LosA, a Key Glycosyltransferase Involved in the Biosynthesis of a Novel Family of Glycosylated Acyltrehalose Lipooligosaccharides from Mycobacterium marinum*

Members of the genus Mycobacterium are characterized by cell envelopes rich in unusual free lipids, interacting with a covalently anchored mycolyl-arabinogalactan matrix. Previous studies have shown that Mycobacterium marinum produces large amounts of a diacylglycosylphenolphthiocerol, “phenolic” glycolipid. When cultivated on liquid Sauton medium, traces of a polar lipooligosaccharide (LOS) glycolipid antigen were also previously indicated. In this study, it was found that growth of the type strain of M. marinum on solid Sauton or Middlebrook 7H10 agar gave substantial, but different, amounts of a family of four major trehalose-based LOSs. The core pentasaccharide LOS-I was a rhamnosyl diglucosyl-acylated trehalose. The heptasaccharide, LOS-II, was derived from LOS-I by adding xylose accompanied by a novel sugar (X); repeated addition of this sugar unit X gave the octasaccharide LOS-III. LOS-IV has a decasaccharide component with two additional unusual sugar units, YZ. In a recent study (Alexander, D. C., Jones, J. R., Tan, T., Chen, J. M., and Liu, J. (2004) J. Biol. Chem. 279, 18824-18833), chromatographically similar glycolipids were assigned to the family of phosphatidylinositol mannosides (PIMs) and a “PimF” (Rv1500) glycosyltransferase implicated in the conversion of a supposed “PIM5” to a “PIM7.” The present study indicates that these putative PIMs are in fact members of the phosphorus-free LOS family of glycolipids and that the protein product of Rv1500, which we have now termed LosA, is a glycosyltransferase involved in transferring sugars to LOS-III to form LOS-IV of M. marinum.

Members of the genus Mycobacterium include the major human pathogens Mycobacterium tuberculosis and Mycobacterium leprae but also some less common opportunists, such as Mycobacterium malmoense, Mycobacterium marinum, Mycobacterium kansasii, Mycobacterium gastri, Mycobacterium gordonae, and Mycobacterium szulgai.
All these mycobacteria possess a cell envelope structure based on longchain mycolic acids esterified to an arabinogalactan polysaccharide, which is attached to a peptidoglycan backbone. This mycolyl-arabinogalactan-peptidoglycan complex intercalates with an array of unusual free lipids, resulting in an effective external permeability barrier (1). This organelle also contributes to the intrinsic resistance of mycobacterial pathogens to antimicrobial drugs (2).
The free lipids of mycobacterial cell envelopes include a selection of species and subspecies-specific antigenic glycolipids, which may contribute to the external relationships of the organism (1-7). There are three major classes of these extractable glycolipids. First, the "phenolic glycolipids" (PGLs) 4 possess similar aglycone structures, formed from polymethyl-branched fatty acids esterified to a phenolphthiocerol diol unit (7). Variations in the PGL sugar moieties allow the expression of the inherent antigenicity of these glycolipids. Second, the glycopeptidolipids found in the members of the Mycobacterium avium complex as well as in Mycobacterium smegmatis (4 -6) can display subspecies specificity because of subtle variations in their oligosaccharide units. Finally, there is a broad class of trehalose-based lipids (7), ranging from apolar pentaacyl trehaloses, through to amphiphilic tri-and diacyltrehaloses and sulfolipids to highly polar lipooligosaccharides (LOSs) (8). LOSs were found and described in M. kansasii (9 -11), M. gastri (11,12), M. szulgai (13), M. malmoense (14), M. gordonae (15), and certain representatives of M. tuberculosis (16).
