Journal of Bacteriology, February 2003, p . 1181-1189, Vol . 185, No . 4

CDP-2,3-Di-O-Geranylgeranyl-sn-Glycerol:L-Serine O-Archaetidyltransferase (Archaetidylserine Synthase) in the Methanogenic Archaeon Methanothermobacter thermautotrophicus

Hiroyuki Morii and Yosuke Koga^*

Department of Chemistry, School of Medicine, University of Occupational and Environmental Health, Kitakyushu 807-8555, Japan

Received 8 August 2002/ Accepted 18 November 2002

 
Studying archaeal polar lipid biosynthesis, in particular the step of the attachment of the polar head group, one may expect to encounter several questions concerning the specific archaeal lipid structures . One of the questions is how archaetidylserine synthase contributes to the establishment and selection of the specific features of archaeal polar lipid structure . The enzymes that catalyze reactions 1 to 3 in Fig . 1 are specific for G-1-P and its derivatives of the same stereoconfiguration . By contrast, CDP-archaeol synthase, which catalyzes reaction 4, does not recognize the structure of the GP backbone or the ether or ester bonds between GP and hydrocarbon chains but mainly targets a substrate possessing geranylgeranyl chains . The second question deals with the relationship between archaetidylserine synthase and bacterial or eukaryal phosphatidylserine synthases . Because homologies of the gene encoding phosphatidylserine synthase (pssA) in Bacillus subtilis, but not in Escherichia coli, were found in several archaeal species whose whole genome sequences are known, the enzymatic properties which cannot be inferred from their genome sequences should be compared with those of the analogous enzymes . This might give a clue to the origin of amino group-containing phospholipids in Archaea . The other problem is the exact sequence of polar group attachment and hydrogenation of unsaturated hydrocarbon chains, which contrasts with completely saturated chains in the final products .

The present work reports some properties and the substrate specificity of archaetidylserine synthase in M . thermautotrophicus and compares them to those of bacterial phosphatidylserine synthase . The nomenclature of archaeal lipids proposed by Nishihara and Koga (15) is used throughout this paper .

Chemical synthesis of CDP-archaeol and CDP-diacylglycerol. CDP-2,3-di-O-geranylgeranyl-sn-glycerol and CDP-2,3-di-O-phytanyl-sn-glycerol were chemically synthesized with cytidine 5'-monophosphomorpholidate from corresponding archaetidic acid as previously described (9, 14) . Other substrates, CDP-1,2-di-O-geranylgeranyl-sn-glycerol and CDP-1,2-di-O-oleyl-sn-glycerol (diether type) and CDP-diacylglycerols (diester type, CDP-2,3-di-O-oleoyl-sn-glycerol and CDP-1,2-di-O-acyl-sn-glycerol) were synthesized as previously described (9, 14) . L--phosphatidic acid from egg yolk lecithin (Nacalai, Kyoto, Japan) was used as a starting material for the synthesis of CDP-1,2-di-O-acyl-sn-glycerol .

Enzymatic preparation of [³H]CDP-unsaturated archaeol. [Cytosine-5-³H]CDP-unsaturated archaeol was enzymatically synthesized as described in the previous paper (14) except that 15 µCi of [5-³H]CTP (0.625 Ci/mol) was used in 1.2 ml of the reaction mixture . After incubation for 5 h, a chloroform-soluble fraction was obtained from the reaction mixture and then [cytosine-5-³H]CDP-unsaturated archaeol was purified by acidic-alkaline partitioning (14) .

TLC. Thin-layer chromatography (TLC) gel was developed on a Silica Gel 60 plate (Merck) with the following solvents: solvent A, chloroform-methanol-7 M ammonia (60:35:8); solvent B, chloroform-methanol-acetic acid-water (80:30:15:5) . Spots of amino group-containing lipids were visualized by spraying with ninhydrin reagent . Authentic archaetidylserine was isolated from M . thermautotrophicus cells as previously described (17) . Water-soluble products were developed on a thin-layer cellulose plate (Merck 5716) with phenol-water (100:38) . Standard CMP was purchased from Kohjin, Tokyo, Japan . Radioactive spots were recorded by a Fujifilm Fluor image analyzer (model FLA-2000) with an imaging plate: Fujifilm type BAS-TR for ³H-labeled lipids and type BAS-MS for ³²P- and ³³P-labeled lipids .

