Phospholipids of Nocardia coeliacaIKUYA YANO, YOSHIYA FURUKAWA, AND MASAMICHI KUSUNOSE
Research Laboratory ofBiochemistry, Toneyama Hospital, National Sanatorium, Toyonaka, Osaka, and ToneyamaInstitute for Tuberculosis Research, Osaka City University Medical School, Toyonaka, Osaka 560, Japan
Received for publication 21 January 1969
The lipids of Nocardia coeliaca were separated into at least 10 components by theuse of thin-layer chromatography. Phosphatidylcholine was the most abundantphospholipid in this organism, accounting for 25 to 40% of the total phospholipids.The major fatty acid components of the phosphatidylcholine were 14-methyl-pentadecanoic acid (41 %), the other C15 and C17 iso- and anteiso-fatty acids (29%),and palmitic acid (13.5%). The next most abundant phospholipid was phospha-tidylethanolamine (25 to 30%), followed by phosphatidylinositol (11 to 14%) andcardiolipin (7 to 15%). Phosphatidylethanolamine and phosphatidylinositol werevery similar to the phosphatidylcholine in fatty acid composition, whereas cardio-lipin was characterized by a higher content of palmitic acid (30%). In all of the phos-pholipids examined, only trace amounts of monounsaturated fatty acids werepresent. When washed cells of N. coeliaca were incubated with methionine-methyl-"4C for 1 to 3 hr, the radioactivity was mainly incorporated into the choline moietyof the phosphatidylcholine. In contrast, acetate-1-14C or glycerol-1-14C was incorpo-rated much more slowly into the phosphatidylcholine than into the other phospho-lipids and neutral lipids. No phosphatidylcholine was detected in 10 other species ofNocardia examined.
There have been several investigations on thelipids of mycobacteria (2, 3). In contrast, rela-tively little information is available concerningthe lipids of Nocardia, the bacterium most closelyrelated to the mycobacteria (5, 20). In a previouspaper (32), we reported that N. polychromogenescontained four major phospholipids (cardiolipin,phosphatidylethanolamine, phosphatidylinositol,and phosphatidylinositol monomannoside) hav-ing 10-methylstearic acid residue as the majorfatty acid component. However, later studiesrevealed that not all the species of Nocardiapossess identical lipid and fatty acid compositions.In this paper, we show that N. coeliaca containsphosphatidylcholine as the most abundant phos-pholipid. To our knowledge, this report is thefirst to demonstrate the existence of phosphatidyl-choline in the Actinomycetales.
MATERIALS AND METHODSGrowth of organism. A strain of N. coeliaca kindly
supplied by M. Mayama, Shionogi Research Labora-tories, Osaka, Japan, was used. N. polychromogenes,N. asteroides, N. erythropolis, N. leishmanii, N.corallina, N. rubra, N. transvalensis, N. flava, andN. madurae were also kindly donated by M. Mayama,and N. lutea, by S. Fukui, Faculty of Engineering,Kyoto University, Kyoto, Japan. The medium usedcontained 1% polypeptone (Daigo-eiyo Chemical
Co., Osaka, Japan), 0.5% yeast extract (Difco), and1% glucose; the pH was adjusted to 7.0. The cellswere inoculated from a 3-day subculture of the samemedium, and were incubated at 30 C on a rotaryshaker for 25 to 100 hr.
Separation of cellular lipids. Lipids were extractedwith 10 volumes of chloroform-methanol (2:1, v/v)and washed by the method of Folch et al. (7); theywere then concentrated with a rotary evaporator be-low 50 C, and finally were chromatographed on thin-layer plates (0.5 mm thick) of Silica Gel H (MerckCo., Darmstadt, Germany), in chloroform-methanol-acetic acid-water (85:15:10:4, v/v). Total lipids weredemonstrated by charring the plates at 250 C for 15min, after spraying them with 18 N H2SO4. Phos-phorus, amino groups, reducing sugars, and cholinewere detected with Dittmer's reagent (6), ninhydrin,anthrone, and Dragendorff's reagent (30), respec-tively. Each phospholipid band was detected with2',7'-dichlorofluorescein, scraped off with a razorblade, and then recovered from the plates withchloroform-methanol (1:2, v/v).
