Structured Review

MiddleBrook Pharmaceuticals 7h9 liquid medium
Growth of M. tuberculosis strains in liquid and solid media. (A) Wild-type M. tuberculosis H37Rv, Δ cuvA Mt , and complemented Δ cuvA (Δ cuvA Mt /C) strains were grown in <t>7H9-ADC-Tw</t> liquid medium with 0.2% glucose, and the OD 600 was measured daily. (B) Growth of the same strains on agar plates containing MM plus 0.2% glucose or MM plus 0.01% cholesterol. Serial 10-fold dilutions were spotted and incubated at 37°C, and photographs were taken after 1 month of incubation. In both panels, data shown are from one experiment that was repeated at least twice with similar results. Glc, 0.2% glucose; Chol., 0.01% cholesterol.
7h9 Liquid Medium, supplied by MiddleBrook Pharmaceuticals, used in various techniques. Bioz Stars score: 99/100, based on 22 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "Mycobacterial Gene cuvA Is Required for Optimal Nutrient Utilization and Virulence"

Article Title: Mycobacterial Gene cuvA Is Required for Optimal Nutrient Utilization and Virulence

Journal: Infection and Immunity

doi: 10.1128/IAI.02207-14

Growth of M. tuberculosis strains in liquid and solid media. (A) Wild-type M. tuberculosis H37Rv, Δ cuvA Mt , and complemented Δ cuvA (Δ cuvA Mt /C) strains were grown in 7H9-ADC-Tw liquid medium with 0.2% glucose, and the OD 600 was measured daily. (B) Growth of the same strains on agar plates containing MM plus 0.2% glucose or MM plus 0.01% cholesterol. Serial 10-fold dilutions were spotted and incubated at 37°C, and photographs were taken after 1 month of incubation. In both panels, data shown are from one experiment that was repeated at least twice with similar results. Glc, 0.2% glucose; Chol., 0.01% cholesterol.
Figure Legend Snippet: Growth of M. tuberculosis strains in liquid and solid media. (A) Wild-type M. tuberculosis H37Rv, Δ cuvA Mt , and complemented Δ cuvA (Δ cuvA Mt /C) strains were grown in 7H9-ADC-Tw liquid medium with 0.2% glucose, and the OD 600 was measured daily. (B) Growth of the same strains on agar plates containing MM plus 0.2% glucose or MM plus 0.01% cholesterol. Serial 10-fold dilutions were spotted and incubated at 37°C, and photographs were taken after 1 month of incubation. In both panels, data shown are from one experiment that was repeated at least twice with similar results. Glc, 0.2% glucose; Chol., 0.01% cholesterol.

Techniques Used: Incubation, Gas Chromatography

2) Product Images from "Mycobacterial Gene cuvA Is Required for Optimal Nutrient Utilization and Virulence"

Article Title: Mycobacterial Gene cuvA Is Required for Optimal Nutrient Utilization and Virulence

Journal: Infection and Immunity

doi: 10.1128/IAI.02207-14

Growth of M. tuberculosis strains in liquid and solid media. (A) Wild-type M. tuberculosis H37Rv, Δ cuvA Mt , and complemented Δ cuvA (Δ cuvA Mt /C) strains were grown in 7H9-ADC-Tw liquid medium with 0.2% glucose, and the OD 600 was measured daily. (B) Growth of the same strains on agar plates containing MM plus 0.2% glucose or MM plus 0.01% cholesterol. Serial 10-fold dilutions were spotted and incubated at 37°C, and photographs were taken after 1 month of incubation. In both panels, data shown are from one experiment that was repeated at least twice with similar results. Glc, 0.2% glucose; Chol., 0.01% cholesterol.
Figure Legend Snippet: Growth of M. tuberculosis strains in liquid and solid media. (A) Wild-type M. tuberculosis H37Rv, Δ cuvA Mt , and complemented Δ cuvA (Δ cuvA Mt /C) strains were grown in 7H9-ADC-Tw liquid medium with 0.2% glucose, and the OD 600 was measured daily. (B) Growth of the same strains on agar plates containing MM plus 0.2% glucose or MM plus 0.01% cholesterol. Serial 10-fold dilutions were spotted and incubated at 37°C, and photographs were taken after 1 month of incubation. In both panels, data shown are from one experiment that was repeated at least twice with similar results. Glc, 0.2% glucose; Chol., 0.01% cholesterol.

Techniques Used: Incubation, Gas Chromatography

3) Product Images from "Chlorine Disinfection of Atypical Mycobacteria Isolated from a Water Distribution System"

Article Title: Chlorine Disinfection of Atypical Mycobacteria Isolated from a Water Distribution System

Journal: Applied and Environmental Microbiology

doi: 10.1128/AEM.68.3.1025-1032.2002

Effects of pH on the rate of inactivation of M. gordonae . Experimental conditions were a temperature of 25°C and an initial chlorine concentration of 0.5 mg/liter. Cells were grown in Middlebrook 7H9-Tween medium. No, initial number of CFU; N, number of CFU at each time point. Experiments were conducted with different phosphate (0.05 M) buffers within the pH range of 6.0 to 8.0. Slopes were calculated as follows: pH 6, y = 0.11 x ; pH 7, y = 0.09 x ; and pH 8, y = 0.02 x . R 2 , correlation coefficient values.
Figure Legend Snippet: Effects of pH on the rate of inactivation of M. gordonae . Experimental conditions were a temperature of 25°C and an initial chlorine concentration of 0.5 mg/liter. Cells were grown in Middlebrook 7H9-Tween medium. No, initial number of CFU; N, number of CFU at each time point. Experiments were conducted with different phosphate (0.05 M) buffers within the pH range of 6.0 to 8.0. Slopes were calculated as follows: pH 6, y = 0.11 x ; pH 7, y = 0.09 x ; and pH 8, y = 0.02 x . R 2 , correlation coefficient values.