Preparation of cell-free homogenates. Frozen M . thermautotrophicus cells (about 20 g wet weight) suspended in 25 ml of 10 mM Bicine buffer (pH 8.0) containing 1 mM MgCl₂, 5 mM 2-mercaptoethanol (buffer M), and 1 mg of DNase I (Sigma) were passed through a French pressure cell operated at 1,400 kg/cm² . This process was repeated twice . Cell debris and unbroken cells were removed by centrifugation (10,000 x g) for 10 min . The homogenate was stored at -20°C until further use . The membrane fraction was obtained by centrifugation at 100,000 x g for 2 h . The pelleted membrane fraction was resuspended in buffer M . Crude cell extracts of B . subtilis and E . coli were prepared as previously described (5, 6) .

Enzyme assay. The complete assay mixture (final volume, 0.2 ml) for archaetidylserine synthase of M . thermautotrophicus contained 0.5 mM CDP-archaeol, 10 mM [3-³H]L-serine (1.25 Ci/mol; Amersham Pharmacia Biotech), the cell-free homogenate of M . thermautotrophicus (568 µg of protein), 0.125 M Bicine buffer (pH 8.5), l% Triton X-100, and 10 mM MnCl₂ . After incubation at 60°C for 10 min, the reaction was stopped by the addition of 1 ml of 0.1 M HCl in methanol, 2 ml of chloroform, and 3 ml of 1 M MgCl₂ . Chloroform-extractable ³H material was separated from water-soluble components by phase partitioning, and radioactivity was counted . In the case of determination of stereospecificity to serine of archaetidylserine synthase, [3-³H]L-serine was replaced by nonradioactive D- or L-serine in 0.8 ml of the reaction mixture . After incubation at 60°C for 30 min, phospholipid was precipitated by the addition of acetone to a chloroform-soluble fraction in order to remove Triton X-100 (14) . The precipitate of phospholipids was developed on TLC with solvent B . A spot corresponding to archaetidylserine was visualized by spraying with acid molybdate reagent, and the phosphorus content of the spot scraped off from the plate was determined (1) . The activity of archaetidylserine synthase was calculated based on the formation of archaetidylserine over 30 min measured by phosphate determination . The phosphorus content of the spot corresponding to archaetidylserine in a control experiment without D- or L-serine was subtracted from the total phosphorus of archaetidylserine determined for the TLC spot .

Phosphatidylserine synthase activity was measured in B . subtilis and E . coli as previously described (5, 6) . Protein content was determined by the bicinchoninic acid method (22) .

Identification of the reaction product. To obtain a large amount of the enzyme reaction product for structural analysis, 20 times more reactants were incubated for 30 min . Nonradioactive L-serine replaced L-[³H]serine . The product was extracted and purified by acetone precipitation and TLC as described above . The fast atom bombardment-mass spectrum of the product was recorded with a mass spectrometer (JEOL JMS DX-303) with a matrix of m-nitrobenzyl alcohol containing a small amount of NaI in a positive mode . The presence of an allyl ether linkage was checked by the lability to the treatment of 5% HCl-methanol at 80°C for 1 h . For the identification of water-soluble product, [cytosine-5-³H]CDP-archaeol (290,000 dpm) was reacted with unlabeled L-serine in a standard reaction mixture . The water-soluble radioactive product was recovered in the aqueous phase after Bligh-Dyer partitioning and was developed by cellulose-TLC .

Detection of allyl ether containing archaetidylserine in M . thermautotrophicus cells. M . thermautotrophicus was grown successively twice in 50 ml of low-phosphate medium (17) containing 100 µCi of ³³P_i (5 Ci/mol) under a pressurized atmosphere of H₂ + CO₂ + H₂S (78:22:0.2) in a 500-ml flask with shaking for 24 h . Finally, 50 ml of the same medium containing the same specific radioactivity of ³³P_i was inoculated with 5 ml of the last subculture . At the early logarithmic phase of growth (after incubation for 7 h), 2 mCi of ³²P_i was added and the culture was allowed to continue to grow . After 10 min, cells were harvested and total lipid was extracted . The radioactive phospholipid corresponding to archaetidylserine was purified from the total lipid by two-dimensional TLC . The isolated archaetidylserine was treated with 5% HCl-methanol at 80°C for 1 h to degrade allyl ether archaetidylserine . The degradation products were partitioned into chloroform-soluble and water-soluble fractions . ³²P and ³³P radioactivities in the chloroform-soluble products, aqueous products, and the untreated archaetidylserine were counted using a liquid scintillation analyzer (Packard TRI-CARB 2700TR) with Aquasol-2 (Packard) as a scintillator .

