Bacterial triterpenoids of the hopane series from the methanotrophic bacteria Methylocaldum spp.: phylogenetic implications and first evidence for an unsaturated aminobacteriohopanepolyol


  • Jelena H. Cvejic,

    1. Université Louis Pasteur/CNRS, Institut Le Bel, 4 rue Blaise Pascal, 67070 Strasbourg Cedex, France
    Search for more papers by this author
  • Levente Bodrossy,

    1. Institute of Biophysics, Biological Research Centre, Hungarian Academy of Sciences, P.O. Box 521, Szeged, Hungary
    2. Department of Biotechnology, József Attila University of Szeged, Temesvári krt. 62, Szeged, Hungary
    Search for more papers by this author
  • Kornél L. Kovács,

    1. Institute of Biophysics, Biological Research Centre, Hungarian Academy of Sciences, P.O. Box 521, Szeged, Hungary
    Search for more papers by this author
  • Michel Rohmer

    Corresponding author
    1. Université Louis Pasteur/CNRS, Institut Le Bel, 4 rue Blaise Pascal, 67070 Strasbourg Cedex, France
    Search for more papers by this author

*Corresponding author. Tel.: +33 3 88 41 61 02; Fax: +33 3 88 60 75 50, E-mail address:


The hopanoid content of the two methanotrophic bacteria Methylocaldum szegediense and Methylocaldum tepidum was investigated. 35-Aminobacteriohopane-30R,31R,32R,33S,34S-pentol and its 3β-methyl homologue were present in both strains. In M. tepidum, they were accompanied by 35-aminobacteriohopane-31R,32R,33S,34S-tetrol and its 3β-methyl homologue. The side chain structure was identical to those previously reported from two other obligate methanotrophs, Methylococcus capsulatus and Methylomonas methanica. The two Methylocaldum species shared with the Methylococcus species the presence of 3β-methylhopanoid as well as of a hopanoid releasing adiantol upon H5IO6/NaBH4 treatment. A rare feature was in addition found in M. szegediense. The saturated hopanoids were accompanied by an unsaturated aminobacteriohopanepentol with a Δ11 double bond. Comparison of the hopanoid fingerprints was in accordance with the close phylogenetic relationship of Methylococcus and Methylocaldum. The major difference was the absence of sterols in Methylocaldum which were always detected in the Methylococcus species.


Methanotrophic bacteria use methane as sole carbon and energy source. They belong to the group of the methylotrophic bacteria capable of utilising one-carbon compounds, which are more reduced than formic acid, and of assimilating formaldehyde for their carbon metabolism [1]. Two strains of obligate methanotrophs were recently described: the thermophilic Methylocaldum szegediense OR2 was isolated from the effluent of an underground hot spring, and the thermotolerant Methylocaldum tepidum LK6 from an agricultural soil [2]. M. szegediense grows at a temperature optimum of ca. 52°C, M. tepidum at ca. 43°C. The Methylocaldum species represent a novel group of type I methanotrophs [2], which already included the genera Methylomonas, Methylobacter, Methylomicrobium and Methylococcus[1]. From the 16S rRNA and the methane monooxygenase gene sequences, the two above-mentioned species were shown to represent, together with ‘Methylomonas gracilis’ VKM-14LT and ‘Methylococcus thermophilus’ IMV-B3122, only three distinct species belonging to the genus Methylocaldum gen. nov.: Methylocaldum szegediense, Methylocaldum tepidum and Methylocaldum gracile[2]. These analyses also indicated that Methylocaldum is a monophyletic clade within the γ-subgroup of the Proteobacteria.

Triterpenoids of the hopane series are widely distributed amongst prokaryotes, where they fulfil the function of membrane stabilisers, much like sterols do in membranes of eukaryotes [3,4]. All investigated methanotrophic bacteria are good hopanoid producers [5]. In this contribution, the hopanoid content of the two obligate methanotrophs Methylocaldum szegediense OR2 and Methylocaldum tepidum LK6 was investigated and compared with those of other obligate methanotrophs.

