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Journal of Bacteriology, August 2007, p. 5515-5522, Vol. 189, No. 15
0021-9193/07/$08.00+0     doi:10.1128/JB.00344-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Characterization of the Fucosylation Pathway in the Biosynthesis of Glycopeptidolipids from Mycobacterium avium Complex

Yuji Miyamoto,¹ Tetsu Mukai,^1* Yumi Maeda,¹ Noboru Nakata,¹ Masanori Kai,¹ Takashi Naka,² Ikuya Yano,² and Masahiko Makino¹

Department of Microbiology, Leprosy Research Center, National Institute of Infectious Diseases, 4-2-1 Aobacho, Higashimurayama, Tokyo 189-0002, Japan,¹ Japan BCG Central Laboratory, 3-1-5 Matsuyama, Kiyose, Tokyo 204-0022, Japan²

Received 3 March 2007/ Accepted 2 May 2007

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The cell envelopes of several species of nontuberculous mycobacteria, including the Mycobacterium avium complex, contain glycopeptidolipids (GPLs) as major glycolipid components. GPLs are highly antigenic surface molecules, and their variant oligosaccharides define each serotype of the M. avium complex. In the oligosaccharide portion of GPLs, the fucose residue is one of the major sugar moieties, but its biosynthesis remains unclear. To elucidate it, we focused on the 5.0-kb chromosomal region of the M. avium complex that includes five genes, two of which showed high levels of similarity to the genes involved in fucose synthesis. For the characterization of this region by deletion and expression analyses, we constructed a recombinant Mycobacterium smegmatis strain that possesses the rtfA gene of the M. avium complex to produce serovar 1 GPL. The results revealed that the 5.0-kb chromosomal region is responsible for the addition of the fucose residue to serovar 1 GPL and that the three genes mdhtA, merA, and gtfD are indispensable for the fucosylation. Functional characterization revealed that the gtfD gene encodes a glycosyltransferase that transfers a fucose residue via 13 linkage to a rhamnose residue of serovar 1 GPL. The other two genes, mdhtA and merA, contributed to the formation of the fucose residue and were predicted to encode the enzymes responsible for the synthesis of fucose from mannose based on their deduced amino acid sequences. These results indicate that the fucosylation pathway in GPL biosynthesis is controlled by a combination of the mdhtA, merA, and gtfD genes. Our findings may contribute to the clarification of the complex glycosylation pathways involved in forming the oligosaccharide portion of GPLs from the M. avium complex, which are structurally distinct.

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Mycobacteria have a unique multilayer cell envelope composed of peptidoglycan, arabinogalactan, mycolic acids, and an outer layer that contains abundant species-specific glycolipids. It is thought that these cell wall characteristics allow mycobacteria to survive in host cells (8, 11). Among the glycolipids present in the outer layer of the cell envelope, glycopeptidolipids (GPLs) are recognized as highly antigenic surface molecules in several species of nontuberculous mycobacteria, including the Mycobacterium avium complex (29). The common structure in which the fatty acyl-tetrapeptide core is glycosylated with both 6-deoxy-talose (6-d-Tal) and O-methyl-rhamnose (O-Me-Rha), termed core GPLs, is present in all types of GPLs (2, 4, 12). The core GPLs are further glycosylated with a Rha residue or various oligosaccharides linked to the 6-d-Tal residue. These glycosylation events give rise to structural variations, especially in the GPLs from the M. avium complex, that are responsible for the creation of serotypes, and they are also known as serovar-specific GPLs (ssGPLs). The ssGPLs define approximately 30 serotypes, some of which are found predominantly in isolates from patients. For example, the serotypes 1, 4, 6, and 8 are frequently isolated from AIDS patients coinfected with the M. avium complex (17, 30). In addition, ssGPLs are associated with host responses to infection and function as agonists of cell surface receptors such as Toll-like receptor 2 (28).