Techniques Used: Concentration Assay

Effects of temperature on chlorine inactivation of M. gordonae . Experimental conditions were pH 7 and an initial chlorine concentration of 0.5 mg/liter. Cells were grown in Middlebrook 7H9-Tween medium. The chlorine susceptibility of M. gordonae was analyzed at 4, 16, and 25°C. No, initial number of CFU; N, number of CFU at each time point. Symbols: ▪, 4°C ( y = 0.01 x ); ▴, 16°C ( y = 0.02 x ); ⧫, 25°C ( y = 0.09 x ). R 2 , correlation coefficient values.
Figure Legend Snippet: Effects of temperature on chlorine inactivation of M. gordonae . Experimental conditions were pH 7 and an initial chlorine concentration of 0.5 mg/liter. Cells were grown in Middlebrook 7H9-Tween medium. The chlorine susceptibility of M. gordonae was analyzed at 4, 16, and 25°C. No, initial number of CFU; N, number of CFU at each time point. Symbols: ▪, 4°C ( y = 0.01 x ); ▴, 16°C ( y = 0.02 x ); ⧫, 25°C ( y = 0.09 x ). R 2 , correlation coefficient values.

Techniques Used: Concentration Assay

4) Product Images from "The Mycobacterium tuberculosis relBE toxin:antitoxin genes are stress-responsive modules that regulate growth through translation inhibition"

Article Title: The Mycobacterium tuberculosis relBE toxin:antitoxin genes are stress-responsive modules that regulate growth through translation inhibition

Journal: Journal of microbiology (Seoul, Korea)

doi: 10.1007/s12275-015-5333-8

Over expression of relE Mtb inhibits growth and macromolecular synthesis. M. smegmatis was transformed with either pYA1611 (LIX32), pYA- 1611:: relE (LIX33) or pYA1611:: relBE (LIX34). Overnight cultures were diluted to an OD 600 of 0.01 in 7H9-Tw-Hyg. At an OD 600 of 0.1, cultures were split, washed, resuspended in 7H9-Tw-Hyg and to one culture 100 ng/ml ATc was added to induce gene expression. (A) Growth of LIX33 with (solid line) and without (dashed line) ATc inducer. At the indicated time points uninduced LIX33 were diluted and plated on LB-agar, whereas induced LIX33 were plated on LB-ATc media. (B-D) Pulse-chase experiments were conducted to monitor the incorporation of radiolabeled precursors into (B) protein, [35S-Met], (C) RNA, [3H-Urd] or (D) DNA, [3H-Thy] with time. The incorporation rates are expressed as the percent relative to the incorporation of precursor into the uninduced culture at each time point. The values presented are the averages of three independent experiments; error bars represent the standard error of the mean. For statistical analysis, two-way analysis of variance with Bonferroni post tests was used to obtain P values for each time point *, P
Figure Legend Snippet: Over expression of relE Mtb inhibits growth and macromolecular synthesis. M. smegmatis was transformed with either pYA1611 (LIX32), pYA- 1611:: relE (LIX33) or pYA1611:: relBE (LIX34). Overnight cultures were diluted to an OD 600 of 0.01 in 7H9-Tw-Hyg. At an OD 600 of 0.1, cultures were split, washed, resuspended in 7H9-Tw-Hyg and to one culture 100 ng/ml ATc was added to induce gene expression. (A) Growth of LIX33 with (solid line) and without (dashed line) ATc inducer. At the indicated time points uninduced LIX33 were diluted and plated on LB-agar, whereas induced LIX33 were plated on LB-ATc media. (B-D) Pulse-chase experiments were conducted to monitor the incorporation of radiolabeled precursors into (B) protein, [35S-Met], (C) RNA, [3H-Urd] or (D) DNA, [3H-Thy] with time. The incorporation rates are expressed as the percent relative to the incorporation of precursor into the uninduced culture at each time point. The values presented are the averages of three independent experiments; error bars represent the standard error of the mean. For statistical analysis, two-way analysis of variance with Bonferroni post tests was used to obtain P values for each time point *, P

Techniques Used: Over Expression, Transformation Assay, Expressing, Pulse Chase

5) Product Images from "The Twin-Arginine Translocation Pathway of Mycobacterium smegmatis Is Functional and Required for the Export of Mycobacterial ?-Lactamases"

Article Title: The Twin-Arginine Translocation Pathway of Mycobacterium smegmatis Is Functional and Required for the Export of Mycobacterial ?-Lactamases

Journal: Journal of Bacteriology

doi: 10.1128/JB.187.22.7667-7679.2005

Δ tatA and Δ tatC mutants have growth defects. (A) Single colonies of wild-type (mc 2 155, pMB198), Δ tatA mutant (MB692, pMB198), and complemented Δ tatA attB :: tatA (MB692, pJM124) strains are shown on 7H10 agar medium containing 0.1% Tween 80, grown at 37°C. Not shown are the Δ tatC mutant and the complemented Δ tatC strain, which display the same growth phenotypes. (B) Representative growth curves for wild-type (WT) (mc 2 155, pMV261), Δ tatC mutant (JM567, pMV261), and complemented Δ tatC (JM567, pJM120) strains in 7H9 liquid medium containing 0.1% Tween 80, grown at 37°C, are shown as OD 600 . (C) Viable-count measurements from the same cultures shown in panel B, shown here as the log 10 of viable CFU/ml.
Figure Legend Snippet: Δ tatA and Δ tatC mutants have growth defects. (A) Single colonies of wild-type (mc 2 155, pMB198), Δ tatA mutant (MB692, pMB198), and complemented Δ tatA attB :: tatA (MB692, pJM124) strains are shown on 7H10 agar medium containing 0.1% Tween 80, grown at 37°C. Not shown are the Δ tatC mutant and the complemented Δ tatC strain, which display the same growth phenotypes. (B) Representative growth curves for wild-type (WT) (mc 2 155, pMV261), Δ tatC mutant (JM567, pMV261), and complemented Δ tatC (JM567, pJM120) strains in 7H9 liquid medium containing 0.1% Tween 80, grown at 37°C, are shown as OD 600 . (C) Viable-count measurements from the same cultures shown in panel B, shown here as the log 10 of viable CFU/ml.

