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Keywords:

  • Bradyrhizobium;
  • gammacerane;
  • hopanoid;
  • tetrahymanol;
  • squalene

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

The nitrogen-fixing, symbiotic root-nodule forming bacterium Bradyrhizobium japonicum USDA 110 contained gammacerane derivatives next to triterpenoids of the hopane series. Diploptene, diplopterol, 2β-methyldiplopterol, aminobacteriohopanetriol and adenosylhopane were accompanied by tetrahymanol and the corresponding novel methylated homologues 2β-methyltetrahymanol, 20α-methyltetrahymanol, and 2β,20α-dimethyltetrahymanol. Incorporation of [2H3]methyl–l-methionine indicated that the additional methyl groups originated from methionine, probably with S-adenosylmethionine acting as methyl donor, with retention of the three deuterium atoms. The simultaneous presence of hopane and gammacerane derivatives seems a characteristic feature of the genus Bradyrhizobium and the phylogenetically closely related Rhodopseudomonas palustris.

Abbreviations
SAM

S-adenosylmethionine, USDA, United States Department of Agriculture

Triterpenes constitute a group of structurally variable and functionally important natural products belonging to the large class of the isoprenoids. Their structures are based on a C30 skeleton derived from the cyclization of squalene or oxidosqualene. The tetracyclic sterols are found in nearly all eukaryotic cells. This is in contrast to prokaryotes, which are, with rare exceptions, unable to synthesize sterols. Many bacteria however produce pentacyclic triterpenoids that belong to the hopane series (Fig. 1, 15) [1–3]. These hopanoids are widespread among members of the domain Bacteria, but have not been detected in the Archaea[4].

image

Figure 1. Isoprenoids from Bradyrhizobium japonicum USDA 110 (1-9, 15) and derivatives (10-14). (1) diploptene, (2) diplopterol (R=H), (3) 2β-methyldiplopterol (R=CH3), (4) 35-aminobacteriohopanetriol, (5) adenosylhopane, (6) tetrahymanol (1R=2R=H), (7) 2β-methyltetrahymanol (1R=CH3, 2R=H), (8) 20α-methyltetrahymanol (1R=H, 2R=CH3), (9) 2β,20α-dimethyltetrahymanol (1R=2R=CH3), (10) tetrahymanone (1R=2R=3R=H), (11) 2β-methyltetrahymanone (1R=CH3, 2R=3R=H), (12) 20α-methyltetrahymanone (1R=3R=H, 2R=CH3), (13) 2β,20α-dimethyltetrahymanone (1R=2R=CH3, 3R=H), (14) 20β-methyltetrahymanone (1R=2R=H, 3R=CH3), (15) ubiquinone Q10.

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Tetrahymanol (gammaceran-21α-ol) (6) is a triterpene with a pentacyclic carbon skeleton closely related to the hopanoids [5]. In contrast with the hopane series, which possesses a five-membered ring E, the gammacerane skeleton is characterized by a six-membered ring (Fig. 1). Tetrahymanol was first isolated from the ciliate protozoan Tetrahymena pyriformis[6]. Later, it was detected in a number of other eukaryotes, e.g. in ferns, fungi and some other ciliates. Its occurrence was long thought to be restricted to eukaryotes. The frequent presence of gammacerane in the organic matter from sediments pointed out a much more widespread distribution in living organisms, and the finding of tetrahymanol accompanying hopanoids in the purple nonsulfur bacterium Rhodopseudomonas palustris opened new insights into the biochemistry of triterpenoids in bacteria [7].

The biosynthesis of the hopane skeleton involves an acid-catalysed cyclization of the acyclic triterpene squalene via carbocationic intermediates [8], yielding diploptene hop-22(29)-ene (Fig. 1, 1) and diplopterol (hopan-22-ol) (2) as main products. Functionally, hopanoids and tetrahymanol were proposed to stabilize the phospholipid bilayers in plasma membranes [1,2]. They share this function with the sterols of eukaryotes that act, besides their precursor functions for bile acids and steroid hormones, as potent membrane stabilisers. For example, the membrane condensing effect of cholesterol has been also found for bacteriohopanetetrol and other hopanoids [2,9].

Bradyrhizobia are capable of forming symbiotic root nodules with a variety of legume plants, including soybean, and of fixing nitrogen inside these nodules [10,11]. Earlier work revealed in these bacteria the presence of hopanoids and tetrahymanol [12], as well as of several unidentified triterpenoids. This work describes the full structures of the Bjaponicum composite hopanoids and of three new methylated tetrahymanol derivatives. It also intends (a) to contribute to a better understanding of the cyclization processes catalysed by the squalene cyclases; (b) to unravel biological functions of the diverse triterpenoids in bacteria in general, and in rhizobial cells during free-living and symbiotic growth; (c) to yield valuable data for a chemotaxonomic approach of bacterial phylogeny; and (d) finally to give clues for the reconstruction of palaeoenvironments through an improved understanding of bacterial molecular fossils found in the organic matter of sediments.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Culture conditions

Bradyrhizobium japonicum USDA 110 [13] was cultivated at 28 °C for 4 days in a yeast/mannitol medium containing KH2PO4 (0.5 g·L−1), MgSO4.7H2O (0.2 g·L−1), NaCl (0.1 g·L−1), d-mannitol (10 g·L−1) and yeast extract (1 g·L−1) in a 20-L fermenter (Giovanola, Vevey, Switzerland). After cultivation, the cells were harvested by centrifugation and lyophilized, yielding approximately 550 mg·L−1 (dry weight).

