m smegmatis mc  (ATCC)

Journal: bioRxiv

Article Title: Hydrogenase-driven ATP synthesis from air

doi: 10.1101/2025.03.14.643271

Figure Lengend Snippet: Bioenergetic parameters of Mycobacterium smegmatis continuous cultures as a function of dilution rate (controlled by glycerol availability). A) Glycerol consumption rate and cell counts measured by colony forming units (CFU mL −1 ), B) Cell yield per glycerol consumpted (Y glycerol ), C) Cell yield per ATP consumed (Y ATP ), D) Maintenance energy requirements (m ATP ; based on slope of function 1/Y ATP over 1/Dilution rate). The values reported are the means of two to three independent experiments and three technical replicates as each dilution rate and the experimental error associated with the values was less than 20%.

Article Snippet: Middlebrook 7H9 basal medium supplemented with 0.1% (w/v) Tween 80 and 0.2% (w/v) glycerol was used to culture M. smegmatis mc 2 155 continuously in a New Brunswick BioFlo fermenter model C30 using a 350 ml constant culture volume at 37°C.

Techniques:

Journal: bioRxiv

Article Title: Hydrogenase-driven ATP synthesis from air

doi: 10.1101/2025.03.14.643271

Figure Lengend Snippet:

Article Snippet: Middlebrook 7H9 basal medium supplemented with 0.1% (w/v) Tween 80 and 0.2% (w/v) glycerol was used to culture M. smegmatis mc 2 155 continuously in a New Brunswick BioFlo fermenter model C30 using a 350 ml constant culture volume at 37°C.

Techniques:

Journal: BMC Microbiology

Article Title: Involvement of Mycobacterium smegmatis small noncoding RNA B11 in triacylglycerol accumulation and altered cell wall permeability

doi: 10.1186/s12866-025-03826-7

Figure Lengend Snippet: Mutant isolation and phenotype analysis. ( A ) Prediction of the secondary structure of M. tuberculosis B11 using MFold. ( B ) Schematic representation of site-directed mutagenesis in L2 and L3 of B11, with red indicating the mutation sites. ( C ) Colony morphology, ( D ) Sliding motility, and ( E ) Growth curve of recombinant strains. ( F ) Morphology of recombinant strains observed under SEM. ( G ) Calculation of cell lengths from representative fields (approximately 100 cells) as visualized by SEM. The cell length was calculated using ImageJ. A total of 100 cells from multiple fields of view were randomly selected and measured. Lengths of the bacterial cells were calculated from the coordinates of both ends of the cell as measured from representative fields as visualized by SEM. * P < 0.05, ** P < 0.01, *** P < 0.001. Ms_vc, M. smegmatis harboring empty vector as the control. Ms_B11, M. smegmatis expressing B11. Ms_B11 as, M. smegmatis expressing B11 antisense. Ms_B11 L2, M. smegmatis expressing B11 with site-directed mutagenesis in the L2 loop. Ms_B11 L3, M. smegmatis expressing B11 with site-directed mutagenesis in the L3 loop

Article Snippet: M. smegmatis mc 2 155 recombinant strains were cultured in LB medium supplemented with 100 mg/mL Congo Red (Sigma), Sauton liquid medium, and 7H9 medium supplemented with 0.3% agar, respectively, and phenotypes were observed.

Techniques: Mutagenesis, Isolation, Recombinant, Plasmid Preparation, Control, Expressing

Journal: BMC Microbiology

Article Title: Involvement of Mycobacterium smegmatis small noncoding RNA B11 in triacylglycerol accumulation and altered cell wall permeability

doi: 10.1186/s12866-025-03826-7

Figure Lengend Snippet: Transcriptome analysis. ( A ) Volcano plots showing differentially expressed genes between B11-overexpressing and control M. smegmatis strains. ( A , D ) GO terms and KEGG pathways of upregulated genes. ( C , E ) GO terms and KEGG pathways of downregulated genes. ( F ) Identification of glycerolipid metabolism as the most significantly enriched pathway in KEGG analysis. CC, Cellular Component; BP, Biological Process; MF, Molecular Function

Article Snippet: M. smegmatis mc 2 155 recombinant strains were cultured in LB medium supplemented with 100 mg/mL Congo Red (Sigma), Sauton liquid medium, and 7H9 medium supplemented with 0.3% agar, respectively, and phenotypes were observed.

