group at C3P and two stereoisomers at C22 [3-61. This paper describes the isolation, identification and biosynthesis from [methyl-'H,]methionine of a novel series of methylhopanoids with an additional methyl group at C31 from the recently described Acetobacter europaeus which, in contrast to other Acetobacter species, requires acetic acid for growth [7]. of the bacteriohopanetetrols (R, = 0.83) and acetylated tetrol ethers (RF = 0.09). Preparative reverse-phase HPLC on a Dupont Zorbax ODS column (250X21.2 mm) using methanol as eluent (15 mYmin) and a Spectra Physic 6040 differential MATERIALS AND METHODS Culture conditions and isolation of hopanoidsAcetobacter europaeus DSM 6160 (Deutsche Sammlung von Mikroorganismen, Braunschweig) was grown aerobically at 30°C either in a 10-1 fermenter for 96 h (yield 0.2-0.3 g/l, dry mass), or in 2-1 conical flasks on a rotatory shaker for 72 h (yield 0.2 g/l) using the medium described by Sievers et al. [7]. For labelling experiments, ~-[rnethyl-~H,]methionine (CEA, Saclay, France ; isotopic abundance 99%) was added to the culture medium at a 1 mM concentration. Most of the analytical procedures were as described previously [8, 91. The CHCl,/CH,OH (2: 1, by vol.) extract of a small sample of freeze-dried cells (0.1 g) was first treated by our HJOJNaBH, method in order to evaluate the hopanoid content [l]. Bacteriohopanepolyols were isolated from a larger sample (4 g) as acetylated derivatives by TLC (cyclohexane/ethyl acetate, 1 : 1, by vol.) giving the tetraacetates Chimie de Mulhouse,

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Intracellular localization of triterpenic membrane stabilizers of the hopane series is described for the first time for a cyanobacterium. In Synechocystis PCC 6714, a bacteriohopanetetrol derivative (main compound) and diplopterol were detected in cell wall (CW) and thylakoid membrane (TM). Both hopanoids were enriched 4.5‐fold and 9.0‐fold in CW and outer membrane (OM) fractions, respectively, compared to TMs.