The reaction product from CDP-2,3-di-O-geranylgeranyl-sn-glycerol was also chemically and mass-spectrometrically analyzed . The lipid product enzymatically prepared from CDP-2,3-di-O-geranylgeranyl-sn-glycerol was purified by TLC with solvent B . The fast atom bombardment-mass spectrum of the lipid product gave signals of m/z 803 (M)⁺, m/z 827 (M + Na + H)⁺, m/z 762 (M - serine + 2Na+H)⁺, and m/z 784 (M - serine + 3Na)⁺, which were consistent with the structure of archaetidylserine with geranylgeranyl groups as hydrocarbon chains . The presence of allylic ether linkages was also suggested by the acid lability . The product of the enzyme reaction was completely degraded by treatment with 5% HCl-methanol at 80°C for 1 h . These results suggest that the product from CDP-unsaturated archaeol most likely is 2,3-di-O-geranylgeranyl-sn-glycero-1-phosphoserine (unsaturated archaetidylserine), even though the individual components and the stereostructure of the product were not completely determined . We also analyzed the water-soluble product of the reaction with [cytosine-5-³H]CDP-archaeol and unlabeled L-serine as substrates . One radioactive spot (R_f = 0.33) was found that cochromatographed with standard CMP on a cellulose TLC plate .

A nonradioactive by-product was detected on the TLC with an R_f of 0.19 . The compound was positively stained with acid molybdate reagent on the TLC plate . For an analogous reaction, Walton and Goldfine (26) reported that phosphatidyltransferase from Clostridium butyricum catalyzed the transfer of the phosphatidyl moiety of phospholipid to Triton X-100, and in vitro formation of phosphatidyltriton was observed . Therefore, it was assumed to be a Triton X-100 adduct of an archaetidyl group (e.g., archaetidyltriton X-100) and was not further analyzed .

Properties of archaetidylserine synthase. Under the assay conditions used, radioactivity was incorporated into a chloroform-soluble fraction almost linearly for 30 min, and then the rate gradually slowed (Fig . 2) . Approximately 30 nmol of archaetidylserine was formed when 100 nmol of CDP-archaeol was incubated in the reaction mixture for 1 h (Fig . 2) . In routine assays, the incubation time was 10 min . Archaetidylserine synthase activity was roughly linear with protein concentration under the assay conditions (data not shown) . The effect of nonionic detergent Triton X-100 on archaetidylserine synthase activity is shown in Fig . 3A . Triton X-100 was required for archaetidylserine synthase activity and maximum activity was obtained at a concentration of 1% . The enzyme activity was stimulated by the addition of Mn²⁺, with maximum activities occurring at concentrations of 5 mM or more . The addition of Mg²⁺ had much less effect on the enzyme activity (Fig . 3B) . These results show that the enzyme activity was dependent on addition of Triton X-100 and Mn²⁺ ion . The enzyme did not require the addition of K⁺ ion . The enzyme activity was lowered to about 70% of its maximum at 0.6 M K⁺, which corresponds to the intracellular concentration found in M . thermautotrophicus (20) (Fig . 3C) . Maximal enzyme activity was observed at pH 8.0 to 8.5 (Bicine buffer) (data not shown) and at 60°C (Fig . 3D) . The membrane fraction and cell supernatant, respectively, contained 61 and 32% of the total activity found in the cell-free homogenate of M . thermautotrophicus . Specific activities of cell-free homogenates, membrane fraction and cell supernatant were 64, 146, and 19 nmol/h/mg of protein, respectively . Unfractionated cell-free homogenate was usually used for further studies .