Analysis. Lipid phosphorus was determined by themethod of Bartlett et al. (4). The lipids were hydro-lyzed in 3 N HCl overnight, and the resulting water-soluble base was separated by paper chromatographywith the solvent of n-butyl alcohol-phenol-80% formicacid-water saturated with KCI (50: 50:3:10, v/v).
Gas-liquid chromatographic analysis of the methylesters of fatty acids was carried out by use of aPackard instrument with argon ionization detector.
The column packed with 15% ethyleneglycoladipatepolyester on Chromosorb W was operated at 185 Cwith a flow rate of 50 ml/min. The methyl esters offatty acids were obtained by transmethylation of thelipids with 5% HCl-methanol for 3 hr, and wereidentified by comparison with the retention time ofauthentic standards.
Incubation studies. Flasks containing 2 ,uc of 14C-labeled precursor (methionine-methyl-l4C, acetate-1-14C, or glycerol-1-14C), 1 mmole of phosphatebuffer (pH 7.0), and the washed-cell suspension ofN. coeliaca (114 mg, dry weight) in a final volume of5 ml were incubated with vigorous shaking at 30 Cfor 1 to 3 hr. After the reaction was stopped by theaddition of 10 volumes of chloroform-methanol(2:1, v/v), the lipids were extracted, washed tho-roughly with water to remove any free labeled precur-sor, and chromatographed on a thin-layer plate; thenthe radioactivity in the lipids was measured with athin-layer chromatogram scanner (Nippon-musen Co.,Tokyo, Japan). The per cent distribution of radio-activity in each lipid component was calculated fromthe peak area of radioactivity on the thin-layerchromatogram.
Infrared spectrum analysis of phospholipids wasperformed with a Hitachi ETI-G-type infrared spec-trophotometer (Hitachi Co., Tokyo, Japan) as a thinfilm on KBr discs.
Materials. All chemicals used were of the highestpurity commercially available. Organic solvents wereredistilled before use. The lipids and fatty acids foruse as reference standards were purchased from Ap-plied Science Laboratories, State College, Pa.13-Methyltetradecanoic acid was generously donatedby K. Saito, Kansai Medical College, Osaka, Japan.10-Methylstearic acid (tuberculostearic acid) was ob-tained through the courtesy of J. Cason, University ofCalifornia. Radioactive compounds were obtainedfrom Dai-ichi Pure Chemical Co., Tokyo, Japan.
RESULTSChromatographic separation of the lipids of N.
coeliaca on a thin-layer plate. When the totallipids of N. coeliaca were chromatographed on athin-layer plate of silica gel, at least 10 differentcompounds were observed (Fig. 1). Individuallipid components were tentatively identified on thebasis of the specific color reaction and the RFvalues of water-soluble deacylation products onpaper chromatography (Table 1).
Spot a: neutral lipids. This spot did not reactwith any reagent for detection of phosphorus,sugars, and free amino groups. It was shown tocontain glycerides and free fatty acids with traceamounts of pigments, by the use of thin-layerchromatography with the solvent of hexane-ether-acetic acid (90:10:1, v/v).
Spot b: glycolipid. This spot reacted withanthrone reagent, and its RF was the same asthat of monogalactolipid isolated from planttissues (24).
N. coeliaca and N. polychromogenes. The plates weredeveloped with chloroform-methanol-acetic acid-water(85:15:10:4, v/v), and the spots were demonstratedby spraying with 50% H2S04 and charring on blue-linediazopaper. (1) N. coeliaca lipids; (2) phosphatidyl-ethanolamine (upper) and phosphatidylcholine (lower)from egg yolk; (3) N. polychromogenes lipids. (a)Neutral lipid; (b) unknown glycolipid; (c) cardiolipin;(d) phosphatidylethanolamine; (e) phosphatidylglycerol;(f) phosphatidylcholine; (g) phosphatidylserine; (h)phosphatidylinositol; (i) phosphatidylinositol mono-mannoside overlapped with peptide lipid; (j) unknownpeptide lipid; (k) origin.
