0079-6832/78/0701-0301SO5.C!4/0
THE PHYTANYL ISOPRENOID
ETHER-LINKED POLAR LIPIDS NEUTRAL LIPIDS OF EXTREMELY HALOPHILIC BACTERIA M.
Deparrment
o/ Biochemistry,
University
AND
KATES of Ottawa,
Ottawa,
Canada
Kl N 6N5
.
CONTENTS I. INTRODUCTION II.
ISOLATION
301
AND IDENTIFICATION
OF LIPID
COMPONENTS
A. Overall lipid composition B. Identification of the ether-containing lipid moiety as 2,3-di-0-phytanyl-sn-glycerol I. Isolation and characterization of the unsaponifiable material 2. Identification of alkyl group(s) as phytanyl groups 3. Absolute stereochemical configuration of phytanyl group 4. Structure and configuration of the diphytanyl glycerol ether C. Identification of polar lipid components I. Composition and characterization of the polar lipids 2. Phosphatidyl glycerophosphate (diphytanyl ether analog) 3. Phosphatidyl glycerol (diphytanyl ether analog) (PC) 4. Phosphatidyl glycerosulfate (diphytanyl ether analog) (PGS) 5. Glycolipid sulfate (GLS) D. Neutral lipids I. C,,-Isoprenoids (a) Geranylgeraniol (b) Di-0-phytanyl glycerol (c) Retinal 2. C,,-Isoprenolds (Squalenes) 3. C,,-Isoprenoids 4. Vitamin MK-8 5. C,,-Isoprenoids (Bacterioruberins) III.
IV.
SYNTHEW OF PHYTANYL GLYCEROL ETHER DERIVATIVES A. Phytanyl glycerol ethers I. 2,3-di-o-phytanyland I,?-di-0-phytanyl-sn-glycerols 2. l,3-di-0-phytanyl glycerol 3. 3-0-phytanyl-w-glycerol and 2-0-phytanyl glycerol B. Diphytanyl ether analogs of phosphatidic acid and derivatives I. 2.3-di-0-phytanyl-sn-glycero-l-phosphate 2. I-(Cytidine-5’-diphosphate)-2,3-di-O-phytanyI-sn-glycerol C. Phosphatidyl glycerophosphate (diphytanyl ether analog) D. Phosphatidyl glycerols (diphytanyl ether analogs) I. Diasteriomeric I-sn-phosphatidyl-1’(3’)-sn-glycerols (a-PGS) 2. I-sn-Phosohatidyl-2’-glycerol (4-PG) E. Phosphatidil gly&rosuif&es (diphytanyl ether analogs) I. I-srl-Phosphatidyl-3’-sn-glycero-1’-sulfate (PG-1-S) and I-sn-phosphatidyl-3’-sn-glycerol’,2’-disulfate (PG-1,2-di-S) 2. I-swPhosphatidyl-I’-sn-glycero-2-sulfate (PG-2-S) F. Monophytanyl ether analogs of lysophosphatidic acid and derivatives I. 3-0-Phytanyl-sn-glycero-l-phosphate (lyso-PA) 2. 3-O-Phytanyl-sn-glycero-l-phosphoryl-l’-sn-glycerol (lyso-PG)
CHEMICAL
DISTRIBUTION
OF LIPIDS
IN RED AND PURPLE
MEMBRANE
FRACTIONS
303 303 305 305 308 309 310 311 311 313 317 318 320 323 323 323 323 324 325 326 326 326 328 328 328 328 329 330 330 332 332 333 333 334 336 336 337 338 338 339 339 341
REFERENCES
I. INTRODUCTION
The extremely halophilic bacteria are a group of remarkable organisms that require saturated or nearly saturated salt solutions (20-30x NaCl) for optimum growth and survival. 1o-55*72*73These organisms occur naturally in salt lakes such as the Great Salt Lake, and in the Dead Sea. They are also commonly found in concentrated brines and in salt flats formed by evaporation of sea water, as well as in the common salt obtained from these salt flats, to all of which these organisms impart a characteristic 301
302
M. Kates
red or pink color. This coloration is attributed to the presence in most of the extreme halophiles of red carotenoid pigments (see Section II.D.5). Halophilic bacteria may be found also in salted foods and salted hides to which they are introduced with the sea salt used in their preservation. Two distinct groups of extremely halophilic bacteria are known: the rod-shaped Halobacterium genus and the coccoid Halococcus genus, both grouped in the family Halobacteriaceae (see Gibbons28 for a discussion of the taxonomy of halophilic bacteria). Bacteria in the first group are Gram-negative, obligate aerobes, non-spore forming, and require at least 12% salt for growth, the optimal salt concentration being 25-307;. The halophilic cocci are Gram-negative or Gram-variable spheres, l-l.5 pm in diameter, occurring singly, in pairs, or in packets (sarcinae). They also are all obligate aerobes, non-spore forming, and require at least 5-100/d NaCl for growth, the optimal concentration being 2025”,‘,, with growth occurring even in up to 3Oqd salt.72.73 The extremely halophilic bacteria require high concentrations of salt not only for growth but also for preservation of their cellular structure. For example, growth of the halobacteria cells ceases when the salt concentration is decreased below 3 M and the rods gradually lose their regular shape and become spherical; below 1 M NaCl, the cell envelopes begin to disintegrate and