©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Presence of -

D

-Ribosyl-1-monophosphodecaprenol in Mycobacteria (*)

(Received for publication, June 5, 1995)

Beata A. Wolucka (§) Edmond de Hoffmann

From the Department of Chemistry, University of Louvain, Place Pasteur 1, B-1348 Louvain-la-Neuve, Belgium

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Biosynthesis of the cell envelope in mycobacteria is largely unknown; however, several antituberculosis drugs apparently interfere with this process. Recently, we described a lipid intermediate for the biosynthesis of the cell wall arabinogalactan/arabinomannan of Mycobacterium smegmatis: beta-D-arabinofuranosyl-1monophosphodecaprenol (Wolucka, B. A., McNeil, M. R., de Hoffmann, E., Chojnacki, T., and Brennan, P. J.(1994) J. Biol. Chem. 269, 23328-23335). In the present work, by means of gas chromatography-mass spectrometry, fast atom bombardment tandem mass spectrometry, and proton NMR, the major pentose-containing component of the polyprenyl-P-sugar family from M. smegmatis was characterized as beta-D-ribosyl-1-monophosphodecaprenol (decaprenyl-P-ribose). Additionally, the structure of a minor arabinose-containing compound, beta-D-arabinosyl-1-monophosphooctahydroheptaprenol, could be deduced. In vivo labeling experiments with [^14C]glucose demonstrated unequivocally that decaprenyl-P-ribose is actively synthesized in Mycobacterium tuberculosis H37Ra and Mycobacterium avium serovar 4. It is proposed that decaprenyl-P-ribose could be a precursor for the biosynthesis of either some unknown ribose-containing cell envelope polymers of mycobacteria or the arabinan part of the cell wall arabinogalactan/arabinomannan due to the presence of a 2`-epimerase activity at some late stages of the arabinogalactan/arabinomannan biosynthesis.


INTRODUCTION

The cell walls of all mycobacteria, including two important human pathogens: Mycobacterium tuberculosis and Mycobacterium leprae, contain large amounts of an unusual sugar: D-arabinofuranose (D-Ara(f)). (^1)D-Ara(f) is the major component of two mycobacterial polysaccharides: arabinogalactan and arabinomannan (for review, see (1) ). Arabinogalactan, a key element responsible for the integrity of the lipid-rich cell envelope of mycobacteria(2) , is covalently linked to the innermost peptidoglycan layer and serves as a point of attachment for long chain mycolic acids, which, in turn, interact hydrophobically with numerous free lipids and glycolipids of the mycobacterial envelope (for review, see (3) ). The arabinan segments of the membrane-anchored lipoarabinomannan (4) and exocellular arabinomannan (5) are structurally similar to that of the arabinogalactan(6) . Arabinofuranose was also found in a glycolipid isolated from Mycobacterium avium(7) and structurally related to the nonreducing-end motif of the arabinogalactan-mycolate complex(8) .

The biosynthesis of D-Ara(f) residues is largely unknown. Mycobacteria have long been known to contain two unusual polyprenyl phosphates: a C-decaprenyl-P (9) and a partially saturated C-octahydroheptaprenyl-P(10) . Recently, a family of endogenous glycosyl-P-polyprenols of Mycobacterium smegmatis, comprising decaprenyl and octahydroheptaprenyl phosphate derivatives of arabinose, ribose, and mannose, was described and the complete structure of one component of this family, beta-D-arabinofuranosyl-1-monophosphodecaprenol, elucidated(11) . In the same work, we determined another peculiar feature of the mycobacterial polyprenol, namely the presence of only one internal trans-isoprene residue within the decaprenol molecule. In vivo labeling pulse-chase experiments demonstrated that the lipid-linked ribose, arabinose, and mannose of M. smegmatis are active biosynthetic precursors. Additionally, it was shown that ethambutol, a key antituberculosis drug known to inhibit arabinogalactan/arabinomannan biosynthesis(12) , results in the accumulation of decaprenyl-P-arabinose with a concomitant decrease of the lipid-linked ribose content(11) .