In addition to the complex glycolipids outlined above, all mycobacterial species produce families of polar glycophospholipids, the phosphatidylinositol mannosides (PIMs) (1, 4 -6). Four major PIMs are usually expressed, mono-and diacyldimannosides (AcPIM 2 and Ac 2 PIM 2 ) and mono-and diacylhexamannosides (AcPIM 6 and Ac 2 PIM 6 ) (6). In addition, a minor PIM 4 lipid has been shown to be a natural antigen for CD1d-restricted T cells (17). Furthermore, the characteristic mycobacterial lipoglycans, lipoarabinomannan and lipomannan are both multiglycosylated versions of PIMs. We initially proposed the following biosynthetic pathway phosphatidylinositol 3 PIM 3 lipomannan 3 lipoarabinomannan (18), which is now supported by biochemical and genetic evidence (19 -22). PimA catalyzes the addition of Manp provided by GDP-mannose to the 2-position of the myo-inositol of phos-* This work was supported in part by the Darwin Trust of Edinburgh. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1  phatidylinositol to form PIM 1 (21). PimB is responsible for the addition of a second Manp to the 6-position to yield PIM 2 (19). PimC has been recently demonstrated to allow further mannosylation to form PIM 3 (20). It has been proposed that this is the direct precursor of lipomannan, characterized by a linear ␣(136)-linked mannan backbone linked with ␣(132) mannopyranose side chains and that lipomannan is further glycosylated with arabinan to produce lipoarabinomannan (22). Recently, Alexander and co-workers (23) reported that, in M. marinum, a glycosyltransferase, termed "PimF, " was involved in the biosynthesis of higher molecular weight PIMs, specifically PIM 7 from PIM 5 . However, the thin layer chromatographic (TLC) behavior of the putative PIM 7 was reminiscent of a highly antigenic polar phosphorus-free glycolipid isolated previously from M. marinum (24). In this study, we have reinvestigated the PIMs and other highly polar glycolipids of M. marinum and show that the proposed PIM 5 and PIM 7 (23) are in fact members of a novel family of LOSs. In addition, analyses based on M. marinum strains with a reported genetic disruption of Rv1500 and subsequent complementation with a plasmid expressing Rv1500, allows us to draw the conclusion that the product of this open reading frame, which we have termed LosA, is involved in the final assembly of the M. marinum LOS-IV glycolipid.

MATERIALS AND METHODS
Strains and Culture Conditions-The type strain of M. marinum 1218R (ATCC 927, MS425, GA 411) and strain MRS2521 (Rv1500::MycoMar) generated by transposon mutagenesis of 1218R (23) were grown at 30°C both on Sauton agar and Middlebrook 7H11 agar, supplemented with 10% oleic acid/albumin/dextrose/catalase (Difco). Escherichia coli DH5␣ was used for standard manipulations and propagation of plasmid DNA. E. coli DH5␣ pir116 was used for isolation of transposon-containing plasmid. Antibiotics were added as required: kanamycin, 50 g/ml for E. coli and 25 g/ml for M. marinum 1218R; hygromycin B, 150 g/ml for E. coli and 75 g/ml for M. marinum 1218R.
Generation and Screening of M. marinum MycoMar Insertion Library-Propagation of the MycoMar transposon phage and preparation of phage lysates were carried out as previously described (25). For phage infection, M. marinum 1218R cells were washed and resuspended in 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10 mM MgSO 4 , and 2 mM CaCl 2 . Phage were added at a multiplicity of infection of 10:1 and incubated at 37°C for 3 h to allow infection to occur. Bacteria were then plated on Middlebrook 7H11 agar supplemented with kanamycin and incubated at 30°C. Kanamycin-resistant (i.e. transposon-containing) M. marinum colonies were patched onto Middlebrook 7H11 agar to obtain a library of 7680 (i.e. 80 plates ϫ 96 colonies/plate) clones. Colonies with unusual morphology were identified by visual inspection as reported previously (23). One colony, M. marinum MRS2521, was chosen for further characterization.
Localization of M. marinum MRS2521 MycoMar Insertion-M. marinum MRS2521 chromosomal DNA was isolated as described previously (23). BamHI, a restriction endonuclease, which does not cut within the MycoMar element, was used to cleave total chromosomal DNA. Such digestion generates a restriction fragment containing the kanamycin resistance cassette and R6K ␥ ori of the MycoMar element plus flanking chromosomal DNA. Self-ligation of this restriction fragment generates a plasmid that can replicate in E. coli strains containing the pir gene. Digested DNA was self-ligated with T4 DNA ligase and transformed into competent E. coli DH5␣ pir116 and plasmid DNA was isolated (23). Oligonucleotide primers MAR1 (5Ј-CCCGAAAAG-TGCCACCGTGAAAAGCCC-3Ј) and MAR2 (5Ј-CGCTTCCTCGT-GCTTTACGGTATCG-3Ј) were used to determine the position of the transposon, which was inserted at a TA dinucleotide, 412 bp downstream, from the ATG start codon of an open reading frame previously termed pimF (23). The DNA sequence was compared with the genome sequences of M. marinum and M. tuberculosis at the Sanger Institute and analyzed with NTI Suite software (Informax) revealing that the predicted protein was homologous to the Rv1500 of M. tuberculosis H37Rv.