2Materials and methods

2.1Cell cultures

The methanotrophs were grown on a nitrate mineral salts medium [6] in a 4-l fermenter (BioFlo IIc, New Brunswick Scientific) fed with a continuous flow of methane/air (1:2, v/v). Oxygen concentration was followed with a dissolved oxygen probe, and the agitation speed was regulated to maintain the dissolved oxygen level below 5%. M. szegediense OR2 was grown at 52°C with an agitation speed of 250–400 rpm and harvested at 1.7 OD540, and M. tepidum LK6 was grown at 43°C with an agitation speed of 100–150 rpm, and harvested at 1.6 OD540. Cells were harvested by centrifugation (15 000×g, 60 min) and lyophilised.

2.2Analytical methods

Most analytical procedures and spectroscopic identifications were carried out as previously described [7]. 1H NMR spectra were recorded on a Bruker WP 400 spectrometer in [2H]chloroform solution at 300 K. GC was performed on a Carlo Erba Strumentazione Fractovap Series 4160 chromatograph on a fused silica column coated with a DB5 stationary phase (30 m×0.25 mm, 0.1 μm film thickness). GC/MS spectra were recorded on a Fisons Instruments MD 800 spectrometer.

2.3Detection and isolation of hopanoids

Lyophilised cells were extracted under reflux with CHCl3/CH3OH (3×50 ml, 2:1, v/v). An aliquot of the crude extract (1/10) was treated with H5IO6/NaBH4, in order to cleave the side chain of the hopanoids [5]. The relative amounts of the hopanoids were determined on the resulting derivatives from the integration of the GC peak areas. The remaining part of the crude extracts was acetylated. TLC separation (ethyl acetate/cyclohexane, 7:3, v/v, Rf=0.24) afforded a mixture of the acetates of the aminobacteriohopanepentols 1, 2 and 2a from M. szegediense (9 mg from 1.4 g lyophilised cells), and of the acetates of aminobacteriohopanetetrols 1 and 2 and aminobacteriohopanepentols 3 and 4 from M. tepidum (4 mg from 0.6 g lyophilised cells).

2.4Analytical data

1H NMR of the mixture of the hexa-acetates of 1 (24%), 2 (68%) and 2a (8%) isolated from M. szegediense (400 MHz, CDCl3): δ(ppm)=*0.628 (s, 18α-CH3), *°0.643 (broad s, *4α- and °18α-CH3), *0.774 (s, 4β-CH3), °0.786 (s, 4β-CH3), *0.807 (d, J=6.7, 3β-CH3), °0.808 (s, 4α-CH3); °0.840 (s, 10β-CH3), *0.857 (s, 10β-CH3), 0.921 and 0.931 (2 broad s, °8β- and °14α-CH3; *8β- and *14α-CH3), 0.944 (d, J=6.5 Hz, 22-CH3), 1.948 (3H, s, CH3CONH-), 2.073 (3H, s, CH3COO-), 2.078 (3H, s, CH3COO-), 2.103 (3H, s, CH3COO-), 2.105 (3H, s, CH3COO-), 2.117 (3H, s, CH3COO-), 3.33 (1H, ddd, J35a,35b=14.7 Hz, J34,35a=7.2 Hz, J35a,NH=6.2 Hz, 35-Ha), 3.62 (1H, ddd, J35a,35b=14.7 Hz, J35b,NH=6.2Hz, J34,35b=3.8 Hz, 35-Hb), 5.10 (1H, dt, J34,35a=7.2 Hz, J33,34=4.2 Hz, J34,35b=3.8 Hz, 34-H), 5.24 (3H, m, 30-H, 31-H, 32-H), 5.31 (1H, dd, J33,34=4.2 Hz, J33,32=6.4 Hz, 33-H), +5.53 and +5.64 (2 dd, J11,12=10.8 Hz, J=3.2 Hz, J=2.4 Hz, 11-H and 12-H), 5.69 (1 H, t, J35a,NH=J35b,NH=6.0 Hz, NH). Spectroscopic data labelled with a degree symbol (°) correspond to the spectrum of the acetylated hopanoid (1) and those labelled with an asterisk (*) correspond to the spectrum of its 3β-methyl homologue (2). Those without superscript are common to the spectra of both compounds. Those labelled with a plus symbol (+) correspond to the signals of the vinylic protons of acetylated 2a. Integration was only given for the side chain signals, which was identical in the three investigated hopanoids 1, 2 and 2a.