In the present report, we describe the isolation and structural characterization of the ribose-containing lipid of M. smegmatis (decaprenyl-P-ribose) and demonstrate that this compound is actively synthesized in M. tuberculosis H37Ra and M. avium. The presence of polyprenyl-P-ribose in mycobacteria is puzzling, since ribose-containing cell envelope components, such as polymers or glycolipids, have not been found in these organisms until now. The possible functions of decaprenyl-P-ribose in mycobacteria are discussed.


EXPERIMENTAL PROCEDURES

Bacteria

M. smegmatis, strain 1515 from the Trudeau Mycobacterial Collection, was grown at 37 °C on Nutrient Broth (Gibco Ltd., Paisley, Scotland) with shaking for 20 h. M. tuberculosis H37Ra strain 7417 from the National Collection of Type Cultures (United Kingdom) and M. avium serovar 4 were grown with shaking for 1 week on GAS medium (pH 6.6) containing (per liter): 0.3 g of Bacto-Casitone (Difco), 0.05 g of ammonium iron (III) citrate, 4 g of potassium phosphate (monobasic), 2 g of citric acid, 1 g of L-alanine, 1.2 g of magnesium chloride hexahydrate, 0.6 g of potassium sulfate, 2 g of ammonium chloride, and 1% glycerol (v/v), supplemented with 1 mM glucose.

Isolation of Ribosyl Monophosphodecaprenol

The polyprenyl-P-sugar pool from untreated M. smegmatis cells was prepared as described previously(11) , and resolved by preparative silica TLC in chloroform-methanol-concentrated aqueous ammonia-water (65:25:2:2). Spots were located with iodine vapors and the second of the fastest migrating, alpha-naphthol-positive bands (band B, R 0.43) was scraped off, eluted with choroform-methanol (2:1), and reapplied to DEAE-cellulose, which was developed with 0.05 M ammonium formate in methanol. Salts were removed by partitioning the material in chloroform-methanol-water (8:4:3). The organic phase was evaporated to dryness yielding the purified ribosyl monophosphodecaprenol.

Preparation of ^14C-Labeled Polyprenyl-P-sugars from M. tuberculosis and M. avium

M. tuberculosis and M. avium cells grown on GAS medium supplemented with glucose were centrifuged, washed, suspended in 100 ml of GAS medium devoid of glucose, and subsequently incubated at 37 °C with 50 µCi of D-[^14C]glucose (specific activity: 292 mCi/mmol) (Amersham International plc, United Kingdom) for 2 h and 45 min, respectively. Radiolabeled cells were harvested by centrifugation and extracted with hot 95% ethanol at 70 °C for 15 min. After further centrifugation, supernatants (ethanolic extracts) were withdrawn, evaporated to dryness, and partitioned into a mixture of chloroform-methanol-water (8:4:3). The organic layers were washed once with the theoretical upper phase, evaporated to dryness, and submitted directly to mild alkaline hydrolysis to destroy mild alkali-labile lipids. Alkali-stable lipids were applied to a small column of DEAE-cellulose. The column was washed with five volumes of a chloroform-methanol (2:1) solvent, and polyprenyl-P-sugars were eluted with three volumes of 50 mM ammonium formate in methanol. The eluate was desalted and hydrolyzed in 10 mM HCl at 100 °C for 5 min. ^14C-Labeled sugars were recovered in the water phase and analyzed by TLC, followed by autoradiography.

Analytical Procedures

GC/MS of alditol acetates (13) was conducted on a RSL 200 bonded FSOT capillary column (30 m 0.32 mm) (Alltech Associates, Deerfield, IL). The injector temperature was 250 °C. The initial temperature was 150 °C, and the temperature program involved a 1.5 °C/min rise to 175 °C, followed by a 12 °C/min rise to 192 °C, a 0.2 °C/min rise to 193 °C, and a final 7 °C/min rise to 300 °C. Alternatively, sugars were analyzed as trimethylsilyl ethers of the corresponding methyl glycosides, as described(14) . The absolute configuration of monosaccharides was determined by the method of Gerwig et al.(15) .