Molecular Cloning-The complementing pPMT1-Rv1500 plasmid was generated as described previously (23) by cloning DNA fragments containing the homologous Rv1500 gene from a M. tuberculosis H37Rv BAC library (obtained from S. Cole, Institut Pasteur, Paris) (26) into the E. coli-Mycobacterium shuttle vector pNBV1, which contains a hygromycin resistance cassette (27).
Lipid Extraction and Analysis-Polar lipids and apolar lipids were extracted from freeze-dried M. marinum cells grown either on Sauton or Middlebrook 7H11 agar according to the procedures of Dobson et al. (28) by stirring in 220 ml of methanolic saline (20 ml of 0.3% NaCl and 200 ml of CH 3 OH) and 220 ml of petroleum ether for 2 h. The cells were centrifuged at 3000 ϫ g for 5 min. The resulting biphasic solution was separated and the upper layer containing apolar lipids was recovered. An additional 220 ml of petroleum ether was added, mixed, and harvested as described above. The two upper petroleum ether fractions were combined and dried under reduced pressure.
To extract polar lipids, 260 ml of chloroform, methanol, 0.3% NaCl (9:10:3, v/v/v), were added to the lower aqueous methanol layer and the solution stirred for 4 h. This mixture was filtered and the filter cake re-extracted twice with 85 ml of chloroform, methanol, 0.3% NaCl (5:10:4, v/v/v). Chloroform (145 ml) and 0.3% NaCl (145 ml) were added to the combined filtrates. This mixture was stirred for 1 h, allowed to settle, and the lower layer containing the polar lipids recovered and dried under reduced pressure. The polar lipid extract was examined by two-dimensional TLC on aluminum backed plates of Silica Gel 60 F254 (Merck 5554), using chloroform/methanol/water (60:30:6, v/v/v) in the first direction and chloroform/acetic acid/methanol/water (40:25:3:6, v/v/v) in the second direction (28). Glycolipids were visualized by either spraying plates with ␣-naphthol/sulfuric acid followed by gentle charring of the plates, or visualized using the Dittmer and Lester reagent that is specific for phospholipids and glycophospholipids.
Sugar Compositional Analysis-Samples were hydrolyzed in 1 M methanolic hydrogen chloride at 80°C for 16 h and the reagent was removed under a stream of nitrogen. Hexosamines were re-N-acetylated in 500 l of methanol/pyridine/acetic anhydride (500:1:5, v/v/v) for 15 min at room temperature, then dried under nitrogen. Trimethylsilyl derivatization was performed in 200 l of Tri-Sil "Z " (Pierce) at room temperature for 30 min, after which the reagent was removed under nitrogen. Derivatized monosaccharides were resuspended in 1 ml of hexanes, centrifuged at 3000 ϫ g for 10 min, and the supernatant was transferred and dried under nitrogen for analysis by gas chromatography-mass spectrometry (GC-MS). Samples were analyzed by GC-MS using temperature program A (see below).
Fatty Acid Methyl Ester Analysis-Samples were hydrolyzed in 200 l of methanolic hydrogen chloride at 80°C for 16 h and the reagent was removed under a stream of nitrogen. The products were dissolved in CHCl 3 and washed several times with water before drying under a stream of nitrogen, and analyzing by GC-MS using temperature program B (see below).