Techniques: Control

Journal: BMC Microbiology

Article Title: Involvement of Mycobacterium smegmatis small noncoding RNA B11 in triacylglycerol accumulation and altered cell wall permeability

doi: 10.1186/s12866-025-03826-7

Figure Lengend Snippet: Overexpression reduces biofilm formation and alters cell wall permeability. ( A ) Effect of B11 on biofilm formation of M. smegmatis . Recombinant strains were cultured in Sauton’s medium without shaking. ( B ) Quantification of biofilm formation after crystal violet staining. Values are shown as mean ± SD ( n = 12). ( C ) Mid-log-phase cultures of recombinant strains incubated in phosphate-Buffered Saline containing Tween 20 (PBST) containing 2 µg/ml ethidium bromide (EtBr). ( D ) Mid-log-phase cultures of recombinant strains incubated in PBST containing 20 µM Nile red. RFI, relative fluorescence intensity. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: M. smegmatis mc 2 155 recombinant strains were cultured in LB medium supplemented with 100 mg/mL Congo Red (Sigma), Sauton liquid medium, and 7H9 medium supplemented with 0.3% agar, respectively, and phenotypes were observed.

Techniques: Over Expression, Permeability, Recombinant, Cell Culture, Staining, Incubation, Saline, Fluorescence

Journal: BMC Microbiology

Article Title: Involvement of Mycobacterium smegmatis small noncoding RNA B11 in triacylglycerol accumulation and altered cell wall permeability

doi: 10.1186/s12866-025-03826-7

Figure Lengend Snippet: B11 indirectly regulates target genes without direct binding to proteins. ( A ) Identification of B11-binding proteins. Left: silver staining of pulled-down proteins. Right: mass spectrometry showing the top 10 proteins pulled-down by the B11 probe. ( B ) Heat map illustrating differentially expressed genes in RNA-seq dataset. Red indicates highly expressed genes; blue, lowly expressed genes. ( C ) qRT-PCR analysis verifying the expression of genes corresponding to B11-binding proteins. * P < 0.05, ** P < 0.01, *** P < 0.001. ( D ) Expression of hspX, rpiL, and MSMEG_6092 in M. smegmatis . Western blot images showing the expression of flag-tagged protein. ( E ) Biotinylated B11 or antisense RNA incubated with whole-cell lysates from hspX, rpiL, or MSMG_6092 overexpression strains. Membranes immunoblotted for hspX, rpiL, and MSMG_6092. Input, total protein group

Article Snippet: M. smegmatis mc 2 155 recombinant strains were cultured in LB medium supplemented with 100 mg/mL Congo Red (Sigma), Sauton liquid medium, and 7H9 medium supplemented with 0.3% agar, respectively, and phenotypes were observed.

Techniques: Binding Assay, Silver Staining, Mass Spectrometry, RNA Sequencing, Quantitative RT-PCR, Expressing, Western Blot, Incubation, Over Expression

Journal: Scientific Reports

Article Title: Repurposing of apoptotic inducer drugs against Mycobacterium tuberculosis

doi: 10.1038/s41598-025-91096-8

Figure Lengend Snippet: Antimycobacterial activity of apoptotic inducer drugs against M. smegmatis mc 2 155 and M. tuberculosis H37Rv .

Article Snippet: This was followed by a host-directed evaluation of compounds to stimulate clearance of M. smegmatis mc 2 155 in infected THP-1 macrophage cells and the production of cytokines during treatment, which were measured using Luminex.

Techniques: Activity Assay

Journal: Scientific Reports

Article Title: Repurposing of apoptotic inducer drugs against Mycobacterium tuberculosis

doi: 10.1038/s41598-025-91096-8

Figure Lengend Snippet: Cytotoxicity profile of apoptotic inducer drugs against differentiated THP-1 macrophage cells. The healthy THP-1 macrophages were treated with various concentrations of the drugs corresponding to previously recorded MIC values against M. smegmatis mc 2 155 and M. tuberculosis H37Rv. Data represents three technical and biological replicates where treated macrophages were analyzed against untreated controls. Data was interpreted as viable percentages and analyzed through ANOVA where p ≤ 0,05*.

Article Snippet: This was followed by a host-directed evaluation of compounds to stimulate clearance of M. smegmatis mc 2 155 in infected THP-1 macrophage cells and the production of cytokines during treatment, which were measured using Luminex.

Techniques:

Journal: Scientific Reports

Article Title: Repurposing of apoptotic inducer drugs against Mycobacterium tuberculosis

doi: 10.1038/s41598-025-91096-8

Figure Lengend Snippet: Percentage survival of M. smegmatis mc 2 155 ( M.smeg ) within THP-1 macrophages relative to untreated control at 6 and 12 h post-treatment with apoptotic inducer drugs. Data is a representative of three biological replicates and shown as mean ± SD. Two-way ANOVA followed by Dunnett’s multiple comparison test was used for data analysis where significant difference is indicated as p ≤ 0.01**, p ≤ 0.001***, p ≤ 0.0001****.

Article Snippet: This was followed by a host-directed evaluation of compounds to stimulate clearance of M. smegmatis mc 2 155 in infected THP-1 macrophage cells and the production of cytokines during treatment, which were measured using Luminex.

