Figure 1.
The antimutator role of MazG in Msm (A) and Mtb (B).
Both the bacterial culture conditions and the methods for determination of mutation frequencies were illustrated in Materials and Methods in detail. The frequencies conferring resistance to rifampicin in wild-type (wt), mazG-null (ΔmazG) and the complemented mutant (compl) strains were determined in exponential phase (OD600∼0.5) with or without oxidative stress and in the stationary growth phase. Oxidative stress was induced by treating exponential phase cultures with 10 mM H2O2 for 5 h (Msm) or 24 h (Mtb). Stationary phase was at the 5th-day or 28th-day of culture for Msm or Mtb, respectively. (C) Survival rate of Msm and Mtb strains after exposure to H2O2. The numbers shown are mean ± S.E. of 3 independent experiments totaling 15 cultures of Msm and 6 of Mtb.
Table 1.
mazG-null Msm exhibited elevated CG to TA mutation under oxidative stress conditions and in the stationary phase of growth.
Figure 2.
The NTP-PPase activity of mycobacterial MazG against 5-OH-dCTP.
(A) Time- and enzyme concentration-dependent hydrolysis of 5-OH-dCTP. 5-OH-dCTP (200 µM) was incubated with 1 µg or varied amounts (from 1 µg to 4 µg) of heterogeneously expressed MazG purified to nearly SDS-PAGE homogeneity. The reaction was carried out at 37°C and terminated after 10 min or at the time points as indicated. PPi, pyrophosphate. Shown are mean ± S.E. of 3 repeats. (B) Michaelis-Menten curves of MazG with 5-OH-dCTP or dCTP as substrate. The hydrolytic product PPi is shown as µM/10 min. Data shown are mean ± S.E. of 3 independent experiments.
Table 2.
Kinetic constants of Mtb MazG.
Figure 3.
5-OH-dCTP is an in vivo substrate of mycobacterial MazG proved by chemical genetic analysis.
The Msm competent cells were prepared as described in Materials and Methods, the nucleotides were incorporated by transformation. (A) mazG-null Msm (ΔmazG) exhibited a dose-dependent 5-OH-dCTP induced mutagenesis. wt, wild-type Msm. The data shown are mean ± S.E. of four repeats. (B) The rifampicin-resistance mutation frequencies of wild-type Msm, the mazG-null mutant and the complemented mutant treated with 5-OH-dCTP and dCTP. Nucleotides were added at 100 µM final concentration. Mean ± S.E of 12 independent transformations. **p<0.01 vs wt, * p<0.05 vs wt. (C) The rifampicin-resistance mutation frequencies of wild-type and mazG-null Msm treated with 100 µM 2-OH-dATP, 5-CHO-dUTP and normal dNTPs. Mean ± S.E of 8 independent transformations. (D) mazG-null Msm is susceptible to killing by 5-OH-dCTP. Strains were treated with 100 µM 5-OH-dCTP for 5 hours at 37°C. Shown is percent survival compared to an untreated control (100%). The data shown are mean ± S.E. of four repeats.
Figure 4.
Mtb MazG is required for resistance to RNS shown in vitro and ex vivo.
(A) mazG-null Mtb is susceptible to killing by acid nitrite. Exponential phase cultures (OD600∼0.5) were suspended in 7H9-OADC pH4.5 with or without 2.5 mM NaNO2 and treated for 20 hours. wt, wild-type Mtb; ΔmazG, mazG-null Mtb; compl, the complemented mutant. Data shown are mean ± S.E. of triplicates. * p<0.05 vs wt. (B) Quantitative real-time PCR analyses of mazG and dosR from Mtb treated with DETA/NO or acid nitrite. All data were normalized to the levels of sigA. The dosR gene was used as a positive control [70]. Results are expressed as the changes in expression levels compared to untreated samples. Mean ± S.E of three independent repeats. C to E, Survival of wild-type Mtb, the mazG-null mutant and the complemented mutant in resting (C), activated (D) or NMMA treated (E) cells of murine macrophage cell line RAW264.7. Shown is one representative result of two independent experiments.
Figure 5.
MazG is required for Mtb survival in vivo and the corresponding lung pathogenesis.
A to B, Bacterial loads in spleen (A) and lung (B) of mice infected with wild-type Mtb (wt), the mazG-null mutant (ΔmazG) and the complemented mutant (compl). Data shown are mean ± S.E from 4 mice per group. (C) Lung sections taken from mice at 8-wk after infection and stained with hematoxylin and eosin. Inserted column shows mean ± S.E of lung inflammation of each group.