the cells lyse rapidly.7’.73 Concentrations of 0.1-0.5 M Mg2+ and 1.3-2.5 x 10e2 M Kf are also required in addition to 3-5 M NaCl for optimal growth. No salt has been found to replace NaCl to any significant extent for optimal growth.55 The extreme halophiles possess very high internal salt concentrations, as high as that of their external environment. However, these cells are not freely permeable to all ions but are able to actively concentrate K+ and to exclude Na+. For example, the internal concentrations of Na+ and K+ in H. salinarium are 1.37 M and 4.57 M, respectively, whereas the external concentrations are 4.0 M and 0.03 M, respectively.20 Such high internal concentrations of K+ (as well as Mg2+) are required for maintaining the ribosomes in their physiologically active 70 S form,’ and most, if not all, of the known enzymes in extreme halophiles require high concentrations of salts for optimum activity, K+ being more effective than Na+.9.55.72 These bacteria thus appear to have evolved biochemical reactions and macromolecular structures that are capable of functioning at intracellular and extracellular salt concentrations approaching saturation. It also seems reasonable to suppose that the extreme halophiles have evolved membrane lipids functioning optimally in high salt concentrations. Investigation of the lipids of halophilic bacteria was begun at the National Research Council, Ottawa, at the suggestion of Dr N. E. Gibbons, then head of the Microbiology Section of the Division of Biosciences. Dr Gibbons had become interested in these bacteria during World Ward II when he was called in to help solve the “red spot” problem in the cod fish industry, which was caused by infection of the salt cod by extremely halophilic bacteria. Subsequently, he and his group became involved in physiological and biochemical studies of halophilic bacteria, the results of which led him to suspect that these organisms might possess an unusual membrane structure to help them cope with high salt concentrations. A previous study by this group 9o had dealt only with the total lipid content of the cell envelopes of several species of halophile. It was felt desirable to carry out a detailed analysis first on the lipids of a moderate halophile, Micrococcus halodenitrificarzs, grown in salt concentrations ranging from 0.55 to 1.0~. The results of this study41 were rather disappointing since the lipids turned out to be not very different from those of non-halophiles (e.g. Bacillus cereus (see Kates36)) and little or no influence of salt concentration on lipid composition, and hence on membrane composition, was detected. We then proceeded, early in 1959, to extract and analyze the lipids of a typical extremely halophilit bacterium Halobacteriunr cutirubrum; it was soon realized that these lipids were most unusual.s9 First the total lipids extracted had a deep red color due to the presence of large amounts of red carotenoid pigments, but the polar lipids could, however, be
Phytanyl
ether-linked
polar
lipids
303
easily separated from these pigments by precipitation with acetone. Secondly, chromatography showed the presence of several phosphatide components, none of which corresponded to any of the commonly known bacterial phosphatides.36 Finally, after methanolysis and saponification of the lipids, large amounts of unsaponifiable material were obtained, but only traces of fatty acids could be detected. The first positive clue to the identity of these lipids came from the I.R. spectrum of the unsaponifiable material which showed a strong C-O-C ether band at 1110 cm-’ and an isopropyl band (doublet) at 13651385 cm- ’ (see below). These observations led to the hypothesis that H. cutirubrum cells contain a new class of lipids having ether-linked phytanyl groups instead of ester-bound fatty acids, and this was fully validated by subsequent investigations. This article will deal largely with the chemistry, structure determination and synthesis of the phytanyl ether-derived polar lipids of extremely halophilic bacteria. The neutral lipids of the extreme halophiles are also of interest, because they are virtually all isoprenoid compounds, and their chemistry will also be presented here. A previous review3’ dealing extensively with the structural determination of the phytanyl ether-linked lipids and their metabolism should be consulted for further details. II.
