Thomas G. Tornabene

Squalenes, phytanes and other isoprenoids as major neutral lipids of methanogenic and thermoacidophilic “archaebacteria”

SummaryThe neutral lipids of nine species of methanogenic bacteria including five methanobacilli, two methanococci, a methanospirillum, one methanosarcina as well as two thermoacidophilic bacteria, Thermoplasma and Sulfolobus, were analyzed. The major components were C30, C25 and/or C20 acyclic isoprenoid hydrocarbons with a continuous range of hydroisoprenoid homologues. The range of acyclic isoprenoids detected were from C14 to C30. Apart fromMetbanosarcina barkeri, squalene and/or hydrosqualene derivatives were the predominant components in all species studied. The components ofMetbanosarcina barkeri were a family of C25 homologues. The distribution of the neutral lipid components and their specItIc variations in relative intensities emphasized the differences between the test organisms while the generic nature of the isoprenoid hydrocarbons demonstrated similarities between the diverse bacteria.The neutral lipid compositions from these bacteria, many of which exist in evironmental conditions like those described for the various evolutionary stages of the archean ecology, resemble the isoprenoid distribution isolated from ancient sediments and petroleum. Therefore, these findings may have major implications to biological and biogeochemical evolution.

Phytanyl-glycerol ethers and squalenes in the archaebacterium Methanobacterium thermoautotrophicum

SummaryThe lipids of a thermophilic chemolithotroph,Metbanobacterium thermoautotropbicum, have been analyzed by chromatographic techniques and identified by infrared spectrometry and combined gas chromatography-mass spectrometry. Of the total chloroform soluble lipids 79% and 21% are polar and non-polar lipids, respectively. The major components of the polar lipids are dialkyl ethers of glycerol or its derivatives. The nature of the glycerol ether alkyl groups was found to be that of the saturated tetraisoprenoid hydrocarbon phytane. The non-polar lipids of the chloroform soluble fraction consist principally of three series of C20, C25 and C30 acyclic isoprenoid hydrocarbons, the major components being squalene and a continuous range of hydrosqualene derivatives, from dihydrosqualene up to and including decahydrosqualene. These data establish thatM. tbermoautotropbicum contains predominantly non-sapo-nifiable lipids as doHalobacterium, Halococcus, Sulfolobus andTbermoplasma. In particular, the composition of the chloroform soluble lipids ofM. tbermoautotropbicum is quite similar to that ofHalobacterium cutirubrum. The results strongly support the recent proposal, based on 16S rRNA sequence homologies, that the extreme halophiles and methanogens share a common ancestor. In addition, it is pointed out that the occurrence of phytane and related polyisoprenoid compounds in ancient sediments can no longer be considered unequivocally as indicative of past photosynthetic activity. Finally, speculations are made concerning the possible role of and evolutionary significance of the presence of squalene and hydrosqualenes in these organisms. To our knowledge this is the first report of squalene and hydrosqualenes in a strictly anaerobic microorganism.

Lipid composition of selected strains of Yersinia pestis and Yersinia pseudotuberculosis

Abstract The nature and quantity of the lipids of selected strains of Yersinia pestis and Yersinia pseudotuberculosis have been examined. The lipid compositions of the organisms studied are relatively similar with the exception of minor variations in the relative proportions of the major phospholipids and the presence of additional minor components in Y. pseudotuberculosis . The phospholipids detected in strains and species of Yersinia have been identified as phosphatidylethanolamine, phosphatidylmonomethylethanolaniine, phosphatidyldimethylethanolamine, phosphatidic acid, phosphatidylglycerol, diphosphatidylglycerol, phosphatidylserine and phosphatidylinositol by chromatography of the glycerol phosphate esters derived from the lipids after mild alkaline hydrolysis and by chromatography of the products of acid hydrolysis of the esters. No phosphatidylcholine was detected. Two of the minor components detected in Y. pseudotuberculosis were identified as mono- and diglucosyl diglyceride. No glycolipids were detected in Y. pestis strains. The fatty acid compositions generally consist of 12:0, 14:0, 16:0, 16:1, cyclopropane(cyc)-17:0, 18:0, 18:1, hydroxy(OH)-16:0, cyc-19:0 and 20:0. Relatively small quantities of 10:0 and 14:1 and trace quantities of OH-14:0 and OH-18:0 were also detected. The lipid composition of Y. pestis and Y. pseudotuberculosis are quite similar to those reported for members of the Enterobacteriaceae .

Non-aerated cultivation of Halobacterium cutirubrum and its effect on cellular squalenes

SummaryHalobacterium cutirubrum was successfully cultivated under aerobic and microaerobic conditions. The early stationary phase of growth was obtained at 2.2 days and 45–55 days for aerated and non-aerated cultures, respectively. The dry cell yields were 0.7–1.2 gm/l in all preparations grown to early stationary growth phase. The cellular ratio of squalene to dihydro- and tetra-hydrosqualene decreased proportionately with decreased aeration rates.

