m smegmatis wild type strain mc  (ATCC)

Journal: Microbiology Spectrum

Article Title: Selective Pressure by Rifampicin Modulates Mutation Rates and Evolutionary Trajectories of Mycobacterial Genomes

doi: 10.1128/spectrum.01017-23

Figure Lengend Snippet: MA experimental evolution with increasing concentrations of rifampicin. The MA assay comprised 40 independent lines, generated from the M. smegmatis wild type and its nucS -null (Δ nucS ) mutant derivative (20 each) and evolved in parallel with increasing antibiotic concentrations. The experimental evolution was carried out for 40 weeks, from 0.25 μg mL −1 rifampicin (subinhibitory concentration) until 32 μg mL −1 rifampicin (inhibitory concentration), doubling the concentration every 5 weeks. MA lines able to grow at week 35 with 16 μg mL −1 rifampicin (18 wild type-derived and 20 Δ nucS mutant-derived) were sequenced by WGS. The figure also shows the number of lines that survived at each rifampicin concentration (below the plates) and the number of passages (black arrows). Created with BioRender.com .

Article Snippet: M. smegmatis wild-type strain mc 2 155 (American Type Culture Collection; 700084) and its noncanonical MMR-deficient (Δ nucS mutant) derivative ( ) were grown at 37°C in Middlebrook 7H9 broth or 7H10 agar (Difco) with 0.5% glycerol and Tween 80 (0.05% in 7H10 agar and 0.5% in 7H9 broth).

Techniques: Generated, Mutagenesis, Concentration Assay, Derivative Assay

Journal: Microbiology Spectrum

Article Title: Selective Pressure by Rifampicin Modulates Mutation Rates and Evolutionary Trajectories of Mycobacterial Genomes

doi: 10.1128/spectrum.01017-23

Figure Lengend Snippet: Comparison of the mutational spectra of the MA lines evolved in the presence and absence of rifampicin. (A) Mutational spectra for M. smegmatis wild-type lines evolved with rifampicin (dark blue) and without antibiotic (light blue); (B) mutational spectra for M. smegmatis Δ nucS lines evolved in the presence (dark orange) and absence of rifampicin (light orange). Bars represent the mutation rate per nucleotide per generation for each type of DNA mutation obtained by WGS data of MA experiments from this study and our previous work . Error bars indicate 95% confidence intervals (CIs). *, P < 0.05 ( t test). t tests were complemented with Mann-Whitney U tests (see Table S5), as data for some types of mutations are nonparametric, although they are close to normality.

Article Snippet: M. smegmatis wild-type strain mc 2 155 (American Type Culture Collection; 700084) and its noncanonical MMR-deficient (Δ nucS mutant) derivative ( ) were grown at 37°C in Middlebrook 7H9 broth or 7H10 agar (Difco) with 0.5% glycerol and Tween 80 (0.05% in 7H10 agar and 0.5% in 7H9 broth).

Techniques: Comparison, Mutagenesis, MANN-WHITNEY

Journal: Microbiology Spectrum

Article Title: Selective Pressure by Rifampicin Modulates Mutation Rates and Evolutionary Trajectories of Mycobacterial Genomes

doi: 10.1128/spectrum.01017-23

Figure Lengend Snippet: Mutations in rpoB in the MA lines: identification and effect on antibiotic resistance. (A) Types of RNAP β substitutions generated by rpoB mutations. M. smegmatis RNAP β is represented with amino acid numbering. Gray regions indicate the rifampicin resistance clusters described in E. coli ( , ). Amino acid substitutions generated by rpoB mutations in the MA lines are shown in blue (wild-type lines) and orange (Δ nucS mutant lines). Mutations associated with resistance in M. tuberculosis by the WHO catalogue are marked with asterisks. Below are shown protein alignments of RNAP β of M. smegmatis ( Msm ), M. tuberculosis ( Mtb ), and E. coli , with the amino acid positions where mutations were detected (red). (B) Effect of RNAP β substitutions on rifampicin resistance. Bars show the rifampicin MIC of the MA lines before (light purple) and after (dark purple) the acquisition of each rpoB mutation. When the rpoB mutation was present in more than one line (H442R and G450D), a representative example is shown. *, S438L substitution appeared together with V167M at the same week of the evolution.