Spot c: cardiolipin. This spot contained phos-phorus. The RF value of the water-soluble productobtained after mild alkaline hydrolysis was thesame as that of the sample obtained by hydrolysisof Mycobacterium tuberculosis polyglycerophos-phatide.
Spot d: phosphatidylethanolamine. This spotgave a positive ninhydrin reaction and containedphosphorus. Mild alkaline hydrolysis yielded aproduct with the same RF value as that of eggyolk glycerylphosphorylethanolamine on paperchromatography.
unidentified component was very close to that ofphosphatidylglycerol.
Spot f: phosphatidylcholine. As indicated inFig. 1 and 2, the RF of this spot coincided withthat of egg yolk phosphatidylcholine. The occur-rence of phosphatidylcholine in Actinomycetaleshas not been demonstrated previously (13, 19).To determine whether the presence of phosphati-dylcholine is common to genus Nocardia, 10other strains (N. polychromogenes, N. asteroides,N. erythropolis, N. leishmanii, N. corallina, N.rubra, N. transvalensis, N. flava, N. madurae,and N. lutea) were also examined. However,phosphatidylcholine was not detected in any ofthese species. A thin-layer chromatogram of thelipids of N. polychromogenes (32) is shown inFig. 1, in comparison with that of the lipids ofN. coeliaca. Spot f contained phosphorus andreacted with Dragendorf reagent. The water-soluble deacylation product of this spot gave thesame RF as that of egg yolk phosphatidylcholineon paper chromatography. Acid hydrolysis of thisspot yielded choline. The infrared spectrum ofspot f showed good agreement with that of eggyolk phosphatidylcholine, in absorption at 2,920,2,840, 1,030, and 970 cm-'; a slight shift of esterabsorption at 1,740 cm-' presumably suggests theoccurrence of iso- and anteiso-fatty acid esters inthe molecule (27).
Spot g: glycolipid and phosphatidylserine.Further identification of these components isneeded. A compound similar to glycolipid wasfound to be abundant in N. polychromogenes,and its characterization will be described in asubsequent paper.
Spot h: phosphatidylinositol. The chromato-graphic behavior of this spot was the same asthat of phosphatidylinositol from N. polychromo-genes (32).
Spot i: phosphatidylinositol monomannoside andpeptidolipid. This spot gave positive reactions foranthrone and ninhydrin reagents, with faintcoloration for phosphorus, possibly suggestingthe overlapping of phosphatidylinositol mono-mannoside and lipids which contain amino acids.On hydrolysis, this spot gave mannose and aminoacids including leucine and isoleucine.The relative proportion of the individual phos-
pholipids was as follows: cardiolipin, 7 to 15%;phosphatidylethanolamine, 25 to 30%; phos-phatidylcholine, 25 to 40%; phosphatidylinositol,11 to 14%; phosphatidylserine, 2 to 6%; phos-phatidylinositol monomannoside, 2 to 4%. Inconclusion, phosphatidylcholine was proved tobe the most abundant phospholipid in N. coeliaca,although the amounts of the individual phospho-lipids varied considerably at different stages andunder different growth conditions.
