Sulfolipid I of Mycobacterium tuberculosis, strain H37Rv I. Purification and properties

116

BIOCHIMICA

ET BIOPHYSICA

ACTA

BBA 55702

I OF MYCOBACTERIUM

SULFOLIPID

I. PURIFICATION

MAYER

TUBERCULOSIS,

STRAIN

H37Rv

AND PROPERTIES

B. GOREN

Division of Research, National Jewish Hospital and Research Center, and Department Microbiology, University of Colorado School of Medicine, Denver, Cola. (U.S.A.) (Received

December

of

2nd. 1969)

SUMMARY

A sulfur-containing lipid extracted from virulent human terium tuberculosis and previously described by MIDDLEBROOK

strains of Mycobacand co-workers has

been shown to consist of several families of sulfolipids, many of which are structurally related. The most abundant class, sulfolipid I, is obtained by DEAE-cellulose column chromatographic procedures. This substance is a complex glycolipid ester of average empirical formula C,,,H,,,O,,NS, molecular weight 2400; the carbohydrate moiety bears a single sulfuric acid half ester substituent, in a secondary equatorial position. The ammonium salt undergoes spontaneous desulfation under such innocuous circumstances as mere dissolution in ether. Strains of Mycobacterium bovis which have been examined elaborate only minute amounts of similar substances; none appear to be identical

with the principal

sulfolipid

of human

strains.

INTRODUCTION

MIDDLEBROOK

et al.1 have described an anionic sulfur-containing

from strains of virulent Mycobacterium decylamine. Hexane solutions containing

lipid extracted

tuberculosis, var. hominis by hexane-o.rO/ this sulfolipid extracted neutral red from an

aqueous phase into the organic layer and the amount of dye fixed was proportional to the specific radioactivity of lipids labeled with 3. Similar extracts of avirulent or attenuated strains showed little neutral red activity and correspondingly little radiosulfur in the lipids extracted by the very mild solvent. In short, the data suggested that the sulfolipid had a role in the cytochemical neutral red-fixing activity of viable, cord-forming virulent tubercle bacilli, in cord formation, and in the pathogenesis of tuberculosis. Subsequent investigations of IT0 et ak2 dealt with a partial purification of sulfolipid from H37Rv. GANGADHARAM et aL3 established a correlation between the levels of sulfolipid elaborated by different strains of M. tuberculosis and their order of infectivity for the guinea pig. Biochim. Baophvs. Acta, 1x1 (1970) 116-126

M. t&YCulOSiS SULFOLIPID1: PURIFICATION

117

As part of a continuing program on the surface chemistry of mycobacteria begun in collaboration with MIDDLEBROOK,we have extended the work of the earlier investigators in an effort to determine the chemical structures and the relationships between what has now been characterized as a family of sulfolipids. This report deals with the isolation and purification, and with certain unusual properties of the most abundant sulfolipid class from the H37Rv strain. The succeeding paper deals with structural studies which show that this principal sulfolipid is a complex 2,3,6,6’tetraester of trehalose. The average molecular weight is very near 2400, and the carbohydrate moiety is sulfated at the z’-position. The suggested gross molecular structure of ammonium sulfolipid I from H37Rv is pictured in Fig. I.

Fig’. I. Tentative gross molecular structure of ammonium sulfolipid I from M. t~~eyc~Zos~s,Strain H37Kv: approximate molecular formula C &t&&,NS. (Cf. NOTE ADDED IN PROOF)

MATERIALS Solvents were purified by appropriate distillation. Thin-layer chromatographic plates of silica gel-magnesium silicate were prepared and activated according to ROUSER et al.&. Plates spotted with sulfolipid samples were developed with chloroform-acetone-methanol-water-acetic acid (158 : 83 : I : 6: 32, by vol.) (referred to as the “s-component” system). Plates were sprayed with sulfuric acid-dichromate and charred at 180’. METIIODS For lipid chromatography, Carl Schleicher and Schuell Co. DEAE-cellulose Type 40 was conditioned, and columns were packed, by the method of ROUSER et &.a (‘lot. cit.). Minute quantities of lipids in column effluents are detectable as follows: a few ,ul are sampled in thin capillary tubing which has been pulled to a needle point, and the point is applied to a frosted glass surface. A circle of solvent spreads and evaporates to leave a peripheral ring of nonvolatile lipid if this is present. About 0.25 pg of lipid is readily detected. Infrared spectra were obtained on a Baird KM-I Infrared spectrophotometer. Noncrystalline lipid samples were thinly spread on a NaCl window; crystalline substances (I-1.5 mg) were usually examined after pelletization in KBr (125 mg).