Sugar Derivatization-Per-O-methylation was performed as described previously (29). Briefly, 1 ml of a dimethyl sulfoxide/NaOH slurry was added followed by 0.5 ml of iodomethane (trideuterioiodomethane for per-O-deuterio-methylation). The reaction mixture was vigorously shaken for 10 min at room temperature and the reaction quenched with ϳ1 ml of water. Per-O-methylated samples were then extracted into chloroform (1 ml) and washed several times with water before drying under a stream of nitrogen. After derivatization, the reaction products were purified on a Sep-Pak C 18 cartridge (Waters), as described previously (29), and eluted successively with 35, 50, 75, and 100% acetonitrile in water and finally chloroform.
To establish the location of acyl functions on the oligosaccharide backbone, the native samples were subjected to the neutral alkylating conditions of Prehm (30), followed by de-O-acylation, then per-O-ethylation using the sodium hydroxide procedure. Samples were dissolved in 100 l of trimethyl phosphate, then 100 l of 2,6-di-(tert)-butylpyridine and 50 l of methyltrifluoromethanesulfonate were added. The mixture was stirred and incubated at 50°C for 2 h, following which 1 ml of water was added. The mixture was loaded onto a Sep-Pak C 18 cartridge and the sample eluted as described above. After analysis, the relevant Sep-Pak fractions were dried and the samples de-O-acylated in 200 l of methanol, 35% aqueous NH 3 (1:1, v/v) solution at 37°C for 16 h and then dried. The samples were then per-O-ethylated and purified on a Sep-Pak C 18 cartridge as described above.
Characterization of the trehalose core was performed by first de-Oacylating samples as described above. Dried samples were then reduced with 200 l of 10 mg/ml sodium borodeuteride in 2 M aqueous ammonium hydroxide at room temperature for 2 h, and the reaction was terminated with a few drops of acetic acid. Excess borates were removed by loading directly onto Dowex columns (Merck) and samples were eluted with 2 column volumes of 5% acetic acid (ϳ2 ϫ 2 ml). Samples were dried and per-O-methylated using the sodium hydroxide procedure and purified on a Sep-Pak C 18 cartridge as described above.
Linkage Analysis-Per-O-methylated (or per-O-deuteriomethylated) samples were hydrolyzed with 2 M trifluoroacetic acid for 2 h at 121°C, reduced with 10 mg/ml sodium borodeuteride in 2 M aqueous ammonium hydroxide at room temperature for 2 h, and the reaction terminated with a few drops of acetic acid. Samples were dried and then acetylated with acetic anhydride at 100°C for 1 h. The reagent was removed under a stream of nitrogen, then the samples were dissolved in chloroform and washed several times with water before drying under nitrogen. Samples were analyzed by GC-MS using temperature program C (see below).
Order of Linkages-Samples were incubated in 100 l of 50 mM sodium periodate in 50 mM ammonium acetate buffer, pH 4.5, for 48 h at 4°C in the dark. The reaction was terminated by adding 4 l of ethylene glycol and incubating for a further 60 min in the dark and the reaction mixtures were freeze-dried. Samples were reduced with 10 mg/ml sodium borohydride in 2 M aqueous ammonium hydroxide at room temperature for 2 h, then the reaction was terminated with a few drops of acetic acid. Excess borates were removed by loading directly onto Dowex columns (Merck) and samples were eluted with 2 column volumes of 5% acetic acid (ϳ2 ϫ 2 ml). Samples were dried and per-Omethylated using the sodium hydroxide procedure described above.
Absolute Configuration of Sugars-Samples were prepared as described for trimethylsilyl derivatives, except hydrolysis was performed in 1 M (S)-(ϩ)-2-butanolic-HCl prior to re-N-acetylation and trimethylsilyl derivatization. Samples were analyzed by GC-MS using temperature program D (see below).
GC-MS Analysis-This was carried out using a PerkinElmer Clarus 500 instrument, fitted with a RTX-5 (30 m ϫ 0.25-mm internal diameter, Restek Corp.) for sugar analysis or a Stabilwax (30 m ϫ 0.32 mm internal diameter, Restek Corp.) for lipid analysis. For temperature program A the oven was held at 65°C for 1 min before being increased to 140°C at a rate of 25°C/min, then to 200°C at a rate of 5°C/min, and finally to a temperature of 300°C at a rate of 10°C/min. For temperature program B the oven was held at 90°C for 1 min before being increased to 150°C at a rate of 20°C/min, then to 250°C at a rate of 3°C/min. For temperature program C the oven was held at 60°C for 1 min before being increased to 300°C at a rate of 8°C/min. For temperature program D the oven was held at 65°C for 1 min before being increased to 160°C at a rate of 25°C/min, then to 250°C at a rate of 3°C/min, finally to a temperature of 300°C at a rate of 25°C/min.