 
Substrate specificity of archaetidylserine synthase. One of the major questions concerning archaeal lipid biosynthesis is how and at which step the specific structural characteristics of archaeal lipids are formed . The substrate stereospecificity of archaetidylserine synthase was, therefore, examined using a variety of chemically synthesized substrate analogs listed in Table 1 . The list includes CDP-archaeol (substrates 1 to 4)/CDP-diacylglycerol (substrates 5 and 6) analogs with both stereoisomers of the GP backbone, ether and ester bonds between GP and hydrocarbons, unsaturated and saturated isoprenoid hydrocarbon chains, and straight-chain hydrocarbons . Archaetidylserine synthase of M . thermautotrophicus showed similar activities when either enantiomer of CDP-archaeol possessing geranylgeranyl chains (substrates 1 and 2) was used as a substrate . The substrate analogs with saturated ones (substrate 3) or straight hydrocarbon chains (substrate 4) showed slightly lower activities, but the activity range was between less than ±50% of the activity on the natural substrate (substrate 1) . Interestingly, when ester-lipids containing straight aliphatic chains (substrates 5 and 6) were used, activities were two to three times higher than those observed with an ether-type substrate (substrate 1) (Table 1) .

 
The stereospecificity of archaetidylserine to L- or D-serine was determined using nonradioactive substrates . Archaetidylserine synthase preferred L-serine to D-serine . The relative activity for D-serine was 32% ± 6% (n = 2) of what was observed with L-serine .

Substrate specificity of bacterial phosphatidylserine synthase. Because archaetidylserine synthase is known to be homologous to phosphatidylserine synthase from B . subtilis (12), the substrate specificities of phosphatidylserine synthase from B . subtilis and another type of phosphatidylserine synthase from E . coli were compared to that of archaetidylserine synthase (Table 1) . B . subtilis phosphatidylserine synthase showed almost similar activities in every case when the substrates listed in Table 1 were used . That is, the enzyme did not discriminate between the stereostructures of the GP backbone, ether or ester linkage, and hydrocarbon chains of the analogs of CDP-archaeol . The substrate specificity of the B . subtilis enzyme was quite similar to that of the methanogen's archaetidylserine synthase described above . By contrast, E . coli phosphatidylserine synthase appeared to distinguish between such differences in the substrate structures . A drastic decrease in activity was observed when the mirror image isomer (CDP-2,3-diacyl-sn-glycerol [Table 1, substrate 5]) of the natural substrate (substrate 6) or the ether analogs with isoprenoid chain (substrates 1 to 3) were incubated with E . coli cell extracts . An ether-type substrate with natural GP stereostructure and straight-chain hydrocarbons (CDP-1,2-dioleyl-sn-glycerol [substrate 4]) revealed low but significant activity (41%) when compared with the ester-type natural substrate (substrate 6) . In other words, substitution of ester linkages in the substrate structure showed a significant effect on the activity of E . coli phosphatidylserine synthase . Thus, a difference between phosphatidylserine synthases from both bacterial species was demonstrated also in substrate specificity .

Intracellular occurrence of allyl ether-type archaetidylserine in M . thermautotrophicus cells. In order to elucidate whether unsaturated (or allyl ether-type) archaetidylserine is really formed in the cells, we tried to detect it on the basis of the acid lability of allyl ether lipids . An allyl ether bond is degraded with 5% HCl-methanol at 80°C for 1 h (14) . On the other hand, saturated archaetidylserine is stable to acid treatment because nonallyl ether bonds are not readily hydrolyzed and the phosphodiester bond cannot be hydrolyzed by cyclic phosphodiester formation due to the lack of a free hydroxyl group on serine residues (11) . M . thermautotrophicus was continuously labeled with ³³P_i and pulse-labeled with ³²P_i for 10 min in the presence of ³³P_i . In this experiment, ³³P in archaetidylserine must represent the amount of mature archaetidylserine (the final product of the biosynthetic pathway) with saturated hydrocarbons, while ³²P must express the amount of newly synthesized archaetidylserine . Labeled archaetidylserine was purified from the total lipid of the cells . The purified archaetidylserine was decomposed with 5% HCl-methanol at 80°C for 1 h . The ratio of radioactivities of ³²P versus ³³P (³²P/³³P) of chloroform-soluble products and water-soluble products after acid treatment of archaetidylserine was compared to that of untreated archaetidylserine . The ³²P/³³P ratio (1.26) of untreated archaetidylserine decreased by 17% to 1.04 in chloroform-soluble products (acid-stable lipids) after acid treatment . On the other hand, the ratio (2.17) in the aqueous fraction after acid treatment (acid-labile degradation products) was 1.7 times higher than the ratio (1.26) observed in untreated archaetidylserine . These results suggest that only a trace amount of newly synthesized allyl ether-type archaetidylserine is present in the cells . Almost all archaetidylserines have fully saturated hydrocarbon chains, as shown by chemical analysis (17) .