Techniques Used: Mutagenesis

6) Product Images from "A rheostat mechanism governs the bifurcation of carbon flux in mycobacteria"

Article Title: A rheostat mechanism governs the bifurcation of carbon flux in mycobacteria

Journal: Nature Communications

doi: 10.1038/ncomms12527

Loss of ICD2 results in glutamate auxotrophy and impaired viability. ( a ) Growth (OD 600 ) of wild-type, Δ icd and Δ icd attB ::P np icd -complemented strains of M. smegmatis in Middlebrook 7H9 medium. Solid lines, culture density (OD 600 ). Dashed lines, glutamate concentration in culture medium. ( b ) Growth (OD 600 ) of wild-type, Δ icd and Δ icd attB ::P np icd -complemented strains of M. smegmatis in minimal medium supplemented with glucose and devoid of glutamate. Data are means±s.d. ( n =3 independent experiments). ( c ) Intracellular metabolites in wild-type, Δ icd and Δ icd attB ::P np icd -complemented strains of M. smegmatis 24 h after transferring cells into minimal medium supplemented with glucose and devoid of glutamate. Data are means±s.d. ( n =3 independent experiments). CIT/ICT, citrate/isocitrate; α-KG, alpha-ketoglutarate; GLU, glutamate. ( d , e ) Growth (OD 600 ) of wild-type, Δ icd1 , Δ icd2 and Δ icd2 attB ::P np icd2 -complemented strains of M. bovis BCG in minimal medium supplemented with glucose and glutamate ( d ) or without glutamate ( e ). ( f ) Survival (CFU) of wild-type, Δ icd and Δ icd attB ::P np icd -complemented strains of M. smegmatis in minimal medium supplemented with glucose and devoid of glutamate. Data are means±s.d. ( n =3 independent experiments each performed in triplicate). ( g ) Survival (CFU) of wild-type, Δ icd2 and Δ icd2 attB ::P np icd2 -complemented strains of M. bovis BCG in minimal medium supplemented with glucose and devoid of glutamate. Data are means±s.d. ( n =3 independent experiments each performed in triplicate).
Figure Legend Snippet: Loss of ICD2 results in glutamate auxotrophy and impaired viability. ( a ) Growth (OD 600 ) of wild-type, Δ icd and Δ icd attB ::P np icd -complemented strains of M. smegmatis in Middlebrook 7H9 medium. Solid lines, culture density (OD 600 ). Dashed lines, glutamate concentration in culture medium. ( b ) Growth (OD 600 ) of wild-type, Δ icd and Δ icd attB ::P np icd -complemented strains of M. smegmatis in minimal medium supplemented with glucose and devoid of glutamate. Data are means±s.d. ( n =3 independent experiments). ( c ) Intracellular metabolites in wild-type, Δ icd and Δ icd attB ::P np icd -complemented strains of M. smegmatis 24 h after transferring cells into minimal medium supplemented with glucose and devoid of glutamate. Data are means±s.d. ( n =3 independent experiments). CIT/ICT, citrate/isocitrate; α-KG, alpha-ketoglutarate; GLU, glutamate. ( d , e ) Growth (OD 600 ) of wild-type, Δ icd1 , Δ icd2 and Δ icd2 attB ::P np icd2 -complemented strains of M. bovis BCG in minimal medium supplemented with glucose and glutamate ( d ) or without glutamate ( e ). ( f ) Survival (CFU) of wild-type, Δ icd and Δ icd attB ::P np icd -complemented strains of M. smegmatis in minimal medium supplemented with glucose and devoid of glutamate. Data are means±s.d. ( n =3 independent experiments each performed in triplicate). ( g ) Survival (CFU) of wild-type, Δ icd2 and Δ icd2 attB ::P np icd2 -complemented strains of M. bovis BCG in minimal medium supplemented with glucose and devoid of glutamate. Data are means±s.d. ( n =3 independent experiments each performed in triplicate).

Techniques Used: Concentration Assay, Transferring

7) Product Images from "Metabolic Network for the Biosynthesis of Intra- and Extracellular α-Glucans Required for Virulence of Mycobacterium tuberculosis"

Article Title: Metabolic Network for the Biosynthesis of Intra- and Extracellular α-Glucans Required for Virulence of Mycobacterium tuberculosis

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1005768

Compensatory flux of ADP-glucose through GlgA and OtsA links the GlgC-GlgA and TreS-Pep2 routes for α-glucan production. (A) α-Glucan visualization in M . smegmatis mutant strains. Cells were cultivated on Middlebrook 7H10 agar plates for 3 days and exposed to iodine vapor for staining of α-glucans. Branched α-glucans give a pale red-brown color. In the absence of branching enzyme GlgB, long linear glucans are produced resulting in a dark blue color of cells with the intensity of staining correlating with the amount of total cellular α-glucans. (B) Analysis of nucleotide sugar diphosphates in cell extracts of M . smegmatis mutant strains. Cells were cultivated for 24 h in Middlebrook 7H9 liquid medium. Due to trehalose auxotrophy of the M . smegmatis Δ glgA (u) Δ otsA mutant, 50 μM trehalose was added to all cultures. Cell suspensions were normalized to OD 600 nm , washed with PBS, concentrated 50-fold and disrupted by bead beating. Cell-free extracts were heat inactivated for 15 min at 100°C, and 1 H NMR spectroscopy was used to detect the anomeric protons of ADP-glucose.
Figure Legend Snippet: Compensatory flux of ADP-glucose through GlgA and OtsA links the GlgC-GlgA and TreS-Pep2 routes for α-glucan production. (A) α-Glucan visualization in M . smegmatis mutant strains. Cells were cultivated on Middlebrook 7H10 agar plates for 3 days and exposed to iodine vapor for staining of α-glucans. Branched α-glucans give a pale red-brown color. In the absence of branching enzyme GlgB, long linear glucans are produced resulting in a dark blue color of cells with the intensity of staining correlating with the amount of total cellular α-glucans. (B) Analysis of nucleotide sugar diphosphates in cell extracts of M . smegmatis mutant strains. Cells were cultivated for 24 h in Middlebrook 7H9 liquid medium. Due to trehalose auxotrophy of the M . smegmatis Δ glgA (u) Δ otsA mutant, 50 μM trehalose was added to all cultures. Cell suspensions were normalized to OD 600 nm , washed with PBS, concentrated 50-fold and disrupted by bead beating. Cell-free extracts were heat inactivated for 15 min at 100°C, and 1 H NMR spectroscopy was used to detect the anomeric protons of ADP-glucose.

