Figure 7. Acetylation of K134 in MsNrtR determines its role in the homeostasis of the intracellular NAD+ pool.
(A) Genetic assays for the auto-repression of nrtR using PnrtR-lacZ transcriptional fusion. These results suggest that functional impairments in K134 acetylation lead to de-repression of nrtR, as does the removal of nrtR. (B) RT-qPCR analyses of the transcription of the nad operon in the mutant carrying a point-mutation of K134 in nrtR (K134A, K134R and K134Q). Levels of intracellular NAD+ (C) and NADH (D) in the WT nrtR strain and its point-mutants (K134A, K134R and K134Q). All of the experiments were performed at least three times, and the data are presented as means ± SD. The p-values were calculated using one-way ANOVA along with Tukey's test.
Figure 7—figure supplement 1. Determining the number of live cells at OD600 during the exponential phase.
The wild-type and mutant strains were cultured to exponential phase, harvested by centrifugation, and then re-suspended with fresh 7H9 broth. The optical density (OD600) was adjusted to ~1.0. Following series of 10-fold dilution (1 × 104, 1 × 105 and 1 × 106 times) with fresh 7H9 broth, 100 μl of diluted bacterial suspension was plated on LB agar plates. Colony counting was performed after incubation at 37°C for three days. It suggested that the OD600 of M. smegmatis represents around 108 cells (indicated with a dashed line).