A Complex Lipid with a Cyclic Phosphate from the Archaebactefium Natronococcus Occultus

Biochimica et Biophysica Acta, 1001 (1989) 31-34 31 Elsevier

BBA 53013

A complex lipid with a cyclic phosphate from the archaebactefium Natronococcus occultus

Virginia Lanzotfi ~, Barbara Nicolaus ~, Antonio Trincone ~, Mario De Rosa 1,2, William D. Grant 3 and Agata Gambacorta t lstituto per la Chimica di Molecole di lnteresse Biologico, dei Consiglio Nazionale delle Rieerche, Naples, 2 lstituto di Biochimica delle Macromolecole dell'Universita; Naples (Italy) and ~ Department of Microbiology,, University of Leicester, leicester (U.K.)

(Received 14 September 1988)

Key words: Archaebat ~erium; Phospholipid; Cyclized phosphate; Complex lipid; Haloalkaliphile; (N. occultus)

Polar lipid extract from the haloa|kaHphWe archaebacterium Natronococcus oceultus contains a new type of phospholipid. Spectroscopic methods establish file strac~re as a phosphatidylg|ycero! phosphate derivative wieh a cyclic phosphate (2,3.dbO.phytanyl.sn.glyeere-bphosphoryl-3'-sn=glyeero=l',2"=cyelie phosphate). A 2-O-seste~erpanyb3-O- phytanyl (Czs,Cz0) glycerol diether form of ~he novel phospholipid is also present.

Introduction Recently, spectroscopic methods, including 13C- NMR, have been used to unequivocally establish the All halophilic archaebacteria have lipids based on structures of C20,C20 and C25,C,0 derivatives of PG and 2,3-di-O-phytanyl-sn-glycerol (C20,C20) [1], and the PGP from Natsonobacterium and Natronococcus spp. haloalkaliphilic isolates also have substantial amounts [8]. Here, we report a similarly detailed characterization of lipids based on 2-O-sesterterpanyl-3-O-phytanyl-sn- of the polar lipid PL2 from Nc. oecultus. glycerol (C2s,C2o) [2,3]. The complex lipids of these organisms have diphytanyl glycerol linked to different polar groups, such as glycerol phosphate, glyce,ol sulfate, glycerol diphosphate, sugars and sugar sulfates [4-6]. The haloalkaliphilic archaebacteria of the genus Natronobacterium and Natronococcus have a relatively simple polar lipid composition, the major species of polar lipids being C20,C20 and C2~,Cz0 derivatives of phosphatidylglyeerol (PG) and phosphatidylglycerol phosphate (PGP) [7,8]. However, there are also several minor unidentified phospholipids present [7,91 in exam- ples of these genera. In particular, Natronococcus oc- PG cultus has a significant amount of a phospholipid desig- nated PL2 by Morth and Tindall [91. This phospholipid O has a mobility intermediate between those of PG and PGP when analysed by TLC in CHCI3/MeOH/H:O (65 : 25 : 4, v/v.) (Fig. 1).

Abbreviations: PG, phosphatidylglycerol; PGP, phosphatidylglycerol mOrigin phosphate. o b c Correspondence: A. Gambacorta, lstituto per la Chimica di Molecole Fig. l, TLC oi nixture of standards PG and PGP (a); extract of polar di lnteresse Biologico del Consiglio Nazionale delle Ricerche, Via lipid of Natro,~ococcus occultus (b); PL2 (TLC-pure) (Fig. 2) (c). Toiano 6, 80072-Arco Felice, Naples, Italy. Solvent system CHCI 3/MeOH/H :O (65 : 25 : 4, v/v).