Techniques Used: Mutagenesis, Staining, Produced, Nuclear Magnetic Resonance, Spectroscopy

The central importance of the GlgE pathway in intracellular and capsular α-glucan synthesis in M . tuberculosis . Quantification of intracellular (A) or extracellular (i.e. capsular) (B) α-glucan in M . tuberculosis H37Rv mutant strains. Cells were grown in Middlebrook 7H9 liquid medium for 7 days with shaking. Intracellular glucans were measured in hot water extracts of washed cells. Capsular glucans were measured from cell-free culture supernatants. Intracellular and capsular glucans were assayed by sandwich ELISA employing an α-glucan specific monoclonal antibody. Similar results for intracellular glucan content were also obtained using an enzymatic assay with cells from independent biological replicates ( S3 Fig ). Values were normalized based on OD 600 nm of cultures. Values in (A) and (B) represent means of triplicates ± SEM. (C) Visualization of the α-glucan capsule in the M . tuberculosis WT and the Δ glgC (u) Δ treS mutant by immunogold labelling. Cells were grown in liquid medium without shaking, fixed, labelled with an α-glucan specific monoclonal antibody, and analyzed by electron microscopy (scale bar 0.5 μm). (D) Quantitative evaluation of α-glucan capsule visualization as shown in (C), plotted as anti-α-glucan specific gold particles per cell. Values represent means ± SEM (WT n = 27, Δ glgC (u) Δ treS n = 28). Negative controls were not treated with the primary anti-α-glucan antibody (n = 32).
Figure Legend Snippet: The central importance of the GlgE pathway in intracellular and capsular α-glucan synthesis in M . tuberculosis . Quantification of intracellular (A) or extracellular (i.e. capsular) (B) α-glucan in M . tuberculosis H37Rv mutant strains. Cells were grown in Middlebrook 7H9 liquid medium for 7 days with shaking. Intracellular glucans were measured in hot water extracts of washed cells. Capsular glucans were measured from cell-free culture supernatants. Intracellular and capsular glucans were assayed by sandwich ELISA employing an α-glucan specific monoclonal antibody. Similar results for intracellular glucan content were also obtained using an enzymatic assay with cells from independent biological replicates ( S3 Fig ). Values were normalized based on OD 600 nm of cultures. Values in (A) and (B) represent means of triplicates ± SEM. (C) Visualization of the α-glucan capsule in the M . tuberculosis WT and the Δ glgC (u) Δ treS mutant by immunogold labelling. Cells were grown in liquid medium without shaking, fixed, labelled with an α-glucan specific monoclonal antibody, and analyzed by electron microscopy (scale bar 0.5 μm). (D) Quantitative evaluation of α-glucan capsule visualization as shown in (C), plotted as anti-α-glucan specific gold particles per cell. Values represent means ± SEM (WT n = 27, Δ glgC (u) Δ treS n = 28). Negative controls were not treated with the primary anti-α-glucan antibody (n = 32).

Techniques Used: Mutagenesis, Sandwich ELISA, Enzymatic Assay, Electron Microscopy

8) Product Images from "Viability, biofilm formation, and MazEF expression in drug-sensitive and drug-resistant Mycobacterium tuberculosis strains circulating in Xinjiang, China"

Article Title: Viability, biofilm formation, and MazEF expression in drug-sensitive and drug-resistant Mycobacterium tuberculosis strains circulating in Xinjiang, China

Journal: Infection and Drug Resistance

doi: 10.2147/IDR.S148648

Growth profiles of MTB in standard, hypoxic and nutrient starvation conditions at different culture time points. Notes: ( A ) MTB incubated under standard conditions in 7H9 medium. Difference in growth ability became apparent on the eighth day, and resistant strains grew more rapidly than H37Rv ( P
Figure Legend Snippet: Growth profiles of MTB in standard, hypoxic and nutrient starvation conditions at different culture time points. Notes: ( A ) MTB incubated under standard conditions in 7H9 medium. Difference in growth ability became apparent on the eighth day, and resistant strains grew more rapidly than H37Rv ( P

Techniques Used: Incubation

9) Product Images from "Role of Mycobacterium tuberculosis Ser/Thr Kinase PknF: Implications in Glucose Transport and Cell Division"

Article Title: Role of Mycobacterium tuberculosis Ser/Thr Kinase PknF: Implications in Glucose Transport and Cell Division

Journal: Journal of Bacteriology

doi: 10.1128/JB.187.10.3415-3420.2005

Expression of PknF in M. smegmatis . (A). Analysis of PknF in M. smegmatis by Western blotting. Different strains were grown to log phase and harvested, cellular lysates were prepared, and approximately 40 μg protein from each sample was resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Western blotting was performed using anti-PknF antibodies. Lane 1, lysate of M. tuberculosis H 37 Rv; lane, 2 to 4, lysates from MSKM, MS5, and MSF, respectively. (B) Cell growth of M. smegmatis carrying various plasmids. Bacterial cultures were grown for 4 days in 50 ml of 7H9 liquid medium. At various time points, CFU were counted for growth analysis. Each point is the mean ± standard error of two values obtained from two different experiments.
Figure Legend Snippet: Expression of PknF in M. smegmatis . (A). Analysis of PknF in M. smegmatis by Western blotting. Different strains were grown to log phase and harvested, cellular lysates were prepared, and approximately 40 μg protein from each sample was resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Western blotting was performed using anti-PknF antibodies. Lane 1, lysate of M. tuberculosis H 37 Rv; lane, 2 to 4, lysates from MSKM, MS5, and MSF, respectively. (B) Cell growth of M. smegmatis carrying various plasmids. Bacterial cultures were grown for 4 days in 50 ml of 7H9 liquid medium. At various time points, CFU were counted for growth analysis. Each point is the mean ± standard error of two values obtained from two different experiments.

