The Mycobacterium tuberculosis ino1 gene is essential for growth and virulence.
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.
Movahedzadeh
Farahnaz
F
Department of Pathology and Infectious Diseases, Royal Veterinary College, London, UK.
Smith
Debbie A
DA
Norman
Richard A
RA
Dinadayala
Premkumar
P
Murray-Rust
Judith
J
Russell
David G
DG
Kendall
Sharon L
SL
Rison
Stuart C G
SC
McAlister
Mark S B
MS
Bancroft
Gregory J
GJ
McDonald
Neil Q
NQ
Daffe
Mamadou
M
Av-Gay
Yossef
Y
Stoker
Neil G
NG
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
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