Techniques: Control, Comparison

Journal: Scientific Reports

Article Title: Repurposing of apoptotic inducer drugs against Mycobacterium tuberculosis

doi: 10.1038/s41598-025-91096-8

Figure Lengend Snippet: Effect of apoptotic inducer drugs on the secretion of 13 selected cytokines in uninfected and M. smegmatis mc 2 155-infected THP-1 macrophages. Cytokine expression was evaluated at two time points (6 and 12 h) in infected and uninfected THP-1 cells after treatment with apoptotic inducer drugs. Data was expressed in pg/mL and normalized per column where 0% (purple) was indicated by the lowest value and 100% (red) was indicated by the highest value.

Article Snippet: This was followed by a host-directed evaluation of compounds to stimulate clearance of M. smegmatis mc 2 155 in infected THP-1 macrophage cells and the production of cytokines during treatment, which were measured using Luminex.

Techniques: Infection, Expressing

Journal: Scientific Reports

Article Title: Repurposing of apoptotic inducer drugs against Mycobacterium tuberculosis

doi: 10.1038/s41598-025-91096-8

Figure Lengend Snippet: Representative plots showing cytokine changes measured by Luminex multiplex assay in uninfected and M. smegmatis mc 2 155 infected THP-1 macrophages ( A ): IL-1β, ( B ): TNF-α, ( C ): IL-6. Data were analyzed using two-way ANOVA.

Article Snippet: This was followed by a host-directed evaluation of compounds to stimulate clearance of M. smegmatis mc 2 155 in infected THP-1 macrophage cells and the production of cytokines during treatment, which were measured using Luminex.

Techniques: Luminex, Multiplex Assay, Infection

Journal: Scientific Reports

Article Title: Repurposing of apoptotic inducer drugs against Mycobacterium tuberculosis

doi: 10.1038/s41598-025-91096-8

Figure Lengend Snippet: Antimycobacterial activity of apoptotic inducer drugs against M. smegmatis mc 2 155 and M. tuberculosis H37Rv .

Article Snippet: A cryopreserved M. smegmatis mc 2 155 stock was cultured in 10 mL of 7H9 OADC (Difco, Becton, Dickinson and Company, New Jersey, United States) until reaching an OD 600 nm of 1.0.

Techniques: Activity Assay

Journal: Scientific Reports

Article Title: Repurposing of apoptotic inducer drugs against Mycobacterium tuberculosis

doi: 10.1038/s41598-025-91096-8

Figure Lengend Snippet: Cytotoxicity profile of apoptotic inducer drugs against differentiated THP-1 macrophage cells. The healthy THP-1 macrophages were treated with various concentrations of the drugs corresponding to previously recorded MIC values against M. smegmatis mc 2 155 and M. tuberculosis H37Rv. Data represents three technical and biological replicates where treated macrophages were analyzed against untreated controls. Data was interpreted as viable percentages and analyzed through ANOVA where p ≤ 0,05*.

Article Snippet: A cryopreserved M. smegmatis mc 2 155 stock was cultured in 10 mL of 7H9 OADC (Difco, Becton, Dickinson and Company, New Jersey, United States) until reaching an OD 600 nm of 1.0.

Techniques:

Journal: Scientific Reports

Article Title: Repurposing of apoptotic inducer drugs against Mycobacterium tuberculosis

doi: 10.1038/s41598-025-91096-8

Figure Lengend Snippet: Percentage survival of M. smegmatis mc 2 155 ( M.smeg ) within THP-1 macrophages relative to untreated control at 6 and 12 h post-treatment with apoptotic inducer drugs. Data is a representative of three biological replicates and shown as mean ± SD. Two-way ANOVA followed by Dunnett’s multiple comparison test was used for data analysis where significant difference is indicated as p ≤ 0.01**, p ≤ 0.001***, p ≤ 0.0001****.

Article Snippet: A cryopreserved M. smegmatis mc 2 155 stock was cultured in 10 mL of 7H9 OADC (Difco, Becton, Dickinson and Company, New Jersey, United States) until reaching an OD 600 nm of 1.0.

Techniques: Control, Comparison

Journal: Scientific Reports

Article Title: Repurposing of apoptotic inducer drugs against Mycobacterium tuberculosis

doi: 10.1038/s41598-025-91096-8

Figure Lengend Snippet: Effect of apoptotic inducer drugs on the secretion of 13 selected cytokines in uninfected and M. smegmatis mc 2 155-infected THP-1 macrophages. Cytokine expression was evaluated at two time points (6 and 12 h) in infected and uninfected THP-1 cells after treatment with apoptotic inducer drugs. Data was expressed in pg/mL and normalized per column where 0% (purple) was indicated by the lowest value and 100% (red) was indicated by the highest value.