Archaetidylserine synthase showed a loose specificity for CDP-archaeol analogs; that is, the enzyme is able to utilize CDP-archaeol analogs with both enantiomers of the GP backbone, ester and ether linkages, and unsaturated and saturated isoprenoid and straight-chain fatty acid . This means that archaetidylserine synthase is not involved in establishing the specific features of archaeal polar lipid structures . According to knowledge obtained so far, the first three enzymes in the biosynthesis pathway of the archaeal polar lipid appear to play a central role in the formation of the specific features of archaeal polar lipid structures, while the ensuing steps do not . Archaetidylserine synthase preferred L-serine over D-serine . Serine stereospecificity of phosphatidylserine synthase in Bacteria has not been reported .

There are two genetically distinct subclasses of phosphatidylserine synthase . The enzymes classified in subclass I are distributed in gram-negative bacteria (e.g., E . coli) . The subclass II enzymes have widespread distribution in gram-positive bacteria (e.g., B . subtilis), yeast, and Archaea (Methanococcus jannaschii) (12) . It is known that there are some different properties between E . coli-type phosphatidylserine synthase (subclass I) and B . subtilis phosphatidylserine synthase (subclass II) . For example, their intracellular localization, divalent cation requirement, and reaction mechanisms are different (7) . In addition, the enzymes from E . coli and B . subtilis show no sequence homology (19) . An M . jannaschii gene encoding archaetidylserine synthase is known to belong to subclass II (12) . A gene (MT1027) homologous to the B . subtilis phosphatidylserine synthase gene (pssA) has been identified in the M . thermautotrophicus genome (21) . The gene MT1027 was annotated as phosphatidylserine synthase . The amino acid sequence data of phosphatidylserine synthase of B . subtilis (P39823), M . thermautotrophicus (A69004 = MT1027), and Methanocaldococcus jannaschii (Q58609 = MJ1212) were obtained at the NCBI site (www.ncbi.nlm.nih.gov) . A multiple alignment of the three sequences was constructed with an alignment software, CLUSTAL W 1.7 (Fig . 4) . Although the archaetidylserine synthase characterized in this work has not been cloned and sequenced, it is most likely that MT1027 codes for archaetidylserine synthase . The deduced amino acid sequence of MT1027 showed significant homology with B . subtilis phosphatidylserine synthase (23.1% identity and 52.6% similarity), whereas MT1027 showed no homology with that of the E . coli enzyme . In addition to finding homology in the primary structures, this work also demonstrated similarities in some biochemical properties (requirements of Triton X-100 and Mn²⁺ ion) of archaeal and Bacillus enzymes . Substrate specificity is the other characteristic used for comparison of archaeal archaetidylserine synthase with subclass I and II phosphatidylserine synthases . We investigated lipid substrate specificity of archaetidylserine synthase from M . thermautotrophicus and phosphatidylserine synthases from B . subtilis and E . coli . The data described in this paper confirm the results presented by Carman and Dowhan on the lipid substrate stereoisomer specificity of phosphatidylserine synthase from E . coli (3): they showed that the purified enzyme was specific for the sn-glycero-3-phosphate (G-3-P) isomer of the liponucleotide and did not recognize the G-1-P isomer in a kinetic study using CDP-1,2-dipalmitoyl-sn-glycerol and CDP-1,2-dipalmitoyl-rac-glycerol as substrates . In addition, our work showed that E . coli phosphatidylserine synthase recognized ester bonds between GP and the hydrocarbon chains and nonbranched hydrocarbon chain . On the other hand, B . subtilis phosphatidylserine synthase revealed loose substrate specificity like M . thermautotrophicus archaetidylserine synthase . Therefore, we conclude that archaeal archaetidylserine synthase is a member of the Bacillus-type phosphatidylserine synthase family (subclass II) not only on the basis of amino acid sequence homology but also from the enzymatic properties including substrate specificity .