Radioactive sugars were separated by TLC on cellulose-coated glass plates (Merck, Darmstadt, Germany) in tert-butanol-methylethyl ketone-formic acid-water (8:6:3:3). Plates were autoradiographed.

Phosphate determination was performed as described earlier(11) .

^1H NMR and FAB-MS/MS analyses were conducted as described previously(11) .


RESULTS AND DISCUSSION

Our previous studies demonstrated the presence of the mild acid-labile ribose in the lipid extracts of M. smegmatis(11) . About 75% of the lipid-ribose was recovered in the fraction of mild alkali-stable, weakly anionic lipids, which represented, as determined by means of ^1H NMR and FAB-MS/MS, a family of glycosyl monophosphopolyprenols comprising C-decaprenyl-P and C-octahydroheptaprenyl-P derivatives. The mycobacterial glycosyl-P-polyprenols could be resolved by preparative TLC into four major bands, A-D. The fastest migrating band A (R = 0.46) consisted of beta-D-arabinofuranosyl-1-monophosphodecaprenol (11) . Sugar analysis of the slower migrating band B (R = 0.43) revealed the presence of mild acid-labile ribose (340 nmol/100 g of cells) and trace amounts of arabinose (27 nmol/100 g of cells) representing 7% of the total sugar of band B. The mild acid-freed ribose of band B was assigned to the D series by GC/MS analysis of its(-)-2-butylglycoside derivative(15) . It was determined also that the band B-derived pentoses and the organic phosphate were present in equimolar amounts.

Further structural information on band B components was obtained by FAB-MS/MS experiments. The negative-ion FAB-MS spectrum of the purified band B (Fig. 1A) showed the presence of an intense signal at m/z 909 (relative intensity, 100%), corresponding to the deprotonated molecule of decaprenyl-P-ribose (M(r) = 910), and of a less abundant [M - 132 - H] fragment ion at m/z 777, corresponding to C-P. Signals at m/z 925 and 941 are attributable to oxidation products of the decaprenyl-P-ribose. Moreover, a series of fragments differing by 68 mass units was observed (at m/z 567, 499, 431, 363, and 295) derived from the loss of 5-9 isoprene units from the -end of the isoprenoid chain of decaprenyl-P-ribose. A very weak signal at m/z 713 (relative intensity, 8%) indicated for the presence of trace amounts of a C-P-pentose (M(r) = 714).


Figure 1: Negative ion FAB-MS/MS analysis of the purified decaprenyl-P-ribose of M. smegmatis. A, the FAB-MS spectrum; B, the production spectrum of the m/z 909 ion.



The product ion spectrum of the m/z 909 ion (decaprenyl-P-ribose) (Fig. 1B) was clearly different from that of beta-D-arabinosyl-P-decaprenol(11) . The deprotonated molecule of the latter compound was shown to produce, in tandem mass spectrometry, two abundant fragments at m/z 819 and 777, derived from the loss of a part or the entire D-arabinosyl residue and corresponding to [decaprenyl-PO(4)-(CH-CHOH] ion and [decaprenyl-PO(4)] ion, respectively(11) . In contrast, the tandem mass spectrum of the deprotonated molecule of decaprenyl-P-ribose (Fig. 1B) showed the presence of only one abundant fragment of decaprenyl-P at m/z 777 (relative intensity, 100%), whereas the [decaprenyl-PO(4)-(CH-CHOH] ion (at m/z 819) was barely observable (relative intensity, 6%). Recently, we demonstrated that the FAB-MS/MS technique can be applied for the determination of the anomeric configuration of glycosyl esters of nucleoside pyrophosphates (16) (^2)and polyisoprenyl phosphates.^2 In the latter work, it was shown that the fragmentation pattern of polyisoprenyl-P-sugars depends on cis/trans configuration of the phosphodiester and 2`-hydroxyl groups of the glycosyl residue. Compounds with cis configuration, as in beta-D-mannopyranosyl-P-undecaprenol and beta-D-Ara(f)-P-decaprenol, give two abundant fragments corresponding to [polyisoprenyl-PO(4)-(CH-CHOH)] (B) and [polyisoprenyl-HPO(4)] (A), with the relative intensity ratio (B/A) of about 1. In contrast, compounds with trans configuration of the above-mentioned groups, as in alpha-D-mannopyranosyl-P-undecaprenol, give an abundant [polyisoprenyl-HPO(4)] fragment and practically no [polyisoprenyl-PO(4)-(CH-CHOH)] ion in tandem mass spectrometry; consequently, the relative intensity ratio of these two ions (B/A) is very low (close to 0.01) for compounds with trans configuration. Since the mycobacterial decaprenyl-P-ribose produces, in tandem mass spectrometry, only one abundant ion corresponding to [decaprenyl-HPO(4)], and the relative intensity ratio of [decaprenyl-PO(4)-(CH-CHOH)] ion to [decaprenyl-HPO(4)] ion is very low (0.06), it can be deduced that this compound has trans configuration of the phosphodiester and 2`-hydroxyl groups of its D-ribosyl residue. Therefore, the D-ribosyl residue of the mycobacterial decaprenyl-P-ribose must be beta-linked to decaprenyl-P.