M. tuberculosis, Strain H37Rv: Cultwing, harvesting, extraction Surface cultures of H37Rv were grown on Wong-Weinzirl medium as described earlier&, but containing 0.5 g sodium pyruvate, I mg Cu2+ and I mg Zna+ per 1. For radiolabeling when desired, 0.8 mC of Na, sbso, was added per 1 of medium, the Biochim. Biophys. Ada, 210 (1970) 116-126

118

M. B. GOREN

MgSO, content of the medium was halved and 0.41g MgC1,.6H,O per 1 was substituted. 4-week veil growth is killed by autoclaving for 35 min at go”, filtered, washed with distilled water and rubber-dammed to yield 350-400 g of moist cells from 12 1. Lipids are obtained according to MIDDLEBROOKet aL.rwhich involves three hexane-decylamine extractions of the bacilli and citric acid extraction of the organic phase to free it of excess amine. This product is referred to as “crude extract” or “crude lipids” and the sulfolipids at this stage are largely in the form of decylamine salts. The vacuumconcentrated red-brown crude extract solution is dried by percolation through a short column of anhydrous Na,SO,. Total crude extract lipids are about 2.5 g from typical bacterial

harvests

as described.

Fig. 2. Thin-layer chromatographic pattern silica gel, developed IO cm in “5-component”

“Neutral

red activity”

of sulfolipids

is estimated

SOlVent

SyStem

according

(in descending

order) II, I and III:

(SC-2 MATERIALS).

to unpublished

methods

of G.

MIDDLEBROOK AND C. M. COLEMAN: an appropriate portion of sulfolipid-containing material diluted to 4.0 ml in hexane is vigorously contacted with 12 ml of 0.027; aqueous neutral red hydrochloride, centrifuged and the absorbance of the hexane solution determined in a Coleman, Jr. Spectrophotometer at 525 m,u. This absorbance is referred to as the number of neutral red A units contained in the 4 ml of solvent. Typical crude extract from a bacterial harvest as described contains a total of about 1250 neutral red A units. A significant part of this activity is attributable to highly polar, sulfur-free lipids, some of which have been shown to contain phosphorus. The principal sulfolipid of H37Rv has an activity of approx. 1.44 neutral red A units/mg; “crude lipids”, less than 0.5 neutral red A unit/mg. Biochim

Biophys.

Acta,

210 (1970)

116-126

N.

tiLhXdclSiS SULFOLIPID

“9

I: PURIPlCA'llON

RESULTS

Some five or six sulfur-containing

substances

can be recognized in crude extracts

from H37Rv. These were revealed by thin-layer chromatography and subsequent autoradiography of %-labeled lipids found in either column chromatography effluents or directly in crude material. The thin-layer chromatographic pattern of three of these labeled lipids is shown in Fig. 2. These were recovered individually

in column chroma-

tography and portions were recombined for thin-layer chromatographic presentation. In descending order of mobility these are referred to as sulfolipid classes II, I, and (tentatively )I11 (SL-II, SL-I, SL-III)*. SL-I is the most abundant sulfolipid, with

Fig. 3. A. Thin-layer chromatographic plate illustrating labeled lipids in three different crude extracts of H37Kv. The second sample is from an ether-ethanol (‘ANDERSON' type) extract and illustrates the stability of the sulfolipid in this solvent system. B. Autoradiograph of the thinlayer chromatographic plate of A. Solid blocks outline the positions of the (decayed) authentic comparison sulfolipids II and I. The dashed blocks indicate the position of SL-II from the extract samples.