 
It may be inferred from our results and other reports that archaeal archaetidylserine synthase and Bacillus phosphatidylserine synthase originated from a common ancestral enzyme . Because amino group-containing phospholipids are confined only to methanogens and some related Euryarchaeota among the Archaea (11, 24) and widely distributed in bacteria (8), it is speculated that the gene encoding the ancestral enzyme was transferred from a gram-positive bacterium possessing subclass II phosphatidylserine synthase to a group including methanogens after differentiation from other Archaea groups . A symbiotic relationship between methanogens and some kind of hydrolytic fermentative bacteria via interspecies hydrogen transfer in anaerobic environments (2, 25) would give an increasing chance to exchange genes . This speculation is supported by the fact that the archaetidylserine synthase activities of ester lipids are twice (or more) those of ether lipids . Bacillus phosphatidylserine synthase can catalyze the formation of archaetidylserine from CDP-archaeol and L-serine (Table 1, substrate 1) . It could be also speculated that, when the pssA gene was transferred, the loose substrate specificity of the subclass II phosphatidylserine synthase would be the most adequate to catalyze reaction 5 (Fig . 1) in the biosynthesis of the enantiomeric ether phospholipid in an archaeon, if substrate specificity at that time was the same as at present . The subclass I (E . coli-type) phosphatidylserine synthase could not do this task because of the strict specificity . Although the intracellular salt (K⁺) concentration (0.6 M) in M . thermautotrophicus is not optimal for activity, the enzyme is still active at the intracellular K⁺ concentration . This is in contrast to CDP-archaeol synthase, which shows no homology with bacterial enzymes and exhibits maximum activity in the presence of 0.5 M K⁺ (14) . This fact is consistent with the speculation that the gene encoding archaetidylserine synthase was transferred from Bacteria to Archaea .

Although the three preceding enzymes involved in the ether bond formation (Fig . 1, reactions 2 and 3) and the activation of the intermediate by CDP of phospholipid biosynthesis (Fig . 1, reaction 4) have been shown to be specific to geranylgeranyl chains, archaetidylserine synthase (Fig . 1, reaction 5) has not . Archaetidylserine synthase reacted with substrates with both saturated and unsaturated isoprenoid chains . The cellular polar lipids have fully saturated hydrocarbon chains . Therefore, there should be steps of hydrogenation (saturation) of geranylgeranyl chains somewhere before (reaction 3 in Fig . 5) or after (reaction 2 in Fig . 5) the step of archaetidylserine formation . The exact sequence is not known . In order to obtain a clue to clarify this problem, the presence of an unsaturated archaetidylserine intermediate was surveyed based on the acid lability of geranylgeranyl ethers . An in vivo pulse-label experiment with ³²P_i revealed the intracellular presence of newly synthesized acid-labile (probably allyl ether-bonded) archaetidylserine . This result is consistent with the work of Moldoveanu and Kates (13), in which they demonstrated the presence of acid-labile unsaturated ether intermediates of phospholipid biosynthesis in the extremely halophilic archaeon Halobacterium cutirubrum by pulse-labeling and chase experiments . The detection of an acid-labile archaetidylserine intermediate indicates that at least an allyl ether-bonded archaetidylserine intermediate is really present in the cells which are involved in phospholipid biosynthesis . The presence of an allyl ether serine-containing intermediate suggests that the biosynthetic pathway via reactions 1 and 2 in Fig . 5 is probably operated in the cells, although the possibility of another pathway via reactions 3 and 4 is not excluded because archaetidylserine synthase did react with saturated CDP-archaeol and the intracellular absence of saturated CDP-archaeol has not been excluded .

This work was partly supported by Grant-in-Aid for Scientific Research B 11460051 from the Ministry of Education, Science, Sports, and Culture of Japan .