The 500-MHz ^1H NMR spectrum of the decaprenyl-P-ribose preparation from M. smegmatis (Fig. 2) shows all signals characteristic for a glycosylated decaprenyl-P derivative (11) . Proton resonances of the phospholipid part (decaprenyl phosphate) are, as expected, almost identical with those observed for the purified decaprenyl-P-arabinose(11) . However, signals at 0.83 ppm and 1.2 ppm are not derived from the C-P compound and can be assigned to the protons of methyl and methylene groups of the octahydroheptaprenyl-P contaminant. To estimate the molar ratio of the decaprenyl-P-ribose versus the C-P compound in band B, we compared the integration values measured for the proton signals of the trans-methyl groups of the - and the isoprene unit(s) adjacent to it (at 1.55 ppm) with that of the methyl protons of the alpha-isoprene residue (at 1.69 ppm). The mycobacterial C-octahydroheptaprenol has four saturated isoprene units at the -end (10) and, thus, is expected to contain no isoprenyl trans-methyl groups. Indeed, the mycobacterial C-P-mannose recently purified in our laboratory does not give any signal at 1.55 ppm. (^3)Therefore, the signal at 1.55 ppm of the band B spectrum (Fig. 2) (integral 6.0) corresponds to 6 protons of the two isoprenyl trans-methyl groups of the mycobacterial decaprenyl-P derivative (11) . To the signal at 1.69 ppm (integral, 3.5) contribute three methyl protons of the alpha-residue of both, C- and C-P derivatives. Therefore, the molar ratio of the octahydroheptaprenyl-P compound versus decaprenyl-P-ribose in band B was approximately 1 to 6. The position of the individual protons of the ribosyl residue of decaprenyl-P-ribose (Fig. 2) was determined through sequential selective decoupling experiments. The anomeric proton signal of the decaprenyl-P-ribose (a doublet at 5.41 ppm, J = 4.11 Hz, J < 1.5 Hz) is upperfield-shifted in comparison with that measured for the commercially available alpha-D-ribofuranosyl 1-phosphate standard (from Sigma) (a doublet of doublets at 5.77 ppm, J = 6.29 Hz, J = 3.31 Hz). It is known from the literature that the upperfield shift of the anomeric proton signal of D-ribofuranosyl derivatives is characteristic for beta-linked residues(18) . Therefore, it can be concluded that the D-ribosyl residue of the mycobacterial decaprenyl-P-ribose is beta-linked to decaprenyl phosphate. This is in agreement with the above-described results obtained in FAB-MS/MS experiments. Since, in nature, ribose is always found in the furanose ring-form, we propose the following structure for the mycobacterial beta-D-ribosyl-1-monophosphodecaprenol (Fig. 3).


