The Mycobacterium tuberculosis ino1 gene is essential for growth and virulence

Abstract

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.

Genomic identification and in vitro reconstitution of a complete biosynthetic pathway for the osmolyte di- myo -inositol-phosphate

“…The identity of the resonances for these two compounds was confirmed by TOCSY and HMQC experiments (6,26). 13 C chemical shifts were obtained from an HMQC experiment and were consistent with published 13 C and 1 H shifts for these molecules (27). The MS analysis revealed the presence of 421.2 m/z peak consistent with the DIP anion and the 267.2 m/z peak likely corresponding to mannosylglycerate.…”

“…Two of these enzymes, IPS and IMP, are not solely committed to DIP biosynthesis as inositol derivatives play an important role in cell wall biogenesis, signaling, and other pathways in a broad spectrum of species including animals, plants, fungi, and mesophilic bacteria (11)(12)(13). Both enzymes are broadly conserved, and examples of each have been well characterized biochemically and structurally, including IMP and IPS from the hyperthermophiles Methanocaldococcus jannaschii (14), Archaeoglobus fulgidus (15)(16)(17)(18)(19), T. maritima (20), and Thermococcus kodakaraensis (21).…”

The pleiotropic transcriptional response of Mycobacterium tuberculosis to vitamin C is robust and overlaps with the bacterial response to multiple intracellular stresses

“…This gene encodes myoinositol-1-phosphate synthase, which is involved at the first step of inositol biosynthesis (Movahedzadeh et al, 2004) and is a component of cell wall constituents and of the redox buffer mycothiol (Fahey, 2001). Other upregulated genes include accA2 and accD2, which are believed to be involved in mycolic acid biosynthesis (Barry et al, 2007), as well as fadE5, fadE13 and fadD19, having fatty acid degradation function Muñoz-Elías & McKinney, 2006), and putative acyl-CoA dehydrogenases fadE1, fadE6, fadE26, fadE28 and fadE32, indicating that the bacterium might alter its metabolism to utilize fatty acids as a carbon source upon vit C exposure.…”

Probing the Mechanism of the Archaeoglobus fulgidus Inositol-1-phosphate Synthase

“…Additional evidence for significant conformational changes along the pathway and sequestration of the intermediates from dissociation into solution are needed to draw more detailed conclusions about the role of a given residue in catalysis. Indeed, several active site mutants of yeast and M. tuberculosis mIPS have been generated based on the crystal structures (17,18) and shown to be inactive. Exactly at what point in the mechanism the activity has been impaired was difficult to deduce from those experiments.…”

“…NAD ϩ binding to vacant sites decreases the intrinsic fluorescence intensity but not the wavelength of maximum emission of the archaeal mIPS protein. This change in fluorescence intensity can be used to quantify NAD ϩ binding to the proteins by measuring the intrinsic fluorescence intensity at 334 nm compared with that for the protein where only buffer was added over an NAD ϩ concentration range of [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20] M. There are two tryptophans near the active site of A. fulgidus mIPS (13) that could have altered characteristics upon NAD ϩ binding. From the change in fluorescence intensity as a function of added NAD ϩ , we used the concentration for 50% of the maximum change to define an apparent K D for NAD ϩ binding (see Fig.…”