Techniques Used: Expressing, Western Blot, Polyacrylamide Gel Electrophoresis

Expression of antisense RNA in M. tuberculosis . (A) Western blot analysis. Lysates were resolved on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and proteins were electroblotted on nitrocellulose. The blot was probed with anti-PknF antibodies and developed with ECL reagent. Lane 1, lysate of ASF strain; lane 2, lysate of M. tuberculosis H 37 Rv. (B) Growth curve analysis. Mycobacterial cultures were grown for 30 days in 50 ml of 7H9 liquid medium. At each time point CFU were counted for growth analysis. Each point is the mean ± SE of CFU obtained from two cultures of two different experiments.
Figure Legend Snippet: Expression of antisense RNA in M. tuberculosis . (A) Western blot analysis. Lysates were resolved on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and proteins were electroblotted on nitrocellulose. The blot was probed with anti-PknF antibodies and developed with ECL reagent. Lane 1, lysate of ASF strain; lane 2, lysate of M. tuberculosis H 37 Rv. (B) Growth curve analysis. Mycobacterial cultures were grown for 30 days in 50 ml of 7H9 liquid medium. At each time point CFU were counted for growth analysis. Each point is the mean ± SE of CFU obtained from two cultures of two different experiments.

Techniques Used: Expressing, Western Blot, Polyacrylamide Gel Electrophoresis

10) Product Images from "Proteomic Analysis of Drug-Resistant Mycobacteria: Co-Evolution of Copper and INH Resistance"

Article Title: Proteomic Analysis of Drug-Resistant Mycobacteria: Co-Evolution of Copper and INH Resistance

Journal: PLoS ONE

doi: 10.1371/journal.pone.0127788

Growth curve and the susceptibility of M . smegmatis to copper and isoniazid. (a) The growth curve of M . smegmatis mc 2 155 and the copper resistant strain mc 2 155-Cu were measured in 7H9 media. Experiments were performed in triplicate. Squares, mc2155-Cu strains; circle, mc2155 strain; (b) The bacterial growth on 7H10 plates for M . smegmatis mc 2 155 and mc 2 155-Cu that were treated with CuSO 4 at different concentrations for 3 days, respectively. The panels show serial dilution (1:10) of mc 2 155 and mc 2 155-Cu. Diluted M. smegmatis cultures were spotted onto solid 7H10 media in the presence of CuSO 4 ranged from 0 to 500 μM. Images were taken after 3 days incubation at 37°C. Images stand for 3 independent experiments.; and (c) The bacterial survival rate for M . smegmatis mc 2 155 and mc 2 155-Cu that were treated with 0.1 mg/ml isoniazid.*** p
Figure Legend Snippet: Growth curve and the susceptibility of M . smegmatis to copper and isoniazid. (a) The growth curve of M . smegmatis mc 2 155 and the copper resistant strain mc 2 155-Cu were measured in 7H9 media. Experiments were performed in triplicate. Squares, mc2155-Cu strains; circle, mc2155 strain; (b) The bacterial growth on 7H10 plates for M . smegmatis mc 2 155 and mc 2 155-Cu that were treated with CuSO 4 at different concentrations for 3 days, respectively. The panels show serial dilution (1:10) of mc 2 155 and mc 2 155-Cu. Diluted M. smegmatis cultures were spotted onto solid 7H10 media in the presence of CuSO 4 ranged from 0 to 500 μM. Images were taken after 3 days incubation at 37°C. Images stand for 3 independent experiments.; and (c) The bacterial survival rate for M . smegmatis mc 2 155 and mc 2 155-Cu that were treated with 0.1 mg/ml isoniazid.*** p

Techniques Used: Serial Dilution, Incubation

11) Product Images from "Unexpected Link between Lipooligosaccharide Biosynthesis and Surface Protein Release in Mycobacterium marinum *"

Article Title: Unexpected Link between Lipooligosaccharide Biosynthesis and Surface Protein Release in Mycobacterium marinum *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M111.336461

LOS biosynthesis mutants show reduced PE_PGRS and EspE release. A , whole cells pellets and supernatant from M. marinum E11 wild-type, ESX-5 mutant 7C1, and PE_PGRS mutants A2, A5, A8, and B1 grown in 7H9 broth without Tween 80 were analyzed by immunoblot for expression and secretion of PE_PGRS, ESAT-6, and EsxN. GroEL2 was used as a negative control for lysis. LOS-deficient mutants A2 and A5 show a clear effect on the secretion of PE_PGRS and EsxN, although LOS-IV-deficient mutants A8 and B1 are unaffected. Results are representative of three independent experiments. B , immunoblot of whole cells pellets and supernatant from M. marinum WT and mutants A5 and A5c shows that the secretion defect of mutant A5 can be complemented. C , genapol X-080-treated whole cell pellets ( Gp ) and cell surface extracts ( Gs ) of bacteria grown on solid agar. No reproducible differences between E11 wild type and the tested mutants were observed for both PE_PGRS and EspE extractability. D , selected LOS mutants A2, A4, A5, A6, A8, A9, B2, B7, and B10 were plated on a nitrocellulose filter together with E11 wild-type, ESX-5 mutant 7C1, and a pks12 mutant. All LOS mutants are reproducibly negative for EspE secretion on double filter assay, whereas all the controls are positive for secretion.
Figure Legend Snippet: LOS biosynthesis mutants show reduced PE_PGRS and EspE release. A , whole cells pellets and supernatant from M. marinum E11 wild-type, ESX-5 mutant 7C1, and PE_PGRS mutants A2, A5, A8, and B1 grown in 7H9 broth without Tween 80 were analyzed by immunoblot for expression and secretion of PE_PGRS, ESAT-6, and EsxN. GroEL2 was used as a negative control for lysis. LOS-deficient mutants A2 and A5 show a clear effect on the secretion of PE_PGRS and EsxN, although LOS-IV-deficient mutants A8 and B1 are unaffected. Results are representative of three independent experiments. B , immunoblot of whole cells pellets and supernatant from M. marinum WT and mutants A5 and A5c shows that the secretion defect of mutant A5 can be complemented. C , genapol X-080-treated whole cell pellets ( Gp ) and cell surface extracts ( Gs ) of bacteria grown on solid agar. No reproducible differences between E11 wild type and the tested mutants were observed for both PE_PGRS and EspE extractability. D , selected LOS mutants A2, A4, A5, A6, A8, A9, B2, B7, and B10 were plated on a nitrocellulose filter together with E11 wild-type, ESX-5 mutant 7C1, and a pks12 mutant. All LOS mutants are reproducibly negative for EspE secretion on double filter assay, whereas all the controls are positive for secretion.

