Novel sulfonolipid in the extremely halophilic bacterium Salinibacter ruber

Abstract

Salinibacter ruber is an extremely halophilic bacterium, phylogenetically affiliated with the Flavobacterium/Cytophaga branch of the domain Bacteria. Electrospray mass analyses (negative ion) of the total lipid extract of a pure culture of S. ruber shows a characteristic peak at m/z 660 as the most prominent peak in the high-mass range of the spectrum. A novel sulfonolipid, giving rise to the molecular ion [M-H]- of m/z 660, has been identified. The sulfonolipid isolated and purified by thin-layer chromatography was shown by chemical degradation, mass spectrometry, infrared spectroscopy, and nuclear magnetic resonance analysis to have the structure 2-carboxy-2-amino-3-O-(13'-methyltetradecanoyl)-4-hydroxy-18-methylnonadec-5-ene-1-sulfonic acid. This lipid represents about 10% of total cellular lipids, and it appears to be a structural variant of the sulfonolipids found as main components of the cell envelope of gliding bacteria of the genus Cytophaga and closely related genera (W. Godchaux and E. R. Leadbetter, J. Bacteriol. 153:1238-1246, 1983) and of diatoms (R. Anderson, M. Kates, and B. E. Volcani, Biochim. Biophys. Acta 528:89-106, 1978). Since this sulfonolipid has never been observed in any other extreme halophilic microorganism, we consider the peak at m/z 660 the lipid signature of Salinibacter. This study suggests that this novel sulfonolipid may be used as a chemotaxonomic marker for the detection of Salinibacter within the halophilic microbial community in saltern crystallizer ponds and other hypersaline environments.

FIG. 1.

(A) ESI-MS spectrum (negative ion) of total lipid extract of S. ruber cells (amu, atomic mass unit); (B) TLC profile of total lipid extract in solvent A (chloroform-methanol-acetic acid-water, 85:15:10:3.5 by volume) and rechromatography in solvent B (chloroform-methanol-29.9% [wt/vol] ammonia, 65:35:5, vol/vol/vol) of lipid extract from spot 2. The chromatograms were stained by charring at 120°C after being sprayed with 5% sulfuric acid-H₂O. On the right is shown the low-resolution ESI-MS (negative ion) spectrum of the isolated and purified lipid 2_A.

FIG. 2.

IR spectrum of isolated and purified lipid 2_A (1 mg on KBr disk).

FIG. 3.

High-resolution product ion spectrum of the molecular ion at m/z 660 (upper panel) and of the fragment ion at m/z 418 (lower panel). amu, atomic mass unit.

FIG. 4.

¹H-NMR spectrum of isolated and purified lipid 2_A. Two milligrams of pure sulfonolipid was dissolved in 700 μl of CDCl₃-CD₃OD (4:3, vol/vol). Chemical shifts are given relative to TMS as an internal standard.

FIG. 5.

High-resolution product ion spectrum of the molecular ion at m/z 436, i.e., aminosulfonate, obtained by strong alkaline hydrolysis. amu, atomic mass unit.

FIG. 6.

Proposed structure and fragmentation pathway for lipid 2_A. All molecular formulas reported in the figure were obtained by accurate mass measurements (see Table 1).

FIG. 7.

ESI-MS (negative ion) profile of the lipid extract obtained from the biomass of crystallizer brines of Margherita di Savoia (Italy). amu, atomic mass unit.