S.C. Kushwaha

Novel polar lipids from the methanogen Methanospirillum hungatei GP1

The methanogenic bacterium Methanospirillum hungatei GP1 has been shown to contain two unusual phosphoglycolipids (phosphoglycolipid I and phosphoglycolipid II) that account for 64% (by wt.) of the total cellular lipids. These lipids are derivatives of the dibiphytanyldiglycerol tetraether. One of the free hydroxyls of this tetraether is esterified with glycerophosphoric acid and the other is linked glycosidically to a disaccharide with structure alpha-Glcp-(1 leads to 2)-beta Gal phi in phosphoglycolipid I and beta-Gal phi-(1 leads to 6)-beta Gal phi in phosphoglycolipid II. Smaller amounts of the sn-2,3-diphytanylglycerol analog of phosphatidylglycerol and diglycosyldiphytanylglycerol ethers (DGD-I and DGD-II) containing the same disaccharide residues as in phosphoglycolipid I and phosphoglycolipid II, respectively, were identified, together with very small amounts of diglycosyldibiphytanyldiglycerol tetraethers (DGT-I and DGT-II) containing the same disaccharide residues as in phosphoglycolipid I and phosphoglycolipid II, respectively. A biosynthetic pathway involving head-to-head condensation of phosphatidylglycerol with DGD-I or DGD-II to form phosphoglycolipid I or phosphoglycolipid II, respectively, is proposed.

Isolation and characterization of C50-carotenoid pigments and other polar isoprenoids from Halobacterium cutirubrum

The polar acetone-soluble lipids of Halobacterium cutirubrum were found to contain (in addition to the previously reported vitamin MK-8 and retinal) neo-bacterioruberin U, bacterioruberin, monoanhydrobacterioruberin, bis-anhydrobacterioruberin, an isomer of geranylgeraniol (with one internal cis-isoprene residue), 2,3,-di-O-phytanyl-sn-glycerol and two unidentified polar isoprenoids. All compounds were isolated in pure form by column and thin-layer chromatography, quantitated and characterized by their visible, ultraviolet, infrared, proton magnetic resonance and mass spectra and the spectra of their acetyl or silyl derivatives and/or dehydrated products.

Comparison of purple membrane from Halobacterium cutirubrum and Halobacterium halobium

Direct comparison of purple membrane preparations from Halobacterium cutirubrum and Halobacterium halobium was carried out. Both preparations were found to be essentially identical with respect to their molecular weight, retinal content, lipid composition, fingerprinting of peptides from peptide digestion, electron micrographs and X-ray diffraction patterns, and behaviour as a light-activated proton pump. Thus, there would appear to be no species differences in the purple membranes from these two bacteria.

Isolation and identification of dehydrosqualene and C40-carotenoid pigments in Halobacterium cutirubrum

Abstract 1. 1. The acetone-soluble lipids of Halobacterium cutirubrum were found to consist of about 58% non-polar (hexane-soluble) and 42% polar (95% methanol-soluble) lipids. 2. 2. The non-polar fraction studied here contained, in addition to the previously reported squalenes and vitamin MK-8, the following C 40 -carotenoids: phytoene, cis - and trans -phytofluenes, neo -α-carotene, β-carotene and neo -β-carotene. Small amounts of dehydrosqualene were also detected. 3. 3. All compounds were isolated in pure form by column and thin-layer chromatography and were characterized by their visible, ultraviolet, NMR and mass spectra as well as by their thin-layer Chromatographic mobilities and gas-liquid Chromatographic retention times of their hydrogenated products.

Nutritional control of pigment and isoprenoid compound formation in extremely halophilic bacteria

Summary1.Cells of the extremely halophilic bacteria, Halobacterium cuti-rubrum and H. halobium grown in a chemically defined medium (BSMK) were red due to the presence of bacterioruberins (maxima, 370, 388, 494 and 527 nm). Adding 0.1% glycerol to BSMK stimulated growth, but cells rapidly lost bacterioruberins becoming greyish purple in the stationary phase. Acetone extracts of these cells were yellow with a broad absorption band at 360–390 nm, partly attributable to retinal. In BSMK medium with or without glucose, the bacterioruberin concentration increased until maximal growth was reached, then fell rapidly.2.In complex medium (CM) cells formed less bacterioruberins than in BSMK. Adding 0.1% glycerol to CM stimulated growth but did not change pigmentation; adding glucose only slightly stimulated growth but greatly increased bacterioruberin formation. Exposure to visible light did not affect growth or pigmentation.3.Addition of glycerol or glucose to BSMK increased the formation (relative to squalene) of dihydrosqualene, tetrahydrosqualene, and vitamin MK8. Higher levels of these compounds were found in cells grown in CM than in BSMK. Though glycerol decreased the formation of bacterioruberins it increased the formation of β-carotenes. Glucose increased the formation both of bacterioruberins and β-carotene. A preliminary hypothesis to account for the effects of nutrients on pigmentation is presented.