Matrix-assisted Laser Desorption Ionization-Mass Spectrometry (MALDI-MS) Analysis-MALDI-MS was performed using a PerSeptive
Biosystems Voyager DE STR mass spectrometer in the reflectron mode with Delayed Extraction. Per-O-methylated samples were dissolved in methanol and 1-l aliquots were loaded onto a metal plate with 1 l of the matrix 2,5-dihydroxybenzoic acid. Native glycolipids were analyzed using the matrix 2-(4-hydroxphenylazo)benzoic acid. Sequazyme peptide mass standards were used as external calibrants (Applied Biosystems).
Electrospray Ionization-MS Analysis-Per-O-methylated samples were dissolved in methanol and sequenced by tandem mass spectrometry (MS/MS) using a hybrid quadrupole orthogonal acceleration time of flight mass spectrometer (Micromass, UK). MS and MS/MS spectra were collected in the positive ion mode. Collision energies typically were 50 -90 eV. Data were acquired and processed using Masslynx software (Micromass, UK). The instrument was pre-calibrated using a 1-pmol/l solution of [Glu 1 ]fibrinopeptide B in acetonitrile, 5% aqueous acetic acid (1:3, v/v).

Effect of Growth Conditions on TLC Patterns of M. marinum
Glycolipids-Polar lipids were extracted from M. marinum 1218R bacilli grown on both Sauton or Middlebrook 7H11 agar and glycolipid profiles were recorded by two-dimensional TLC (Fig. 1). It is clearly evident that the growth condition affects the two-dimensional TLC pattern of polar glycolipids as shown by ␣-naphthol-sulfuric acidstained TLCs. The lipid pattern when M. marinum was grown on Sauton agar appears to be more complex than the one obtained for M. marinum grown in Middlebrook 7H11. The two-dimensional TLC obtained under the latter growth conditions and stained with ␣-naph- with either pNBV1 or pPMT1-Rv1500 were grown on Sauton agar and Middlebrook 7H11 agar. Polar lipids were extracted as described under "Materials and Methods" and visualized on two-dimensional TLC. Solvent system for the first direction (arrow 1) was chloroform/methanol/water (60:30:6, v/v/v) and the second direction (arrow 2) was chloroform/acetic acid/methanol/water (40:25:3:6, v/v/v/v). Glycolipids were detected with ␣-naphthol/sulfuric acid, and phospholipids/glycophospholipids with Dittmer-Lester stain. AcPIM 2 and Ac 2 PIM 2 , mono-and diacyl phosphatidyli-nositol dimannosides; AcPIM 6 and Ac 2 PIM 6 , mono-and diacyl phosphatidylinositol hexamannosides; LOS-I-IV, lipooligosaccharides; PI, phosphatidylinositol; DPG, diphosphatidylglycerol; PE, phosphatidylethanolamine; and P, unknown phospholipids. thol/sulfuric acid was similar to the one recorded by Alexander et al. (23) with similar growth conditions on Middlebrook 7H9 or 7H11 agar.
Analysis of M. marinum Polar Lipids by Two-dimensional TLCs Using Stain Specificity-Polar lipids were also extracted from the mutant strain M. marinum MRS2521, and the mutant strain complemented with the empty plasmid pNBV1 and pPMT1-Rv1500 grown on Middlebrook 7H11. Lipid profiles were revealed by two-dimensional TLCs stained with Dittmer and Lester reagent specific for phospholipids and glycophospholipids and ␣-naphthol/sulfuric acid for glycolipids (Fig. 1). Not all the glycolipids gave a positive response with the phosphate reagent (Fig. 1). For all strains, the major lipid phosphate spots corresponded to diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, mono-and diacylphosphatidylinositol dimannosides (AcPIM 2 and Ac 2 PIM 2 ), and mono-and diacylhexamannosides (AcPIM 6 and Ac 2 PIM 6 ).