Analytical methods

TLC was performed on silica gel using commercial Merck 60 F254 plates (0.25 mm thickness) or, for larger quantities, with hand made plates (1 mm thickness, Merck 60 PF254 silica gel). Triterpenoids were visualized under UV light (352 nm) after spaying with a 0.1% berberine hydrochloride solution in ethanol and were recovered from silica with either dichloromethane or chloroform/methanol (2 : 1, v/v), depending on their polarity. HPLC was carried out on a Waters 519 apparatus with reverse phase Dupont Zorbax ODS C8 column. The flow rate was set to 1 mL·min−1 for analytical HPLC or 15 mL·min−1 for preparative HPLC.

GC analysis was carried out on fused silica DB1 or DB5 capillary columns (30 m × 0.25 mm) coated with a grafted DB1 or DB5 stationary phase (0.1 µm) on a Carlo Erba 4160/OO gas chromatograph fitted with an on-column injector and a flame ionization detector, with hydrogen as carrier gas. The temperature program was set from 50 °C to 220 °C with 20 °C·min−1 increase, followed by a 6 °C·min−1 increase to 310 °C and a 30-min isotherm. The injector temperature was set to 50 °C and the detector temperature to 310 °C. Mass spectrometry (direct inlet or GC-MS, 70 eV electron impact) was performed with a Finnigan TSQ700 spectrometer.

1H- and 13C-NMR spectra were recorded on a Bruker AC 200, Bruker AC 250, Bruker AC 400 or Bruker AC 500 spectrometers at 300 K in C2HCl3 solution using CHCl3 (δ = 7.26 p.p.m.) as internal standard for 1H-NMR and 13C2HCl3 (δ = 77.0 p.p.m.) as internal standard for 13C-NMR. Two-dimensional spectra (COSY 1H/1H, COSY 1H/13C, NOESY, HMBC) were recorded on a Bruker AC 500 spectrometer. Tentative signal assignments were made according to literature data for (6) and (7), completed by 2D NMR data for (7) and (8) and for (9) by comparison with the assignments made for (6) and (7). In contrast with the usual numbering of 3-hydroxytriterpenes, which is derived from the cyclization of oxidosqualene, numbering of the gammacerane skeleton (Fig. 1) was adapted from the numbering of the hopane skeleton, considering that both series are phylogenetically related. Both correspond to 3-deoxytriterpenes directly derived from the cyclization of squalene, the hydroxy group of tetrahymanol being derived from water and not from dioxygen.

Gammacerane derivatives were recrystallised from methanol/dichloromethane. Melting points were measured on a Reichert Thermovar microscope with a heating stage and were not corrected.

Isolation of triterpenoids

The lyophilized cells (108 g, dry weight) were extracted 6 times under reflux with toluene (10 mL·g−1). After evaporation of the solvent, the extract (56 mg·g−1) was separated by TLC (CH2Cl2, two migrations) on preparative silica plates (1 mm thickness) yielding diploptene (1) (Rf = 0.95, 1 mg·g−1), a mixture of 20α-methyltetrahymanol (8) and 2β,20α-dimethyltetrahymanol (9) (Rf = 0.56, 0.3 mg·g−1), ubiquinone Q10 (15) (Rf = 0.46, 0.2 mg·g−1), a mixture of diplopterol (2) and 2β-methyldiplopterol (3) (Rf = 0.32, 1.7 mg·g−1) and finally a mixture of tetrahymanol (6) and 2β-methyltetrahymanol (7) (Rf = 0.25, 16 mg·g−1). Reverse phase HPLC on a Dupont Zorbax ODS C8 column (25 cm × 4.6 mm, CH3OH/H2O, 93 : 7, 15 mL·min−1) allowed the separation of 20α-methyltetrahymanol (8) (Rt = 62 min, 60 µg·g−1, freeze-dried cells) from 2β,20α-dimethyltetrahymanol (9) (Rt = 70 min, 10 µg·g−1) and of tetrahymanol (6) (Rt = 42 min, 300 µg·g−1) from 2β-methyltetrahymanol (7) (Rt = 47 min, 600 µg·g−1).

Bjaponicum cells contained large amounts of poly β-hydroxybutyric acid, which is extracted with CHCl3/CH3OH (2 : 1, v/v), the usual solvent for hopanoid extraction, and which hampered the isolation of bacteriohopane derivatives. After toluene extraction, the bacterial cells were directly treated at room temperature for 41 h with an acetic anhydride/pyridine/toluene mixture (1 : 1 : 2, 4 mL·g−1) [14]. After removal under vacuum of the excess of reagents, the residue was extracted under reflux with tetrahydrofuran (THF) (4 × 100 mL). After filtration at room temperature and evaporation of the solvent, the extract (2.4 g) was fractionated by flash chromatography [15] using cyclohexane/ethyl acetate (3 : 7, v/v, 300 mL) and ethyl acetate/cyclohexane (9 : 1, v/v, 500 mL). The hopanoid containing fraction (identified according to the methyl singlets of the hopane skeleton observed in the 1H-NMR spectra) was further separated by preparative TLC (ethyl acetate/cyclohexane, 9 : 1, v/v), affording the tetraacetate of aminobacteriohopanetriol (4) (0.4 mg·g−1) and the diacetate of adenosylhopane (5) (0.8 mg·g−1).

