Production of DHA in procaryotes was first reported in deep-sea isolates, including barophilic bacteria (4
). The present study showed that the DHA observed in the barophilic strain 16C1 was 22:6n-3, which contained methylene-interrupted double bonds and was normally observed in lipids of eucaryotes (17
). Furthermore, it was found that the double-bond positions of monounsaturated fatty acids were n-9 and n-7 in the strain. Generally, unsaturated fatty acids in bacteria have been considered to be produced by two possible mechanisms: an oxygen-independent (anaerobic) pathway and an oxygen-dependent (aerobic) pathway (7
). In the anaerobic pathway, which is catalyzed by a fatty acid synthetase, palmitoleic acid (16:1n-7) and cis
-vaccenic acid (18:1n-7) are produced. In the aerobic pathway, which involves fatty acid desaturation analogous to that of eucaryotes, palmitoleic acid (16:1n-7) and oleic acid (18:1n-9) are commonly produced from saturated fatty acids by position-specific desaturase. A production mechanism for PUFA, such as DHA, however, is unknown in bacteria. In procaryotic algae producing 18:4 (28
) and eucaryote-synthesized DHA (17
), it was reported that C18
PUFAs of the n-3 and n-6 series were present in their lipids. Also in strain 16C1, those PUFAs were shown to be contained in the lipids, although no previous reports have been made of the presence of C20
PUFAs, even EPA, in bacteria containing DHA (4
). Furthermore, according to the known elongation system of fatty acid, it is difficult to imagine that 22:6n-3 containing methylene-interrupted double bonds is formed by means of an anaerobic pathway. Thus, we suppose that DHA in the bacterial strain 16C1 may be also synthesized via C18
PUFAs of the n-3 and n-6 series, as is the case in eucaryotes. However, C22
PUFAs (with the except of DHA) were not detected in strain 16C1 or in other bacteria containing DHA. In fish, DHA is considered to be formed by the addition of C2
to EPA, followed by desaturation (13
). However, in rat hepatocytes, it has been reported that elongation of 22:5n-3 to 24:5n-3 is followed by desaturation to 24:6n-3, and then this is metabolized, via β-oxidation, to 22:6n-3 (27
). Thus, further investigations are needed to clarify the synthetic pathway of DHA in bacteria.
Phospholipids in strain 16C1 were mainly PE and PG, as described previously (34
). PE was found to be abundant in monounsaturated fatty acids (74% of total fatty acids) and lacking in PUFAs, including DHA (5.4% of total fatty acids), compared with PG. In PG, monounsaturated fatty acids were present at a lower level (47% of total fatty acids) and DHA accounted for 29.6% of the total fatty acids. This tendency was also observed in strain 2D2, as shown in Table , and the proportion of DHA in the PG accounted for about 50% of the total fatty acids. Although there have been no reports on the fatty acid composition of phospholipids in bacteria containing DHA, it has been reported that PG has higher levels of EPA than PE in two EPA-producing bacterial strains isolated from freshwater fish and shallow-sea fish (10
The change in phospholipid composition in relation to growth pressure was not distinct, except that the proportion of PE became high when strain 16C1 underwent pressurized incubation. It is well known that the phospholipid compositions of bacteria and the changes related to incubation conditions vary with bacterial species (24
). In a bacterium containing EPA, which has PE and PG as the major lipids, the proportion of PE was reported to increase at a lower growth temperature (10
), although what the change meant was unknown.
With increasing growth pressure, DHA levels increased in the fatty acids of the total lipids in strains 16C1 and 2D2. This indicates that DHA was more necessary at high pressures and confirms that DHA plays some roles in the growth and biological functions of barophilic bacteria at high pressures, suggested by DeLong and Yayanos (4
). Furthermore, the present study found that the increases in the level of DHA occurred in phospholipids, especially PG. This finding suggests that the role of DHA may be closely related to the functions of the membrane.
In PE, in the present study, the proportions of 14:0 and 14:1 were reduced with an increase in pressure, and these decreases were mainly balanced by a increase in 16:1. In PG, the decrease of 14:0 was mainly balanced by an increase in DHA. Generally speaking, with increasing growth pressure, saturated fatty acids decreased and unsaturated fatty acids, including DHA, increased in major phospholipids of barophilic strains. It is well known that the increase in the level of unsaturation of fatty acids occurs with the lowering of growth temperature in bacteria and poikilotherms (15
). In the present study also, the lowering of growth temperature caused the increase in unsaturated fatty acids. These results suggest that the barophilic strains compensate for pressure increases through homeoviscous adaptation in a fashion similar to the response to lowering of temperature. Thus, the present study further confirms the suggestion of DeLong and Yayanos (3
). That is to say, one of the roles of DHA may be in maintaining the fluidity of lipids at high pressure.
In the present study, the changes in fatty acid composition in phospholipids were slight or ambiguous between medium pressure and high pressure. It has also been reported that in the barophilic strain MT41, there was a decrease in the relative amount of DHA at the highest growth pressure compared with that at the medium pressure (4
). If these strains always regulate their lipid states when pressure increases, it is difficult to understand slight or no change in the fatty acid composition at high pressure, as observed in the present study. Furthermore, although it is known that a shortening of carbon length in fatty acids is observed at lower temperatures (9
), the proportions of 14:0 and 14:1 were reduced at higher pressures in the present study. These results may suggest that there were changes in the lipid composition which were not detected by the analysis of fatty acid composition. This indicates the need for more detailed analysis of the molecular species of phospholipids.