Structure elucidation and biosynthesis of 31‐methylhopanoids from Acetobacter europaeus

Abstract

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,

Characteristic fragmentation of bacteriohopanepolyols during atmospheric pressure chemical ionisation liquid chromatography/ion trap mass spectrometry

“…In the preliminary paper we proposed a series of ions which should be characteristic of pentafunctionalised components other than those with an amino group at C‐35 ( m/z 713, 653, 593, 533 and 473) 4. Currently, the best‐known bacterial sources of composite pentafunctionalised BHPs are the cyanobacteria,10 although they have also been observed in nitrogen‐fixing bacteria11 and acetic acid bacteria 12. Accordingly, we screened a number of cyanobacterial cultures for the presence of pentafunctionalised components (H. M. Talbot, R. Summons, L. Jahnke, P. Farrimond, unpublished results).…”

Euphotic zone bacterioplankton sources major sedimentary bacteriohopanepolyols in the Holocene Black Sea

“…Trace amounts of a ring-monounsaturated bishomohopanol -most likely a product from the cleavage of vicinal diols of the unsaturated aminotriol -were also found. High fragments with m/z 367, 189 and especially 334, 307 and 217 after GC/MS of the TMSderivative indicated the double bond position of the bishomohopenol at D11 (see also Simonin et al, 1994, who reported respective fragments for the acetate derivative of D11 hopenols). However, due to the lack of reference compounds, the uncertainties from the GC/MS identification, and the coelution of the unsaturated aminotriol with other components during LC-MS analyses, we were not able to unambiguously identify the double bond position of the unsaturated aminotriol.…”

Methylhopanoids: Molecular indicators of ancient bacteria and a petroleum correlation tool

“…2␤-MethylBHPs occur in many hopanoid-producing cyanobacteria, for which they seem to be good marker compounds , although 2-methylhopanoids lacking the C 5 side chain (i.e., 2␤-methyldiploptene and/or 2␤-methyldiplopterol) have been found in a Type II methanotrophic bacterium (Methylobacterium organophilum; Renoux and Rohmer, 1985) and in three nitrogen-fixing bacteria (Vilchèze et al, 1994;Bravo et al, 2001). 3␤-MethylBHPs are absent from these bacteria, but have been reported in many Type I methanotrophs (Neunlist and Rohmer, 1985;Cvejic et al, 2000), and in some acetic acid bacteria (Simonin et al, 1994). Type I and II methanotrophic bacteria are phylogenetically distinct groups of methane-oxidizing bacteria that tend to predominate in different environments; Type I methanotrophs are dominant at low methane concentrations, such as in aquatic environments, with Type II methanotrophs generally being favored in soils and ground waters (Hanson and Hanson, 1996).…”