Identification of the triterpenoids

Diploptene (1), diplopterol (2), 2β-methyldiplopterol (3), tetrahymanol (6), aminobacteriohopanetriol (4) tetraacetate and adenosylhopane (5) diacetate were identified by 1H- and 13C-NMR spectroscopy and comparison of the data with those previously published for reference compounds [7,16–18]. This identification was completed by GC coelution with standards, directly for (1) and (6) or on the trimethylsilyl ethers for (2) and (3) and by measuring a mixed melting point of (6) with a tetrahymanol standard isolated from T. pyriformis (270–272 °C).

Each hydroxylated gammacerane derivative (a few mg, 69) in dichloromethane solution (1 mL) was oxidized with pyridinium dichromate (100 mg) by stirring for 12 h at room temperature in the presence of 4 Å molecular sieves. Purification on preparative TLC plates (CH2Cl2) led to the isolation of the pure ketones (10) or (11) (Rf = 0.31), or (12) or (13) (Rf = 0.48).

The structure of 20α-methyltetrahymanol (8) was confirmed by chemical correlation. Tetrahymanone (10) (20.5 mg) dissolved in THF (0.5 mL) was added to a solution of lithium diisopropylamide in dimethoxyethane (1.6 mL), prepared from diisopropylamine (48 µL) and a 1.6-m solution of n-butyl lithium in THF (180 µL). After 15 min at room temperature, methyl iodide (128 µL) was added, and the reaction mixture was stirred for additional 50 min. After quenching with water, the reaction mixture was evaporated to dryness and separated by TLC (CH2Cl2), resulting in the isolation of 20β-methyltetrahymanone (14) (Rf = 0.52, 10 mg).

20α-Methyltetrahymanone (12) (3 mg), obtained by oxidation of 20α-methyltetrahymanol (8), was treated for 2 h at room temperature with a solution of concentrated sulfuric acid (200 µL) in ethanol/water (1 : 1, 800 µL). After neutralization with a 1-m solution of NaOH and hexane extraction, the 20β-methyltetrahymanone (14) (1 mg) was purified by TLC (CH2Cl2) and was identical (1H-NMR, GC, mixed melting point) with the corresponding ketone obtained by methylation of tetrahymanone.

2β-Methyltetrahymanol (7). M.p. = 244.5–245 °C. 1H-NMR: δ (p.p.m.) = 0.746 (3H, s, 22α-CH3), 0.805 (3H, s, 18α-CH3), 0.825 (3H, s, 2β-CH3), 0.827 (3H, s, 4β-CH3), 0.882 (3H, s, 4α-CH3), 0.889 (3H, s, 10β-CH3), 0.940 (3H, s, 8β-CH3), 0.942 (3H, s, 14α-CH3), 0.954 (3H, s, 22β-CH3), 3.18 (1H, dd, 11.2 and 5.1 Hz, 21β-H). 13C-NMR: δ (p.p.m.) = 15.4 (C-29), 16.09 and 16.14 (C-26 and C-28), 16.5 (C-27), 18.4 (C-16), 19.9 (C-7), 21.5 (C-12), 21.8 (C-25), 22.1 (C-11), 23.2 (C-31), 24.8 (C-2), 26.1 (C-24), 27.4 (C-20), 28.0 (C-30), 31.1 (C-23), 32.4 (C-6), 33.2 (C-15), 37.1 (C-18), 37.77a (C-10), 38.78 (C-19), 38.9 (C-22), 41.8a (C-8), 42.0a (C-14), 45.2 (C-3), 46.5 (C-4), 49.70 and 49.73 (C-1 and C-5), 50.7 (C-13), 50.9 (C-9), 55.2 (C-17), 79.0 (C-21). Assignments of signals bearing the same superscripts may be interchanged. MS: m/z (%) = 442 (M+, 26), 427 (M+-Me, 8), 424 (M+-H2O, 7), 207 (b, 66), 205 (a, 100), 189 (b-H2O, 27).

20α-Methyltetrahymanol (8). M.p. = 233.5–234 °C. 1H-NMR: δ (p.p.m.) = 0.783 (3H, s, 10β-CH3), 0.806 (3H, s, 4β-CH3), 0.837 (3H, 2 s, 8β-CH3), 0.868 (3H, s, 22β-CH3), 0.899 (3H, s, 22α-CH3), 0.948 (3H, s, 4α-CH3), 0.966 (3H, s, 14α-CH3), 0.966 (3H, d, 6.9 Hz, 20α-CH3), 1.026 (3H, s, 18α-CH3), 3.29 (1H, d, 2.2 Hz, 21β-H). 13C-NMR: δ (p.p.m.) = 16.0 (C-25 and C-26), 16.6 (C-27), 18.6a (C-29), 18.7 (C-2 and C-6), 18.9 (20α-CH3), 19.8a,b (C-28 and C-15), 21.3 and 21.9 (C-11 and C-12), 21.6 (C-27), 29.0 (C-20), 31.0a (C-30), 32.7b (C-16), 33.0 (C-7), 33.4 (C-4), 36.8 (C-18), 37.3 (C-10), 40.3 (C-1), 42.0 (C-14), 42.2 (C-3 and C-8), 44.0b (C-19), 33.3 (C-23), 50.7 and 50.9 (C-9 and C-13), 51.1 (C-17), 53.4 (C-22), 56.2 (C-5), 80.2 (C-21). Assignments of signals bearing the same superscripts may be interchanged. MS: m/z (%) = 442 (M+, 22), 427 (M+-Me, 5), 409 (M+-H2O-Me, 7), 424 (M+-H2O, 22), 221 (2, 100), 203 (b-H2O, 33), 191 (a, 100).