1. Bartlett, G . R. 1959 . Phosphorus assay in column chromatography . J . Biol . Chem . 234:466-468.
2. Bonchosmolovskaya, E . A., and K . O . Stetter. 1991 . Interspecies hydrogen transfer in cocultures of thermophilic Archaea . Syst . Appl . Microbiol . 14:205-208.
3. Carman, G . M., and W . Dowhan. 1979 . Phosphatidylserine synthase from Escherichia coli—the role of Triton X-100 in catalysis . J . Biol . Chem . 254:8391-8397.
4. Dowhan, W. 1997 . CDP-diacylglycerol synthase of microorganisms . Biochim . Biophys . Acta 1348:157-165.
5. Dowhan, W. 1992 . Phosphatidylserine synthase from Escherichia coli. Methods Enzymol . 209:287-298.
6. Dutt, A., and W . Dowhan. 1981 . Characterization of a membrane-associated cytidine diphosphate-diacylglycerol-dependent phosphatidylserine synthase in bacilli . J . Bacteriol . 147:535-542.
7. Dutt, A., and W . Dowhan. 1985 . Purification and characterization of a membrane-associated phosphatidylserine synthase from Bacillus licheniformis . Biochemistry 24:1073-1079.
8. Goldfine, H. 1982 . Lipids of procaryotes—structure and distribution . Curr . Top . Membr . Transport 17:1-43.
9. Kates, M., C . E . Park, B . Palameta, and C . N . Joo. 1971 . Synthesis of diphytanyl ether analogues of phosphatidic acid and cytidine diphosphate diglyceride . Can . J . Biochem . 49:275-281.
10. Kates, M. 1978 . The phytanyl ether-linked polar lipids and isoprenoid neutral lipids of extremely halophilic bacteria . Prog . Chem . Fats Other Lipids 15:301-342.
11. Koga, Y., M . Nishihara, H . Morii, and M . Akagawa-Matsushita. 1993 . Ether polar lipids of methanogenic bacteria . Structures, comparative aspects, and biosynthesis . Microbiol . Rev . 57:164-182.
12. Matsumoto, K. 1997 . Phosphatidylserine synthase from bacteria . Biochim . Biophys . Acta 1348:214-227.
13. Moldoveanu, N., and M . Kates. 1988 . Biosynthetic studies of the polar lipids of Halobacterium cutirubrum . Formation of isoprenyl ether intermediates . Biochim . Biophys . Acta 960:164-182.
14. Morii, H., M . Nishihara, and Y . Koga. 2000 . CTP:2,3-di-O-geranylgeranyl-sn-glycero-1-phosphate cytidyltransferase in the methanogenic archaeon Methanothermobacter thermoautotrophicus . J . Biol . Chem . 275:36568-36574.
15. Nishihara, M., and Y . Koga. 1987 . Extraction and composition of polar lipids from the archaebacterium, Methanobacterium thermoautotrophicum: effective extraction of tetraether lipids by an acidified solvent . J . Biochem . 101:997-1005.
16. Nishihara, M., and Y . Koga. 1995 . sn-Glycerol-1-phosphate dehydrogenase in Methanobacterium thermoautotrophicum: key enzyme in biosynthesis of the enantiometric glycerophosphate backbone of ether phospholipids of archaebacteria . J . Biochem . 117:933-935.
17. Nishihara, M., H . Morii, and Y . Koga. 1989 . Heptads of polar ether lipids of an archaebacterium, Methanobacterium thermoautotrophicum: structure and biosynthetic relationship . Biochemistry 28:95-102.
18. Nishihara, M., T . Yamazaki, T . Oshima, and Y . Koga. 1999 . sn-Glycerol-1-phosphate-forming activities in Archaea: separation of archaeal phospholipid biosynthesis and glycerol catabolism by glycerophosphate enantiomers . J . Bacteriol . 181:1330-1333.
19. Okada, M., H . Matsuzaki, I . Shibuya, and K . Matsumoto. 1994 . Cloning, sequencing, and expression in Escherichia coli of Bacillus subtilis gene for phosphatidylserine synthase . J . Bacteriol . 176:7456-7461.