ISOLATION OF
AND LIPID
IDENTIFICATION
COMPONENTS
A. Overall Lipid Composition The total lipid material extracted from H. cutirubrum, the first extremely halophilic bacterium examined, was deep red in color, due to the presence of carotenoid pigments (chiefly bacterioruberin); it accounted for about 2.5-4.0x of the cell dry weight and the total lipid-P amounted to 3.9-4.6”/, total lipid, 35*44*89 indicating a high phosphatide content. Precipitation of the lipids with acetone in the cold yielded an acetone-insoluble tan-colored precipitate of polar lipids accounting for about 90% by weight of the total lipids, and an acetone-soluble red-colored “neutral lipid” fraction amounting to about 10% of the total lipids.89.95 The total lipids and the polar lipid fraction had low nitrogen contents, N/P atomic ratio, 0.13 and 0.09, respectively, 89 indicating the presence of only traces of nitrogenous lipids. A lipopeptide has, however, been detected in small amounts in H. halobium, along with traces of two unidentified nitrogenous phosphatides.‘* The low content or virtual absence of nitrogenous lipids in extreme halophiles is unusual because most non-halophilic Eubacteria examined contained major amounts of nitrogenous phosphatides, usually phosphatidyl ethanolamine or lipoamino acids.36 Most unusual was the finding 89 that no fatty acids or water-soluble phosphate esters were released after mild alkaline hydrolysis or “deacylation” of the lipids of H. cutirubrum by Dawson’s procedure.23 Even after more drastic hydrolysis, such as heating in boiling 0.7 N methanolic-HCl for 2-4 hr and then in boiling 0.1 N NaOH for 1 hr (Fig. l), only traces of fatty acids and long-chain aldehydes could be detected. Surprisingly, after this hydrolysis procedure, about 800/, of the lipid was recovered as phosphorusfree unsaponifiable matter in the ether extract of the alkaline hydrolysate, and 96% of the lipid-P was recovered in the methanol/water phase as organically-bound phosphorus. 44.89 These findings indicated that the lipids of this extreme halophile were not derivatives of diacyl glycerols, as in normal bacteria, but were probably derived from the long-chain “unsaponifiable” compound (I, Fig. 1). The absence of fatty acid ester groups was confirmed by the negative hydroxamate-FeCl, test for ester groups and by the I.R. absorption spectrum of the total lipids (Fig. 2, Spectrum 1) which showed no ester absorption in the expected range 173CL1750cm-‘. However, the spectrum did show strong absorption bands indicative of long-chain groups (2920, 2850 and 1460 cm- ‘), OH groups (3300 cm- ‘, broad) and phosphate ester groups (P=O,
304
M. Kates 0 II
0 II
CH,-OH I CH-OR I CH,-OR
CH2-o--P-o-x {.,,
o-
- HCL,
Methonolic NaOH
CH,-OR Diether
cH,-O-sugar
CH20H
I CH-0-R I
I CH-OR I CH2-OR
NaOH
cH,-OR
glycolipids
I
THE PHYTANYL ISOPRENOID
ETHER-LINKED POLAR LIPIDS NEUTRAL LIPIDS OF EXTREMELY HALOPHILIC BACTERIA M.