Materials and Methods Quantitative analysis Phosphorus was determined by the method of Ames Microorganism and culture conditions [14] and glycerol was assayed enzymatically [15]. Natronococcus occultus (NCMB 2192) was obtained Glycerol diethers were determined gravimetrically after from the National Collection of Marine Bacteria, Terry drying to constant weight. Research Station PO Box 31, Aberdeen AB9 8DG, U.K., and was grown as described by TindaU et al. [7]. I ~C.NMR spectroscopy Cells were harvested in the late exponential phase by Samples for NMR analysis were prepared by dissolv- centrifugation, washed with a basal salts solution, and ing each sample in 0.5 ml C2HCI3/C2H302H (7:3, iyophilized. v/v). All the 13C-NMR spectra were run at 125 MHz on a Extraction and isolation of lipids Bruk¢~'r WH-500 spectrometer. ~3C Fourier transform Lipids were extracted from wet cells (30 g) with NMR spectra, with sweep widt~ and transmitter fre- CHCI3/MeOH (1 : 1, v/v) and MeOH/H20 (1 : 1, v/v) quencies optimized for the lowest possible digital reso- as described elsewhere [8], purified on a silica gel col- lution (Hz/data point), were obtained from 32 K free unto (70 8, Kieselgei, 70-230 mesh, Merck) (40 cm × 10 inducl:ion decay signals. Exponential multiplication was nun, i.d.). The column was eluted with 3 I of a step applied previously by Fourier transformation, and line gradient of 0-50~$ (v/v) MeOH in CHCi 3 in 5~ incre- broade, ning constants were adapted for the digital reso- ments (250 ml for each gradient step). The minor com. lution, pound PL2 was e!uted with CHCI3/MeOH (95 : 5, v/v) AnAlysis of multiplicities was achieved by distortion- and the major polar lipids PGP and PG were eluted, less enhancement by the polarization transfer (DEPT) respectively, with CHCI3/MeOH (90:10, v/v) and technique, using a commercially available micropro- CHCI3/MeOH (85:i5, v/v), and all were weighed to gramme. Two DEPT experiments were performed using evaluate their percentages [8]. polarization transfer pulses of 90 and 135 ° , respectively, obtaining in the first case only signals for methine Chemical procedures groups and in the other case positive signals for methine The lipids were permethylated with dimethylform- and methyl groups, and negative signals for methylene amide/NaH/Mel using a standard procedure [10]. groups. Acid methanolysis of lipids was done in dry methaaolic 2 M HCI. The reaction mixture was heated at II0°C in a stoppered reaction tube for 16 h [11]. Mass spectrometry After being cooled, the hydrolysis products were dried MS analyses were done with a Kratos MS-50 instru- under vacuum and then extracted with CHCI3/H~O ment equipped with a Kratos fast atom bombardment (1:1, v/v). After thorough mixing, the CHCI~ phase (FAB) source. The negative FAB mass spectra were was analysed by HPLC using a Waters Associates ap- obtained by dissolving the samples in a glycerol matrix paratus equipped with a differential refractometer. 2,3- on the probe prior to bombardment with Argon atoms Di-O-phytanyi-sn-glycerol and 2-O-sesterterpanyl.3-O- having a ldnetic energy equivalent to 2-6 keV. phytanyl-sn-glyeerol were detected in the CHCI 3 phase by elution with n-hexane/diethyl ether (8: 2, v/v) using Res~ and Discussion a MieroporasU column (flow rate 1 ml/min) [3]. Glycerol was identified in the aqueous phase by elution with The new phospholipid PL2, eluted with CHCi3/ acetonitrile/H20 (9: 1, v/v) on a carbohydrate column MeOH (95 : 5, v/v), represents approx. 10~ of the lipid (flow rate 2 mi/min). extract and has an R F --- 0.3 on TLC (CHCI3/MeOH / H20, 65 : 25 : 4, v/v) (Fig. 1). Thin-layer chromatography Acid methanolysis yielded glycerol, phosphate and Polar lipids were analysed by two-dimensional thin- glycerol diether in the molar ratio shown by PGP, i.e., layer chromatography on silica gel (0.25 ram, F 254, 1:2:1. Glycerol and phosphate were quantified and Merck) using as solvent system CHCi~/MeOH/H20 identified as described in Materials and Methods; (65:25:4, v/v) in the first dimension, and CHCl3/ glycerol diethers were quantified and identified by com- McOH/CH~COOH/H20 (80: 12:15 : 4, v/v) in the parison with authentic samples [2]. The proportions of second dimension [12]. the C25,C20 and C20,C20 forms of PL2 were determined Compounds were detected by exposure to 12 vapour, by HPLC as 38:62, respectively, after hydrolysis to or by spraying with 0.1~ Ce(SO4)2 (w/v) in 2 M release the core structure similar to those reported for H2SO4, followed by heating at 150°C for 5 rain. Ditt- PG and PGP derivatives [8]. mer and Lester reagent [13] was also used for phos- Negative fast atom bombardment-mass spectra of pholipids. PL2 showed the same peaks as PG, i.e., identical to 33 those of PGP following the loss of the additional phos- O- i phate group [8]. ~CH2-O-P--O--R ~3C-NMR assignments are reported in Table l. ~3C ~ 0 data were obtained from a proton noise decoupled spectrum and from DEPT experiments that allowed us to determine the multiplicity of each signal. ~3C-NMR spectra of POP and PL2 showed practically identical PGP PL2 resonances, except that in PL2, carbon 2' was shifted by 4.3 ppm and appeared as a doublet with Jc-o-P = 1 0-- I H,~ in the proton noise decoupled L~C-NMR spectrum. ~'CH2-O--P-O- ~'CH2--O ,/0- II "" These results suggested that the structure of PL2 ° ,, H.... OH ~H--o/P%0 differed from PGP only in an additional substitution on / carbon 2' of the second glycerol molecule. Since the R = --a 2 R = --3'C H 2 quantitative analyses indicated a phosphate composition identical to PGP, the most likely explanation was Fig. 2. Chemical structures of C20,C2o (n =1) and C25,C20 (n = 2) that a single terminal phosphate group was linked both PGP and PL2. PGP, 2,3-di-O-phytanyl-sn-giycero-l-phosphoryl-3'- sn-glycero-l'-phosphate. PL2, 2,3-di-O-phy~anyl-sn-glyccro-l-phos- to carbon 1' and to calbon 2' of the second glycerol phoryl-3'-s,-glycero-l',2'-cyclic phosphate. C25,C~0 forms have molecule. sesterterpanyi chains replacing phytanyl chains on C-2 of the glycerol moiety.