The biosynthesis of GPLs, including methylation, acetylation, and peptide synthesis, has been investigated mainly with M. smegmatis, but it has not been fully elucidated (6, 15, 21, 23). We have recently identified the glycosyltransferase genes involved in the formation of core GPLs in M. smegmatis and M. avium (19), but the glycosylation pathways of ssGPLs have not been clarified, except for that of serovar 1 GPL (14). The presence of a Rha residue linked to 6-d-Tal, as observed with serovar 1 GPL, is structurally required for the formation of all types of ssGPLs. The Rha residue is further extended by the subsequent addition of one of the three different sugars, Rha, fucose (Fuc), or glucose, to form the structural group of ssGPLs (9). In these groups, the Fuc-containing ssGPLs, such as serovar 2, 3, 4, and 9 GPLs, shown in Table 1, comprise the major group in the ssGPLs of the M. avium complex, and serovar 2 GPL is the basic structure for these ssGPLs (9). The biosynthetic pathway for the Fuc-containing ssGPLs is still unclear. In the M. avium complex, a 22- to 25-kb chromosomal region with the ability to synthesize the serovar 2 GPL has been identified and cloned from two strains (4, 5). However, the key genes responsible for the formation of the Fuc residue of serovar 2 GPL have not been identified in this chromosomal region. In this study, we have focused on a 5.0-kb segment that includes the genes predicted to be associated with Fuc synthesis and have characterized their functions in the biosynthesis of GPLs in the M. avium complex.

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TABLE 1. Oligosaccharide structures of Fuc-containing ssGPLs

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Bacterial strains, culture conditions, and DNA manipulation. The bacterial strains and vectors used and constructed in this study are listed in Table 2. M. avium, used for the isolation of chromosomal DNA, was grown in Middlebrook 7H9 broth (Difco) with 0.05% Tween 80 supplemented with 10% Middlebrook ADC enrichment broth (BBL). Recombinant M. smegmatis strains for GPL production were cultured in Luria-Bertani broth with 0.2% Tween 80. The isolation of DNA, transformation, and PCR were carried out as previously described (20). Escherichia coli strain DH5 was used for the routine manipulation and propagation of plasmid DNA. The following antibiotics were added as required: kanamycin, 50 µg/ml for E. coli and 25 µg/ml for M. smegmatis; hygromycin B, 150 µg/ml for E. coli and 75 µg/ml for M. smegmatis. Oligonucleotide primers used for PCR are available on request.

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TABLE 2. Bacterial strains and vectors used in this study

Construction of the integrating mycobacterial vector (pYM301). The site-specific integrating mycobacterial vector pYM301 was constructed from parts of pYUB854, pMV306kan, and pMV261 (3, 24, 27). To replace the oriM region of pMV261 with the region needed for integration, a fragment containing attP and int was amplified from pMV306kan DNA using the primers INT-S and INT-A, digested with the respective restriction enzyme, and cloned into the XbaI-MluI site of pMV261 to give pMV301kan. The hygromycin-resistant cassette, a selective marker of integration, was excised from pYUB854 with XbaI and NheI and inserted into the NheI-SpeI site of pMV301kan to obtain hyg instead of kan. Because the resulting plasmid had restriction sites for EcoRI, BamHI, and PstI outside of the multicloning site, it was disrupted in turn by PCR to create pYM301 by using the following primers: 301ECO-D and 301ECO-U for disruption of the EcoRI site, 301PST-D and 301PST-U for disruption of the PstI site, and 301BAM-D and 301BAM-U for disruption of the BamHI site.

Construction of expression vectors. Previous studies have shown that the clustered gtfC-gtfD region is about 5.0 kb long and contains five genes, designated gtfC, mdhtA, merA, mtfF, and gtfD (13). To express these five genes as one operon, the 5.0-kb segment was obtained as a PstI-EcoRI fragment to be inserted into the expression cassette of pMV261. Prior to cloning into pMV261, it was necessary to clone the 5.0-kb segment into pUC18 to confirm the DNA sequences. Since we could not directly clone the 5.0-kb segment as one PCR-amplified fragment, three divided fragments were amplified from the genomic DNA of M. avium JATA51-01 by using following primers: GTFC-S and HA for the 2.0-kb PstI-HindIII fragment, HS and KA for the 1.0-kb HindIII-KpnI fragment, and KS and GTFD-A for the 2.0-kb KpnI-EcoRI fragment (Fig. 1A). These three fragments then were combined in pUC18 as a PstI-EcoRI fragment to give pUCgtfCD (Fig. 1A). A 5.0-kb PstI-EcoRI fragment was excised from pUCgtfCD and inserted into the PstI-EcoRI site of pMV261 to create pMVgtfCD, which expressed the above-described five genes (Fig. 1B).