Article Snippet: M. smegmatis wild-type strain mc 2 155 (American Type Culture Collection; 700084) and its noncanonical MMR-deficient (Δ nucS mutant) derivative ( ) were grown at 37°C in Middlebrook 7H9 broth or 7H10 agar (Difco) with 0.5% glycerol and Tween 80 (0.05% in 7H10 agar and 0.5% in 7H9 broth).

Techniques: Generated, Mutagenesis

Journal: Heliyon

Article Title: Mycobacterium tuberculosis Rv2617c is involved in stress response and phage infection resistance

doi: 10.1016/j.heliyon.2024.e27400

Figure Lengend Snippet: Heterologous expression of M. tuberculosis Rv2617c in M. smegmatis . (A) PCR amplification of Rv2617c from M. tuberculosis H37Rv genome. (B) The constructed Ms_Rv2617c strain was cultured in a 7H9 medium supplemented with acetamide and then subjected to PCR amplification to detect the Rv2617c gene with specific primers. (C) The cell lysates prepared from acetamide-induced Ms_pAL and Ms_Rv2617c cultures were analyzed by SDS-PAGE and visualized under UV after Coomassie Brilliant Blue staining to identify the His-Tag-associated Rv2617c protein. (D) Western Blot analysis of cell wall lysates of both strains. The His-tag-associated Rv2617c protein was targeted with anti-His-tag antibodies. See original and uncropped images in supplementary material.

Article Snippet: M. smegmatis , Wild type M. smegmatis mc 2 155 strain , Invitrogen.

Techniques: Expressing, Amplification, Construct, Cell Culture, SDS Page, Staining, Western Blot

Journal: Heliyon

Article Title: Mycobacterium tuberculosis Rv2617c is involved in stress response and phage infection resistance

doi: 10.1016/j.heliyon.2024.e27400

Figure Lengend Snippet: Rv2617c overexpression affects M. smegmatis phenotypes. (A) Biofilm formation of Ms_pAL and Ms_Rv2617c in acetamide-induced 7H9 medium. (B) Single-cell colony of strains on MB 7H10 plates. Few Ms_Rv2617c colonies are observed on the plate and show a less compact shape in the center. (C) Sliding motility assay of Ms_pAL and Ms_Rv2617c on 0.3% 7H9 agar. Ms_Rv2617c increases in size. Induced bacterial cultures (OD 600 = 0.5, ∼4*10 8 ) were used for the above assays. (D) Sizes of colonies on the culture dishes were measured after 3 days of incubation at 37 °C. The Figure scale is indicated where required. Data are shown as the means ± SD of 3 independent experiments (* p < 0.05, ** p < 0.01, and *** p < 0.001).

Article Snippet: M. smegmatis , Wild type M. smegmatis mc 2 155 strain , Invitrogen.

Techniques: Over Expression, Motility Assay, Incubation

Journal: Science Advances

Article Title: Specificity and mutagenesis bias of the mycobacterial alternative mismatch repair analyzed by mutation accumulation studies

doi: 10.1126/sciadv.aay4453

Figure Lengend Snippet: Total number of mutations ( A ), BPSs ( B ), and indels ( C ) of the evolved wild-type M. smegmatis mc 2 155 (blue circles) and ∆ nucS (red circles) strains after performing the MA experiment. The horizontal straight segment represents the median value of mutations in each case.

Article Snippet: The M. smegmatis wild-type reference strain mc 2 155 (American Type Culture Collection, 700084) and a noncanonical MMR strain (∆ nucS ) were used in this study.

Techniques:

Journal: Science Advances

Article Title: Specificity and mutagenesis bias of the mycobacterial alternative mismatch repair analyzed by mutation accumulation studies

doi: 10.1126/sciadv.aay4453

Figure Lengend Snippet: Rates of spontaneous mutations per cell per generation conferring kanamycin resistance for M. smegmatis mc 2 155 (wild type) (blue) and its Δ nucS derivative (red) are shown. Ninety-five percent confidence intervals are shown. The limit of detection was around 10 −10 , as inferred by observed mutation rates of A:T>T:A transversion.

Article Snippet: The M. smegmatis wild-type reference strain mc 2 155 (American Type Culture Collection, 700084) and a noncanonical MMR strain (∆ nucS ) were used in this study.

Techniques: Mutagenesis