Acetate of 3-methylhop-11-en-30-ol (12) from M. szegediense (GC/MS, electron impact, 70 eV): m/z=482 (M+, 13%), 467 (M+-CH3, 4%), 381 (M+-side chain, 2%), 276 (retro Diels-Alder reaction, 100%), 249 (ring C cleavage, 9%), 232 (retro Diels-Alder reaction, 94%), 205 (ring C cleavage, 49%), 189 (ring C cleavage-AcOH, 39%). Acetate of adiantol (6) from M. tepidum (GC/MS, electron impact, 70 eV): m/z=496 (M+-AcOH, 3%), 369 (M+-side chain, 1%), 235 (ring C cleavage, 2%), 191 (ring C cleavage, 100%), 175 (ring C cleavage-AcOH, 21%). Acetate of 3-methyladiantol (7) from M. tepidum (GC/MS, electron impact, 70 eV): m/z=410 (M+-AcOH, 8%), 383 (M+-side chain, 3%), 235 (ring C cleavage, 1%), 205 (ring C cleavage, 100%), 175 (ring C cleavage-AcOH, 57%).

3Results and discussion

35-Aminobacteriohopane-30R,31R,32R,33S,34S-pentol (1) (Fig. 1, 24% of the total hopanoid content), its saturated 3β-methyl homologue (2) (68%) and its unsaturated 3β-methyl homologue with a Δ11 double bond (2a) (8%) were found in M. szegediense, whereas 35-amino-3β-methylbacteriohopane-31R,32R,33S,34S-tetrol (4) was only identified in trace amounts. In M. tepidum in contrast, 35-aminobacteriohopane-31R,32R,33S,34S-tetrol (3) (32%) and its 3β-methyl homologue (4) (7%) represented major hopanoids and were accompanied by the aminobacteriohopanepentol (1) (40%) and its 3β-methyl homologue (2) (21%). According to the 1H NMR spectra of the intact hopanoids and of the GC/MS data of the derivatives obtained after side chain oxidation, the aminobacteriohopanepentol (1) and aminobacteriohopanetetrol (3) as well as their 3β-methyl homologues (2 and 4) found in the two Methylocaldum species were identical to the hopanoids previously detected in Methylococcus capsulatus, Methylomonas methanica[8] and Methylococcus luteus[9,10].

Figure 1.

Hopanoids from methanotrophic bacteria. A: Isolated from Methylocaldum szegediense (1, 2, 2a) and Methylocaldum tepidum (1, 2, 3, 4). B: Obtained after side chain degradation (612). 1: 35-aminobacteriohopane-30R,31R,32R,33S,34S-pentol; 2: 35-aminobacteriohopane-30R,31R,32R,33S,34S-pentol; 2a: 35-amino-3β-methylbacteriohop-11-ene-30,31,32,33,34-pentol; 3: 35-aminobacteriohopane-31R,32R,33S,34S-tetrol; 4: 35-amino-3β-methylbacteriohopane-31R,32R,33S,34S-tetrol; 5: 35-aminobacteriohopane-32R,33S,34S-triol; 6: adiantol; 7: 3β-methyladiantol; 8: hopan-29-ol; 9: 3β-methylhopan-29-ol; 10: homohopan-31-ol; 11: 3β-methylhomohopan-31-ol; 12: 3β-methylhop-11-en-29-ol.