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FIG. 1. Schematic presentation of the cloning procedure for the 5.0-kb gtfC-gtfD region (A) and its gene-deleted constructs, which were inserted into an expression cassette made up of the hsp60 promoter and the terminator of pMV261 (B). The primers used for PCR amplification of the three fragments are indicated by filled triangles. In pMVgtfC and pMVgtfD, the genes gtfC and gtfD were completely deleted from the gtfC-gtfD region of pMVgtfCD. In-frame deletions were designed for the construction of the expression cassettes of pMVmdhtA, pMVmerA, and pMVmtfF to prevent the polar effect on each downstream gene. P, PstI; H, HindIII; K, KpnI; E, EcoRI.

Deletion of each gene from the gtfC-gtfD region was performed as follows. The expression vectors were constructed from pUCgtfCD by PCR using following primers: 18PST-U and MDHTA-S for the deletion of gtfC, MDHTA-U and MDHTA-D for the deletion of mdhtA, MERA-U and MERA-D for the deletion of merA, MTFF-U and MTFF-D for the deletion of mtfF, and MTFF-A and 18ECO-D for the deletion of gtfD. The PCR products were digested with each restriction enzyme and were ligated. The PstI-EcoRI fragment was excised from each resulting plasmid and was cloned into the same restriction sites of pMV261 to give pMVgtfC, pMVmdhtA, pMVmerA, pMVmtfF, and pMVgtfD (Fig. 1B).

The two M. avium genes rtfA and gtfD were amplified from genomic DNA of M. avium JATA51-01 by using the following primers: RTFA-S and RTFA-A for rtfA and GTFD-S and GTFD-PA for gtfD. The PCR products were digested with each restriction enzyme and were cloned into the corresponding site of pYM301 and pMV261 to give pYMrtfA-int and pMVgtfD, respectively.

To construct the vector for the simultaneous expression of rtfA, mdhtA, and merA, the HpaI site of pYMrtfA-int was replaced with an AflII site by PCR using AFL-U and AFL-D to give pYMrtfA-int-Afl. The mdhtA and merA genes were amplified as one operon from genomic DNA of M. avium JATA51-01 by using the primers MDHTA-S2 and MERA-A. The PCR product was digested with each restriction enzyme and cloned into the PstI-AflII site of pYMrtfA-int-Afl to give pYMrtfA-mdhtA-merA-int.

Isolation and purification of GPLs. Whole-lipid extracts were isolated from harvested bacterial cells that had been mixed with CHCl₃-CH₃OH (2:1, vol/vol) for several hours at room temperature. The extracts obtained from the organic phase were separated from the aqueous phase and evaporated to dryness. To remove the lipid components except for GPLs, the whole-lipid extracts were subjected to mild alkaline hydrolysis as previously described (20, 21). For analytical thin-layer chromatography (TLC), crude GPLs from equal amounts of harvested bacterial cells were spotted on silica gel 60 plates (Merck) using CHCl₃-CH₃OH (9:1, vol/vol) as the solvent and were visualized by spraying with 10% H₂SO₄ and then charring. Purified GPLs were prepared from crude GPLs by preparative TLC on the same plates and were extracted from the bands corresponding to each GPL. Perdeuteriomethylation for determination of the linkage position of sugar moieties was carried out as previously described (7, 10, 14).

GC-MS and MALDI-TOF analyses. For the monosaccharide analysis, crude GPLs from equal amounts of harvested cells were hydrolyzed in 2 M trifluoroacetic acid (2 h, 120°C), and the released sugars were reduced with sodium tetradeuterborate and then were acetylated with pyridine-acetic anhydride (1:1, vol/vol) at room temperature overnight. The resulting alditol acetates were separated and analyzed by gas chromatography-mass spectrometry (GC-MS) on a TRACE DSQ (Thermo Electron) equipped with an SP-2380 column (SUPELCO) using helium gas. The temperature program was from 52 to 172°C with 40°C/min increments and then from 172 to 250°C with 3°C/min increments. To determine the total mass of the purified GPLs, matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectra (in the positive mode) were acquired on a QSTAR XL (Applied Biosystems) with a pulse laser emitting at 337 nm. Samples mixed with 2,5-dihydroxybenzoic acid as the matrix were analyzed in the reflectron mode with an accelerating voltage of 20 kV and operating in positive-ion mode.