Modification of phenol-sulfuric acid method for the estimation of sugars in lipids

Tota l sugars in glycol ipids and p h o s p h o glycol ipids have general ly b e e n d e t e r m i n e d by the phenol su l fu r ic acid m e t h o d (1) e i the r on the i n t a c t l ipid or on water so lub le l ipid h y d r o lysates (2). This m e t h o d gives good resul ts w i th water -soluble glycol ipid hyd r o l y s a t e s and wi th i n t ac t acyl es ter glycol ipids (e.g., see ref. 3) b u t was f o u n d to give i ncons i s t en t and i r reproducible resul ts w h e n appl ied to i n t a c t glycol ipids of e x t r e m e l y ha loph i l i c bac te r ia (4) or m e t h a n o g e n i c bac te r i a (5), wh ich are der ived f rom C20-phytany lg lycero l d ie the r or C40glycerol t e t r ae the r , respect ively. I t was f o u n d t ha t m a i n t a i n i n g the t e m p e r a t u r e of the react i on w i th conc. sulfuric acid at 100 C was an i m p o r t a n t f ac to r in ob t a in ing r ep roduc ib le results. The p r o c e d u r e was t h e n mod i f i ed as fol lows: p ipe t an a l iquo t of l ipid so lu t ion or aqueous hyd ro ly sa t e con ta in ing 30-60 /ag sugar (as hexose ) in to a 35-ml Lewis-Benedic t sugar t u b e and evapora te the so lvent to dryness u n d e r a s t ream of n i t r ogen ; to the residue add 2 ml of wa te r and 1.0 ml of 5% p h e n o l so lu t ion , and mix gent ly b y vor tex ing , m a k i n g sure t ha t the fi lm of l ipid at the b o t t o m of t ube is undistu rbed . Add 5 ml of conc. sulfur ic acid wi th vo r t ex ing and t h e n h e a t for 5 m in in a bo i l ing wa te r ba th . V o r t e x the m i x t u r e br ief ly and al low it to cool for 30 min . Read the absorbance of the orange color at 490 n m against a reagen t b lank. Fo r ca l ibra t ion , use s t andards con ta in ing 20, 40 and 80 /ag of hexose or an appropr i a t e m i x t u r e of d i f fe ren t hexoses , depend ing on the k ind of hexoses and the i r mola r ra t ios in the or iginal lipid. Beer s law ho lds in the range 0-80 /lg hexose ; 20 /~g glucose gives an abso rbance of 0 .112. The mod i f i ed m e t h o d has been t e s t ed in this labora to ry on several g lycerol -e ther -der ived glyco-

Structure determination of the squalene, dihydrosqualene and tetrahydrosqualene in Halobacterium cutirubrum

Abstract 1. 1.The structure of the squalene in Halobacterium cutirubrum was shown to be identical with that of authentic all trans-squalene from shark liver. 2. 2 The structure of the bacterial dihydrosqualene has been established as 2,6,10, 15,19,23-hexamethyl-all-trans-2,6,10,l4,18-tetracosapentaene, and that of tetrahydrosqualene as 2,6,10,15,l9,23-hexamethyl-all-trans-6,10,14,18-tetracosatetraene.

Isolation and identification of “bacteriorhodopsin” and minor C40-carotenoids in Halobacterium cutirubrum

Abstract 1. 1. Lycopersene, lycopene and retinal have been identified in cells of Halobacterium cutirubrum . 2. 2. These compounds were isolated in the pure form by column- and thin-layer chromatography and were characterized by their visible-,ultraviolet- and mass spectra, and by their Chromatographic mobilities. Retinal was also characterized by its product from borohydride reduction (retinol). 3. 3. The retinal has been shown to be present in cells of H. cutirubrum in the form of a ‘bacteriorhodopsin” complex which was characterized spectrophotometrically by its bleaching behavior and extractability towards organic solvents and detergents.

Lipid composition ofNeurospora crassa

The lipids ofNeurospora crassa, isolated in pure form from freeze-dried mycelium, were found to contain squalene, sterol esters, triglycerides, free fatty acids, geranylgeraniol, free sterols, carotenoids, cardiolipin, phosphatidyl ethanolamine, phosphatidyl choline, phosphatidyl serine, and phosphatidic acid. The above compounds were isolated in pure form by column and thin layer chromatography and were characterized by infrared spectroscopy and chromatographic mobilities. Fatty acid moieties were characterized by gas liquid chromatographic retention times of their methyl esters relative to those of authentic standards. The fatty acid composition of the triglycerides was found to be similar to that of phosphatidic acid, cardiolipin, and lecithin.

The terpenyl pyrophosphates of wild type and tetraterpene mutants ofNeurospora crassa

Mutations to two albino loci ofNeurospora (al-2 andal-3) are known to block tetraterpene synthesis prior to phytoene but after farnesyl pyrophosphate. Analysis of the terpenyl pyrophosphates labeled with [214C] mevalonic acid revealed the presence of radioactive prephytoene, pyrophosphate in both albino mutants and in the corresponding wild type strain. Theal-2 andal-3 mutations are thus associated with lesions between prephytoene pyrophosphate and phytoene.