The aminopentol with a Δ11 double bond (2a) has, however, never been reported before. Hopanoids with a Δ11 double bond have only been reported from Acetobacter spp. until now [11,12]. The presence of such a double bond at the C-11 position in hopanoid 2a from M. szegediense was supported by GC/MS as well as by 1H NMR data [11,12]. In the mass spectrum of acetylated hopanoid 12 obtained after side chain degradation of 2a, the m/z ratio of all ions corresponding to the molecular ion, the loss of acetic acid and the loss of the side chain were shifted by 2 Da towards the lower masses as compared to those of the corresponding saturated hopanoid. The prominent m/z 276 and 232 fragments derived from a retro Diels-Alder reaction represented the signature of the Δ11 double bond. Indeed, the Δ11 double bond migrates upon electron impact to the Δ9 or Δ12 positions, yielding the m/z 249 and 205 fragments by cleavage of ring C and the above-mentioned even mass fragments by a retro Diels-Alder reaction [12]. In addition, in the 1H NMR spectrum of the mixture of the acetylated hopanoids 1, 2 and 2a, the signals of the vinylic protons at 5.64 ppm and 5.53 ppm confirmed the presence of a Δ11 double bond. The acetylated intact hopanoid 2a coeluted by TLC with the two other major acetylated aminopentols 1 and 2, and upon H5IO6/NaBH4 treatment, it afforded, like hopanoids 1 and 2, a derivative with an isopropyl side chain. A 35-amino-3β-methylbacteriohop-11-ene-30,31,32,33,34-pentol structure was consequently assigned to hopanoid 2a.

Traces of adiantol (6) and 3β-methyladiantol (7) were tentatively identified in M. tepidum after the H5IO6/NaBH4 degradation of the side chain by comparison of the mass spectra with those previously reported [13].

The presence of aminobacteriohopanepolyols is a common feature to all investigated obligate methanotrophs. An aminotriol was the major hopanoid in Methylosinus trichosporium[14]. Hopanoids possessing an aminotetrol or aminopentol side chains were previously detected in two other species of methanotrophic bacteria: Methylomonas methanica and Methylococcus capsulatus[8] and Methylococcus luteus[9,10] (Table 1). The three latter ones are phylogenetically close to the Methylocaldum species. Methylocaldum szegediense, Methylocaldum tepidum and Methylococcus capsulatus share in addition the same phylogenetic subcluster: all contained hopanoids with a 3β-methyl group as well as a hopanoid releasing adiantol after the H5IO6/NaBH4 treatment. In contrast, Methylomonas methanica and Methylosinus trichosporium, which are more distant species from the three above-mentioned bacteria, do not synthesise 3β-methylhopanoids. The hopanoid fingerprint is thus consistent with the presumed phylogeny of these bacteria. The Methylococcus species belong to the few bacteria capable of synthesising sterols [9,15]. This feature is quite exceptional amongst prokaryotes. Neither sterols nor sterol precursors were found in the Methylocaldum strains, which were in this aspect like all other methanotrophs.

Table 1.  Hopanoids from methanotrophic bacteria
Methanotrophic bacteriumHopanoid
Methylocaldum szegediense OR2+++++
Methylocaldum tepidum LK6++++++
Methylococcus capsulatus NCIB 11132 [7]++++++
Methylococcus luteus NCIB 11914 [8]+++++++
Methylomonas methanica NCIB 11130 [7]+++
Methylosinus trichosporium NCIB 11131 [13]++

The detection of 35-amino-3β-methylbacteriohop-11-ene-30,31,32,33,34-pentol (2a) represents the first evidence for the presence of an unsaturated Δ11-hopanoid in a soil bacterium. An unsaturated Δ6-hopanoid was recently identified in a Burkholderia sp. [16]. Introduction of a double bond represents the only modification so far known of the pentacyclic hopane skeleton in bacteria. Such a reaction might represent the initial step for a reaction sequence leading from a biohopanoid to degraded molecular fossils with a polyunsaturated or aromatic skeleton, often present in soils and sediments [17].


We thank Mr. Jean-Daniel Sauer for the NMR measurements and Mr. Jean-Marc Strub for his help in recording the mass spectra. This work was supported by the European Community BIOMASS program (ENV4 CT95 0026), the Hungarian Academy of Sciences OTKA program, a grant to L.B. from the Magyary Zoltán Foundation and a grant to M.R. from the ‘Institut Universitaire de France’.