Article Figures & Data
Figures
- FIG. 1.
Organization of the 6.8-kb genomic segment isolated from MAC serovar 4 strain (ATCC 35767). Filled triangles indicate the primers used for PCR amplification.
- FIG. 2.
TLC analysis of crude GPL extracts from recombinant M. smegmatis strains MS-S2/pYM301a (A), MS-S2/pYM-hlpA (B), MS-S2/pYM-hlpA-orf2 (C), and MS-S2/pYM-orf3-orf4-orf5 (D). GPL extracts were prepared from the total lipid fraction after a mild alkaline hydrolysis step. Each recombinant strain was tested by two samples derived from independent colonies. Samples were spotted and developed in CHCl3-CH3OH-H2O (30:8:1 [vol/vol/vol]).
- FIG. 3.
GC-MS of alditol acetate derivatives from GPL-S2 (A), GPL-S4 (B), and GPL-S4M (C), which were purified from GPL extracts of recombinant M. smegmatis strains MS-S2/pYM301a, MS-S2/pYM-hlpA, and MS-S2/pYM-hlpA-orf2, respectively.
- FIG. 4.
MALDI-TOF MS of GPL-S2 (A), GPL-S4 (B), and GPL-S4M (C), which were purified from GPL extracts of recombinant M. smegmatis strains MS-S2/pYM301a, MS-S2/pYM-hlpA, and MS-S2/pYM-hlpA-orf2, respectively.
- FIG. 5.
GC-MS spectra and fragment ion assignments of 2,3,4-tri-O-Me-Rha (A), Rha (A and B), 6-d-Tal (C), and Fuc (D), which are derived from alditol acetates of sugars released from deuteriomethylated GPL-S4. Ac, acetate; D, deuterium.
- FIG. 6.
Proposed structures and biosynthetic pathways for GPL-S2, GPL-S4, and GPL-S4M. Parentheses indicate structural differences between three compounds, which are deduced from MALDI-TOF MS analyses {pseudomolecular ions ([M + Na]+)}.
Tables
- TABLE 1.
Oligosaccharide structures of serovar 2- and 4-specific GPLs
Serovar Oligosaccharide Reference 2 2,3-di-O-Me-α-l-Fuc-(1→3)-α-l-Rha-(1→2)-l-6-d-Tal 9 4 4-O-Me-α-l-Rha-(1→4)-2-O-Me-α-l-Fuc-(1→3)-α-l-Rha-(1→2)-l-6-d-Tal 24 - TABLE 2.
Bacterial strains and vectors used in this study
Strain or vector Characteristic(s) Source or reference Bacterial strains E. coli DH5α Cloning host TaKaRa M. smegmatis mc2155 Expression host 29 M. intracellulare ATCC 35767 MAC serovar 4 strain 35 M. avium JATA51-01 Source of the rtfA gene 26 Vectors pYM301a Site-specific integrating mycobacterial vector carrying an hsp60 promoter cassette 25 pMV261a E. coli-Mycobacterium shuttle vector carrying an hsp60 promoter cassette with an AflII site This study pMVΔmtfF Source of mdhtA, merA, and gtfD genes 26 pMV-rtfA-mdhtA-merA-gtfD pMV261a carrying rtfA, mdhtA, merA, and gtfD genes This study pYM-hlpA pYM301a carrying the hlpA gene This study pYM-hlpA-orf2 pYM301a carrying the hlpA gene and ORF2 This study pYM-orf3-orf4-orf5 pYM301a carrying ORF3, ORF4, and ORF5 This study - TABLE 3.
Oligonucleotide primers used in this study
Primer Sequencea Restriction site RTFA-S 5′-CGGGATCCCATGAAATTTGCTGTGGCAAG-3′ BamHI RTFA-A 5′-AACTGCAGCTCAGCGACTTCGCTGCGCTTC-3′ PstI MDHTA-S2 5′-GCTCTAGACTGCAGAAAAACCAACTTCTACTGCCTGACCTG-3′ PstI GTFD-A2 5′-GGAATTCTTAAGTCTACGGTTCTGCGCTTCGTTCTTTG-3′ AflII HLPA-S 5′-GGAATTCGTGACAACGACGCCACCAGT-3′ EcoRI HLPA-A 5′-CCATCGATACTACGCTGCCGCGCTAGGCG-3′ ClaI ORF2-A 5′-CCCAAGCTTCTCAGACTCTAACGTACAGTTC-3′ HindIII ORF3-S 5′-CACCTGCAGAAATGACCGCCACAACCAGGGC-3′ PstI ORF5-A 5′-GCAGAATTCCTACGGCGCCAATTCGATGAG-3′ EcoRI GTFB-S1 5′-GGAACTCCTGCACCTTGGGGCCGT-3′ MDHTA-A2 5′-GGTGCGGGTCAACGTAGAGGTG-3′ ↵ a Underlining indicates the restriction site.