Chromatographic studies of the lipid components of Vibrio fetus

Abstract Two different strains of Vibrio fetus , Strain I-17 and Strain V-2673, were grown on solid and liquid media. Both the vibrio- (curved bacillus) and coccoid-type cells of Strain I-17 were studied. The lipid components of both strains in the vibrio form were closely related whereas differences between the lipid components of the vibrio- and coccoid-type cells were observed. A large proportion of the lipids in the vibrio form (32%) and the coccoid form (60%) consisted of neutral lipids, predominantly free fatty acids and glycerides. The major phosphatides in all samples were identified as phosphatidyl ethanolamine, phosphatidyl glycerol and phosphatidyl inositol. A small amount of phosphatidyl serine was detected only in the vibrio cells while phosphatidic acid was found only in the coccoid-type cell. Digalactosyl diglyceride was identified in both vibrio- and coccoid-type cells. The major fatty acids in all samples were C 14:0 , C 16:0 , C 16:1 and C 18:1 . A substantial amount of C 19 -cyclopropane acid was found only in Strain I-17 cells.

Identification of methoxyester artifacts produced by methanolic-hcl solvolysis of the cyclopropane fatty acids of the genus yersinia

Abstract Previously unidentified constituents in fatty acid extracts of strains and species of Yersinia (Tornabene, T.G. (1973) Biochim Biophys Acta, 306, 173–185) have been identified as methanolyzed cyclopropane fatty acid methyl esters. These derivatized open ring acids were artifacts formed by a standard mild acidic-methanolysis procedure. The quantities of artifacts produced in natural mixed lipids were highly significant in comparison to the trace amounts obtained from pure samples of cyclopropane fatty acids. Characterization of the methanolyzed derivatives established the location of the propane ring in the cyclopropane fatty acids as 9,10-methylene hexadecanoic and 11, 12-methylene octadecanoic acids.

Membrane Lipids and the Origin of Life

There are two major types of amphiphilic lipids found in the plasma membranes of all living cells. The acylglycerol ester derivatives of ordinary bacteria and eukaryotes,and the isopranyl ether derivatives of Archaebacteria. Due to the importance of membranes in the origin of life and to the limited work done in this field we have undertaken experiments on the prebiotic synthesis of the building blocks of membrane lipids of prokaryotes and eukaryotes, and we have studied the less known membrane lipid components of Archaebacteria. Concerning the former, we have been successful in synthesizing fatty acids, glycerol, glycerophosphate, mono-, di- and tripalmitoyl glycerol esters and the corresponding phosphatidic acids. Glycerol has been obtained by reduction of the products from the formose reaction. Furthermore, under the prebiotic conditions of an evaporating pond model and using cyanamide as a condensing agent we have synthesized glycerol phosphate and all the other complex lipids. These amphiphilic molecules have the ability to self-assemble into stable bilayered microvesicles which show structure and functions similar to cell membrane lipid bilayers. Concerning the Archaebacteria (Methanogens, Halophiles, Sulfolobus and Thermo-plasma) we have analyzed their membrane lipids and found them to be derivatives of diphytanyl glycerol diethers or di-C40-isopranyl diglycerol tetraethers. Such a unique membrane ether lipid composition is ideally suited for stability to the high temperature and other extreme environmental conditions of archaen times. This and other evidence suggests that the extant Archaebacteria evolved from a common anaerobic ancestral line when the primitive Earth was evolving from anoxidic to oxidic conditions at about 3.7 Aeons ago.

Regulation of bacterial abstraction of lead by cell surface charge and chemical equilibria

Abstract Micrococcus luteus cells greatly enhanced the abstraction of lead contained within pouches made of dialyzing tubing. The amount of soluble lead abstracted and subsequently interacting with the cells intertied with the concentration of negative surface charge of the cells. The chemical form of the soluble lead removed from the pouches by bacterial cells existed within the narrow limits of lead solubility through a cyclic phenomenon involving culture pH and carbonate and phosphate equilibria. The initiation of the transfer of lead by the surface charge of cells with a concomitant shift in the solubility equilibrium exerting control over the system illustrated a metal translocation system that should not be uncommon in nature.

Chemical and ultrastructural examination of lead-induced morphological convertants of Bacillus subtilis

Actively-growing Bacillus subtilis 168 cells, exposed to lead nitrate, had only slightly decreased ability to bind the divalent cation magnesium. The nature and quantity of the major cell wall metal binding components, teichoic and teichuronic acids, and the carbohydrate constituents of the peptidoglycan remained relatively constant. Purified cell walls, isolated from cells exposed to lead for 6 and 13 days, retained 9.9 micrograms Pb+2 and 3.5 micrograns Pb+2/mg cell wall, respectively. The occurrence of this lead in the isolated cell wall did not interfere with its Mg2+-binding capacity. While cationic binding properties, growth rate and cell yield indicated non-interference from lead, light and electron microscopic studies clearly demonstrated morphological alterations in approx. 30--50% of the lead treated cells. These alterations included the conversion to irregular spherical forms, some of which contained thickened cell walls. These anomalies are virtually identical to those reported for mutants of this bacterium derived via the introduction of classical mutagens. Protoplasts, similar to those produced by antibiotic and enzymatic treatments, were also present in the lead-treated cells. Although the most tenable explanation appeared to be cellular mutations, the morphological convertants appear to be derived from leads interference with cell wall biosynthesis process and/or the assemblage of cell wall subunits.