Figure 2: The 500-MHz ^1H NMR spectrum of the decaprenyl-P-ribose. The sample was obtained from 44 g of M. smegmatis cells. The purified decaprenyl-P-ribose (150 nmol based on sugar estimation) was dissolved in a mixture of deacidified CDCl(3) (330 µl) and CD(3)OD (120 µl). Extension of the 3.5-5.5 ppm region (uppercurve).




Figure 3: The proposed structure of the decaprenyl-P-ribose, beta-D-ribofuranosyl-1-monophosphodecaprenol, of M. smegmatis.



The minor C-P-pentose component represented about 15% of the total polyprenyl-P-pentoses of band B, as determined by ^1H NMR. This value matches quite well with the amount of arabinose detected in band B (7% of total sugar). The FAB-MS analysis further confirmed the presence of only small amounts of a C-P-pentose (Fig. 1A). Moreover, the product ion spectrum of the deprotonated C-P-pentose molecule (M(r) = 714) showed the presence of two abundant fragments at m/z 581 and 623(19) , corresponding to an [octahydroheptaprenyl-HPO(4)] ion (relative intensity, A = 96%) and an [octahydroheptaprenyl-PO(4)-(CH-CHOH)] fragment derived from the cleavage across the pentose ring (relative intensity, B = 85%), respectively. The relative intensity ratio B/A was 0.88, thus indicating that the compound has cis configuration of the phosphate and 2`-hydroxyl groups of the pentosyl moiety,^2 as in the case of beta-D-arabinofuranosyl-P-decaprenol. Taken together, the presented data suggest that the minor component of band B is beta-D-arabinosyl-1-monophospho-octahydroheptaprenol. Quantification of the arabinose found in band A (as C-P-Ara; 105 nmol/100 g of cells) and in band B (as C-P-Ara; 27 nmol/100 g of cells) demonstrated that the C-P-arabinose represented about 20% of the total polyprenyl-P-arabinose (C- and C-P-arabinose) isolated from M. smegmatis. Accordingly, the decaprenyl-P-ribose (340 nmol/100 g of cells) was recovered from M. smegmatis cells in amounts almost 3-fold higher than for the polyprenyl-P-arabinose, thus indicating that the former compound is the major pentose-containing lipid intermediate in this organism(11) .

To examine if the lipid-ribose is characteristic only for M. smegmatis or if it is a common lipid intermediate of mycobacteria, in vivo labeling experiments were performed with M. tuberculosis and M. avium cells. In the previous studies(11) , we demonstrated that the family of polyprenyl-P-sugars of M. smegmatis can be rapidly labeled by in vivo incubation of cells with D-[U-^14C]glucose. Preliminary experiments with M. tuberculosis H37Ra and M. avium serovar 4 indicated that the efficiency of incorporation of [^14C]glucose label into the lipid-linked sugars in these organisms is lower than in M. smegmatis, whereby longer incubation time and higher label are needed to obtain similar yields of labeling. This fact is probably related to the existence of an efficient permeability barrier in M. tuberculosis and M. avium(20) or to differences in the biochemical systems responsible for glucose uptake and assimilation in mycobacteria. M. tuberculosis and M. avium cells were labeled in vivo with [^14C]glucose for 2 h and 45 min, respectively, and the fraction of ^14C-polyprenyl-P-sugars was isolated, as described under ``Experimental Procedures.'' The purified ^14C-polyprenyl-P-sugar fractions were submitted to mild acid hydrolysis and the ^14C-sugars released to the water phase were separated by TLC and visualized by autoradiography. Comparison of the TLC patterns revealed that the sugar composition of the glycosyl-P-polyprenol families isolated from M. tuberculosis and M. avium (Fig. 4) is very similar to that of M. smegmatis: ribose, mannose, arabinose, and some still unidentified oligosaccharides are the major ^14C-labeled glycosyl residues. Additionally, in the case of M. avium, an unknown ^14C-sugar component, which migrated just behind ribose and co-migrated with cold xylose/lyxose standard, could be observed (Fig. 4, lane1). These results demonstrate clearly that decaprenyl-P-ribose was actively synthesized in the all mycobacteria tested, including M. tuberculosis H37Ra, M. avium serovar 4, and different strains of M. smegmatis, and, moreover, that it represents one of the predominant lipid-linked intermediates in these organisms.