Deparrment
o/ Biochemistry,
University
AND
KATES of Ottawa,
Ottawa,
Canada
Kl N 6N5
.
CONTENTS I. INTRODUCTION II.
ISOLATION
301
AND IDENTIFICATION
OF LIPID
COMPONENTS
A. Overall lipid composition B. Identification of the ether-containing lipid moiety as 2,3-di-0-phytanyl-sn-glycerol I. Isolation and characterization of the unsaponifiable material 2. Identification of alkyl group(s) as phytanyl groups 3. Absolute stereochemical configuration of phytanyl group 4. Structure and configuration of the diphytanyl glycerol ether C. Identification of polar lipid components I. Composition and characterization of the polar lipids 2. Phosphatidyl glycerophosphate (diphytanyl ether analog) 3. Phosphatidyl glycerol (diphytanyl ether analog) (PC) 4. Phosphatidyl glycerosulfate (diphytanyl ether analog) (PGS) 5. Glycolipid sulfate (GLS) D. Neutral lipids I. C,,-Isoprenoids (a) Geranylgeraniol (b) Di-0-phytanyl glycerol (c) Retinal 2. C,,-Isoprenolds (Squalenes) 3. C,,-Isoprenoids 4. Vitamin MK-8 5. C,,-Isoprenoids (Bacterioruberins) III.
IV.
SYNTHEW OF PHYTANYL GLYCEROL ETHER DERIVATIVES A. Phytanyl glycerol ethers I. 2,3-di-o-phytanyland I,?-di-0-phytanyl-sn-glycerols 2. l,3-di-0-phytanyl glycerol 3. 3-0-phytanyl-w-glycerol and 2-0-phytanyl glycerol B. Diphytanyl ether analogs of phosphatidic acid and derivatives I. 2.3-di-0-phytanyl-sn-glycero-l-phosphate 2. I-(Cytidine-5’-diphosphate)-2,3-di-O-phytanyI-sn-glycerol C. Phosphatidyl glycerophosphate (diphytanyl ether analog) D. Phosphatidyl glycerols (diphytanyl ether analogs) I. Diasteriomeric I-sn-phosphatidyl-1’(3’)-sn-glycerols (a-PGS) 2. I-sn-Phosohatidyl-2’-glycerol (4-PG) E. Phosphatidil gly&rosuif&es (diphytanyl ether analogs) I. I-srl-Phosphatidyl-3’-sn-glycero-1’-sulfate (PG-1-S) and I-sn-phosphatidyl-3’-sn-glycerol’,2’-disulfate (PG-1,2-di-S) 2. I-swPhosphatidyl-I’-sn-glycero-2-sulfate (PG-2-S) F. Monophytanyl ether analogs of lysophosphatidic acid and derivatives I. 3-0-Phytanyl-sn-glycero-l-phosphate (lyso-PA) 2. 3-O-Phytanyl-sn-glycero-l-phosphoryl-l’-sn-glycerol (lyso-PG)
CHEMICAL
DISTRIBUTION
OF LIPIDS
IN RED AND PURPLE
MEMBRANE
FRACTIONS
303 303 305 305 308 309 310 311 311 313 317 318 320 323 323 323 323 324 325 326 326 326 328 328 328 328 329 330 330 332 332 333 333 334 336 336 337 338 338 339 339 341
REFERENCES
I. INTRODUCTION
The extremely halophilic bacteria are a group of remarkable organisms that require saturated or nearly saturated salt solutions (20-30x NaCl) for optimum growth and survival. 1o-55*72*73These organisms occur naturally in salt lakes such as the Great Salt Lake, and in the Dead Sea. They are also commonly found in concentrated brines and in salt flats formed by evaporation of sea water, as well as in the common salt obtained from these salt flats, to all of which these organisms impart a characteristic 301
302
M. Kates
red or pink color. This coloration is attributed to the presence in most of the extreme halophiles of red carotenoid pigments (see Section II.D.5). Halophilic bacteria may be found also in salted foods and salted hides to which they are introduced with the sea salt used in their preservation. Two distinct groups of extremely halophilic bacteria are known: the rod-shaped Halobacterium genus and the coccoid Halococcus genus, both grouped in the family Halobacteriaceae (see Gibbons28 for a discussion of the taxonomy of halophilic bacteria). Bacteria in the first group are Gram-negative, obligate aerobes, non-spore forming, and require at least 12% salt for growth, the optimal salt concentration being 25-307;. The halophilic cocci are Gram-negative or Gram-variable spheres, l-l.5 pm in diameter, occurring singly, in pairs, or in packets (sarcinae). They also are all obligate aerobes, non-spore forming, and require at least 5-100/d NaCl for growth, the optimal concentration being 2025”,‘,, with growth occurring even in up to 3Oqd salt.72.73 The extremely halophilic bacteria require high concentrations of salt not only for growth but also for preservation of their cellular structure. For example, growth of the halobacteria cells ceases when the salt concentration is decreased below 3 M and the rods gradually lose their regular shape and become spherical; below 1 M NaCl, the cell envelopes begin to disintegrate and the cells lyse rapidly.7’.73 Concentrations of 0.1-0.5 M Mg2+ and 