Marine lipids
The
1 H NMR spectrum clearly showed the lack of the lactobacillic acid
moiety, the other signals of the molecule remaining unchanged, except
shielding of the signal for H-2 (δ 5.19 to 4.20 ppm). Hence, we were able
to assign the sn-1 position for the palmitic ester and the sn-2 position for
the lactobacillic ester. This is in accordance with the biosynthesis of
cyclopropane fatty acicls, the cyclopropane ring is built up by methylation on the double bond of the unsaturated fatty acid by the cyclopropane-fatty acid synthetase (Hildebrand & Law, 1964). Most often
in Gram (-) bacteria, saturated fatty acids occupy the 1 position, while
the 2 position is occupied by unsaturated fatty acids.
Finally, the alkaline hydrolysis of the phospholipid was carried on. The
resulting mixture was acidified and extracted with dichloromethane.
The crude extract was subjected to a separation on a silica gel column
to afford after elution with hexane-ethylacetate (9:1, v/v) a mixture of
two methyl esters, the palmitic ester (m/z 270) and the lactobacillic
one (m/z 295). This latter was clearly identified by GC-MS coinjection
with a synthetic sample of the methylester of the lactobacillic acid.
Moreover, the crude methanolic extract and compound 7 were compared
to 1,2-dihexadecanoyl-sn-glycero-3-phosphatidylethanolamine as a
positive control on a TLC plate (Merck Kieselgel 60) elutecl by dichloromethane-methanol (6:4, v/v) and sprayed with Phospray (Zinzade reagent
Supelco, to detect phosphorus) which indicated that 7 was the sole
phospholipid present m the crude extract.
The proportion of phosphatidylethanolamine found in this strain is in
agreement with the data of literature. Oliver & Colwell (1973), showed
that the phospholipid compositions of marine and estuarine bacteria
differed very little from those of non-marine organisms with phosphatidylethanolamine and phosphatidylglycerol derivatives being the
predominant phospholipids, i.e., a phosphatidylethanolamine content
of 95% for Pseudomonas aeruginosa. Large proportion of phosphatidyl
ethanolamine (86%) was found in the marine pseudomonad P perfectomarinus (Sinha & Gaby, 1964). This pattern may be typical of the
genus. However, these lipids are most often encountered as a mixture
of several phosphatidylethanolamines which bear different fatty acid
substituents on the primary and secondary alcohols.
Acknowledgements
We thank J.-P. Brouard and Service central d’analyse du CINRS (Lyon) for mass
measurements, E. Abou-Mansour from the Australian Institute ofMarine Science,
Townsville, Australia, for the sample of methylic ester of lactobacillic acid, J.-L.Nicolas (Ifremer Brest, France) for identification of the bacterial strain and Ifremer for
financial support (IRD/Ifremer agreement n° 93/5 570 093).
200
The
1 H NMR spectrum clearly showed the lack of the lactobacillic acid
moiety, the other signals of the molecule remaining unchanged, except
shielding of the signal for H-2 (δ 5.19 to 4.20 ppm). Hence, we were able
to assign the sn-1 position for the palmitic ester and the sn-2 position for
the lactobacillic ester. This is in accordance with the biosynthesis of
cyclopropane fatty acicls, the cyclopropane ring is built up by methylation on the double bond of the unsaturated fatty acid by the cyclopropane-fatty acid synthetase (Hildebrand & Law, 1964). Most often
in Gram (-) bacteria, saturated fatty acids occupy the 1 position, while
the 2 position is occupied by unsaturated fatty acids.
Finally, the alkaline hydrolysis of the phospholipid was carried on. The
resulting mixture was acidified and extracted with dichloromethane.
The crude extract was subjected to a separation on a silica gel column
to afford after elution with hexane-ethylacetate (9:1, v/v) a mixture of
two methyl esters, the palmitic ester (m/z 270) and the lactobacillic
one (m/z 295). This latter was clearly identified by GC-MS coinjection
with a synthetic sample of the methylester of the lactobacillic acid.
Moreover, the crude methanolic extract and compound 7 were compared
to 1,2-dihexadecanoyl-sn-glycero-3-phosphatidylethanolamine as a
positive control on a TLC plate (Merck Kieselgel 60) elutecl by dichloromethane-methanol (6:4, v/v) and sprayed with Phospray (Zinzade reagent
Supelco, to detect phosphorus) which indicated that 7 was the sole
phospholipid present m the crude extract.
The proportion of phosphatidylethanolamine found in this strain is in
agreement with the data of literature. Oliver & Colwell (1973), showed
that the phospholipid compositions of marine and estuarine bacteria
differed very little from those of non-marine organisms with phosphatidylethanolamine and phosphatidylglycerol derivatives being the
predominant phospholipids, i.e., a phosphatidylethanolamine content
of 95% for Pseudomonas aeruginosa. Large proportion of phosphatidyl
ethanolamine (86%) was found in the marine pseudomonad P perfectomarinus (Sinha & Gaby, 1964). This pattern may be typical of the
genus. However, these lipids are most often encountered as a mixture
of several phosphatidylethanolamines which bear different fatty acid
substituents on the primary and secondary alcohols.
Acknowledgements
We thank J.-P. Brouard and Service central d’analyse du CINRS (Lyon) for mass
measurements, E. Abou-Mansour from the Australian Institute ofMarine Science,
Townsville, Australia, for the sample of methylic ester of lactobacillic acid, J.-L.Nicolas (Ifremer Brest, France) for identification of the bacterial strain and Ifremer for
financial support (IRD/Ifremer agreement n° 93/5 570 093).
200