Article Snippet: A cryopreserved M. smegmatis mc 2 155 stock was cultured in 10 mL of 7H9 OADC (Difco, Becton, Dickinson and Company, New Jersey, United States) until reaching an OD 600 nm of 1.0.

Techniques: Infection, Expressing

Journal: Scientific Reports

Article Title: Repurposing of apoptotic inducer drugs against Mycobacterium tuberculosis

doi: 10.1038/s41598-025-91096-8

Figure Lengend Snippet: Representative plots showing cytokine changes measured by Luminex multiplex assay in uninfected and M. smegmatis mc 2 155 infected THP-1 macrophages ( A ): IL-1β, ( B ): TNF-α, ( C ): IL-6. Data were analyzed using two-way ANOVA.

Article Snippet: A cryopreserved M. smegmatis mc 2 155 stock was cultured in 10 mL of 7H9 OADC (Difco, Becton, Dickinson and Company, New Jersey, United States) until reaching an OD 600 nm of 1.0.

Techniques: Luminex, Multiplex Assay, Infection

Journal: eLife

Article Title: Maintenance of cell wall remodeling and vesicle production are connected in Mycobacterium tuberculosis

doi: 10.7554/eLife.94982

Figure Lengend Snippet: ( A ) Domain structure of LCP proteins in Mtb . Proteins are organized according to the presence of (i) both LCP (green) and LytR_C (slight orange) terminal domain (Rv3267, Rv3484 and Rv0822); (ii) the solo LytR_C domain (VirR and Rv2700); and the solo LCP domain (Rv3840). Additional information is provided for Mtb mutants in the indicated genes. ( B ) Phylogenetic analysis of LCP proteins in Mycobacteria ( M. tuberculosis, M. smegmatis, M. marinum and M. leprae ). The unrooted tree includes seven separate clusters for proteins with both LCP and LytR_C terminal domains (CpsA1, CpsA2 and CpsA3); with the solo LytR_C terminal domain (Solo_LytRC1 and Solo_LytRC2); with the solo LCP domain; and a separate cluster including the characterized LCP proteins from S. pneumonia and S. aureus . Note the clustering of one of the M. smegmatis LCP proteins with this group. VirR is highlighted in blue.

Article Snippet: M. smegmatis mc 2 155 (ATCC 700084) and recombinants derived from this strain were grown at 37 °C in 7H9 medium (Difco) supplemented with 10% albumin-dextrose-catalase (ADC, Difco) and 0.05% Tween-80 (Sigma-Aldrich), or on complete 7H11 solid medium (Difco) supplemented with 10% OADC (Difco).

Techniques:

Journal: eLife

Article Title: Maintenance of cell wall remodeling and vesicle production are connected in Mycobacterium tuberculosis

doi: 10.7554/eLife.94982

Figure Lengend Snippet: ( A ) Blue-native PAGE analysis of recombinant VirR and Lcp1. The molecular mass of markers in kDa are indicated on the left side of the gel. The size of the multimers of VirR and Lcp1 are indicated on the left side of the gel. ( B ) Upper panel . Representative image of a flotation assay of recombinant VirR alone or in combination with Lcp1 performed on an idioxanol density gradient. The presence of VirR was detected in each fraction by dot-blot using murine polyclonal antibodies raised against VirR. Numbers indicate collected fractions from top (1) to bottom (11). Lower panel . Quantification of the dot-blot by measuring the pixel intensity of the dots using ImageJ. Data are mean and standard error of three independent experiments. ( C ) Bipartite split-GFP experiment using M. smegmatis D2 expressing plasmids depicted in , including CtpC Nterm , VirR, VirRsol (Δ1–41), Rv3484, Rv3267 (Lcp1), Rv0822, Rv3484 LytR_C , Rv3267 LytR_C and Rv0822 LytR_C . Bacteria were grown in complete 7H9 and fluorescence was recorded by FACS. Pooled data obtained from four to six independent cultures of each strain are shown. Statistical analysis was performed using non-parametric two-sided Mann-Whitney test. ns: not significant. * and ** refer to p-values <0.05 or 0.01, respectively. Figure 9—source data 1. Pngs containing original Blue-native PAGE analysis gel images, indicating the relevant elements displayed in . Figure 9—source data 2. Original files of Blue-native PAGE analysis gel images displayed in .

Article Snippet: M. smegmatis mc 2 155 (ATCC 700084) and recombinants derived from this strain were grown at 37 °C in 7H9 medium (Difco) supplemented with 10% albumin-dextrose-catalase (ADC, Difco) and 0.05% Tween-80 (Sigma-Aldrich), or on complete 7H11 solid medium (Difco) supplemented with 10% OADC (Difco).

Techniques: Blue Native PAGE, Recombinant, Dot Blot, Expressing, Bacteria, Fluorescence, MANN-WHITNEY