Figure 4: TLC of ^14C-sugars released by mild acid from the purified polyprenyl-P-sugar fraction of M. avium (lane 1) and M. tuberculosis (lane 2) after in vivo labeling with [U-^14C]glucose. Cells of M. avium serovar 4 and M. tuberculosis H37Ra were incubated with [^14C]glucose for 45 min and 2 h, respectively, and extracted with 95% ethanol at 70 °C for 15 min. The dried ethanolic extracts were partitioned into a mixture of chloroform-methanol-water (8:4:3), and the organic layers were washed with theoretical upper phase. The lipid extracts were submitted to mild alkaline hydrolysis, and the alkali-stable lipids were chromatographed on a DEAE-cellulose column. The polyprenyl-P-sugar fraction of M. avium and M. tuberculosis were hydrolyzed in 10 mM HCl at 100 °C for 5 min; free sugars were recovered in the water phase after partitioning and, subsequently, resolved by TLC. The radioactive spots were visualized by autoradiography.



D-Ribose is a common component of cell envelope polysaccharides, such as lipopolysaccharides and capsular polysaccharides, in many Gram-negative bacteria, and it always occurs as the beta-furanosyl residue(21) . For example, beta-D-ribofuranose was found in the lipopolysaccharides of Pseudomonas aeruginosa(22, 23) , Salmonella sp.(24, 25) , Shigella sp.(26) , Proteus sp., and spotted fever group rickettsia strains (27) , and of some myxobacteria(28) ; in capsular K antigens of Escherichia coli strains(29, 30) ; and also in a type-specific, teichoic acid-like capsular polysaccharide (polyribosylribitol phosphate) of Haemophilus influenzae(31) . Surprisingly, the nature of beta-D-ribofuranosyl donor for the biosynthesis of these bacterial polysaccharides is still unknown(32) . Recently, ribosyl units were also found in N-linked oligosaccharides of two eukaryotic organisms, namely in trypanosomatids Blastocrithidia culicis(33) and Endotrypanum schaudinni(34) . However, there is no report in the literature on ribose-containing cell envelope polymers from mycobacteria.

In the present work, we described a second member of the glycosyl-P-polyprenol family of M. smegmatis, beta-D-ribosyl-1-monophosphodecaprenol, and showed that decaprenyl-P-ribose is actively synthesized in M. tuberculosis and M. avium. Paradoxically, the function of decaprenyl-P-ribose in mycobacteria remains obscure. One possibility is that beta-D-ribosyl-P-decaprenol could serve as a ribosyl donor for the synthesis of still unknown cell envelope or extracellular polysaccharides of mycobacteria. Such ribose-containing polysaccharides might be, in fact, overlooked in mycobacteria, since the purification procedures for mycobacterial cell wall polysaccharides, commonly used by previous researchers, involved steps, such as strong alkaline hydrolysis, RNase treatment and extensive extractions, which would lead to the destruction or removal of certain kinds of ribose polymers(31) . It is worthy of notice that, in this case, the mycobacterial beta-D-ribosyl-P-decaprenol would then serve as a donor of unusual alpha-linked ribosyl units of the putative polymer, since glycosyl transfer reactions from a glycosyl-P-polyprenol to a glycosyl acceptor occur with inversion of the configuration at the C-1` position of glycosyl residue(32) . Unexpectedly, indirect evidence for the existence of a D-ribofuranosyl-transferase activity in M. smegmatis was obtained during the studies on the mechanisms of drug resistance in mycobacteria(35) . Dabbs et al. (35) demonstrated that M. smegmatis and some other fast-growing mycobacteria inactivate rifampin, a key drug against tuberculosis and leprosy, through an unusual glycosylation mechanism, namely the addition of an alpha-D-ribofuranosyl unit to the rifampin molecule. We can speculate that the beta-D-ribofuranosyl-P-decaprenol described here could act as a donor of alpha-D-ribofuranosyl units for the modification of rifampin in mycobacteria.