Techniques Used: Mutagenesis, Expressing, Negative Control, Lysis

12) Product Images from "Assessing the role of Rv1222 (RseA) as an anti-sigma factor of the Mycobacterium tuberculosis extracytoplasmic sigma factor SigE"

Article Title: Assessing the role of Rv1222 (RseA) as an anti-sigma factor of the Mycobacterium tuberculosis extracytoplasmic sigma factor SigE

Journal: Scientific Reports

doi: 10.1038/s41598-019-41183-4

Growth curve profiles of M. tuberculosis overexpressing Rv1222. Bacteria were grown in 7H9 ADC at 37 °C in rolling bottles; OD 540 was recorded at regular intervals up to 96 hrs. Results represent the average of three independent experiments.
Figure Legend Snippet: Growth curve profiles of M. tuberculosis overexpressing Rv1222. Bacteria were grown in 7H9 ADC at 37 °C in rolling bottles; OD 540 was recorded at regular intervals up to 96 hrs. Results represent the average of three independent experiments.

Techniques Used:

Growth of M. tuberculosis mutant strains in liquid and solid medium. ( A ) Growth curves in liquid medium. Bacteria were grown in 7H9 ADC at 37 °C in rolling bottles; OD 540 was recorded at regular intervals up to 96 hrs; results represent the average of three independent experiments; ( B ) Growth in solid medium. Bacteria were inoculated in Middlebrook 7H10 plates and incubated at 37 °C for 21 days.
Figure Legend Snippet: Growth of M. tuberculosis mutant strains in liquid and solid medium. ( A ) Growth curves in liquid medium. Bacteria were grown in 7H9 ADC at 37 °C in rolling bottles; OD 540 was recorded at regular intervals up to 96 hrs; results represent the average of three independent experiments; ( B ) Growth in solid medium. Bacteria were inoculated in Middlebrook 7H10 plates and incubated at 37 °C for 21 days.

Techniques Used: Mutagenesis, Incubation

13) Product Images from "Methionine Antagonizes para-Aminosalicylic Acid Activity via Affecting Folate Precursor Biosynthesis in Mycobacterium tuberculosis"

Article Title: Methionine Antagonizes para-Aminosalicylic Acid Activity via Affecting Folate Precursor Biosynthesis in Mycobacterium tuberculosis

Journal: Frontiers in Cellular and Infection Microbiology

doi: 10.3389/fcimb.2018.00399

Disruption of bioB is growth inhibitory and potentiates drug action. (A,B) M. bovis BCG bioB :: himar1 was grown to an OD 600 of ~0.5, washed three times to remove residual biotin with biotin-free 7H9 medium, and resuspended in biotin-free 7H9 medium to a starting OD 600 of 0.01. Cultures were then supplemented with biotin and incubated for 14 days with OD 600 readings taken at the given time points. Error bars denote standard deviation and are representative of 2 separate experiments. (B) M. bovis BCG and bioB :: himar1 were grown to an OD 600 of ~0.5, washed three times to remove residual biotin, and resuspended in biotin-free 7H9 medium to a starting OD 600 of 0.01. Cultures were then supplemented with biotin (0.05 and 5) and incubated for 14 days with OD 600 readings taken at the given time points. MIC 90 is defined as the minimum concentration of inhibitor required to restrict at least 90% of growth relative to growth seen in the no-drug control cultures. PAS, para -aminosalicylic acid; SMX, sulfamethoxazole; RIF, rifampin; INH, isoniazid. Results shown are representative of 3 separate experiments.
Figure Legend Snippet: Disruption of bioB is growth inhibitory and potentiates drug action. (A,B) M. bovis BCG bioB :: himar1 was grown to an OD 600 of ~0.5, washed three times to remove residual biotin with biotin-free 7H9 medium, and resuspended in biotin-free 7H9 medium to a starting OD 600 of 0.01. Cultures were then supplemented with biotin and incubated for 14 days with OD 600 readings taken at the given time points. Error bars denote standard deviation and are representative of 2 separate experiments. (B) M. bovis BCG and bioB :: himar1 were grown to an OD 600 of ~0.5, washed three times to remove residual biotin, and resuspended in biotin-free 7H9 medium to a starting OD 600 of 0.01. Cultures were then supplemented with biotin (0.05 and 5) and incubated for 14 days with OD 600 readings taken at the given time points. MIC 90 is defined as the minimum concentration of inhibitor required to restrict at least 90% of growth relative to growth seen in the no-drug control cultures. PAS, para -aminosalicylic acid; SMX, sulfamethoxazole; RIF, rifampin; INH, isoniazid. Results shown are representative of 3 separate experiments.

Techniques Used: Incubation, Standard Deviation, Concentration Assay

Methionine can affect but not bypass essentiality of upstream folate biosynthetic pathways in M. tuberculosis . (A) New working model of methionine-mediated PAS antagonism. (B) M. tuberculosis Δ pabB was grown to an OD 600 of ~0.5, washed three times with PABA-free 7H9 medium to remove residual PABA and resuspended in PABA-free 7H9 medium to a starting OD 600 of 0.01. Cultures were then supplemented with the indicated metabolites and incubated for 14 days with OD 600 readings taken at the given time points.
Figure Legend Snippet: Methionine can affect but not bypass essentiality of upstream folate biosynthetic pathways in M. tuberculosis . (A) New working model of methionine-mediated PAS antagonism. (B) M. tuberculosis Δ pabB was grown to an OD 600 of ~0.5, washed three times with PABA-free 7H9 medium to remove residual PABA and resuspended in PABA-free 7H9 medium to a starting OD 600 of 0.01. Cultures were then supplemented with the indicated metabolites and incubated for 14 days with OD 600 readings taken at the given time points.

