title: 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.
authors.affiliation: Department of Pathology and Infectious Diseases, Royal Veterinary College, London, UK.
authors.forename: Farahnaz
authors.initials: F
authors.lastname: Movahedzadeh
authors.forename: Debbie A
authors.initials: DA
authors.lastname: Smith
authors.forename: Richard A
authors.initials: RA
authors.lastname: Norman
authors.forename: Premkumar
authors.initials: P
authors.lastname: Dinadayala
authors.forename: Judith
authors.initials: J
authors.lastname: Murray-Rust
authors.forename: David G
authors.initials: DG
authors.lastname: Russell
authors.forename: Sharon L
authors.initials: SL
authors.lastname: Kendall
authors.forename: Stuart C G
authors.initials: SC
authors.lastname: Rison
authors.forename: Mark S B
authors.initials: MS
authors.lastname: McAlister
authors.forename: Gregory J
authors.initials: GJ
authors.lastname: Bancroft
authors.forename: Neil Q
authors.initials: NQ
authors.lastname: McDonald
authors.forename: Mamadou
authors.initials: M
authors.lastname: Daffe
authors.forename: Yossef
authors.initials: Y
authors.lastname: Av-Gay
authors.forename: Neil G
authors.initials: NG
authors.lastname: Stoker
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D003545:Cysteine; D004252:DNA Mutational Analysis; D004187:Disaccharides / Q000032:analysis; D017353:Gene Deletion; D005798:Genes, Bacterial; D020043:Genes, Essential; D006020:Glycopeptides; D007294:Inositol / Q000096:biosynthesis; D008070:Lipopolysaccharides / Q000032:analysis; D008264:Macrophages / Q000382:microbiology; D051379:Mice; D016513:Mice, SCID; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009169:Mycobacterium tuberculosis / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development / Q000472:pathogenicity; D007296:Myo-Inositol-1-Phosphate Synthase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D010716:Phosphatidylinositols / Q000032:analysis; D011720:Pyrazoles / Q000032:analysis; D013438:Sulfhydryl Compounds / Q000032:analysis; D014376:Tuberculosis / Q000382:microbiology; D014774:Virulence / Q000235:genetics
chemical_list: D004187:Disaccharides; D006020:Glycopeptides; D008070:Lipopolysaccharides; D010716:Phosphatidylinositols; D011720:Pyrazoles; D013438:Sulfhydryl Compounds; C050016:lipoarabinomannan; C064351:lipomannan; C089265:mycothiol; C034154:phosphatidylinositol mannoside; D007294:Inositol; D007296:Myo-Inositol-1-Phosphate Synthase; D003545:Cysteine