As observed by Alexander et al. (23), the M. marinum MRS2521 mutant accumulated large amounts of one lipid (labeled LOS-III) and failed to produce another (labeled LOS-IV). In contrast to the previous report, our analysis here shows that these two lipids are in fact phosphorous-free glycolipids (Fig. 1). These and the two remaining phosphorus-free glycolipids observed in all strains had the chromatographic behavior of LOSs characteristic of, for example, M. kansasii (9 -11) and the Canetti variants of M. tuberculosis (31). The major components of these glycolipids have been labeled LOS-I-IV, in order of increasing polarity (Fig. 1). The LOS-IV corresponds to the previously isolated glycolipid antigen from M. marinum (24) and also to the putative PIM 7 described by Alexander et al. (23), whereas LOS-III corresponds to the putative PIM 5 observed after disruption of Rv1500 (23). Treatment of the wild type polar lipid extracts with mild base and subsequent twodimensional TLC demonstrated that these unique glycolipids grown either in Sauton or Middlebrook agar were degraded. Hence these glycolipids were alkali-labile and phosphate-negative and similar in this respect to the LOS class of antigens from mycobacteria (8 -16). Moreover, the complementation of the M. marinum MRS2521 mutant strain with plasmid pPMT1-Rv1500 confirmed that the disruption of Rv1500 was responsible for the altered glycolipid profile of MRS2521, restoring the wild type chromatographic behavior, whereas the profile of the mutant transformed with the empty vector remained the same as the mutant (Fig. 1). As a result, the protein product of Rv1500, which we have termed LosA, is a glycosyltransferase involved in the transfer of sugar residues to LOS-III to form LOS-IV.
Characterization of LOS-I-IV-The LOS-I-IV glycolipids were isolated from M. marinum bacilli grown on Sauton agar; LOS-IV was also isolated from M. marinum grown on Middlebrook 7H10 agar. Ionexchange chromatography through a DEAE cellulose column gave total glycolipids in the chloroform/methanol eluate, which were then subjected to preparative TLC to afford LOS-I, LOS-II, LOS-III, and LOS-IV from bacilli grown on Sauton agar and LOS-IV from Middlebrook 7H10 agar.
Analysis of the released fatty acid methyl esters indicated that each LOS had a similar lipid profile (TABLE ONE), with the predominating component being a branched chain acid, tentatively identified as 2,4dimethylhexadecanoate (C18:0; 2,4-dimethyl); this is in accordance with previous fatty acid analyses of M. marinum (29,30). Other acids are present in minor amounts ranging from C15 through to C20, including two 2,4-dimethyl fatty acids (C17 and C19). These dimethyl-branched acids were partly identified from their distinctive fragment ions at m/z 88 and 101, indicating the presence of a 2-methyl substituent. The equivalent chain lengths of these fatty acid methyl esters (TABLE ONE) showed that the three branched acids were a homologous series.
The sugar composition of the LOSs was determined by GC-MS of trimethylsilyl derivatives. LOS-I-IV were shown to be composed predominately of the hexose (Hex) sugar D-glucose (Glc), with a small amount of the 6-deoxyhexose (dHex) sugar, L-rhamnose (Rha). In addition, the pentose sugar L-xylose (Xyl) was present in LOS-II, -III, and -IV.
Per-O-methylated LOSs were analyzed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (Fig. 2). Per-Omethylated LOS-I gave a prominent signal at m/z 1059 [M ϩ Na] ϩ , which is consistent with a glycan of the composition dHexHex 4 (Fig. 2,  A and B). Further evidence for this assignment was obtained by collisionally activated decomposition (CAD) ES-MS/MS of the signal at m/z 1059 [M ϩ Na] ϩ (Fig. 2B). The resulting data show a series of product ions that are consistent with the glycan composition dHexHex 4      The existence of a trehalose core was established as follows. LOS-I was de-O-acylated prior to being reduced by sodium borodeuteride, and then per-O-methylated. MALDI-MS analysis of the derivatized product retained the signal at m/z 1059. The absence of a reduced product is indicative of an oligosaccharide core lacking a free reducing end.