Techniques Used: Incubation

14) Product Images from "Rapid detection of Mycobacterium tuberculosis in sputum with a solvatochromic trehalose probe"

Article Title: Rapid detection of Mycobacterium tuberculosis in sputum with a solvatochromic trehalose probe

Journal: Science translational medicine

doi: 10.1126/scitranslmed.aam6310

DMN-Tre detects Mtb in sputum samples from patients with tuberculosis, similar to the auramine stain ( A ) Illustration of sputum sample labeling protocol: 16 sputum samples were collected from diagnosed treatment-naïve patients with tuberculosis and were decontaminated with N -acetyl-L-cysteine (NalC)/NaOH mixture following recommended standards. Decontaminated sputum samples were split in two equal aliquots, each incubated with 1 mM DMN-Tre or smeared with Auramine O stain, followed by imaging. ( B ) Microscopy analysis of four decontaminated sputum samples incubated with 1 mM DMN-Tre in 7H9 liquid medium for 2 hours in a 37°C atmospheric incubator. ( C ) Microscopy analysis of decontaminated sputum samples either treated with 1 mM DMN-Tre for 30 min at 37°C (top) or directly fixed onto microscope slide for Auramine O staining following standard kit protocol (bottom, orange pseudocolor). ( D ) Bar graph depiction of the total Mtb cell number detected over eight fields of view per sample with either DMN-Tre or Auramine O in the 16 sputum samples treated as in (C). Images were collected in the DIC and FITC (for DMN and auramine fluorescence) channels of a Zeiss Observer Z1-inverted fluorescent microscope. Scale bars, 5 μm.
Figure Legend Snippet: DMN-Tre detects Mtb in sputum samples from patients with tuberculosis, similar to the auramine stain ( A ) Illustration of sputum sample labeling protocol: 16 sputum samples were collected from diagnosed treatment-naïve patients with tuberculosis and were decontaminated with N -acetyl-L-cysteine (NalC)/NaOH mixture following recommended standards. Decontaminated sputum samples were split in two equal aliquots, each incubated with 1 mM DMN-Tre or smeared with Auramine O stain, followed by imaging. ( B ) Microscopy analysis of four decontaminated sputum samples incubated with 1 mM DMN-Tre in 7H9 liquid medium for 2 hours in a 37°C atmospheric incubator. ( C ) Microscopy analysis of decontaminated sputum samples either treated with 1 mM DMN-Tre for 30 min at 37°C (top) or directly fixed onto microscope slide for Auramine O staining following standard kit protocol (bottom, orange pseudocolor). ( D ) Bar graph depiction of the total Mtb cell number detected over eight fields of view per sample with either DMN-Tre or Auramine O in the 16 sputum samples treated as in (C). Images were collected in the DIC and FITC (for DMN and auramine fluorescence) channels of a Zeiss Observer Z1-inverted fluorescent microscope. Scale bars, 5 μm.

Techniques Used: Staining, Labeling, Incubation, Imaging, Microscopy, Fluorescence

DMN-Tre labeling is inhibited by tuberculosis drugs ( A and B ) Flow cytometry MFI analysis (A) and no-wash imaging (B) of control and drug-treated (37°C for 3 hours) Msmeg cells labeled with 100 μM DMN-Tre for 30 min. Drug cocktail contents: ethambutol (1 μg/ml), rifampicin (0.2 μg/ml), SQ109 (20 μg/ml), and isoniazid (20 μg/ml) in 7H9 medium. ( C ) Flow cytometry MFI analysis of wild-type (WT) Msmeg or KatG mutant cells treated with isoniazid (INH) at the indicated final concentrations (0, 3, 6, or 10 μg/ml of isoniazid) for 3 hours, followed by incubation with DMN-Tre for 30 min. Images were collected in the DIC, FITC/GFP (for DMN fluorescence), or RFP (for mCherry fluorescence) channels of a Nikon A1R confocal microscope. Scale bars, 5 μm. In (A) and (C), data are means ± SEM from at least two independent experiments. Data were analyzed by one-way ANOVA test (**** P
Figure Legend Snippet: DMN-Tre labeling is inhibited by tuberculosis drugs ( A and B ) Flow cytometry MFI analysis (A) and no-wash imaging (B) of control and drug-treated (37°C for 3 hours) Msmeg cells labeled with 100 μM DMN-Tre for 30 min. Drug cocktail contents: ethambutol (1 μg/ml), rifampicin (0.2 μg/ml), SQ109 (20 μg/ml), and isoniazid (20 μg/ml) in 7H9 medium. ( C ) Flow cytometry MFI analysis of wild-type (WT) Msmeg or KatG mutant cells treated with isoniazid (INH) at the indicated final concentrations (0, 3, 6, or 10 μg/ml of isoniazid) for 3 hours, followed by incubation with DMN-Tre for 30 min. Images were collected in the DIC, FITC/GFP (for DMN fluorescence), or RFP (for mCherry fluorescence) channels of a Nikon A1R confocal microscope. Scale bars, 5 μm. In (A) and (C), data are means ± SEM from at least two independent experiments. Data were analyzed by one-way ANOVA test (**** P

Techniques Used: Labeling, Flow Cytometry, Cytometry, Imaging, Mutagenesis, Incubation, Fluorescence, Microscopy

