Cell wall enrichment unveils proteomic changes in the cell wall during treatment of Mycobacterium smegmatis with sub-lethal concentrations of rifampicin
“…To examine how cobalamin availability in M. smegmatis affected MetE protein content, we adapted a targeted MS method (21) to measure MetE peptide levels in wild type and Δ cobK strains grown in the presence or absence of exogenous CNCbl. This analysis indicated that MetE protein levels were 3× more abundant in the Δ cobK mutant than in the parental wild type strain (Figure 4B).…”
Section: Resultsmentioning
confidence: 99%
“…Triplicate cultures of M. smegmatis were grown to OD 600 ~1.2 in 7H9-OADC with or without 10μM CNCbl. Cell lysis, fractionation and the generation of tryptic peptides was done as described (21). Selected Reaction Monitoring (SRM) assays were developed in Skyline (version 4.1) using a spectral library generated from previous discovery MS data (21) with a cut-off score of 0.9.…”
Section: Methodsmentioning
confidence: 99%
“…Total RNA was extracted using the FastRNA® Pro Blue kit (MP Biomedicals) and DNase-treated with TURBO DNAse (Ambion) after which 0.5g was used as template for cDNA synthesis, using the High Capacity RNA to cDNA kit (Thermofisher). Primers and minor groove binder (MGB) Taqman probes (Table S2) using a spectral library generated from previous discovery MS data (21) with a cutoff score of 0.9. Skyline was set up to select two peptides per input protein, with the highest picked MS1 intensity in the discovery data, and then the top 5 most intense fragment ions for each of those peptides.…”
Section: Quantitative Gene Expression Analysis By Ddpcr Droplet Digimentioning
Cobalamin is an essential co-factor in all domains of life, yet its biosynthesis is restricted to some bacteria and archaea. Mycobacterium smegmatis, an environmental saprophyte frequently used as surrogate for the obligate human pathogen, M. tuberculosis, carries approximately 30 genes predicted to be involved in de novo cobalamin biosynthesis. M. smegmatis also encodes multiple cobalamin-dependent enzymes, including MetH, a methionine synthase which catalyses the final reaction in methionine biosynthesis. In addition to metH, M. smegmatis possesses a cobalamin-independent methionine synthase, metE, suggesting that enzyme selection – MetH or MetE – is regulated by cobalamin availability. Consistent with this notion, we previously described a cobalamin-sensing riboswitch controlling metE expression in M. tuberculosis. Here, we apply a targeted mass spectrometry-based approach to confirm de novo cobalamin biosynthesis in M. smegmatis during aerobic growth in vitro. We also demonstrate that M. smegmatis transports and assimilates exogenous cyanocobalamin (CNCbl; a.k.a. vitamin B12) and its precursor, dicyanocobinamide ((CN)2Cbi). Interestingly, the uptake of CNCbl and (CN)2Cbi appears restricted in M. smegmatis and dependent on the conditional essentiality of the cobalamin-dependent methionine synthase. Using gene and protein expression analyses combined with single-cell growth kinetics and live-cell time-lapse microscopy, we show that transcription and translation of metE are strongly attenuated by endogenous cobalamin. These results support the inference that metH essentiality in M. smegmatis results from riboswitch-mediated repression of MetE expression. Moreover, differences observed in cobalamin-dependent metabolism between M. smegmatis and M. tuberculosis provide some insight into the selective pressures which might have shaped mycobacterial metabolism for pathogenicity.
“…To examine how cobalamin availability in M. smegmatis affected MetE protein content, we adapted a targeted MS method (21) to measure MetE peptide levels in wild type and Δ cobK strains grown in the presence or absence of exogenous CNCbl. This analysis indicated that MetE protein levels were 3× more abundant in the Δ cobK mutant than in the parental wild type strain (Figure 4B).…”
Section: Resultsmentioning
confidence: 99%
“…Triplicate cultures of M. smegmatis were grown to OD 600 ~1.2 in 7H9-OADC with or without 10μM CNCbl. Cell lysis, fractionation and the generation of tryptic peptides was done as described (21). Selected Reaction Monitoring (SRM) assays were developed in Skyline (version 4.1) using a spectral library generated from previous discovery MS data (21) with a cut-off score of 0.9.…”
Section: Methodsmentioning
confidence: 99%
“…Total RNA was extracted using the FastRNA® Pro Blue kit (MP Biomedicals) and DNase-treated with TURBO DNAse (Ambion) after which 0.5g was used as template for cDNA synthesis, using the High Capacity RNA to cDNA kit (Thermofisher). Primers and minor groove binder (MGB) Taqman probes (Table S2) using a spectral library generated from previous discovery MS data (21) with a cutoff score of 0.9. Skyline was set up to select two peptides per input protein, with the highest picked MS1 intensity in the discovery data, and then the top 5 most intense fragment ions for each of those peptides.…”
Section: Quantitative Gene Expression Analysis By Ddpcr Droplet Digimentioning
Cobalamin is an essential co-factor in all domains of life, yet its biosynthesis is restricted to some bacteria and archaea. Mycobacterium smegmatis, an environmental saprophyte frequently used as surrogate for the obligate human pathogen, M. tuberculosis, carries approximately 30 genes predicted to be involved in de novo cobalamin biosynthesis. M. smegmatis also encodes multiple cobalamin-dependent enzymes, including MetH, a methionine synthase which catalyses the final reaction in methionine biosynthesis. In addition to metH, M. smegmatis possesses a cobalamin-independent methionine synthase, metE, suggesting that enzyme selection – MetH or MetE – is regulated by cobalamin availability. Consistent with this notion, we previously described a cobalamin-sensing riboswitch controlling metE expression in M. tuberculosis. Here, we apply a targeted mass spectrometry-based approach to confirm de novo cobalamin biosynthesis in M. smegmatis during aerobic growth in vitro. We also demonstrate that M. smegmatis transports and assimilates exogenous cyanocobalamin (CNCbl; a.k.a. vitamin B12) and its precursor, dicyanocobinamide ((CN)2Cbi). Interestingly, the uptake of CNCbl and (CN)2Cbi appears restricted in M. smegmatis and dependent on the conditional essentiality of the cobalamin-dependent methionine synthase. Using gene and protein expression analyses combined with single-cell growth kinetics and live-cell time-lapse microscopy, we show that transcription and translation of metE are strongly attenuated by endogenous cobalamin. These results support the inference that metH essentiality in M. smegmatis results from riboswitch-mediated repression of MetE expression. Moreover, differences observed in cobalamin-dependent metabolism between M. smegmatis and M. tuberculosis provide some insight into the selective pressures which might have shaped mycobacterial metabolism for pathogenicity.
“…1). We have successfully applied this method to quantify the changes in the cell wall proteome of M. smegmatis after exposure to rifampicin [4]. Fig.…”
Section: Methods Detailsmentioning
confidence: 99%
“…We have successfully used glycosides such as 1% dodecyl-D-maltoside, non-ionic and anionic detergent mixtures such as 0.1% Triton X100, 0.05% Tween-20 and 0.2% CHAPS or 0.15% deoxycholate and 0.1% SDS, as well as 2% Triton X114. We have shown that each detergent favours the extraction of specific protein classes – glycosides such as DDM are good solubilisers of porins, whereas the Triton family detergents are good at solubilising more hydrophobic proteins such as lipoproteins [4]. Therefore, the choice of detergent will be dependent on the experimental question.…”
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