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Sterols in a psychrophilic methanotroph, Methylosphaera hansonii

Stefan Schouten, John P. Bowman, W. Irene C. Rijpstra, Jaap S. Sinninghe Damsté
DOI: http://dx.doi.org/10.1111/j.1574-6968.2000.tb09103.x 193-195 First published online: 1 May 2000

Abstract

A suite of six sterols, lanosterol, lanost-8(9)-en-3β-ol, 4,4-dimethylcholesta-8(14),24-dien-3β-ol, 4,4-dimethylcholest-8(14)-en-3β-ol, 4-methylcholesta-8(14),24-dien-3β-ol and 4-methylcholest-8(14)-en-3β-ol, were identified in the psychrophilic methanotrophic bacterium, Methylosphaera hansonii. Their presence suggests that the capacity for sterol biosynthesis in methanotrophic bacteria is limited to the family Methylococcaceae but which have widely different optimal growth temperatures.

Keywords
  • 4,4-Dimethylsterol
  • 4-Methylsterol
  • Methylosphaera hansonii

1 Introduction

Sterols are membrane lipids that occur ubiquitously in eukaryotic organisms (e.g. [13]). Only in relatively few bacteria the occurrence of sterols has been reported and usually at very low levels making it uncertain whether they are contaminants or biosynthesized by these organisms. One exception is the aerobic methanotrophic bacterium Methylococcus capsulatus which has been reported to contain relatively high levels of sterols, i.e. 4,4-dimethylcholesta-8(14),24-dien-3β-ol (I), 4,4-dimethylcholest-8(14)-en-3β-ol (II), 4-methylcholesta-8(14),24-dien-3β-ol (III) and 4-methylcholest-8(14)-en-3β-ol (IV) [4,5]. They are thought to be biosynthesized by demethylation and hydrogenation of lanosterol (V). Indeed, Bouvier et al. [5] detected very low levels of lanosterol (V), and lanost-8(9)-en-3β-ol (VI) in one culture experiment. Sterols V and I were reported by Jahnke et al. [6] in relatively high levels in Louisiana Slope mussels that are known to contain an aerobic methanotrophic endosymbiont. A suite of different sterols has been reported in very low levels in Methylobacterium organophilum[7] of which only lanosta-8,22,24-trienol (VII) was identified. Other natural occurrences of these or similar sterols have not been reported either in cultures or in field samples.

Here we report the presence of sterols I–VI in a psychrophilic methanotrophic bacterium, Methylosphaera hansonii, isolated from an Antarctic Lake. The results suggest that a subclass of phylogenetically related methanotrophic bacteria with widely different optimal growth temperatures possess the capability to biosynthesize specific 4,4-dimethylated and 4-methylated sterols.

2 Materials and methods

Cells of M. hansonii (strain ACAM 549 [8]) were grown in several 60 ml flasks at 10°C for 2 weeks in nitrate mineral salts seawater medium [8] under an air/methane (1:1) mixture.

The freeze-dried cell material was ultrasonically extracted with methanol (2×), methanol/dichloromethane (1:1, v/v; 2×) and dichloromethane (2×). Alcohols present in the extract were isolated by column chromatography using a silica gel column and ethyl acetate as eluent. The alcohol fraction was subsequently silylated using BSTFA/pyridine and analyzed by gas chromatography (GC) and GC-mass spectrometry (GC-MS).

GC was performed using a Hewlett Packard 5890 series II chromatograph equipped with an on-column injector and fitted with a 25 m×0.32 mm fused silica capillary column coated with CP-Sil 5 (film thickness 0.12 μm). Helium was used as carrier gas and the oven was programmed from 70°C to 130°C at 20°C min−1, followed by an increase of 4°C min−1 to 320°C (15 min hold time). Compounds were detected using a flame ionization detector.

GC-MS analyses were performed using the GC conditions described above. The column was directly inserted into the electron impact ion source of a VG-Autospec Ultima mass spectrometer, operated with a mass range of m/z 40–800, a cycle time of 1.8 s and ionization energy of 70 eV.

