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. 2019 Oct 9;8:e51603. doi: 10.7554/eLife.51603

Figure 4. NrtR is a repressor for the nadABC operon that is responsible for NAD+ and NADH concentration in M. smegmatis.

(A) Genetic organization and transcriptional analyses of the nrtR and its neighboring de novo NAD+ synthesis genes. The arrows represent open reading frames, and the numbered short lines (1 to 7) represent the specific PCR amplicons that were observed in the following PCR and RT-PCR assays (in the bottom panels). PCR and RT-PCR were applied to analyze the transcription of the putative NAD+ de novo synthesis loci. The primer numbering was identical to that shown in the top panel. CK (control) denotes the 16S rDNA. (B) RT-qPCR analyses of nad operon expression in the wild-type strain and in the ΔnrtR mutant and nrtR complementary strains. RT-qPCR experiments were performed at least three times and the data were expressed as means ± standard deviations (SD). The p-value was calculated using one-way ANOVA along with Tukey's test. *p<0.05 and **p<0.01. Comparison of the intra-cellular level of NAD+ (C) and NADH (D) among the WT, ΔnrtR and CΔnrtR strains. Each dark circle or triangle represents an independent experiment. The data are shown as means ± SD. The statistical significance of differences among WT, ΔnrtR and CΔnrtR was determined by Student’s t test and by ANOVA with heterogeneous variances. ***p<0.001; ns, no significant difference.

Figure 4.

Figure 4—figure supplement 1. In vivo evidence that MsNrtR is an auto-repressor.

Figure 4—figure supplement 1.

(A) LacZ-based visualization of the auto-regulation of NrtR in M. smegmatis. Left column: schematic representation of promoter-lacZ transcriptional fusions. A promoter-less LacZ refers to the blank control (abbreviated as ‘B’); the hsp60 promoter-fused LacZ acts as the negative control (indicated by ‘–'); and the nrtR promoter-driven LacZ is used to evaluate the regulatory role of the NrtR repressor (highlighted with ‘R’). Right column: the exponentially growing M. smegmatis cultures of the wild-type and ΔnrtR strains were diluted appropriately and spotted onto 7H10 plates containing 50 μg/ml kanamycin and 50 mg/ml X-gal. The plates were incubated at 37°C for 48 hr. (B) Growth curves of WT and nrtR deletion strains carrying a transcriptional fusion plasmid, pMV261-promoter-lacZ. Cultures were grown in LB medium supplemented with 0.5% glycerol, 0.05% tween 80, and 50 mg/ml kan at 37°C, 220 rpm, and absorbance at 600 nm was recorded at 2 hr intervals for 28 hr. (C) Transcriptional levels of nrtR in lag-phase cultures of the wild-type and in the ΔnrtR mutant of M. smegmatis, evaluated using lacZ-transcriptional fusions. A LacZ controlled by the hsp60 promoter acts as the negative control. Results are expressed as an average ± standard deviation (SD) from no less than three independent tests. (D) Transcriptional levels of nrtR in exponential-phase cultures of wild-type and nrtR-deleted strains of M. smegmatis, evaluated using lacZ-transcriptional fusions (E) Transcriptional levels of nrtR in stationary-phase cultures of wild-type M. smegmatis and its nrtR deletion mutant, evaluated using lacZ-transcriptional fusions. Data are presented as mean ± SD. The p-value was measured using one-way ANOVA along with Tukey's test. **, p<0.01; ***, p<0.001.