GlnR-Mediated Regulation of Short-Chain Fatty Acid Assimilation in Mycobacterium smegmatis

Abstract

Assimilation of short-chain fatty acids (SCFAs) plays an important role in the survival and lipid biosynthesis of Mycobacteria. However, regulation of this process has not been thoroughly described. In the present work, we demonstrate that GlnR as a well-known nitrogen-sensing regulator transcriptionally modulates the AMP-forming propionyl-CoA synthetase (MsPrpE), and acetyl-CoA synthetases (MsAcs) is associated with SCFAs assimilation in Mycobacterium smegmatis, a model Mycobacterium. GlnR can directly activate the expression of MsprpE and Msacs by binding to their promoter regions based upon sensed nitrogen starvation in the host. Moreover, GlnR can activate the expression of lysine acetyltransferase encoding Mspat, which significantly decreases the activity of MsPrpE and MsAcs through increased acylation. Next, growth curves and resazurin assay show that GlnR can further regulate the growth of M. smegmatis on different SCFAs to control the viability. These results demonstrate that GlnR-mediated regulation of SCFA assimilation in response to the change of nitrogen signal serves to control the survival of M. smegmatis. These findings provide insights into the survival and nutrient utilization mechanisms of Mycobacteria in their host, which may enable new strategies in drug discovery for the control of tuberculosis.

Keywords: GlnR; acylation; nitrogen metabolism; post-translational modification; propionyl-CoA/acetyl-CoA synthetase; short-chain fatty acid.

FIGURE 1

GlnR regulates the expression of Msacs and MsprpE in Mycobacterium smegmatis. (A) Acetyl-CoA synthetase activity of MsAcs. (B) Propionyl-CoA synthetase activity of MsPrpE and MsAcs. (C) Transcriptional levels of the Msacs genes in wild type (WT) strain grown in glucose or acetate as carbon sources. (D) Transcription levels of MsprpE and Msacs in WT grown in glucose or propionate as carbon sources. (E,F) Electrophoretic mobility shift assay (EMSA) identification of GlnR binding with the promoter region of MsprpE and MsacsA1. GlnR protein labeled with His tag incubate with probe (5 ng, MsprpE or MsacsA1). Unlabeled specific probe (S) (200-fold excess) or non-specific competitor DNA (sperm DNA) (N) were used as controls. (G) Differential expression analysis of glnR regulating MsprpE and MsacsA1. WT, ΔglnR (glnR knocked out mutant) and ΔglnR::glnR (glnR complementary) were cultured at 30°C to the middle exponential phase. (H) WT was cultured in Sauton medium supplemented with N⁺ and N^-, following comparative analysis of glnR, MsprpE, or MsacsA1 gene in the excess (N⁺) or limited (N^-) nitrogen condition. Error bars represent the standard error, n = 3. P-values were determined by two-tailed unpaired t-test. ^∗P < 0.05; ^∗∗P < 0.01; ^∗∗∗P < 0.001.

FIGURE 2

The activity of MsPrpE and MsAcsA1 are regulated by propionylation. (A) MsAcsA1 (2 μM) was incubated independently or with MsPat (2.7 μM), Propionyl-CoA (20 μM), and cAMP (1 mM) within a total volume of 100 μL at 37°C for 2 h, followed by SDS-PAGE analysis. The propionylation extent was verified using Western blot assay (Upper). Meanwhile, another PAGE gel was stained with Coomassie brilliant blue as control (Lower). (B) Enzymatic activity of MsAcsA1 using same condition with (A). (C) LC-MS/MS spectrum of a tryptic peptide (m/z = 419.3) obtained from propionylated MsAcsA1. This spectrum matched the peptide sequence (boxed) in MsAcsA1, where a mass shift occurred between b3 and y4 ions consistent with propionylation at the lysine residue (amino acid position 906). (D) MsPrpE was incubated alone or in the presence of MsPat with the same condition of (A), followed by SDS-PAGE analysis. The propionylation extent was verified by Western blot (Upper). (E) Enzymatic activity of MsPrpE and MsAcsA1 using same condition with (D). (F) LC-MS/MS spectrum of a tryptic peptide (m/z = 401.3) obtained from propionylated MsPrpE. This spectrum matched the peptide sequence (boxed) in MsPrpE, where a mass shift occurred between b3 and y4 ions consistent with propionylation at the lysine residue (amino acid position 586). Error bar represent the standard error, and all experiments replicate three times. ^∗∗∗P < 0.001.

