The role of the embA and embB gene products in the biosynthesis of the terminal hexaarabinofuranosyl motif of Mycobacterium smegmatis arabinogalactan

The emb genes are conserved among different mycobacteria. In Mycobacterium smegmatis and Mycobacterium tuberculosis, they belong to an operon comprising three genes, embC, embA, and embB. The EmbB protein has been proposed to be the target of ethambutol, a drug which is known to inhibit the synthesis of the arabinan portion of the mycobacterial cell wall arabinogalactan (AG). To further define the role of EmbB protein in arabinan biosynthesis, embA, -B, and -C genes were inactivated individually by homologous recombination in M. smegmatis. All three mutants were viable, and among the three, the slowest growing embB(-) mutant encountered profound morphological changes and exhibited a higher sensitivity to hydrophobic drugs and detergents, presumably due to an increase in cell wall permeability. Furthermore, chemical analyses showed that there was a diminution in the arabinose content of arabinogalactan from the embA(-) and embB- mutants. Specific-ally, in comparison with the wild-type strain, the crucial terminal hexaarabinofuranosyl motif, which is a template for mycolylation, was altered in both embA- and embB-mutants. Detailed nuclear magnetic resonance studies coupled with enzyme digestion, chromatography, and mass spectrometry analyses revealed that the disaccharide beta-D-Ara(f)(1-->2)-alpha-D-Ara(f) extension from the 3-position of the 3,5-linked alpha-D-Ara(f) residue is markedly diminished. As a consequence, a linear terminal beta-D-Ara(f)(1-->2)-alpha-D-Ara(f)-(1-->5)-alpha-D-Ara(f)-(1-->5)-alpha-D-Ara(f) is formed, a motif which is a recognized, nonreducing terminal feature of lipoarabinomannan but not of normal AG. Upon complementation with the embB and embA wild-type genes, the phenotype of the mutants reverted to wildtype, in that normal AG was resynthesized. Our results clearly show that both EmbA and EmbB proteins are involved in the formation of the proper terminal hexaarabinofuranoside motif in AG, thus paving the way for future studies to identify the complete array of arabinosyltransferases involved in the synthesis of mycobacterial cell wall arabinan.