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Identification of the genes involved in the formation of the Fuc residue in serovar 2 GPL. To reveal the genes responsible for the fucosylation pathway that lead to the formation of serovar 2 GPL, we focused on the 5.0-kb segment designated the gtfC-gtfD region (GenBank accession no. AF125999.1). This region contains five genes: mdhtA and merA, whose deduced amino acid sequences show a high level of similarity to those of enzymes involved in Fuc synthesis; mtfF, previously identified as the fucosyl 2-O-methyltransferase gene; and gtfC and gtfD, putative glycosyltransferase genes whose functions remain unknown (13, 18). Since M. smegmatis only produces core GPLs, we introduced the chromosomal integrating vector pYMrtfA-int possessing the M. avium gene rtfA, whose gene product transfers the Rha residue to 6-d-Tal of core GPLs, and obtained the recombinant strain rtfA-int, which produces GPL with a terminal Rha residue (termed GPL-S1) that could be a substrate for the synthesis of serovar 2 GPL. The expression vector pMVgtfCD, harboring the gtfC-gtfD region (Fig. 1B), then was introduced into the GPL-S1-producing strain (rtfA-int) and wild-type mc²155, and GPL production was analyzed by TLC (Fig. 2A). Although there were no differences between the TLC profiles of Wt/pMV261 and Wt/pMVgtfCD (Fig. 2A, lanes c and d), new spots appeared in rtfA-int/pMVgtfCD (Fig. 2A, lane b), indicating that the gtfC-gtfD region contains genes with the ability to convert GPL-S1 into structurally different compounds, but it can do so only in the presence of the rtfA gene. To confirm that the products formed in rtfA-int/pMVgtfCD contained Fuc, a characteristic of serovar 2 GPL, alkaline-stable extracts from rtfA-int/pMVgtfCD were hydrolyzed, and the released monosaccharides were analyzed by GC-MS (Fig. 2B). The results showed that 2-O-Me-Fuc, which is structurally related to serovar 2 GPL, was present together with Rha, 6-d-Tal, 3-O-Me-Rha, 3,4-di-O-Me-Rha, and 2,3,4-tri-O-Me-Rha in rtfA-int/pMVgtfCD (Fig. 2B, graph b). 2-O-Me-Fuc was not detected in strain rtfA-int/pMV261 (vector control) (Fig. 2B, graph a) or recombinant wild-type mc²155 (data not shown). These results indicated that the gtfC-gtfD region is responsible for the transfer of the Fuc residue to serovar 1 GPL. Additionally, for identification of the individual genes involved in this fucosylation, we constructed various plasmids that have one of the genes deleted from the gtfC-gtfD region, as shown in Fig. 1B, and examined the sugar moieties of the alkaline-stable GPL extracts from each recombinant strain by GC-MS analysis (Fig. 3). The results show that the profile of rtfA-int/pMVgtfC was the same as that of rtfA-int/pMVgtfCD, indicating that the gtfC gene does not participate in the formation of 2-O-Me-Fuc (Fig. 2B, graph b, and 3A). In rtfA-int/pMVmtfF, Fuc was detected instead of 2-O-Me-Fuc, demonstrating that the mtfF gene encodes fucosyl 2-O-methyltransferase (Fig. 3D). On the other hand, no Fuc derivatives were detected in the recombinant strains rtfA-int/pMVmdhtA, rtfA-int/pMVmerA, and rtfA-int/pMVgtfD (Fig. 3B, C, and E). These results indicated that the three genes mdhtA, merA, and gtfD are all indispensable for the formation of the Fuc residue in serovar 2 GPL.

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FIG. 2. Functional analyses of the gtfC-gtfD region. (A) TLC analysis of crude GPL extracts from rtfA-int/pMV261 (lane a), rtfA-int/pMVgtfCD (lane b), Wt/pMV261 (lane c), and Wt/pMVgtfCD (lane d). The total lipid fraction after mild alkaline hydrolysis was spotted on plates and was developed in CHCl3-CH3OH (9:1 [vol/vol]). (B) GC-MS analyses of alditol acetates of sugars released from crude GPL extracts of rtfA-int/pMV261 (a) and rtfA-int/pMVgtfCD (b). Alditol acetate derivatives were prepared from the total lipid fraction after mild alkaline hydrolysis. Asterisks indicate noncarbohydrates.