2β,20α-Dimethyltetrahymanol (9). M.p. = 214–215 °C. 1H-NMR: δ (p.p.m.) = 0.827 (3H, d, 6.9 Hz, 2β-CH3), 0.828 (3H, s, 4β-CH3), 0.875 (3H, s, 22β-CH3), 0.882 (3H, s, 4α-CH3), 0.892 (3H, s, 10β-CH3), 0.903 (3H, s, 22α-CH3), 0.943 (3H, s, 8β-CH3), 0.965 (3H, s, 14α-CH3), 0.973 (3H, d, 6.9 Hz, 20α-CH3), 1.036 (3H, s, 18α-CH3), 3.29 (1H, d, 2.1 Hz, 21β-H). 13C-NMR: δ (p.p.m.) = 16.2 (C-26), 16.5 (C-27), 18.7 and 18.9 (C-29 and 20α-CH3), 19.8a (C-15 and C-28), 19.9 (C-7), 21.9 (C-25), 22.2 and 22.4 (C-11 and C-12), 23.2 (2β-CH3), 24.8 (C-2), 26.1 (C-24), 29.0 (C-20), 31.0 and 31.1 (C-23 and C-30), 32.2 (C-6), 32.5 (C-4), 32.7a (C-16), 36.8 (C18), 37.5 (C-10), 41.7 (C-8), 42.2 (C-14), 44.0a (C-19), 45.2 (C-3), 49.7 (C-5), 49.8 (C-1) 50.8, 50.9 and 51.0 (C-9, C-13 and C-17), 55.6 (C-22), 80.4 (C-21). Assignments of signals bearing the same superscripts may be interchanged. MS: m/z (%) = 456 (M+, 15), 441 (M+-Me, 4), 438 (M+-H2O, 17), 423 (M+-H2O-Me, 6), 221 (b, 48), 205 (a, 100), 203 (b-H2O, 28).

2β-Methyltetrahymanone (11). M.p. = 235–236 °C. 1H-NMR: δ (p.p.m.) = 0.828 (3H, d, 6.5 Hz, 2β-CH3), 0.830 (3H, s, 4β-CH3), 0.886 (3H, s, 10β-CH3), 0.896 (3H, s, 4α-CH3), 0.950 (3H, s, 8β-CH3), 0.962 (3H, s, 18α-CH3), 0.995 (3H, s, 14α-CH3), 1.021 (3H, s, 22α-CH3), 1.071 (3H, s, 22β-CH3), 2.42 (1H, dd, 7.1 Hz and 9.8 Hz, 20α-H), 2.47 (1H, dd, 5.9 Hz and 7.6 Hz, 20β-H).

20α-Methyltetrahymanone (12). M.p. = 198–198.5 °C. 1H-NMR: δ (p.p.m.) = 0.660 (3H, s, 22α-CH3), 0.795 (3H, s, 4β-CH3), 0.830 (3H,s, 10β-CH3), 0.845 (3H, s, 4α-CH3), 0.950 (3H, s, 18α-CH3), 0.975 (3H, d, 6.3 Hz, 20α-CH3), 1.000 (3H, s, 8β-CH3), 1.025 (3H, s, 22β-CH3), 1.055 (3H, s, 14α-CH3), 1.20 and 1.96 (2H, 2 m, 19α- and 19β-H), 2.83 (1H, m, 20β-H).

20β-Methyltetrahymanone (14). M.p. = 197–197.5 °C. 1H-NMR: δ (p.p.m.) = 0.736 (3H, s, 4β-CH3), 0.753 (3H, s, 10β-CH3), 0.793 (3H, s, 4α-CH3), 0.880 (3H, s, 8β-CH3), 0.958 (3H, d, 6.9 Hz, 20β-CH3), 0.965 (3H, s, 14α-CH3), 0.979 (3H, s, 22α-CH3), 1.049 (3H, s, 18α-CH3), 0.95 and 1.98 (2H, 2 m, 19α- and 19β-H), 2.70 (1H, m, H-20).

2β,20α-Dimethyltetrahymanone (13). M.p. = 199–200 °C. 1H-NMR: δ (p.p.m.) = 0.662 (3H, s, 22α-CH3), 0.831 (3H, d, 6.2 Hz, 2β-CH3), 0.838 (3H, s, 4β-CH3), 0.887 (3H, s, 10β-CH3), 0.914 (3H, s, 4α-CH3), 0.947 (3H, s, 8β-CH3), 0.977 (3H, d, 4.9 Hz, 20α-CH3), 1.027 (3H, s, 22β-CH3).

