Article Figures & Data
Figures
- FIG. 1.
Overexpression of the proteins in M. smegmatis confirmed by SDS-PAGE analysis. Lanes: M, marker; 1, vector; 2, Rv0155 (PntAA, 40 kDa); 3, Rv1484 (InhA, 29.5 kDa); 4, Rv2763c (DfrA, 17.4 kDa); 5, Rv1996 (34.9 kDa); 6, Rv2623 (32.7 kDa); 7, Rv2766c (FabG5, 30.8 kDa); 8, Rv2858 (AldC, 50 kDa); 9, uninduced Rv2763c (DfrA); 10, induced Rv2763c (DfrA).
- FIG. 2.
Growth of the wild-type M. smegmatis strain and transformed strains overexpressing DHFR or InhA on media containing the known DHFR inhibitor trimethoprim (50 or 150 μg/ml) or INH (10 or 120 μg/ml).
- FIG. 3.
Growth of M. tuberculosis H37Rv strains overexpressing DHFR or InhA on media containing INH (0.2 μg/ml) or kanamycin (100 μg/ml). Spots: A, pMV261::inhA (InhA overexpresser); B, pMV261 (plasmid only); C to F, four independent transformants of pMV261::dfrA (DHFR overexpresser).
Tables
- TABLE 1.
MICs determined in 7H9 broth by serial dilution method after overexpressing the INH-NAD binding proteins from M. tuberculosis H37Rv in M. smegmatis
Overexpressed protein MIC (μg/ml)a INH ETH None (mc24517 parental strain) 4.7 9.4 Rv1484 (InhA) 300 150 Rv2763c (DfrA) 4.7 9.4 Rv3248c (SahH) 4.7 9.4 Rv0753c (MmsA) 4.7 9.4 Rv1187 (RocA) 4.7 9.4 Rv0155 (PntAA) 4.7 9.4 Rv2623 (universal stress protein) 4.7 9.4 Rv1996 (universal stress protein) 4.7 9.4 Rv0468 (FadB2) 4.7 9.4 Rv2691 (CeoB/TrkA) 4.7 9.4 Rv0091 (Mtn/Sah) NA NA Rv2858c (AldC) 4.7 9.4 Rv1059 (unknown) 4.7 9.4 Rv0926c (unknown) 4.7 9.4 Rv3777 (probable oxidoreductase) 4.7 9.4 Rv2971 (probable oxidoreductase) NA NA Rv2766 (FabG5) 4.7 9.4 Rv2671 (RibD) 4.7 9.4 ↵a NA, not available (overexpression was not successful).
- TABLE 2.
Mutations in genes identified as potential INH-NAD(P) bindersa
Protein Mutation in strain: 5071 5072 5297 5324 5358 5400 M. bovisBCG Rv2763c/dfrA None None None None None None None Rv3248c/sahH None None None None None None None Rv0753c/mmsA None None None None None None None Rv1187/rocA None None None None None None None Rv0155/pntAa A274A None None None None None None Rv2623 None None None None None None None Rv1996 None None None None None None None Rv0468/fadB2 None None None None L87L None L87L Rv2691/ceoB T117A T117A T117A T117A T117A T117A T117A Rv0091/mtn None A155P None Q174Q None 931-bp deletion None Rv2858c/aldC None None T21A None P77P None P77P Rv1059 a-64cb 1-bp deletion in P99 None None None None None Rv3777 None None P101A None None None None Rv3777 V160A V160A V160A V160A V160A V160A V160A Rv3777 L63L L63L L63L L63L L63L L63L L63L Rv0926c None None None None None None None Rv2766c/fabG5 None None None None None None None Rv2671/ribD None None None None None None None Rv2971 None None None None N152H None N152H - TABLE 3.
Mutations in genes traditionally associated with INH resistancea
Protein Mutation(s) in strain: 5071 5072 5297 5324 5358 5400 M. bovisBCG Rv1484/inhA None None None None None None None Rv1908c/katG None None None G285V R463L F129S R463L Rv1909c/furA None None None None A46V None A46V Rv1854c/ndh None None None None G313R None G313R Rv0342/iniA None None None None N88S, H481Q None N88S, H481Q Rv0341/iniB None None None None None None None Rv0343/iniC None None None None None None None Rv3139/fadE24 None None None None None None None Rv2245/kasA None None None None None None None Rv2246/kasB None None None None None None None Rv2428/ahpC None None None None None None None Rv2242 None None None None A363T None A363T Rv0340 None None None None None None None Rv1592c I322V I322V I322V I322V I322V I322V I322V Rv1772 None None None None Deletion None Deletion Rv2846c/efpA None None None Q513R T15R None T15R Rv0486/mshA N111S N111S None N111S None N111S None ↵a Mutations are indicated relative to the amino acid in the H37Rv reference sequence. Synonymous mutations are excluded.