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FIG. 3. GC-MS analyses of alditol acetates of sugars released from crude GPLs. GPLs were extracted from rtfA-int/pMVgtfC (A), rtfA-int/pMVmdhtA (B), rtfA-int/pMVmerA (C), rtfA-int/pMVmtfF (D), and rtfA-int/pMVgtfD (E). Alditol acetate derivatives were prepared from the total lipid fraction after mild alkaline hydrolysis. Asterisks indicate noncarbohydrates.

Functional analysis of the gtfD gene. Although the gtfD gene was predicted to encode a type of glycosyltransferase based on a homology search of its deduced amino acid sequences, it is not clear whether the product of gtfD functions as the glycosyltransferase that transfers the Fuc residue via 13 linkage to the Rha residue of serovar 1 GPL to form the oligosaccharide part of serovar 2 GPL. To elucidate the function of gtfD, we constructed a recombinant strain by introducing a chromosomal integrating vector expressing rtfA, mdhtA, and merA (pYMrtfA-mdhtA-merA-int). We then characterized the product formed when gtfD was expressed solely by the plasmid vector (pMVgtfD). TLC analysis of the recombinant strains showed that the gtfD-expressing strain (rtfA-mdhtA-merA-int/pMVgtfD) caused the appearance of a new spot, termed GPL-S2, in connection with the disappearance of GPL-S1 (Fig. 4, lane A), while the vector control (rtfA-mdhtA-merA-int/pMV261) did not produce GPL-S2 (Fig. 4, lane B). To determine the structure of sugar moieties of GPL-S2, perdeuteriomethylation was performed on purified GPL-S2, and derived alditol acetates were analyzed by GC-MS. The GC-MS profile yielded four peaks corresponding to 6-d-Tal, Rha, Fuc, and 2,3,4-tri-O-Me-Rha (data not shown). The characteristic spectra of Fuc, Rha, and 6-d-Tal are shown in Fig. 5. The spectrum of Fuc gave fragment ions at m/z of 121, 134, and 168, which represent the presence of deuteriomethyl groups at positions C-2, C-3, and C-4 (Fig. 5A). In contrast, the detection of fragment ions at m/z of 121, 134, 193, and 240 from Rha indicated that a deuteriomethyl group was introduced at positions C-2 and C-4 of Rha, whose C-3 position was acetylated (Fig. 5B). Additionally, positions C-3 and C-4 of 6-d-Tal were found to be deuteriomethylated, with the detection of fragment ions at m/z of 134, 181, and 193 (Fig. 5C). These observations demonstrated that position C-1 of Fuc is linked to position C-3 of Rha but not position C-2 of 6-d-Tal, because it was previously determined that position C-1 of Rha is linked to position C-2 of 6-d-Tal in the oligosaccharide of serovar 1 GPL through the catalytic reaction of RtfA (14). Furthermore, we compared the molecular mass of GPL-S2 to that of GPL-S1 purified from the vector control strain by MALDI-TOF (mass spectrometry). The main pseudomolecular ion [M + Na]⁺ from both compounds revealed that the difference between GPL-S2 (m/z, 1,479.9) and GPL-S1 (m/z, 1,333.8) was 146 mass units, suggesting that a Fuc residue was further added to the GPL-S1 (data not shown). Accordingly, the structure of GPL-S2 was determined to have Fuc-(13)-Rha-(12)-6-d-Tal at D-allo-Thr and 2,3,4-tri-O-Me-Rha at D-alaninol (Fig. 6), demonstrating that gtfD encodes the glycosyltransferase that transfers a Fuc residue via 13 linkage to the Rha residue of serovar 1 GPL.

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FIG. 4. TLC analyses of crude GPL extracts from rtfA-mdhtA-merA-int/pMVgtfD (lane A) and rtfA-mdhtA-merA-int/pMV261 (lane B). The total lipid fraction after mild alkaline hydrolysis was spotted onto plates and developed in CHCl3-CH3OH (9:1 [vol/vol]).

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FIG. 5. GC mass spectra and fragment ion assignment of Fuc (A), Rha (B), and 6-d-Tal (C), which are derived from alditol acetates of sugars released from deuteriomethylated GPL-S2. Ac, acetate; D, deuterium.