Incorporation of [2H3]methyl–l-methionine

The labelling experiment was carried out in 10 × 2-L conical flasks each containing 500 mL of the same yeast-mannitol medium supplemented with 100 µm[2H3]methyl-l-methionine. The lyophilized cells were extracted under reflux with toluene (6 × 50 mL) for 40 min. The extract was separated by TLC (CH2Cl2, two migrations) yielding three fractions: a mixture of 20α-methyltetrahymanol (9) and 2β,20α-dimethyltetrahymanol (10) and ubiquinone Q10 (15) (Rf = 0.74), a mixture of diplopterol (3) and 2β-methyldiplopterol (4) (Rf = 0.57) and a mixture of tetrahymanol (7) and 2β-methyltetrahymanol (8) (Rf = 0.48). Because of the small amounts, the losses occurring by HPLC isolation and the close GC retention times of a triterpene and its 2β-methyl homologues, these fractions were analysed directly by direct inlet electron impact mass spectrometry.

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Next to already known hopanoids (15) and tetrahymanol (6) (Fig. 1), which were identified by comparison with reference material previously isolated in our laboratory from other microorganisms, Bjaponicum contained three novel triterpenoids: these compounds were easily extracted with toluene and proved to be methylated gammacerane derivatives (79). Their mass spectra were quite similar to that of tetrahymanol (6), with shifts of the m/z ratio of some fragments indicating the presence of an additional methyl group either on the rings A or B for (7), D or E for (8) or of two additional methyl groups on both moieties for (9) (Fig. 1). Similar to tetrahymanol (6), these new triterpenoids were secondary alcohols: they could be acetylated at room temperature with acetic anhydride in pyridine in conditions, which do not allow the acetylation of tertiary alcohols such as diplopterol (2), and, according to the 1H-NMR spectrum presented a single proton in the α position of the hydroxy group. Furthermore, pyridinium dichromate oxidation afforded for each of them a ketone (1013). Their full identification was supported by 2D-NMR studies, comparison of spectroscopic data with those of related known compounds such as tetrahymanol (6), diplopterol (2) or 2β-methyldiplopterol (3), and for (8) by chemical correlation with tetrahymanol (6). Incorporation of l-methionine with deuterium labelling on the methyl group indicated in addition that SAM was most probably the methyl donor for the methylations at C-2 or C-20.

20α-Methyltetrahymanol (8)

The 1H-NMR spectrum of compound (8) was characterized by nine methyl signals (eight singlets and one doublet). According to the inline imageC-NMR data (DEPT, inline imageH/inline imageC COSY), six quaternary, 10 methylene and six methine groups were in addition characterized. No signals of sp2 carbons were found. With a m/z 442 molecular ion, this triterpenoid most likely corresponded to a methyltetrahymanol (C31H54O). The mass spectrum of triterpenoid (8) was similar to that of tetrahymanol and presented the same fragmentation pattern. From the presence of the two m/z fragments 221 and 203 resulting from ring C cleavage (Fig. 2), the presence of the additional methyl group could be localized on the ring D or E, similar to the hydroxy group. In the 1H-NMR spectrum of tetrahymanol, the H-21 proton signal appeared as a doublet of doublets, being coupled with the two H-20 protons. In the spectrum of (8), the proton vicinal to the hydroxy group appeared as a doublet, indicating that it is only coupled to a single proton and suggesting that the methyl group appearing as a doublet is located at C-20. The structure of a 20-methyltetrahymanol was thus the most likely one. This conclusion was supported by the pyridinium dichromate oxidation of (8), which afforded a ketone, whose spectroscopic data were compatible with this structure assignment. In the 1H-NMR spectrum of the ketone (12), a multiplet (δ = 2.83 p.p.m.) corresponded to a single proton in α position of the carbonyl group. Definitive proof for the structure of (8) was obtained by chemical correlation. 20β-Methyltetrahymanone was synthesized by methylation of the tetrahymanone (10) enolate with methyl iodide [19], and the 20α-methyltetrahymanone (12), obtained by oxidation of the alcohol (8) isolated from Bjaponicum, was isomerized in acidic conditions into 20β-methyltetrahymanone (14), which was fully identical (1H-NMR, GC, mixed melting point) with the ketone (14) obtained by hemisynthesis. Only the configuration of the C-21 carbon bearing the hydroxy group remained to be determined. In the NOESY spectrum, NOE were observed between H-21, the protons of the two methyl groups at C-22 as well as the methyl group at C-20 and an additional proton, which can only be the H-20β proton at 1.86 p.p.m. (Fig. 3). This shows that H-21 is in β position, and that the ring E is in a distorted boat-like conformation, which allows simultaneous interactions of H-21 with the two substituents at C-20 as well as with those at C-22 (Fig. 3). Such interactions are impossible in any conformation of ring E, when the H-21 is in the α position. A distorted-boat or twisted conformation for such a substituted cyclohexane is most likely, as shown by the conformational analysis of ring A in 3-oxo-2β-bromo-4,4-dimethyltriterpenes [20], avoiding thus steric interactions between substituents otherwise in axial position in the chair conformation. Accordingly, the structure of (8) is that of 20α-methyltetrahymanol.

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Figure 2. Mass spectrometry fragmentations of the hopane (right) and gammacerane (left) skeletons. a, b and c refer to fragments with m/z values listed in Table 1.