GC-MS analysis of fatty acid methyl esters from LOS-I, -II, -III, and -IV
Finally, periodate oxidation of vicinal hydroxyl groups was used to ascertain the order of the linkages within the oligosaccharide. Periodate oxidation of the native glycolipid resulted in the cleavage of one C-C bond as shown by a mass shift of the molecular ion from m/z 1059 to 1061 consistent with C-C bond breakage between C2 and C3 of the terminal trehalose Glc residue. In a second experiment LOS-I was de-Oacylated prior to oxidation and per-O-methylation. Analysis of the resulting sample by ES-MS revealed a series of new signals at m/z 1061, 1017, 827, and 651, suggesting the core oligosaccharide had been oxidized and partially degraded. The nature of these signals was further investigated by CAD MS/MS and assignments for each are shown schematically in Fig. 3, panels A-D. The sequence data derived from CAD MS/MS of m/z 1061 and 1017 was consistent with the breakage of one C-C bond and two adjacent C-C bonds on the terminal Hex residue of the glycan dHexHex 4 , respectively. These data are consistent with the loss of the protective acyl groups. CAD MS/MS of m/z 651 indicated that this oxidation product had the composition dHexHex 2 and had not been oxidized. The data are consistent with the linkage sequence dHex(133)Hex(133)Hex, which lacks vicinal hydroxyl groups and would therefore not be expected to oxidize. CAD MS/MS of m/z 783 gave sequence data that can only be attributable to the sequence dHex(133)Hex(133)Hex-(134)Hex, consistent with C-C bond breakage between C2 and C3 of the internal Hex residue of the trehalose core.
In summary, it is proposed that the sequence of LOS-I is

DISCUSSION
Many of the mycobacteria examined to date are characterized by species-or type-specific glycolipid antigens, which fall into three broad categories: the phenolic glycolipids, glycopeptidolipids, and trehalosebased lipids, extending in complexity from simple diacyltrehaloses (7) to LOS (8 -16). The glycolipids (Fig. 1, LOS-I to LOS-IV) characterized from M. marinum, in the present study, belong to the latter class of alkaline-labile phosphorus-free glycolipids. Representatives of M. marinum fall into a select group of mycobacteria, which produce both LOSs and PGLs (32). In addition to M. marinum, this group is limited to M. kansasii, M. gastri, and the Canetti variants of M. tuberculosis. It is interesting that LOSs have not yet been characterized from Mycobacterium ulcerans, a close relative of M. marinum (33).    One of the polar glycolipids (LOS-IV), identified here, corresponds to a lipid antigen described previously (24), but only found as a variable minor component in M. marinum grown in liquid Sauton medium. In the recent study of Alexander et al. (23), a family of glycolipids was apparent in M. marinum, in addition to the expected major amounts of the ubiquitous phosphatidylinositol di-and hexamannosides (PIM 2 and PIM 6 ). These authors designated two of these lipids as PIM 5 and PIM 7 and proposed that a glycosyltransferase PimF was instrumental in the biosynthetic transformation of the former to the latter. No investigation of the phosphorus content (or glycosyl compositional analysis) of these glycolipids was reported. The two-dimensional TLC system used by Alexander et al. (23), and in the present study, is that devised by Dobson et al. (28) to clearly distinguish PIMs from phosphorus-free LOS glycolipids; the LOSs run as a series of spots below, but parallel to, the PIMs. The essential character (Fig. 4) of four novel LOSs (I-IV) from M. marinum has been determined, but further studies will be needed to elucidate the fine structure and stereochemistry of some of the unknown components, such as the novel sugars X, Y, and Z. However, the structures exhibit structural features in common with the previously characterized LOS antigens of M. kansasii (9 -11), M. gastri (11,12), M. szulgai (13), M. malmoense (14), and M. gordonae (15), and M. tuberculosis Canetti (16). All uniformly possess an acylated trehalose terminus, which is further glycosylated by several variable sugar residues. M. marinum LOS-I appears to be superficially the same as a LOS from M. kansasii (9 -11). The principal fatty acyl substituents in both these