Ornithine-containing Lipid

The presence of ornithine in crude phospholipids of mycobacteria was demonstrated in 1955 14), This amino acid was isolated after hydrolysis of the lipids and identified as L-ornithine by its chromatographic behaviour and the properties of derivatives 14), 

Several years later, in the course of a study of mycobacterial lipids, it was shown that ornithine is not a component of the phospholipids, but is part of a new kind of compound devoid of phosphorus and containing fatty acids (75). Amide bands in its I.R. spectrum (1625 and 1550 cm ) made it likely that it was a derivative of N-acylornithine 16). Similar results were obtained in the course of a study of the lipids of Rhodo-pseudomonas sphaeroides (77). 

The carboxyl group in the ornithine-containing lipid of Rh. sphaeroides seemed to be esterified, leading to an incorrect formula 18). In fact, the lipid behaved as a zwitterion. Acid hydrolysis gave a mixture of 3-hydroxy fatty acids, the major components of the mixture containing 16 and 18 carbon atoms, and non-hydroxylated fatty acids, the major components again containing 16 and 18 carbon atoms. Small peaks at m/z 688, 692 and 700 in the E.I.-mass spectrum considered to be the molecular peaks showed that hydroxylated fatty acids, non-hydroxylated 

The lipid was mainly located in the chromatophores and membrane material of Rh. sphaeroides (20). Similar results on its location were found in a reinvestigation of the lipids of subcellular fractions of Rh. sphaeroides (27) besides the ornithine-containing lipid a minor peptidolipid, designated as aminolipid X , was observed. Hydrolysis of this lipid gave ornithine, an unidentified amine and fatty acids. 

A large concentration of ornithine-containing lipid was found in a basic membrane material prepared from a mutant strain of Rhodo-spirillum rubrum (22). Studies on the structure of a similar lipid isolated from van Niels strain SI of R. rubrum (heterotrophically or auto-trophically grown) were in agreement with structure (9) (23). In heterotrophically grown cells a second ornithine-containing lipid was found it was stated that the two lipids seem to be quite dissimilar in structure (23). 

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A few years ago, we began a research program to develop methods of analysis which would involve the use of FAB and a high performance tandem mass spectrometer. The tandem instrument was the first triple sector mass spectrometer to be designed and built by a commercial instrument company (Kratos of Manchester, U.K.). The first mass spectrometer of the combination is a double focussing Kratos MS-50 which is coupled to a low resolution electrostatic analyzer, which serves as the second mass spectrometer U). This FAB MS-MS combination has been used to verify the structures of an unknown cyclic peptide (2), a new amino acid modified by diphtheria toxin (3), and an ornithine-containing lipid (4). A number of methods have also been worked out which rely on this instrumentation. They Include the structural determination of cyclic peptides (5), nucleosides and nucleotides (6), and unsaturated fatty acids (7) and the analysis of mixtures of both anionic (8) and cationic surfactants (9). 

Other lipid types which have been found in bacteria include wax esters, hydrocarbons, sphingolipids, steroids and terpenoids. These occur sporadically, however, and are seldom major components. An obvious exception is the presence of carotenoids and the ornithine-containing lipid of purple photosynthetic bacteria. Further details of minor lipid components of bacteria can be found in Razin and Rottem (1982), Shaw (1974) and Goldfine (1972). 

Tables 1 and 2 show that ornithine-containing lipids are widely distributed in Gram negative bacteria. Many species of Pseudomonas have been studied and ornithine-containing lipids have been characterized in thirteen out of nineteen species (see Table 2). Such bacteria have two membrane layers, a cytoplasmic and an outer membrane. In the case of Thiobacillus thiooxidans, 1.9% of the polar lipids were ornithine-containing lipids (12). After disruption of the cells the two membranes...

Table 1. Distribution of Ornithine-containing Lipids and a Lysine-containing Lipid) in Various Species of Gram Negative Bacteria...

Ornithine-containing lipid with Structure of Type (9) Type (13) References... 

Table 3. Occurrence of Ornithine-containing Lipids in Gram Positive Bacteria... 

Bacterial species Ornithine-containing lipids Amide-linked fatty acids Ester linked fatty acids References... 

No ornithine-containing lipid could be detected in strain H37 Ra. 

No ornithine-containing lipid was found in M. phlei 24, 38) another kind of lipid, devoid of phosphorus and containing ester-bound lysine was identified (2). 

While ornithine-containing lipids are widely distributed in Gram negative bacteria, so far they have only been found in a small number of Gram positive species (Table 3), all of them belonging to the Actino-mycetales order. 