1.3-2.5 x 10e2 M Kf are also required in addition to 3-5 M NaCl for optimal growth. No salt has been found to replace NaCl to any significant extent for optimal growth.55 The extreme halophiles possess very high internal salt concentrations, as high as that of their external environment. However, these cells are not freely permeable to all ions but are able to actively concentrate K+ and to exclude Na+. For example, the internal concentrations of Na+ and K+ in H. salinarium are 1.37 M and 4.57 M, respectively, whereas the external concentrations are 4.0 M and 0.03 M, respectively.20 Such high internal concentrations of K+ (as well as Mg2+) are required for maintaining the ribosomes in their physiologically active 70 S form,’ and most, if not all, of the known enzymes in extreme halophiles require high concentrations of salts for optimum activity, K+ being more effective than Na+.9.55.72 These bacteria thus appear to have evolved biochemical reactions and macromolecular structures that are capable of functioning at intracellular and extracellular salt concentrations approaching saturation. It also seems reasonable to suppose that the extreme halophiles have evolved membrane lipids functioning optimally in high salt concentrations. Investigation of the lipids of halophilic bacteria was begun at the National Research Council, Ottawa, at the suggestion of Dr N. E. Gibbons, then head of the Microbiology Section of the Division of Biosciences. Dr Gibbons had become interested in these bacteria during World Ward II when he was called in to help solve the “red spot” problem in the cod fish industry, which was caused by infection of the salt cod by extremely halophilic bacteria. Subsequently, he and his group became involved in physiological and biochemical studies of halophilic bacteria, the results of which led him to suspect that these organisms might possess an unusual membrane structure to help them cope with high salt concentrations. A previous study by this group 9o had dealt only with the total lipid content of the cell envelopes of several species of halophile. It was felt desirable to carry out a detailed analysis first on the lipids of a moderate halophile, Micrococcus halodenitrificarzs, grown in salt concentrations ranging from 0.55 to 1.0~. The results of this study41 were rather disappointing since the lipids turned out to be not very different from those of non-halophiles (e.g. Bacillus cereus (see Kates36)) and little or no influence of salt concentration on lipid composition, and hence on membrane composition, was detected. We then proceeded, early in 1959, to extract and analyze the lipids of a typical extremely halophilit bacterium Halobacteriunr cutirubrum; it was soon realized that these lipids were most unusual.s9 First the total lipids extracted had a deep red color due to the presence of large amounts of red carotenoid pigments, but the polar lipids could, however, be
Phytanyl
ether-linked
polar
lipids
303
easily separated from these pigments by precipitation with acetone. Secondly, chromatography showed the presence of several phosphatide components, none of which corresponded to any of the commonly known bacterial phosphatides.36 Finally, after methanolysis and saponification of the lipids, large amounts of unsaponifiable material were obtained, but only traces of fatty acids could be detected. The first positive clue to the identity of these lipids came from the I.R. spectrum of the unsaponifiable material which showed a strong C-O-C ether band at 1110 cm-’ and an isopropyl band (doublet) at 13651385 cm- ’ (see below). These observations led to the hypothesis that H. cutirubrum cells contain a new class of lipids having ether-linked phytanyl groups instead of ester-bound fatty acids, and this was fully validated by subsequent investigations. This article will deal largely with the chemistry, structure determination and synthesis of the phytanyl ether-derived polar lipids of extremely halophilic bacteria. The neutral lipids of the extreme halophiles are also of interest, because they are virtually all isoprenoid compounds, and their chemistry will also be presented here. A previous review3’ dealing extensively with the structural determination of the phytanyl ether-linked lipids and their metabolism should be consulted for further details. II.