Fatty acid composition of the individual lipids.The fatty acid composition in N. coeliaca wasdetermined by use of gas-liquid chromatography.The logarithms of the retention times of knownmixtures of saturated, monoenoic, iso, anteiso,and tuberculostearic acid-type branched andcyclopropanoic fatty acid methyl esters wereplotted against the number of carbon atoms, andthe points were connected by straight lines. Theunknown bacterial fatty acids were tentativelyidentified by their position on these graphs.From these relationships, it was revealed thatat least four series of fatty acid homologues,saturated, iso, anteiso, and tuberculostearic acid-type fatty acids, occurred in N. coeliaca lipids.Table 2 lists the distribution of fatty acids in theindividual lipids. The major fatty acids werenormal saturated (C,6 and Ci8) fatty acids and isoand anteiso branched-chain (C,5 to C,9) fattyacids. Tuberculostearic acid-type fatty acids (C,7
___q ~ coeliaca, incubation studies with radioactive pre-cursors were carried out. After the resting cellswere incubated with methionine-methyl-14C, thelipids were separated on a thin-layer plate, fol-lowed by the measurement of radioactivity.Figure 3 shows that the radioactivity of methio-nine-methyl-14C was mainly incorporated intophosphatidylcholine and to a lesser extent intoother phospholipids. As illustrated in Fig. 4, theradioactivity in phosphatidylcholine increasedcontinuously during 3 hr of incubation. After
0_ acid hydrolysis of the labeled lipids, the radio-activity was exclusively recovered in the water-soluble phase. A paper radiochromatogram of thewater-soluble product thus obtained indicatedthat the major radioactive peak was associatedwith the spot of authentic choline. These resultsindicate that N. coeliaca is able to synthesizephosphatidylcholine from methionine. For pur-poses of comparison, acetate-1-14C or glycerol-1IC was used as the common precursor of lipidbiosynthesis. When the washed cells were incu-bated with these precursors, higher levels ofradioactivity were found in phosphatidylethanol-amine, cardiolipin, and neutral lipids; incorpora-tion into phosphatidylcholine and phosphatidyl-inositol occurred to a much lesser extent, in
ll) (2) (3) (4) (5) contrast to the pattern of incorporation of methio-FIG. 2. Thin-layer chromatogram of the phos- nine-methyl-14C. At the initial stage of the incuba-
phatidylethanolamine and phosphatidylcholine from tion, the proportion of incorporation was higherN. coeliaca. Spots d andfshown in Fig. I were scrapedout, eluted with chloroform-methanol (1:2, v/v), andrechromatographed on a thin-layer plate. (1) Spot d inFig. 1; (2) phosphatidylethanolamine from yeast; (3)phosphatidylcholine from yeast; (4) phosphatidylcholinefrom egg yqlk; (S) spotf in Fig. 1. The developing con-ditions were the same as in Fig. 1.
and C18) were found as the minor fatty acids inphospholipids. Only trace amounts of monoenoicfatty acids were detected by thin-layer chromatog-raphy on AgNO8-impregnated silica gel (14).However, no polyunsaturated fatty acids werefound.
These results preclude the possibility that theculture of N. coeliaca might have been con-taminated with fungus-like organisms containingphosphatidylcholine, because fungi, like animalsand plants, contain polyunsaturated fatty acids.Further, it is noted that, in N. coeliaca, phos-phatidylcholine, phosphatidylethanolamine, andphosphatidylinositol were very similar in fattyacid composition whereas cardiolipin was char-acterized by much higher contents of palmiticacid (Table 2).
Incorporation of the radioactive precursors intothe individual lipids. To demonstrate the biosyn-thetic activity of phosphatidylcholine in N.
TABLE 2. Fatty acid composition of the individuallipid classes from N. coeliaca"
FIG. 3. Radioactive scan of the lipid fromcoeliaca after incubation with methionine-methyl-'for 3 hr at 30 C. Experimental details are describedMaterials and Methods. Thin-layer plate, Silica Gel(0.5 mm); solvent, chloroform-methanol-acetic acwater (85:15:10:4, v/v). (A) Neutral lipids;unidentified; (C) cardiolipin; (D) phosphatidylethanomine; (E) unidentified; (F) phosphatidylcholine;phosphatidylinositol; (H) origin.
Nocardia in the absence of chromogenesis. Re-cently, Ikawa (13), reviewing bacterial phospho-lipids, observed that lecithin is present in bacteriarequiring highly efficient electron transport, andhe also speculated that lecithin-containing bac-teria might be the more advanced form in the
2 evolution. Hagen et al. (11) suggested the correla-c tion between bacterial lecithin and intracytoplas-
mic membrane structure, and demonstratedlecithin to be present in Hyphomicrobiwn andNitrocystis oceanus, organisms which have suchcomplex structures (9, 11). Therefore, it may beof great interest to determine whether there are
K significant differences between N. coeliaca andother species of Nocardia in electron-transportsystem or intracellular structure.