FIGURE 3

The activity of MsPrpE and MsAcsA1 are regulated by acetylation. (A) MsPrpE (2 μM) was incubated independently or with MsPat (2.7 μM), AcCoA (100 μM), and cAMP (1 mM) within a total volume of 100 μL at 37°C for 2 h, followed by SDS-PAGE analysis. The acetylation extent was verified using Western blot assay (Upper). Meanwhile, another PAGE gel was stained with Coomassie brilliant blue as control (Lower). (B) Enzymatic activity of MsPrpE using same condition with (A). (C) LC-MS/MS spectrum of a tryptic peptide (m/z = 401.3) obtained from acetylated MsPrpE. This spectrum matched the peptide sequence (boxed) in MsPrpE, where a mass shift occurred between b3 and y4 ions consistent with acetylation at the lysine residue (amino acid position 586). (D) MsAcsA1 was incubated alone or in the presence of MsPat with the same condition of (A), followed by SDS-PAGE analysis. The acetylation extent was verified using Western blot (Upper). (E) Enzymatic activity of MsAcsA1 using same condition with (A). Error bar represent the standard error, and all experiments replicate three times. ^∗∗∗P < 0.001.

FIGURE 4

GlnR regulate the expression of Mspat in M. smegmatis. (A) EMSA identification of GlnR binding with the promoter region of Mspat. GlnR protein labeled with His tag with different gradient was incubated with probe (5 ng, Mspat). A 200-fold excess of unlabeled specific probe (S) or non-specific competitor DNA (sperm DNA) (N) were conducted as controls. (B) Comparative analysis of Mspat gene in the excess (N⁺) or limited (N^-) nitrogen condition. (C) Differential expression analysis of glnR regulating Mspat. WT (wild type), ΔglnR (glnR knocked out mutant) and ΔglnR::glnR (glnR complementary) were cultured at 30°C to the middle exponential phase. Error bar represent the standard error, and all experiments replicate three times. ^∗∗P < 0.01; ^∗∗∗P < 0.001.

FIGURE 5

GlnR regulates acylation of the enzymes associated with short-chain fatty acid (SCFA) assimilation in M. smegmatis. (A) Equal His-PrpE or His-Acs protein (2 mg) purified from WT and ΔglnR strains of M. smegmatis cultured in LB medium were used to determine the propionylation level of MsPrpE (Upper) or acetylation level of MsAcs enzymes (Middle). Equal non-acylation proteins were used as controls (Lower). (B) Equal His-PrpE or His-Acs protein (1 mg) purified from M. smegmatis WT strain cultured in minimal Sauton medium with excess/limited nitrogen (N⁺/N^-) were used to determine the Propionylation level of MsPrpE (Upper) or acetylation level of MsAcsA1 enzymes (Middle). Equal non-acylation proteins were used as controls (Lower).

FIGURE 6

glnR gene mutation increase the growth and viability of M. smegmatis. (A–D) growth curves of the M. smegmatis WT, ΔglnR and ΔglnR::glnR strains growing in minimal medium with 10 mM propionate, acetate, cholesterol, or glucose, respectively. (E) the 96-well plate of 10 h incubation after resazurin addition. the metabolically active cells were indicated with pink color, whereas, the blue color represented resazurin reducing less and indicated low metabolic activity. (F) Quantification of resazurin fluorescence for cells growth. The fluorescence value at 10 h post-resazurin addition was normalized to the control of wild-type in different carbon condition. For (A–D) and (F), error bars denote the SD of three replicates. ^∗∗P < 0.01; ^∗∗∗P < 0.001.

FIGURE 7

GlnR-mediated regulation of SCFAs assimilation revealed a tight connection of carbon and nitrogen metabolism in M. smegmatis. high concn., high concentration; low concn., low concentration; cycle with brown line: TCA cycle; cycle with yellow line: Methylcitrate cycle; cycle with pink line: Methylmalonyl CoA pathway; OAA, oxaloacetate; 2MC, 2 methylcitrate; MMI-CoA, Methylmalonyl-CoA; PDIM, phthiocerol dimycocerosate.