whose isolation, properties and part structure the present work is concerned. Related lipids with somewhat greater thin-layer chromatographic mobility than SL-I are designated types SL-II. Though present in small amounts in the lipid mixture, as the most persistent contaminants of SL-I these played the most prominent role in defining the requirements of the separation scheme. Figs. 3A and 3B show that still other, more polar radiolabeled lipids are present in the crude lipid extract and are separated in thin-layer chromatography (as well as in column processes). Fig. 3A also compares * To avoid awkwardness of construction these will henceforth be referred to as single entities, e.g. “sulfolipid I”, etc., although a very closely related group of substances is implied. The distinctions between classes are based in part upon column and thin-layer chromatographic mobilities, infrared data, and chemical degradation studies currently in progress. Biochim. Biophys.

Acta, 210 (1970)

116-126

M. B. GOREN

I20

the behavior of three different solvents as extractants plate heavily

for H37Rv.

loaded (left to right) with three different

SL-I and SL-II methanol-acetic

extracts

It shows a silica gel of H37Rv

and with

for comparison. The plate was developed 15 cm in chloroformacid-water (95 : I : 5 : 0.3, by vol.), dried and rechromatographed for

IO cm with the “5-component” solvent (MATERIALS). Fig. 3B is a radioautograph of the same thin-layer chromatographic plate. The radiolabel of authentic SL-I and -11 (old samples) had largely radiodecayed but still gave a recognizable darkening in the radioautograph (boxes). The figures show that SL-II (highest mobility) is present in only minute amounts; SL-I is by far the most abundant member; at least four additional more polar sulfur-containing lipids can be distinguished in significant amounts. The three extracts (left to right) were obtained from similar quantities of harvested H37Rv extracted, respectively, with hexane-0.1% decylamine, with ethanolether (50:50, v/v), according to the classical methods of ANDERSON~, and with hexane alone. Hexane alone is a poor solvent, effecting only incomplete extraction of the sulfolipids. SL-II was not directly detectable in the hexane extract but was present in the other two, being most abundant in the hexane which contained decylamine. Incomplete evidence suggests that most of the SL-II may arise from a partial degradation of the principal sulfolipid. The Anderson solvent dissolves, along with SL-I, so much of more polar phosphorus-containing lipids that separation of SL-I becomes very difficult. Accordingly, only hexane-decylamine extraction is used routinely for preparation

TABLE

of crude extract.

1

SINGLE DEAE-CELLULOSE

CHROMATOGRAPHY

OF CRUDE

EXTRACT

Charge: 175 neutral red absorbance units, 6.106 counts/min, 375 mg on 3 g DEAE-cellulose (acetate form). Regenerate column with 75 ml chloroform-methanol-NH,OH (70 : 30: I, by vol.) ; 75 ml methanol ; 75 ml acetic acid ; methanol to neutrality. Fraction

Solvent

Iv0

2

3 4 5 5’ 6 ”

8 9 IO II I2 I3 ‘4 ‘5 16 17 18 19 20

Chloroform-methanol-(7 : 3) Chloroform-methanol-( I : I) Chloroform-methanol-formic acid (80 : 20 : 5) Acetic acid Methanol Chloroform-methanol (8 : 2) Chloroform-methanol-NH,OH (So: 20: I) Chloroform-methanol-NH,OH (So: 20: I) Chloroform-methanol-NH,OH (80: 20 : I) Chloroform-methanol-NH&OH (80 : 20: I) Chloroform-methanol-NH,OH (80 : 20: I) Chloroform-methanol-NH,OH (80 : 20: I) Chloroform-methanol-NH,OH (80 : 20: I) Chloroform-methanol-NH,OH (80 : 20: I) Chloroform-methanol-NH,OH (80 : 20: I) Chloroform-methanol-NH,OH (80 : 20: 3) Chloroform-methanol-NH,OH (So : 20 : 3) Chloroform-methanol-NH,OH (80: 20: 3) Chloroformpmethanol-NH,OH (80 : 20: 3) Chloroform-methanol-NH,OH (80 : 20:3) Chloroform-methanol-NH,OHformic acid (70:30:1:0.6)

* See DEAE-cellulose Biochim.

chromatography

Biophys.