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FIG. 6. Proposed structure and biosynthetic pathway of fucosylated GPL (GPL-S2).

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The gene cluster involved in synthesizing ssGPLs has been cloned from M. avium strains. In this cluster, the functions of several genes responsible for the biosynthesis of serovar 1 GPL, such as rtfA, gtfA, gtfB, mtfB, mtfC, and mtfD, have been elucidated (14, 16, 19). On the other hand, the genes associated with the conversion of serovar 1 GPL to other serotypes, including serovar 2 GPL, have not been identified. In this study, we focused on the five genes assumed to encode the enzymes associated with fucosylation in the biosynthesis of serovar 2 GPL and experimentally showed that GtfD is responsible for the transfer of the Fuc residue to the Rha residue of serovar 1 GPL (Fig. 6). Gene deletion experiments revealed that mdhtA and merA also contribute to the formation of the Fuc residue in serovar 2 GPL. The deduced amino acid sequences of mdhtA and merA showed high levels of similarity to GDP-D-mannose-4,6-dehydratase and GDP-6-deoxy-4-keto-D-mannose-3,5-epimerase-4-reductase, respectively. These are enzymes involved in the synthesis of L-Fuc from D-mannose and are highly conserved among other bacteria (1, 26). For mycobacteria, there are no homologues of mdhtA and merA in the genome databases for M. bovis, M. leprae, and M. smegmatis. However, for M. tuberculosis, the deduced amino acid sequences of Rv1511 and Rv1512 show 89 and 84% homology to those of mdhtA and merA, respectively. This observation is supported by the fact that several strains of M. tuberculosis produce the Fuc-containing phenolic glycolipid, whereas M. bovis and M. leprae lack the Fuc residue, and other carbohydrate components having the Fuc residue have not been reported from the above-mentioned three species. Thus, it is strongly suggested that mdhtA and merA encode synthetases involved in the conversion of D-mannose to L-Fuc that can be transferred by GtfD to form the Fuc residue of serovar 2 GPL (Fig. 6). Before performing the functional analyses of these genes, we speculated that the glycosyltransferase involved in the fucosylation was encoded by gtfC, but not gtfD, from the observations that gtfC includes a putative glycosyltransferase motif and its homologue in M. tuberculosis, Rv1514c, is adjacent to Rv1511 and Rv1512, which are predicted to be responsible for the Fuc synthesis, while a gtfD homologue, Rv2957, is located far from Rv1511, Rv1512, and Rv1514c. However, the deletion analysis demonstrated that gtfC does not contribute to the fucosylation of the GPLs. This result raises the possibility that gtfC is involved in the transfer of another sugar moiety, such as glucuronic acid and Rha, followed by fucosylation, as observed for the serovar 3, 9, and 4 GPLs (9). As for gtfD, Rv2957 is reported to be one of the glycosyltransferase genes involved in the biosynthesis of phenolic glycolipid in M. tuberculosis, but its catalytic functions, such as sugar substrate and glycosidic linkage, are not clear (22). Thus, our findings implied that Rv2957 is the fucosyltransferase gene responsible for the transfer of the Fuc residue in the phenolic glycolipid. Moreover, morphological observations showed that the surface of colony of the GPL-S2-producing strain was rougher than that of the vector control strain, suggesting that the presence of a Fuc residue in the GPL structure affected the cell surface properties (data not shown). Taking the results together, our study is the first report identifying the genes involved in the fucosylation pathway in mycobacteria and might provide a clue to understanding its role in the biosynthesis of glycolipids, including GPLs.

 
This work was supported in part by a grant in aid for research on emerging and reemerging infectious diseases from the Ministry of Health, Labor, and Welfare of Japan.

 
* Corresponding author. Mailing address: Department of Microbiology, Leprosy Research Center, National Institute of Infectious Diseases, 4-2-1 Aobacho, Higashimurayama, Tokyo 189-0002, Japan. Phone: 81-42-391-8211. Fax: 81-42-394-9092. E-mail: tmukai{at}nih.go.jp

Published ahead of print on 25 May 2007.

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Journal of Bacteriology, August 2007, p. 5515-5522, Vol. 189, No. 15
0021-9193/07/$08.00+0     doi:10.1128/JB.00344-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

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