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Figure 3. Selected NOE correlations observed for 2β-methyltetrahymanol (8) (top) and for 20α-methyltetrahymanol (9) (bottom).

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2β-Methyltetrahymanol (7)

The same set of analytical data as those used for the identification of (8) also assigned a C31H54O methyltetrahymanol structure to compound (7). From the presence in the mass spectrum of the m/z 205 and 207 or 189 fragments resulting from ring C cleavage (Fig. 2), the additional methyl group could be localized in the ring A or B and the hydroxy group on the ring D or E. The chemical shifts of the methyl protons and those of the 13C from the rings A, B, C, and D were quite similar to the corresponding signals of the previously identified 2β-methyldiplopterol (3) [19–21]. Full structure was determined after NMR studies using homonuclear 1H/1H and heteronuclear 1H/13C COSY correlation including 1J as well as long range coupling studied by HMBC experiments. Compound (7) was a secondary alcohol with the same Rf as tetrahymanol (6). It yielded after oxidation a ketone (11). Furthermore, the chemical shifts (in the 1H and 13C-NMR spectra) of the methyl protons, the H-21 proton and the carbon atoms from ring E of tetrahymanol (6) or tetrahymanone (10) were, respectively, nearly identical with the corresponding signals in the spectra of the alcohol (7) and the ketone (11). From the presence of a m/z 205 fragment in the mass spectrum, an additional carbon atom had to be present in the ring A or B, if the hydroxy group would be located in ring D or E as suggested by the m/z 207 fragment. From the close GC retention times of tetrahymanol and compound (8), of their trimethylsilyl ethers and their acetates, and by analogy with those of diplopterol, 2β-methyldiplopterol and 3β-methyldiplopterol, it was rather likely that compound (8) was 2β-methyltetrahymanol. Indeed, 3β-methylhopanoids are characterized by much longer GC retention times than those of their nonmethylated homologues, whereas those of 2β-methylhopanoids are very close to those of the corresponding nonmethylated hopanoids [23]. 2D-NMR correlations allowed to assign all 13C signals via HMBC long range and NOE correlations, to propose a twisted boat-like conformation for ring A (Fig. 3) similar to the conformation of ring E of 20α-methyltetrahymanol, and finally to confirm the 2β-methyltetrahymanol (7) structure. Indeed, the two C-4 methyl groups were correlated by NOE. One of them, the C-4β methyl group, was also correlated with the C-10β methyl group, which was itself correlated with the C-8β methyl group. Furthermore the methyl group at C-2 was only correlated with the 2-H proton. Such correlations were only consistent with a β position for the C-2 methyl group and a distorted boat-like conformation for ring A.

2β,20α-Dimethyltetrahymanol (9)

2β,20α-Dimethyltetrahymanol (9) could only be obtained in tiny amounts, which did not allow 2D NMR analyses. The mass spectrum was typical for a gammacerane derivative. A 456-m/z molecular ion indicated the presence of two additional carbon atoms on a tetrahymanol skeleton: a first one on the ring A or B according to the presence of a m/z 205 fragment and a second one on the ring D or E as indicated by the m/z 221 fragment (Fig. 2). Comparison of the 1H- and 13C-NMR spectra with those of the two methylated tetrahymanol derivatives (7) and (8) was however, in accord with a 2β,20α-dimethyltetrahymanol (9) structure. In the NMR spectra, the chemical shifts of protons and 13C from the rings A and B or D and E were, respectively, identical or quite similar to those of the corresponding atoms from the rings A and B of 2β-methyltetrahymanol (7) or from the rings D and E of 20α-methyltetrahymanol (8). Those of the atoms from ring C were similar in the spectra of tetrahymanol (6) and of all methylated tetrahymanol derivatives. This conclusion was corroborated by the identical TLC Rf of (9) and (8) and by the chemical shift of the H-21 proton (3.29 p.p.m.), which appeared as a doublet (J = 2.1 Hz), as in the 1H-NMR spectrum of 20α-methyltetrahymanol (8).

Incorporation of [2H3]methyl-L-methionine

Methylation reactions of bacterial hopanoids were formerly studied by feeding of [2H3]methyl-l-methionine [24,25]. A similar experiment was performed with B. japonicum, and the deuterium incorporation was examined in the methylated gammacerane derivatives (79) and 2β-methyldiplopterol (3) by GC-MS and in ubiquinone Q10 (15) by direct inlet MS. Next to the unlabelled isoprenoids derived from the methionine synthesized from the unlabeled carbon source, only isotopomers possessing either three, six or nine deuterium atoms and synthesized from exogenous labelled methionine were detected (Table 1). This indicated that no methionine catabolism, leading to label scrambling, occurred, and that no deuterium was lost when the methionine methyl group was transferred onto the isoprenoids. The deuterium incorporation yields were quite good. From the relative intensities of the signals of the M+ and (M + 3)+ ions for the methyltetrahymanols (7) and (8) and for 2β-methyldiplopterol (3) and of the M+ (M + 3)+ and (M + 6)+ ions for the 2β,20α-dimethyltetrahymanol (9), a ≈ 70% incorporation was evaluated, whereas deuterium incorporation was around 50%, according to the relative intensities of the signals of the M+, (M + 3)+, (M + 6)+, and (M + 9)+ ions, for ubiquinone Q10 (15), which presents three methylation sites.