Acta.

section.

210 (1970) 116-126

V/01.

Radioactiuzty

(ml)

(counts/min x IO-6)

50 40 65 IO 20

0.035 None 0.021

Identity of sulfolipid*

50 5 5 5 5 5 5 5 5 5 IO IO IO IO IO

0.013 0.581 0.984 0.522 0.332 0.217 0.172 0.135 0.104 0.174 0.460 0.503 0.237 0.122

II 1+11 1+11 I I

35

I.37

I’+III,

1

I I I I I I 1 I’(?)* etc.

121

M. tubenxdosis SULFOLIPIDi : PURIFICATION

DEAE-cellulose chromatography DEAE-cellulose was the most versatile adsorbent found for separation of the mycobacterial sulfolipids; some half dozen procedures for obtaining SL-I and small amounts of SL-II and III were devised. The general DEAE-cellulose n~ethodology for lipid separations and its rationale have been elaborately detailed by ROUSER et aL4. Our most direct process is summarized in Table I. The first solvents for loading and washing (through 5’) separate the bulk of nonpolar and moderately polar substituents and prepare the DEAE-cellulose for a gradual elution of sulfolipids by the ammoniacal solvent. SL-II is enriched in the early fractions and later SL-I is obtained. Occasionally an early fraction containing only SL-II and a late fraction of almost homogeneous SL-III are gotten. The process for a small column detailed in Table I, affords about 45 mg of SL-I directly from 375 mg crude extract*. With larger columns this scheme is less satisfactory, but a variant, applicable to a ro-g column * * yields satisfactory NH,SL-I product : sulfolipid is recovered by eluting the column with a slightly acidic elutrient rather than with an alkaline one (chloroform-methanol-acetic acid-NH ,OH (70 : 30: I : 0.03, by vol.)). The elution is prolonged; ro-ml fractions are collected after radioactivity is seen in the effluent. After thin-layer chromatography, the purest SL-I fractions are combined. From 650 neutral red A units (1.4 g of “crude lipids”) 290 neutral red A units of NH,SL-I (2.03 mg) are obtained; total sulfolipids comprise about 0.75% of the dry bacterial mass.

Ammonium (or sodium) sulfolipid is a colorless, viscous oil. It appears to be homogeneous as judged by silica gel thin-layer chromatography in four different WAVE

NUMBERS

MKXONS Fig. 4. Infrared spectra. Thin film on NaCl plate. A. NH,SL-I. NH absorption is reflected in bands at 3220 and r4oo-1450 cm-i; equatorial secondary sulfate absorption at 1250 and 824 cm-l; a specific band (“Type 3”) associated with trehalose at 808 cm-i.

solvent systems. However, chemical studies show that SL-I nevertheless comprises a mixture of very closely related substances (cJ DISCUSSION). Interpretation of infrared * Fraction ro of Table I contained a product designated SL-I’, au incompletely characterized substance of somewhat lower mobility in thin-layer chromatography than SL-I. ** All solvent volumes of Table I are multiplied by 3.33 up to the ammonium acetate-acetic acid elution. BioGhim. Biophys.

Acta.

210

(1970)