An almost identical lipid in which ornithine is completely replaced by lysine has been isolated from Agrobacterium tumefaciens. Here again the a-amino group of lysine was linked by an amide bond to the usual 3-acyloxyhexadecanoic acid (52). In siolipin, an ornithine-containing lipid produced by Streptomyces sioyaensis and S. toyocaensis (Table 3), lysine was found together with ornithine in the ratio 9 91 (55). 

It is sometimes very difficult to isolate pure ornithine-containing lipids from accompanying lipids, in particular from phosphatidylethanolamine, perhaps because of ionic linkages. Hence they are often... 

Purification and fractionation of ornithine-containing lipid homo-logues has been performed by gas-chromatography of their TMS derivatives. Because of the low volatility of these compounds, a short column has to be used (13). 

The general structure (9) ascribed to the ornithine-containing lipids with two hydrophobic acyl chains and a zwitterionic group shows the amphiphilic character of the molecule. This structure is quite similar to that of phosphatidylethanolamine (Fig. 1). 

When the culture medium of Pseudomonas fluorescens was made phosphate-limited, phosphatidylethanolamine disappeared and the ornithine-containing lipids increased (29). Similar observations were made in the case of the lipids of Streptomyces strains (40). These observations on the similarity of structure and the easy interchange... 

Fig. 1. Conformations of ornithine-containing lipid (left) and phosphatidylethanolamine (right), at the interface air water...

Cells of Paracoccus denitrificans grown in a complex medium deficient in divalent cations exhibited a higher ratio of ornithine-containing lipid to phospholipids than that observed in cells grown in the same medium supplemented with Mg " and Ca ". It has been suggested that the zwitterionic ornithine-containing lipid was less dependent than acidic phospholipids on divalent cations for their incorporation in the outer membrane (41). 

Membrane vesicles prepared from Thiobacillus ferrooxidans contained three enzymes of the iron oxidation system. Delipidation of these vesicles by aqueous acetone decreased the enzymatic activities. They were restored by incubation of the lipid-depleted vesicles with a dispersion of ornithine-containing lipid together with coenzyme Qg. A possible role of the ornithine-containing lipid in the iron oxidation system has been suggested (25). 

During a study of the lipids of Gluconobacter cerinus it was observed that an ornithine-containing lipid was labelled with sulphur-35 when [ S]-sulphate was added to the culture medium. 

A particulate fraction prepared from Gluconobacter cerinus cells was able to catalyze the condensation of ornithine-containing lipid and taurine to produce cerilipin, when the medium was supplemented with ATP and Mn 46). 

During the first analyses of ornithine-containing lipids, the use of mass spectrometry was limited to the characterization of hydrolysis products because of the state of the technique at the time. Gorchein 19)... 

The protonated molecular ions of ornithine-containing lipids isolated from Ermnia aroideae were observed by chemical ionization mass spectrometry with ammonia of the derivatives prepared by treatment with dimethylformamide dimethylacetal (20) (49). 

Similar results were obtained by CID-MIKE (collision induced dissociation) mass spectrometry of an ornithine-containing lipid (57). Homologues were detected by FAB mass spectrometry and the ions were distinguished by their CID spectra (52). 

Fig. 2. MIKE mass spectrum of the ion m/z 625 generated by Fast Atom Bombardment (FAB) from the ornithine-containing lipid of Flavobacterium meningosepticum (50)... 

Siolipin, the hydroxylated ornithine-containing lipid of Streptomyces sioyaensis, was found to specifically inhibit the growth of a strain of Bacillus subtilis when a synthetic culture medium was used. No inhibition was observed when the bacteria were grown on complex medium, as l-histidine and to a lesser degree L-cysteine were able to antagonize the effect of siolipin (55). 

Flavolipin was found to be more active than ornithine-containing lipids in the hemagglutination test the activity was observed at concentrations lower than 1 pg per milliliter (75). 

Ornithine-containing lipid was shown to accelerate the coagulation of blood in a thrombine-fibrinogen system, by using a concentration of 24 pg per ml (55). 

In conclusion, few biological properties have so far been detected for ornithine-containing lipids in spite of their amphiphilic character. 

Gorchein, A. Studies on the Structure of an Ornithine-Containing Lipid from Nonsulfur Purple bacteria. Biochim. Biophys. Acta 152, 358 (1968). 

Rivas, E.A., N.L. Kerber, A.A. Viale, and A.F. Garcia Isolation of a Basic Membrane Fraction Enriched in an Ornithine-Containing Lipid, from a Blue-green Mutant of Rhodospirillum rubrum. FEBS-Letters 11, 37 (1970). 

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