Furthermore, the fatty acid composition of N.coeliaca is very characteristic. The most com-
N. monly occurring iso- and anteiso-fatty acids in14C gram-positive bacteria are 12- and 13-methyltetra-f in decanoic acids (15, 23, 28, 29), whereas that inH N. coeliaca is 14-methylpentadecanoic acid. Inid- the lecithin of N. coeliaca, 14-methylpentadeca-(B) noic acid accounted for 41 % of the total fattyPla-(G)
in neutral lipids than in phospholipids. As theincubation time became longer, the radioactivityin phosphatidylethanolamine and cardiolipinincreased markedly (Fig. 5). After hydrolysis, theradioactivity incorporated from acetate--1-4C orglycerol--1 4C was recovered in the ether- andwater-soluble phases, respectively.
DISCUSSIONLecithin (phosphatidylcholine) is known to be
the most abundant phospholipid in animals andplants. By contrast, this phospholipid appears tooccur in only relatively limited groups of bacteria(8, 9, 17, 21, 26). There have been no reportssupporting the occurrence of lecithin or its re-lated biosynthetic intermediates (19) in theActinomycetales. Laneelle et al. (20) reportedthat the phospholipids separated from Nocardiadid not contain choline. Kataoka and Nojima(18) found that the phospholipids ofStreptomycesgriseus, N. polychromogenes, and Microbispora,as well as those of Mycobacterium, consisted ofcardiolipin, phosphatidylethanolamine, and phos-phatidylinositol mannoside, but not lecithin. Wealso found that 10 different species of Nocardiahad no lecithin, unlike N. coeliaca. At present, wehave no satisfactory explanation for such distinc-tive features of the lipid composition of N.coeliaca, although this organism, in the seventhedition of Bergey's Manual published in 1957,was described to differ from the other species of
50
I-
-J4c
0I-i
IL0U.
40
30
20
to
30 60
TIME (mitt)FIG. 4. Distribution of radioactivity among the
individual lipid classes during incubation with methio-nine-methyl-14C. Experimental details are described inMaterials and Methods. Symbols: 0, phosphatidyl-choline; A, phosphatidylethanolamine; 0, neutrallipids; *, cardiolipin; A, phosphatidylinositol; 0,
FIG. 5. Distribution of radioactivity among theindividual lipid classes during incubation with acetate-l-14C. Experimental details and symbols are the same as
those described in Fig. 4.
acids, but 13-methyltetradecanoic and 15- and14-methylhexadecanoic acids comprised only9.5, 6.5, and 12.6%, respectively (Table 2). Thelecithin in animals (1), plants (10), algae (25),and yeast (I. Yano, Y. Furukawa, and M.Kusunose, unpublished data) has been recognizedto contain generally highly unsaturated fattyacids as the major components. Further, thelecithin of Agrobacterium (12) and photosyntheticbacteria (31) was reported to contain a largeamount of 18-carbon monoenoic acid. However,in the lecithin of N. coeliaca, only trace amountsof unsaturated fatty acids were detected; on theother hand, branched-chain fatty acids repre-
sented 87% of the total fatty acids (Table 2).These findings lead us to suggest that the lecithinin N. coeliaca might be substantially differentfrom its counterparts in other organisms, inchemical or physical properties.The results obtained from the incorporation of
methionine-methyl-'4C into lecithin suggest thatN. coeliaca possesses a pathway for the biosyn-
thesis of lecithin by stepwise methylation (16,22). It should be noted that the incorporation ofacetate-1-P4C or glycerol-1-_4C was much slower inlecithin than in other lipids. It has been reportedthat in animals and plants lecithin had a veryhigh turnover rate among various phospholipids(1, 10, 25). Therefore, it may be suggested thatthe lecithin in N. coeliaca plays a more importantrole with regard to the structure of the cells.Similar results were found by Lascelles andSzilagyi (21) in studies of Rhodopseudomonas, inwhich phosphatidylglycerol, rather than lecithin,was the most metabolically active phospholipid.
ACKNOWLEDGMENTS
We express our gratitude to M. Mayama, Shionogi ResearchLaboratories, Shionogi Co., Osaka, Japan, for his valuable sug-gestions and his generous gifts of many strains of Nocardia, andalso to Y. Noda, Laboratory of Chemistry, Osaka City UniversityMedical School, Osaka, Japan, for his valuable performance ininfrared spectra.
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