116-126

M. B. GOREN

I22

spectra of sulfolipid is facilitated when viewed in terms of its now known structure: a tetraacyltrehalose z-sulfate (see following paper). The infrared spectrum of NH,SL-I is shown in Fig. 4A. Peaks at 3220 cn-1 and the broadened band between 1400and 1450 cm-l are attributable to NH absorption. The carbonyl region between 1660 and 1740 cm-l exhibits shouldering and band broadening that is attributed to a multiplicity of ester and keto-ester functions, as indicated from an examination of substances cleaved from the sulfolipid by gentle alcoholysis or hydrolysis and also from methylation studies (see follo~~ing paper). Absorption due to the sulfate ester group is seen at about 1250 and 824 cm-l, the latter peak being shifted to 829 cm-1 in the sodium sulfolipid; attributable to a sulfate group occupying an equatorial secondary position7 it accords with our findings that the trehalose moiety of the sulfolipid bears a sulfate at a single 2 position. This band disappears when NH,SL-I is desulfated (Fig. 4B). A weak band is seen at about 808 cm-l; designated in an earlier study of BARKER et aL8 as a “Type 3” absorption of a,a-trehalose, this band has been valuable in the present work for distinguishing trehalose derivatives. The infrared patterns of small samples of relatively homogeneous SL-II and SL-III suggest that these, too, are structured on the trehalose core sulfated in an equatorial secondary position. Results of microanalysis are given below. Phosphorus was not detectable. @znz5= 40.5O (c 0.0111 in toluene). Found*: C, 73.1; 13, 11.73; N. (Dumas), 0.54; N (Kjeldahl), 0.59; S**, 1.52. C,,,H,,,O,,NS (mol.wt. 2384) requires C, 73.0; H, 11.61; N. 0.59; S, 1.347;). Molecular

weight:

By desdfatiolt

SL-I as the ammonium salt is remarkably sensitive to desulfation: complete loss of radiolabeled sulfur occurs spontaneously in a short time at room temperature when NH,SL-I is merely dissolved in reagent grade “anhydrous” ether. The sulfur-free lipid is nonionic; the released labeled fragment is water soluble, acidic, and is entirely precipitable with Ba 2+. It is thus clear that all of the sulfur in SL-I is present as a sulfate ester. The degradation is seen as a spontaneous hydrolysis, the moisture content of reagent grade ether being sufficient to provide the necessary water for the reaction. Practically, the spontaneous desulfation was employed as a more precise method for determining the molecular w-eight of NH,SL-I and for preparing desulfated lipid for structural studies: 315.2 mg of radiolabeled NH&L-I was dissolved in IOO ml of Baker’s anhydrous ether and left at room temperature for 44 h, when radiometric analysis for solvent-soluble % indicated the hydrolysis to be 97% complete. The NH,HSO, was quantitatively recovered by appropriate aqueous extractions and percolated through Dower: 50 WX-4 (H+ form) ; the effluent H,SO, was determined by titration. With correction for material sampled, the molecular weight of NH,SL-I so determined was 2398. Desulfated

szalfoli$id;

mechan.isnz

of desulfatim

The desulfated lipid was recovered: 300.5 mg (Calc. 301 mg). This was separated from a small amount of colored impurity by dissolving it in ether and precipitating the product as a colorless oil by addition of methanol. In thin-layer c~lromatography in * Analysis * * Rnalvsis

by Huffman Laboratories. by Schwarzkopf Laboratories.

Riochim. Biophys.

Ada,

ZIO (xg?o)

II&120

M. tubercdosis

SULFOLIPID

I: PURIFICATION

123

the “5-component” system, the de&fated lipid moves to the solvent front. (Found* : requires C, 76-r; H, 11.95; N, C, 76.23; H, 11.91; N (Dumas), 0.04 **. C,,,R,,,O,,

oq/o.) The empirical formula derived for the desulfated SL is in accord with that for sulfolipid itself: C, 4sHa720,, = C,,,H,,,O,,NS + H,O-NH,HSO,. The best agreement for the average empirical formulas derived for the two lipids from the microanalytical and molecular weight data is for a substance C,,-C,,. The suggested formulations are expected to be correct to about fi: 5C, & IO H, with possibly one additional oxygen. Some of the apparent unsaturation suggested by the empirical formula is due in part to ketonic functions. (Cf. NOTE ADDED IN PROOF) Figs. 4A and B compare the infrared spectrograms of NH,SL-I and its desulfated product: the NH and sulfate ester bands at 3220, r400-1450 and 824 cm-l (Fig. 4A) which characterized NH,SL-I have disappeared (Fig. 4B) ; the former also shows a deep broad absorption band at about 1240 cm-1 which is in part attributable to S = 0 stretching vibration in the sulfolipid’, and which is much shallower in the desulfated product. Because of its solubility the NH,SL-I may behave as a strong acid in aprotic but sufficiently basic solvents such as ether. In a separate communication (in preparation) it is suggested that NH,+ may yield a proton to coordinate or hydrogen bond with the ester oxygen which links the sulfur to the carbohydrate and thereby provides the