ISOLATION OF
AND LIPID
IDENTIFICATION
COMPONENTS
A. Overall Lipid Composition The total lipid material extracted from H. cutirubrum, the first extremely halophilic bacterium examined, was deep red in color, due to the presence of carotenoid pigments (chiefly bacterioruberin); it accounted for about 2.5-4.0x of the cell dry weight and the total lipid-P amounted to 3.9-4.6”/, total lipid, 35*44*89 indicating a high phosphatide content. Precipitation of the lipids with acetone in the cold yielded an acetone-insoluble tan-colored precipitate of polar lipids accounting for about 90% by weight of the total lipids, and an acetone-soluble red-colored “neutral lipid” fraction amounting to about 10% of the total lipids.89.95 The total lipids and the polar lipid fraction had low nitrogen contents, N/P atomic ratio, 0.13 and 0.09, respectively, 89 indicating the presence of only traces of nitrogenous lipids. A lipopeptide has, however, been detected in small amounts in H. halobium, along with traces of two unidentified nitrogenous phosphatides.‘* The low content or virtual absence of nitrogenous lipids in extreme halophiles is unusual because most non-halophilic Eubacteria examined contained major amounts of nitrogenous phosphatides, usually phosphatidyl ethanolamine or lipoamino acids.36 Most unusual was the finding 89 that no fatty acids or water-soluble phosphate esters were released after mild alkaline hydrolysis or “deacylation” of the lipids of H. cutirubrum by Dawson’s procedure.23 Even after more drastic hydrolysis, such as heating in boiling 0.7 N methanolic-HCl for 2-4 hr and then in boiling 0.1 N NaOH for 1 hr (Fig. l), only traces of fatty acids and long-chain aldehydes could be detected. Surprisingly, after this hydrolysis procedure, about 800/, of the lipid was recovered as phosphorusfree unsaponifiable matter in the ether extract of the alkaline hydrolysate, and 96% of the lipid-P was recovered in the methanol/water phase as organically-bound phosphorus. 44.89 These findings indicated that the lipids of this extreme halophile were not derivatives of diacyl glycerols, as in normal bacteria, but were probably derived from the long-chain “unsaponifiable” compound (I, Fig. 1). The absence of fatty acid ester groups was confirmed by the negative hydroxamate-FeCl, test for ester groups and by the I.R. absorption spectrum of the total lipids (Fig. 2, Spectrum 1) which showed no ester absorption in the expected range 173CL1750cm-‘. However, the spectrum did show strong absorption bands indicative of long-chain groups (2920, 2850 and 1460 cm- ‘), OH groups (3300 cm- ‘, broad) and phosphate ester groups (P=O,
304
M. Kates 0 II
0 II
CH,-OH I CH-OR I CH,-OR
CH2-o--P-o-x {.,,
o-
- HCL,
Methonolic NaOH
CH,-OR Diether
cH,-O-sugar
CH20H
I CH-0-R I
I CH-OR I CH2-OR
NaOH
cH,-OR
glycolipids
I