3 Results and discussion

The alcohol fraction of the cell material extract of M. hansonii was dominated by a cluster of compounds consisting of sterols I–VI (Fig. 1). Their identification was based on published mass spectra and retention times [4,5]. Furthermore, the identification of 4,4-dimethylcholest-8(14)-en-3β-ol (II) and 4-methylcholest-8(14)-en-3β-ol (VI) was rigorously established by coinjection experiments with a sterol fraction obtained from M. capsulatus. This is the first time that all these sterols are found in such relatively high amounts in a methanotrophic bacterium. Bird et al. [4] only observed sterols I–IV, whilst Bouvier et al. [5] detected sterols VI and VII in only one of a number of cultures of M. capsulatus. The presence of sterols I–VI in M. hansonii confirms the proposed biosynthetic pathway for sterols I–IV and VI by hydrogenation, demethylation and double bond shift of their precursor lanosterol (V) [5].

1

Partial gas chromatogram of the alcohol fraction of cell material extract of M. hansonii.

In lower abundance, squalene and a suite of di- and triunsaturated sterenes were observed with mass spectra similar to those of the sterols present in M. hansonii (Fig. 1). Based on this it is suggested that they represent dehydration products of the sterols I–VI. Since we did not use any acid in the extraction procedure it seems unlikely that our analytical procedure induced the dehydration of sterols, suggesting that the bacterium may biosynthesize the sterenes as such.

Recently in an investigation of surface sediments from Ace Lake, where M. hansonii thrives [8], we encountered sterol VI in relatively high abundance [9]. Its structure and depleted 13C content of ca. −57‰, a value diagnostic for methane-consuming organisms [10], suggests that it is derived from methanotrophic bacteria. It is very likely that M. hansonii is the source for sterol VI, although we can not exclude that other uncultivated methanotrophic bacteria in Ace Lake biosynthesize this sterol. It does suggest that M. hansonii not only biosynthesizes sterols in culture but also in its natural setting, i.e. in Antarctic lakes. Interestingly, the other sterols that were detected in the culture were not found in the sediments. This may be due to the different growth conditions for M. hansonii since the culture temperature was 10°C whilst the water temperature in Ace Lake is typically between 0–8°C. A similar phenomenon has been noted for M. capsulatus. Jahnke [11] found varying concentrations and distributions of sterols with growth temperature, i.e. higher sterol contents with higher percentages of sterols III and V at lower growth temperatures. Summons et al. [10] also reported different sterol compositions for M. capsulatus at different growth conditions. Alternatively, other yet uncultivated methanotrophic bacteria living in Ace Lake specifically produce sterol VI and are the main source for sterols in sediments of Ace Lake.

It is interesting to note that the closest phylogenetically related organism of M. hansonii is the methanotrophic endosymbiont in Louisiana Slope mussels [8]. As mentioned before, Jahnke et al. [6] found 4-methylated sterols, including sterols VI and I, in these mussels and suggested that they were derived from the methanotrophic endosymbiont. This indicates that both M. hansonii and the methanotrophic endosymbiont are capable of sterol biosynthesis and produce similar sterols. M. capsulatus is phylogenetically somewhat more distantly related to both M. hansonii and the methanotrophic symbiont in Louisiana Slope mussels but all belong to the family of Methylococcaceae. Interestingly, the aforementioned methanotrophic bacteria all have different optimal growth temperatures. For example, M. hansonii is psychrophilic and grows optimally at 10–13°C [8] whilst M. capsulatus grows optimally at 45°C [11]. Thus, the capability of sterol biosynthesis is not dependent on temperature adaptation. Although further research is needed, the results obtained thus far suggest that methanotrophic bacteria in the family Methylococcaceae have the rather rare capability for prokaryotes of biosynthesizing steroids in significant amounts.

Acknowledgements

Dr. R.E. Summons is gratefully acknowledged for donation of the sterol fraction of M. capsulatus. This is NIOZ contribution no. 3437.

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