H

!a)

!b)

Fig. 5. Proposed NH,SL-I desulfation mechanism. a. Interaction of p-electrons from the anomeric oxygen with the sulfur (d-orbital) facilitates attack of the sulfate ester oxygen by a proton from NH,+. b. attack of the transition state by a molecule of H,O completes the transformation.

hydrolysis-susceptible transition state (Fig. 5a). With the intervention of a water molecule, 0 to S scission occurs to release a mole of NH,HSO, and the desulfated lipid (Fig. 5b). A molecular model suggests that the energy barrier to the initial attack may be lowered considerably by the proximity of the anomeric oxygen to the sulfate through interaction of the oxygen p-electrons with the sulfur (d-orbital). This would relieve the polarization on the (sulfate) esteroxygen and facilitate the initial attack by the proton. This model should exhibit certain characteristics: the rate for spontaneous desulfation should accelerate with generation of protons (catalyzed by mineral acid) ; select solvents (acetone) should promote the desulfation, but sufficiently basic solvents such as alcohols or water should tend to inhibit it. Finally “onium” nitrogen salts bearing a proton should desulfate spontaneously, but simple metalsalts shouldnot. * Analysis by Huffman Laboratories. ** Nitrogen-free lipids of high molecular weight have been found to “show” when assayed according to the Pumas method (see ref. 9).

traces of nitrogen

Biochim. Bio$dsys. Acta, ZIO (1970) 116-126

124

M. B. GOREN

Appropriate experimental tests yielded data showing the sulfolipid to behave in conformity with these expectations: pyridinium sulfolipid is rapidly desulfated whereas the sodium salt is stable; acetone supports complete desulfation; very small amounts of water (0.5% based on ether) completely inhibit this hydrolysis reaction. The expected acceleration in the rate of the spontaneous desulfation and its catalysis by small amounts of added mineral acid are illustrated in Fig. 6. DISCUSSION

Although the present communication is concerned almost wholly with the principal sulfolipid class of Strain H37Rv, four or five sulfur-containing lipid classes are in fact recognized in the “crude lipid’ extracted from this organism. Whether the whole constitutes a family of sulfolipids which are related as precursors and final I

10 20

30

40

53

60

70

MINUTES

Fig. 6. Kinetics of NH&&I

desuifation in reagent grade anhydrous ether.

product (SL-I ?) or whether SL-I is in part degraded to the others is not clear. Both may be true. Studies in progress suggest that the lipids of higher thin-layer chromatographic mobility (which we term SL-II) may arise from SL-I, possibly by hydrolytic loss of a carboxylic substituent and/or by acyl migrations. SL-II has not yet been directly detected in simple hexane (a poor solvent) extracts of viable H37Rv, but was recognizable as a trace component in ethanolether extracts of living bacilli. It is considerably more abundant in hexane-decylamine extracts of steam-killed organisms and becomes concentrated in specific fractions during DEAE-cellulose chromatography. The more polar sulfur-containing lipids are seen in any of the extracts described. Accordingly, these are not artifacts arising in the recovery processes; solvolytic degradative evidence implies however that they can be obtained from SL-I (Part II). Pe~ethylation analyses (cf- Part II) and hydrolysis studies in progress show that at least four positions of the trehalose core are substituted in desulfated SL-I ; on hydrolysis, carboxylic substituents, separatable by thin-layer chromatography into four distinct substances are obtained. One of these has already been characterized as a mixture consisting principally of palmitic and stearic acids along with lesser amounts of perhaps six other carboxylic substances. The remaining three major carboxylic substituents may also be groups of closely related acids. The complexity of structures thus posed suggests then that total purification of any individual member of even SL-I, in significant quantities, is unlikely. Biochim. Bio$hys.