Another possible explanation for the existence of decaprenyl-P-ribose and the absence of ribose-containing cell envelope polymers in mycobacteria is that decaprenyl-P-ribose could be a precursor for the synthesis of some of the D-arabinofuranosyl residues of arabinogalactan and lipoarabinomannan in mycobacteria due to the presence of a hypothetical 2`-epimerase activity. Such 2`-epimerase could act either at the level of the lipid-linked oligosaccharides, as it was proposed in archaebacteria (36) or at the level of the final polymer, as it occurs in the biosynthesis of heparin (37) and bacterial alginic acid(38) , but apparently not at the level of decaprenyl-P-pentose, since our in vitro enzymatic trials for the conversion of ^14C-labeled decaprenyl-P-ribose into decaprenyl-P-arabinose and vice versa were unsuccessful.^3 This hypothesis would help to explain the observation that polyprenyl-P-ribose is present in severalfold higher amounts than polyprenyl-P-arabinose in mycobacteria and the fact that antimycobacterial drugs (ethambutol and isoniazid), known to inhibit the formation of arabinogalactan-mycolate complex of the mycobacterial cell walls, result in the inhibition of decaprenyl-P-ribose synthesis and the accumulation of decaprenyl-P-arabinose(11) .

There are several reports in the literature suggesting that the proposed 2`-epimerization of D-ribofuranose to the corresponding D-arabinofuranose derivative probably occurs in other bacteria. For example, in Streptomyces antibioticus, which belongs to the same order of Actinomycetales as mycobacteria, a 2`-epimerase is apparently responsible for the conversion of the ribosyl residue of adenosine (or its derivative) into D-arabinofuranosyl residue and the production of D-Ara-A nucleoside analogue(39) . Additionally, both pentofuranoses, D-Ara(f) and D-Rib(f), were found in the lipopolysaccharides of Pseudomonas maltophilia(40) . And finally, it was suggested that an NAD/NADP-dependent sugar epimerase-like nolK gene of Azorhizobium caulidans(41) might be involved in the biosynthesis of D-arabinosyl residues present in the Nod factors of this symbiotic bacterium(17) .

We propose that, for the synthesis of decaprenyl-P-ribose, mycobacteria could employ the same beta-D-ribofuranosyl donor as other bacteria known to produce beta-D-ribofuranosyl-containing cell envelope polysaccharides. The nature of this compound is still unknown(32) . Biosynthetic studies aiming at the identification of the beta-D-ribofuranosyl donor in mycobacteria are now in progress.


FOOTNOTES

*
This work was supported by a grant (to B. A. W.) from the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases (Project 920454) and a grant (to E. d. H.) from the Fonds National de la Recherche Scientifique, Belgium. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom all correspondence should be addressed. Tel.: 32-10-47-30-75; Fax: 32-10-47-30-75.

(^1)
The abbreviations used are: Ara(f), arabinofuranose; C-P, decaprenyl phosphate; C-P-Ara, decaprenyl-P-arabinose; C-P-Rib, decaprenyl-P-ribose; C-P, octahydroheptaprenyl phosphate; C-P-Ara, octahydroheptaprenyl-P-arabinose; GC/MS, gas chromatography/mass spectrometry; FAB-MS/MS, fast atom bombardment-tandem mass spectrometry.

(^2)
B. A. Wolucka, J. S. Rush, C. J. Waechter, and E. de Hoffmann, submitted for publication.

(^3)
B. A. Wolucka, unpublished results.


ACKNOWLEDGEMENTS

We thank Prof. Françoise Portaels (Institute of Tropical Medicine, Antwerp) for the gift of the mycobacterial strains and Pierre-Alain Fonteyne (Institute of Tropical Medicine, Antwerp) for help in experiments with M. tuberculosis. We also thank Prof. Jean-Marie Dereppe (Department of Chemistry, University of Louvain) for the proton NMR measurements.


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