Table 1.  Incorporation of [2H3]methyl-l-methionine into the isoprenoids of Bradyrhizobium japonicum.
  m/z
IsoprenoidMolecular ionM+ –H2OM+ –H2O–CH3abb-H2Oc-2H
2β-Methyldiplopterol (2)       
 Nondeuterated442  205207189381
 Deuterated445  208207189384
2β-Methyltetrahymanol (7)       
 Nondeuterated442424409205207189 
 Deuterated445427412208207189 
20α-Methyltetrahymanol (8)       
 Nondeuterated442424409191221203 
 Deuterated445427412191224206 
2ß,20α-Dimethyltetrahymanol (9)       
 Nondeuterated456  205221203 
 Deuterated459  208224206 
 462444429    
Ubiquinone Q10 (15)       
 Nondeuterated862      
 Deuterated865      
 868      
 871      

Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

A first approach of the hopanoid content in Bradyrhizobia was performed after derivatization of the bacteriohopanepolyols, involving H5IO6 oxidative side-chain cleavage followed by NaBH4 reduction and yielding primary alcohols readily analysed by GC [4]. It revealed in several strains of Bradyrhizobium and of related bacteria (e.g. B. japonicum, B. elkanii, Bradyrhizobium sp. nodulating Lupinus and the closely related, photosynthetic, nitrogen-fixing Photorhizobium sp. BTAiI, nodulating the tropical legume Aeschynomene indica) the occurrence of diploptene (1) and diplopterol (2), which are both nearly ubiquitous in hopanoid producers, as well as dihomohopan-32-ol, which represented the signature of the C35 bacteriohopanepolyol derivatives [12,26]. According to the GC analyses, all these hopanoid producing strains [e.g. B. japonicum ATCC 10324, USDA 59, USDA 123 and 61-A-101, Belkanii USDA 31 and USDA 76, Bradyrhizobium sp. (Lotus) NZP 2257 and Bradyrhizobium sp. (Arachis) NC92] also contained tetrahymanol (6). In addition, unidentified alcohols, most probably corresponding to methylated pentacyclic triterpenic alcohols, accompanied in significant amounts the above mentioned triterpenoids.

B. japonicum USDA 110 presented the typical Bradyrhizobium triterpenoid fingerprint and produced significant amounts of the unknown alcohols. It was therefore chosen for a detailed investigation of the intact biohopanoids. The widespread aminobacteriohopanetriol (4), which affords dihomohopan-32-ol after H5IO6/NaBH4 treatment, was the main bacteriohopane derivative. It was accompanied by significant amounts of the rather rare adenosylhopane (5), which can not be detected by the former derivatization method. So far, adenosylhopane has been only found as minor hopanoid in Rhacidophila[17] and Nitrosomonas europaea[27]. In this triterpenoid, the carbon/carbon linkage between a d-ribose derivative and the triterpenic moiety of the hopanoids is evident. Adenosylhopane might be the precursor or closely related to the precursor of the bacteriohopane skeleton. Indeed, the corresponding lactol was proposed to yield by reduction bacteriohopanetetrol or by reductive amination aminobacteriohopanetriol, the two basic bacterial hopanoids [28,29]. No other composite bacteriohopane derivative was found in Bjaponicum.

The unknown triterpenic alcohols revealed as novel methylated gammacerane derivatives, which accompanied tetrahymanol (6): 2β-methyltetrahymanol (7), 20α-methyltetrahymanol (8) and 2β,20α-dimethyltetrahymanol (9). The simultaneous occurrence of the hopane and the gammacerane series seems to be a common feature of Bradyrhizobium bacteria, which is also shared with Rh. palustris. Indeed, the latter bacterium contained aminobacteriohopanetriol as only C35 hopanoid [17], which was nearly always accompanied by significant amounts of tetrahymanol [7,30]. According to 16S rRNA analysis, Bradyrhizobium bacteria and the purple nonsulfur bacterium Rh. palustris are closely related and cluster together in the phylogenetic tree of the α-subgroup of proteobacteria [31]. This close relationship is now also reflected by the triterpenoid composition of B. japonicum USDA 110 and Rh. palustris.

Molecular fossils of the gammacerane series are widespread in the organic matter from many sediments [32]. They were first interpreted as molecular fossils from ciliate protozoans. Their repeated finding in Bacteria members, such as Rhpalustris, Bradyrhizobium spp. and related species [33], suggests, however, a much broader distribution amongst prokaryotes.

The 2-deoxyhopanoids from Bacteria species and tetrahymanol from the ciliate protozoon T. pyriformis directly result from the cyclization of squalene [5,34]. In contrast, the formation of tetrahymanol in Bradyrhizobium and Rh. palustris is an as yet unsolved problem. Is it the same enzyme, which produces the two triterpene series, or are two different enzymes required? A squalene cyclase gene has been cloned from both organisms and expressed in Escherichia coli[26,35]. In addition, in Rhpalustris, the squalene cyclase has been isolated and purified [30]. In all cases, the recombinant proteins as well as the purified proteins produced only diploptene (1) and diplopterol (2) and did not synthesize tetrahymanol (6) from squalene. This is in sharp contrast with the activity of the purified squalene cyclase of the protozoon Tpyriformis, which simultaneously produces tetrahymanol (6) and diplopterol (2) [36], indicating that in this organism the two different, but related, cyclization processes are catalysed by the same enzyme.