Confirmation of such a structure was obtained by TABLE I comparing the permethylated derivative of PL2 with SsC.NM R assignments for phospholipid PL2 that of PGP. Permethylation of PGP produced a deriva- 13C-NMR spectra were recorded in C2HCI3/C2H302H (7:3, v/v) tive in which the chemical shift of C-2' was shifted from at 125 MHz; the chemical shifts (8) are in ppm with respect to 8 70.3 to 8 78.4, thus indicating that this position was tetramethylsilane. Chenfical shifts data on carbons 21-25 of the isopranic unit in the C2s chain were the same as carbons 16-20 in the methylated. A new signal also appeared at 8 58.4 due to C20 chain. Multiplicities were obtained from DEPT experiments: the presence of the corresponding methoxyl group [16]. s = singlet; d -- doublet; t = triplet; q = quartet. The corresponding derivatized PL2 preparation showed no change in any of the assignments. Accordingly, we Position Carbon atom a PGP b PL2 believe that these results indicate that the structure of PL2 is as shown in Fig. 2. Glycerols 1 65.7 ¢ 65.5 c t This is the first report of a cyclized phosphate in an 2 78.3 78.1 d archaebacterial complex lipid. Such compounds are pro- 3 71.2 71.5 t 1' 66.6 c,a 66.8 ¢,d t duced as intermediates during the mild acid hydrolysis 2' 70.3 74,6 c d of glycerol ester phospholipid derivatives that possess a 3' 66.7 c.d 66.9 c.d t free hydroxyl adjacent to phosphoric acid [17], but we lsopranoid chains 1 69.4 69.5 t are not aware of their participation in the biosyntheses la 70.7 70.8 t or bioconversions of such phospholipids. It is unlikely 2 37.3 37.4 t that PL2 is an intermediate in phospholipid biosynthe- 2a 37.8 37.9 t sis or turnover in Nc. occultus, since it is present in 3 30.4 30.3 d significant amounts at various stages during the growth 3a 30.6 30.6 d 4,6,8,10,12 38.0 38.1 t of the organism. Furthermore, we have not detected tiffs 5 25.0 25.1 t phospholipid in lipid extracts from other halophilic 7,11 33.3 33.4 d archaebacteria (all of which contain PG and PGP). The 9 24.9 25.0 t function of this structure in relation to those of PG and 13 25.3 25.5 t PGP remains to be established. 14 39.9 40.1 t 15 28.5 28.6 d 16 20.1 20.2 q Acknowledgements 17,18 20.2 20.3 q 19 22.9 ~ 23.1 ~ q We thank Mr. E. Esposito and Mr. G. Pinch for their 20 23.0 ~ 23.2 e q technical assistance, and Mr. R. Turco for the art work. a The carbon atom numbers refer to those given in Fig. 2. b De Rosa ¢t al. [8]. Reierences c In the proton noise decoupled 13C-NMR spectrum, signals are doublets with Jc-o.v = 1 Hz. 1 Kates, M. (1978) Pro~. Chem. Fats Other Lipids 15, 301-342. d Assignments are interchangeable with one and other. 2 De Rosa, M., Oambacorta, A., Nicolaus, B., Ross, H.N.M., Orant, e Assignments are interchangeable with one and other. W.D. and Bu'Lock, J.D. (1982) J. Gen. Microbiol. 128, 343-348. 34

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