m smegmatis  (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: mBio

Article Title: FhaA plays a key role in mycobacterial polar elongation and asymmetric growth

doi: 10.1128/mbio.02526-24

Figure Lengend Snippet: FhaA interactome in the living cell. ( A ) Scheme of the strategy used to identify FhaA interacting proteins. Cultures of M. smegmatis overexpressing M. tuberculosis FhaA fused to Streptag were incubated with formaldehyde. FhaA covalently linked to its protein partners was purified using Strep-Tactin columns, and the recovered proteins were digested and identified by nano-liquid chromatography-tandem mass spectrometry (LC-MS/MS). ( B ) Venn diagram showing the number of proteins identified in Msmeg_fhaA and control strains after affinity chromatography. Using the probability mode of PatternLab Venn diagram module, 25 proteins were statistically identified as exclusive of FhaA interactome ( P < 0.01; ; ). (C) Volcano plot showing proteins identified in at least 7 replicates of the 10 replicates analyzed, plotted according to its P -value -(log2 P ) and fold change (log2 Fold change). Proteins statistically enriched in FhaA complexes ( q -value ≤ 0.05) with a fold change greater than 2 are displayed in green, and those related to cell elongation/cell envelope biosynthesis are labeled. Fold changes and P -values for each of the 31 differential proteins are depicted in .

Article Snippet: M. smegmatis strains were maintained on Middlebrook 7H10 agar plates (Difco) plus 10% albumine/dextrose/catalase (ADC) (0.2% dextrose, 0.5% bovine serum albumin, and 0.085% NaCl).

Techniques: Incubation, Purification, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Control, Affinity Chromatography, Labeling