Based on the cyclization mechanism and on the tridimensional structure of the squalene-hopene cyclase from Alicyclobacillus acidocaldarius[37,38], C30 hopanoids as well as tetrahymanol are expected to fit the catalytic cavity of this enzyme. However, in tetrahymanol biosynthesis, the formation of the six-membered ring E requires a C-21 secondary cationic intermediate, whereas a tertiary C-22 cation leads to the formation of the five-membered ring E of diploptene or diplopterol. Thus, structural flexibility is probably necessary if a single cyclase has to synthesize both triterpenic skeletons. In the case of Bradyrhizobium and Rhpalustris, it remains to be established whether in vivo tetrahymanol formation depends on an additional tetrahymanol-specific cyclase or whether tetrahymanol is a by-product of a hopene cyclase functioning differently in vitro and in a peculiar cell environment.

The most striking feature of the Bjaponicum triterpenoids is the presence of methylated gammacerane derivatives (79). Their equivalents in the hopane series were already known. 2β-Methyldiplopterol (3) is probably the most frequent methylhopanoid: it is present as major hopanoid in all Methylobacterium species and in the related pink pigmented facultative, methylotrophs [16,39,40] as well as in Beijerinckia indica and Bmobilis[41]. In M. organophilum, 2β-methyldiplopterol (3) was accompanied by minute amounts of 2β-methylbacteriohopanetetrol. 2β-methylbacteriohopanepolyols are rather frequently found in the cyanobacteria [4] and were proposed to serve as specific biomarkers for these prokaryotes [42]. 2β-Methydiplopterol (3) also accompanied diplopterol (2) in Bjaponicum. The simultaneous presence of 2β-methylated analogues of the hopane (3) and the gammacerane (79) series reflects probably a common feature in the biosynthesis of the two triterpenic skeletons. Interestingly, incorporation experiments with [2H3]methyl-l-methionine and the formation of methylated triterpenoids in Bjaponicum gave similar results to those of previously performed methylation studies on 3β-methylhopanoids in Acetobacter pasteurianus and 2β-methylhopanoids or 2α-methylhopanoids in M. organophilum[19,39] or 31-methylhopanoids in Acetobacter europaeus[25], indicating that SAM is the probable methyl donnor and that the transferred methyl group retained its three deuterium atoms.

The way the additional methyl group is introduced is unknown. The known C-methylations require a double bond or an aromatic ring in the substrate. The methylation at C-2 or C-3 of the ring A in the hopane series was proposed to be performed on a Δ2-hopanoid [24]. Such a mechanism is compatible with the methylation of the rings A or E in the gammacerane skeleton, but would imply in the case of methylation of ring E the methylation of an enol, as postulated in the biosynthesis of indolmycine [43] or of 31-methylbacteriohopane derivatives [25]. The preferential methylation of C30 triterpenes, such as diplopterol or tetrahymanol, is not in contradiction with the often observed absence of detectable amounts of methylated bacteriohopane derivatives. Indeed, in all investigated bacteria, there is no clear correlation between the methylation pattern of the C30 hopanoids (diploptene and diplopterol) and the C35 hopanoids (bacteriohopanepolyols), suggesting an efficient channelling of the metabolism of methylated and nonmethylated hopanoids.

Sterols, hopanoids or tetrahymanol can, to a certain extent, replace each other as stabilisers of the phospholipid bilayer in the plasma membrane [1,2]. In the ciliate T. pyriformis, supplementation with sterols fully inhibits the biosynthesis of tetrahymanol, which is replaced in the membranes by a sterol [44]. Growth of Tpyriformis is blocked by the addition of 2,3-dihydro-2-azasqualene, a squalene cyclase inhibitor, which prevents the biosynthesis of tetrahymanol, and is fully restored by addition of sterols or bacteriohopanetetrols from A. xylinum to the culture medium [45]. In addition, in the case of hopanoid-producing bacteria capable of fixing atmospheric nitrogen (similar to the free-living Azotobacter and Beijerinckia spp. or the symbiotic Frankia spp.), hopanoids were proposed to form a diffusion barrier to protect the oxygen-sensitive nitrogenase complex from oxidative damage [46]. Both series of Bradyrhizobium triterpenoids are capable of fulfilling such membrane stabilizing roles. So far, it remains, however, unclear whether hopanoids and tetrahymanol share the same functions or have other roles besides membrane stabilization.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

We wish to thank Dr R. Graf and Mr J.-D. Sauer for recording the NMR spectra and Mr P. Wehrung and Mrs E. Mastio for the MS measurements. This work was funded by grants to E. K. from the ‘Deutsche Forschungsgemeinschaft’ (PO 117/16-2 and SFB 323) and to M. R. from the European Union (BIOMASS project, contract Nb ENV4-CT95-0026), the Alexander von Humboldt Foundation and the ‘Institut Universitaire de France’. M. P. was a recipient of fellowship from the University of Tübingen.

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
Footnotes
  1. Note: throughout the text, the numbers in bold type refer to the corresponding structures in Fig. 1.