20. Schönheit, P., D . B . Beimborn, and H . Perski. 1984 . Potassium accumulation in growing Methanobacterium thermoautotrophicum and its relation to the electrochemical proton gradient . Arch . Microbiol . 140:247-251.
21. Smith, D . R., L . A . Doucette-Stamm, C . Deloughery, H . M . Lee, J . Dubois, T . Aldredge, R . Bashirzadeh, D . Blakely, R . Cook, K . Gilbert, D . Harrison, L . Hoang, P . Keagle, W . Lumm, B . Pothier, D . Y . Qiu, R . Spadafora, R . Vicaire, Y . Wang, J . Wierzbowski, R . Gibson, N . Jiwani, A . Caruso, D . Bush, H . Safer, D . Patwell, S . Prabhakar, S . McDougall, G . Shimer, A . Goyal, S . Pietrokovski, G . M . Church, C . J . Daniels, J . I . Mao, P . Rice, J . Nolling, and J . N . Reeve. 1997 . Complete genome sequence of Methanobacterium thermoautotrophicum delta H: functional analysis and comparative genomics . J . Bacteriol . 179:7135-7155.
22. Smith, P . K., R . I . Krohn, G . T . Hermanson, A . K . Mallia, F . H . Gartner, M . D . Provenzaro, E . K . Fujimoto, N . M . Goeke, B . J . Olson, and D . C . Klenk. 1985 . Measurement of protein using bicinchoninic acid . Anal . Biochem . 150:76-85.
23. Soderberg, T., A . J . Chen, and C . D . Poulter. 2001 . Geranylgeranylglyceryl phosphate synthase . Characterization of the recombinant enzyme from Methanobacterium thermoautotrophicum . Biochemistry 40:14847-14854.
24. Sprott, G . D. 1992 . Structures of archaebacterial membrane lipids . J . Bioenerg . Biomembr . 24:555-566.
25. Thiele, J . H., and J . G . Zeikus. 1988 . Control of interspecies electron flow during anaerobic digestion: significance of formate transfer versus hydrogen transfer during syntrophic methanogenesis in flocs . Appl . Environ . Microbiol . 54:20-29.
26. Walton, P . A., and H . Goldfine. 1987 . Transphosphatidylation activity in Clostridium butyricum . J . Biol . Chem . 262:10355-10361.
27. Wasserfallen, A., J . Nolling, P . Pfister, J . Reeve, and E . C . de Macario. 2000 . Phylogenetic analysis of 18 thermophilic Methanobacterium isolates supports the proposals to create a new genus, Methanothermobacter gen . nov., and to reclassify several isolates in three species, Methanothermobacter thermautotrophicus comb . nov., Methanothermobacter wolfeii comb . nov., and Methanothermobacter marburgensis sp . nov . Int . J . Syst . Evol . Microbiol . 50:43-53.
28. Zhang, D.-L., L . Daniels, and C . D . Poulter. 1990 . Biosynthesis of archaebacterial membranes . Formation of isoprenyl ethers by a prenyl transfer reaction . J . Am . Chem . Soc . 112:1264-1265.
29. Zhang, D . L., and C . D . Poulter. 1993 . Biosynthesis of archaebacterial ether lipids . Formation of ether linkages by prenyltransferases . J . Am . Chem . Soc . 115:1270-1277.

Free Online Full-text Article

What Is Genetic Engineering?, What Is Cell Biology?, What Is MIC?, What Is Bioremediation?, What Is Bioassay?, c, Bacteriology, o, Bacterium, i, Microbes, o, Microbiology, a, Bacteria, s, Pasteurella, i, Cryptococci, i, Meningococcus, c, Pseudomonas aeruginosa, n, Cryptococci, o, Halophilic bacterium, a, Streptococci, i, Multidrug resistant, s, E coli O157, n, Biological treatment, i, Salmonella, a, Bacteria, s, Antibiotic prophylaxis, o, Escherichia coli, i, Microorganisms, r, Microorganism, i, Bacillus subtilis, o, Microorganism, e, Bacillus anthracis, o, Lactobacillus, n, Pseudomonas aeruginosa




 

© 2005 Transgalactic Ltd (manufacturer of Bioscreen C software) | Privacy Statement | P.O. Box 1393, 00101 Helsinki, Finland, phone: +358 9 85172920, fax: +358 9 8749481, e-mail: microbiology@bionewsonline.com
 

Last modified: May 25, 2005