Acta,

210

(1970) 116-126

M. t’L&?YCU~OsiS SULFOLIPIDI : PURIFICATION

125

Although we have found similar substances in extracts of virulent bovine strains, they are produced only in minute amounts. The principal sulfolipid from some bovine strains is different from SL-I of the present study, as judged from thin-layer chromatography and infrared examinations. A similarity of “core structure” on the other hand is seen in extracts from still other bovine strains : trehalose appears to be present, and the ammonium salts undergo spontaneous desulfation in ether. However, the paucity of products suggests that sulfolipids are not necessary for expression of virulence in Mycobacterium

bovis.

Human strains of tubercle bacilli exhibit a wide range of synthetic abilities with respect to sulfolipid. Studies are currently in progress with ten strains* previously characterized by MITCHISONet UP. and by GANGADHARAM et aL3. The latter found these to vary widely in the levels of a “sulfolipid fraction” which they produce in surface culture. Our own studies confirm these observations with respect to SL-I/II : sulfolipid elaboration varies from almost none up to levels such as are produced by H37Rv. It seems strange that the sulfolipids were not discovered decades ago, as, for example, in the heuristic investigations of ANDERSON6. When the structural fragility, in particular the spontaneous desulfation in ether was first observed, we asumed that sulfolipids simply do not survive the first step of ANDERSONextraction (ethanolether). However, sulfolipids are well extracted, and in fact, are very stable in this mixture, not being desulfated even after many months! We conclude therefore that despite their relative abundance in primary ANDERSONextracts, the sulfolipids were simply overlooked in the 35 years that elapsed from ANDERSON’S~investigations to the original disclosure of MIDDLEBROOKet aL1. NOTEADDEDIN PROOF (Received April 27th) Current studies (in preparation) indicate that the apparent unsaturation of SL-I is not real. Some hydroxy acids, but no ketonic acids are present. A more precise expression for NH,SL-I is C,,,H,,,O,,NS. ACKNOWLEDGMENTS This investigation was supported by the U.S. Japan Cooperative Medical Science Program administered by the National Institute of Allergy and Infectious Diseases of the National Institutes of Health, Department of Health, Education, and Welfare. The excellent technical assistance of Judith Warren is acknowledged with thanks. I am also indebted to Dr. Oscar Reiss for stimulating advice and suggestions. REFERENCES I

;io;;~~~~~~~~~,

C. M.

COLEMAN AND W.B.

SCHAEFER,PVOC.N~~E.

Acad.Sci.

U.S.,45(1g5g)

z I?. ITO, C. M. COLEMAN AND G. MIDDLEBROOK, Kekkaku, 36 (1961) 764. 3 P. R. J. GANGADHARAX, M. L. COHN AND G. MIDDLEBROOK, Tuber&, 44 (1963) * The ten strains

452.

currently under study were kindly supplied by Dr. D. A. Mitchison. Biochim.

Biophys.

Acta,

210

(1970)

116-126

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4 G. ROUSER, G. KRITCHEVSKY, 5 6 7 8 9 IO

D. HELLER

AND E. LIEBER, J. Am.

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Sm., 40 (1963)

425. M. B. GOREN, J. Bacteviol., 94 (1967) 1258. R. J. ANDERSON, J. Biol. Chem., 74 (1927) 525. J. R. TURVEY, in M. S. WOLFROM AND R. S. TIPSON, Advances in Carbohydrate Chemistry, Vol. 20, Academic Press, New York, 1965, p. 193-194. S. A. BARKER,E. J.BouRIu‘E,M. STACEY AND D.H.WHIFFEX,J. Chem. Sm., (1954) 171. H. NOLL, H. BLOCH, J. ASSELINEAU AND E. LEDERER, Biochim. Biophys. Acta, 20 (1956) 305. I>. A. MITCHISON, J. B.SELKON AXII J. LLOYD, /. Pathol. Bactrviol., 86 No. 2 (1963) 377.

Bzochzm.

Bzophys.

Acta,

210 (1970) 116-126