SummaryMycobacterium tuberculosis and other pathogenic mycobacterial species produce large amounts of a glycogen-like a-glucan that represents the major polysaccharide of their outermost capsular layer. To determine the role of the surface-exposed glucan in the physiology and virulence of these bacteria, orthologues of the glg genes involved in the biosynthesis of glycogen in Escherichia coli were identified in M. tuberculosis H37Rv and inactivated by allelic replacement. Biochemical analyses of the mutants and complemented strains indicated that the synthesis of glucan and glycogen involves the a-1,4-glucosyltransferases Rv3032 and GlgA (Rv1212c), the ADP-glucose pyrophosphorylase GlgC (Rv1213) and the branching enzyme GlgB (Rv1326c). Disruption of glgC reduced by half the glucan and glycogen contents of M. tuberculosis, whereas the inactivation of glgA and Rv3032 affected the production of capsular glucan and glycogen, respectively. Attempts to disrupt Rv3032 in the glgA mutant were unsuccessful, suggesting that a functional copy of at least one of the two a-1,4-glucosyltransferases is required for growth. Importantly, the glgA mutant was impaired in its ability to persist in mice, suggesting a role for the capsular glucan in the persistence phase of infection. Unexpectedly, GlgB was found to be an essential enzyme.

M ycobacterial diseases, such as tuberculosis and leprosy, remain serious human public health problems. One critical feature that contributes to the particular pathogenicity and physiology of mycobacteria is the unique, highly hydrophobic and impermeable cell wall (1). Beyond the cytoplasmic membrane, the cell wall of Mycobacterium tuberculosis consists of a core of mycolic acids, arabinogalactan and peptidoglycan, providing the template for insertion of products such as the phthiocerol-containing lipids, the trehalose mycolates, phosphatidylinositol mannosides (PIMs), and their more glycosylated derivatives, lipomannan (LM) and lipoarabinomannan (LAM) (2), all of which contribute to the particular physiology and disease induction capacity of Mycobacterium species. LAM, in its various forms, including those with mannose (Man) ''caps'' (ManLAM), has been implicated in many of the key aspects of the pathogenesis of tuberculosis and leprosy, such as induction of phagocytosis, phagosomal alteration and inhibition of fusion with lysosomes, and induction of innate, humoral, and acquired T cell-mediated immunity (3, 4). However, all of these studies (often conflicting) were conducted with the isolated molecule. Mutants devoid of LAM are crucial to fully resolve its role in disease pathogenesis and bacterial physiology.Although structurally well defined (5), the underlying enzymology and genetics of LAM biosynthesis are unknown. It has been believed, although not empirically demonstrated, that both LM and LAM have their origins in the PIMs, because all contain a phosphatidylinositol (PI) moiety (5, 6). The first step in PIM synthesis involves the transfer of a Manp residue to the 2 position of the myo-inositol ring of PI to form PIM 1 , catalyzed by PimA (7,8). Biosynthesis proceeds by means of the sequential addition of Manp residues to PIM 1 or its acylated counterpart (AcPIM 1 ), § catalyzed in part by PimB and PimC, to produce PIM species having from two (PIM 2 ) to three (PIM 3 ) Manp residues (9, 10). The Manp units at position 6 of the inositol of PIM 3 are further elongated with Manp to generate higher PIMs (PIM 4 , PIM 5 , and PIM 6 ) (11, 12). However, the mannosyltransferases (ManTs) involved in these biosynthetic steps have not been identified. PIM 6 is likely a ''dead-end'' product, because it contains 2-linked Manp, a combination not found in the mannan core of LM͞ LAM (13); PIM 4 is the likely precursor for the subsequent extension of the mannan chain, giving rise to ''linear LM'' (12), which is further mannosylated at the C-2 positions, resulting in mature, branched LM. LAM itself retains the PI end and mannan backbone of LM; the Araf (arabinofuranose) residues originate in decaprenyl-P-arabinofuranose (C 50 -P-Araf ), and addition is partially catalyzed by EmbC (14).The only well characterized glycosyltransferases (GTs) implicated in PIM͞LM͞LAM biosynthesis are the three initial ManTs, PimA, PimB, and PimC, and EmbC (8-10, 14). Apparently, the ManTs that use GDP-Man as sugar donors occur on the cytoplas...

Inositol is utilized by Mycobacterium tuberculosis in the production of its major thiol and of essential cell wall lipoglycans. We have constructed a mutant lacking the gene encoding inositol-1-phosphate synthase (ino1), which catalyses the first committed step in inositol synthesis. This mutant is only viable in the presence of extremely high levels of inositol. Mutant bacteria cultured in inositol-free medium for four weeks showed a reduction in levels of mycothiol, but phosphatidylinositol mannoside, lipomannan and lipoarabinomannan levels were not altered. The ino1 mutant was attenuated in resting macrophages and in SCID mice. We used site-directed mutagenesis to alter four putative active site residues; all four alterations resulted in a loss of activity, and we demonstrated that a D310N mutation caused loss of the active site Zn2+ ion and a conformational change in the NAD+ cofactor.