Transcriptional Self-Regulation of the Master Nitrogen Regulator GlnR in Mycobacteria

Abstract

As a master nitrogen regulator in most actinomycetes, GlnR both governs central nitrogen metabolism and regulates many carbon, phosphate, and secondary metabolic pathways. To date, most studies have been focused on the GlnR regulon, while little is known about the transcriptional regulator for glnR itself. It has been observed that glnR transcription can be upregulated in Mycobacterium smegmatis under nitrogen-limited growth conditions; however, the detailed regulatory mechanism is still unclear. Here, we demonstrate that the glnR gene in M. smegmatis is transcriptionally activated by its product GlnR in response to nitrogen limitation. The precise GlnR binding site was successfully characterized in its promoter region using the electrophoretic mobility shift assay and the DNase I footprinting assay. Site mutagenesis and genetic analyses confirmed that the binding site was essential for in vivo self-activation of glnR transcription. Moreover, based on bioinformatic analyses, we discovered that most of the mycobacterial glnR promoter regions (144 out of 147) contain potential GlnR binding sites, and we subsequently proved that the purified M. smegmatis GlnR protein could specifically bind 16 promoter regions that represent 119 mycobacterial species, including Mycobacterium tuberculosis. Together, our findings not only elucidate the transcriptional self-regulation mechanism of glnR transcription in M. smegmatis but also indicate the ubiquity of the mechanism in other mycobacterial species. IMPORTANCE In actinomycetes, the nitrogen metabolism not only is essential for the construction of life macromolecules but also affects the biosynthesis of secondary metabolites and even virulence (e.g., Mycobacterium tuberculosis). The transcriptional regulation of genes involved in nitrogen metabolism has been thoroughly studied and involves the master nitrogen regulator GlnR. However, the transcriptional regulation of glnR itself remains elusive. Here, we demonstrated that GlnR functions as a transcriptional self-activator in response to nitrogen starvation in the fast-growing model Mycobacterium species Mycobacterium smegmatis. We further showed that this self-regulation mechanism could be widespread in other mycobacteria, which might be beneficial for those slow-growing mycobacteria to adapt to the nitrogen-starvation environments such as within human macrophages for M. tuberculosis.

Keywords: GlnR; mycobacteria; nitrogen metabolism; self-activation.

FIG 1

RT-qPCR analysis of glnR and glnA in the wild-type strain M. smegmatis mc²155 responding to different nitrogen sources. Strain mc²155 was first cultivated for 24 h in Sauton’s medium with 30 mM (NH₄)₂SO₄ as the sole nitrogen source (sample 1); the culture was then harvested, washed, and transferred into nitrogen-limited Sauton’s medium with 5 mM KNO₃ for continued incubation for 15, 30, and 60 min (samples 2, 3, and 4, respectively); finally, 30 mM (NH₄)₂SO₄ was added to the nitrogen-limited Sauton’s medium for continued incubation for another 15, 30, and 60 min (samples 5, 6, and 7, respectively). The relative fold changes of the y axis indicate the ratio of the glnR and glnA transcription level versus that obtained under the original condition [30 mM (NH₄)₂SO₄ as the sole nitrogen source]. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, no statistically significant difference. Primers RT-glnR2-fw/RT-glnR2-rev were used for the transcriptional analysis of glnR. Primers RT-glnA-fw/RT-glnA-rev were used for the transcriptional analysis of glnA.

FIG 2

Characterization of the GlnR binding box in its promoter region. (A) The promoter region sequence of glnR. TSS, the transcriptional start site of glnR revealed by the primer extension assay. The GlnR binding box is indicated by an underlined arrow. The boxed TTG codon is the predicted translational start codon. (B) Competitive EMSA to determine the specific binding ability of the M. smegmatis GlnR protein (MSM_GlnR) to its own promoter region (the probe glnRp). The concentrations of MSM_GlnR are 0, 0.2, 0.4, and 0.8 μM. The binding of MSM_GlnR to glnRp was competed with excess (20×) unlabeled glnRp (S) or part of the glnR coding sequence (NS). (C) Identification of the protected region by MSM_GlnR on its promoter region using the DNase I footprinting assay. (D) Mutation of the GlnR binding box. (E) EMSA analyses of the mutated GlnR binding box. The concentrations of MSM_GlnR are 0, 0.4, and 0.8 μM.

FIG 3

Comparison of glnR and glnA transcription in different M. smegmatis strains responding to different nitrogen sources and the growth phenotype of different M. smegmatis strains. (A) Analysis of the transcription of glnR responding to different nitrogen sources. The nitrogen sources used are Sauton’s medium with 30 mM (NH₄)₂SO₄ or 5 mM KNO₃ as the sole nitrogen source. The strains were first cultivated for 24 h in Sauton’s medium with 30 mM (NH₄)₂SO₄ as the sole nitrogen source; the culture was then harvested, washed, and transferred into nitrogen-limited Sauton’s medium with 5 mM KNO₃ for continued incubation for 30 min. WT/306, the wild-type strain transformed with pMV306; ΔglnR/306, the in-frame deletion mutant strain ΔglnR transformed with pMV306; ΔglnR/C, the in-frame deletion mutant strain ΔglnR transformed with an active glnR (pMV306-MSM_glnR_flag); ΔglnR/pmutC, the in-frame deletion mutant strain ΔglnR transformed with the glnR gene under the control of the mutant promoter (glnRp_mutC). Primers RT-glnR2-fw/RT-glnR2-rev were used for the transcriptional analysis of glnR. (B) Comparison of glnR and glnA transcription in both the wild-type strain and its glnR mutant strain H6 responding to different nitrogen sources. The sample preparation is the same as in panel A. WT/306, the wild-type strain transformed with the empty vector pMV306; H6/306, strain H6 transformed with pMV306; H6/C, strain H6 transformed with an active glnR gene (pMV306-MSM_glnR_flag). The transcription of glnR was detected with primers RT-glnR1-fw/RT-glnR1-rev. Primers RT-glnA-fw/RT-glnA-rev were used for the transcriptional analysis of glnA. (C) The growth phenotype of different M. smegmatis strains. *, P < 0.05; **, P < 0.01; ns, no statistically significant difference.

FIG 4

(A and B) Analysis of the transcription (A) and translation (B) of glnR responding to different nitrogen sources. The nitrogen sources used are Sauton’s medium with 30 mM (NH₄)₂SO₄ or 5 mM KNO₃ as the sole nitrogen source. Strain ΔglnR/C was first cultivated for 24 h in Sauton’s medium with 30 mM (NH₄)₂SO₄ as the sole nitrogen source; the culture was then harvested, washed, and transferred into nitrogen-limited Sauton’s medium with 5 mM KNO₃ for continued incubation for 15 min, 30 min, 1 h, 2 h, 4 h, 6 h, 8 h, and 10 h. Primers of RT-glnR2-fw/RT-glnR2-rev were used for the transcriptional analysis of glnR. WB, Western blot analysis of the FLAG-tagged GlnR protein; CBB, Coomassie brilliant blue staining. *, P < 0.05; **, P < 0.01; ns, no statistically significant difference.