DMN-Tre labeling of Mycobacterium tuberculosis is inhibited by tuberculosis drug cocktail, unlike auramine staining ( A ) Microscopy analysis of Mycobacterium tuberculosis (Mtb) cells incubated with 100 μM DMN-Tre for 2 hours. ( B ) Flow cytometry MFI analysis of Mtb cells incubated with DMN-Tre for the indicated times: 0.5, 1, 2, and 3 hours or overnight (~16 hours). Error bars denote three biological replicates. ( C and D ) Microscopy analysis of control and drug-treated Mtb cells labeled with 100 μM DMN-Tre overnight (C) or stained with the auramine-based Fluorescent Stain kit for mycobacteria (05151, Sigma-Aldrich) (D). Drug cocktail contents: ethambutol (1 μg/ml), rifampicin (0.2 μg/ml), SQ109 (10 μg/ml), and isoniazid (10 μg/ml) in 7H9 medium. Images were collected in the DIC or FITC/GFP (for DMN and Auramine fluorescence) channels of a Nikon A1R confocal microscope. Cells stained with auramine were given an orange pseudocolor. Scale bars, 5 μm. In (B), data are means ± SEM from at least two independent experiments. Data were analyzed by one-way ANOVA test (* P
Figure Legend Snippet: DMN-Tre labeling of Mycobacterium tuberculosis is inhibited by tuberculosis drug cocktail, unlike auramine staining ( A ) Microscopy analysis of Mycobacterium tuberculosis (Mtb) cells incubated with 100 μM DMN-Tre for 2 hours. ( B ) Flow cytometry MFI analysis of Mtb cells incubated with DMN-Tre for the indicated times: 0.5, 1, 2, and 3 hours or overnight (~16 hours). Error bars denote three biological replicates. ( C and D ) Microscopy analysis of control and drug-treated Mtb cells labeled with 100 μM DMN-Tre overnight (C) or stained with the auramine-based Fluorescent Stain kit for mycobacteria (05151, Sigma-Aldrich) (D). Drug cocktail contents: ethambutol (1 μg/ml), rifampicin (0.2 μg/ml), SQ109 (10 μg/ml), and isoniazid (10 μg/ml) in 7H9 medium. Images were collected in the DIC or FITC/GFP (for DMN and Auramine fluorescence) channels of a Nikon A1R confocal microscope. Cells stained with auramine were given an orange pseudocolor. Scale bars, 5 μm. In (B), data are means ± SEM from at least two independent experiments. Data were analyzed by one-way ANOVA test (* P

Techniques Used: Labeling, Staining, Microscopy, Incubation, Flow Cytometry, Cytometry, Fluorescence

15) Product Images from "The Mycobacterium tuberculosis gene, ldtMt2, encodes a non-classical transpeptidase required for virulence and resistance to amoxicillin"

Article Title: The Mycobacterium tuberculosis gene, ldtMt2, encodes a non-classical transpeptidase required for virulence and resistance to amoxicillin

Journal: Nature medicine

doi: 10.1038/nm.2120

Morphology and growth in vitro . ( a ) Morphologies of wild-type M. tuberculosis (WT), Ldt Mt2 mutant (MUT) and the complemented strain (COMP) on solid media after 21 days of growth at 37 °C. (scale = 1 cm).( b ) Growth of wild-type M. tuberculosis (WT), Ldt Mt2 mutant (MUT) and the complemented strain (COMP) in Middlebrook 7H9 liquid medium at 37 °C. The decrease in optical density and CFU at the final time point for WT and COMP is due to clumped cultures.
Figure Legend Snippet: Morphology and growth in vitro . ( a ) Morphologies of wild-type M. tuberculosis (WT), Ldt Mt2 mutant (MUT) and the complemented strain (COMP) on solid media after 21 days of growth at 37 °C. (scale = 1 cm).( b ) Growth of wild-type M. tuberculosis (WT), Ldt Mt2 mutant (MUT) and the complemented strain (COMP) in Middlebrook 7H9 liquid medium at 37 °C. The decrease in optical density and CFU at the final time point for WT and COMP is due to clumped cultures.

Techniques Used: In Vitro, Mutagenesis

16) Product Images from "Mycobacterium tuberculosis PPE32 promotes cytokines production and host cell apoptosis through caspase cascade accompanying with enhanced ER stress response"

Article Title: Mycobacterium tuberculosis PPE32 promotes cytokines production and host cell apoptosis through caspase cascade accompanying with enhanced ER stress response

Journal: Oncotarget

doi: 10.18632/oncotarget.12030

PPE32 enhanced the MS resistance to multiple extracellular stresses ( A ) Survival of MS_Vec and MS_PPE32 upon exposure to 0.05% SDS. Re-suspended 5ml recombinant MS_Vec and MS_PPE32 ( OD600 = 0.5) were exposed to 0.03% SDS for 1, 2, 3, and 4 h. And then the recombinant strains were plated onto 7H10 plates by serially ten-fold dilution ( B ) In vitro growth of recombinant MS_Vec and MS_PPE32 after treatment with different pH gradient for 0, 3, 6, and 9 h. The MS_Vec and MS_PPE32 strains were centrifuged, re-suspended to 5ml MB 7H9 at an OD600 of 0.5, 10-fold serial dilutions of MS_Vec and MS_PPE32 were spotted on MB 7H10 containing Kan. the bacterial numbers were counted after 3–4 days of cultivation at 37°C.
Figure Legend Snippet: PPE32 enhanced the MS resistance to multiple extracellular stresses ( A ) Survival of MS_Vec and MS_PPE32 upon exposure to 0.05% SDS. Re-suspended 5ml recombinant MS_Vec and MS_PPE32 ( OD600 = 0.5) were exposed to 0.03% SDS for 1, 2, 3, and 4 h. And then the recombinant strains were plated onto 7H10 plates by serially ten-fold dilution ( B ) In vitro growth of recombinant MS_Vec and MS_PPE32 after treatment with different pH gradient for 0, 3, 6, and 9 h. The MS_Vec and MS_PPE32 strains were centrifuged, re-suspended to 5ml MB 7H9 at an OD600 of 0.5, 10-fold serial dilutions of MS_Vec and MS_PPE32 were spotted on MB 7H10 containing Kan. the bacterial numbers were counted after 3–4 days of cultivation at 37°C.

Techniques Used: Mass Spectrometry, Recombinant, In Vitro

17) Product Images from "A Thiolase of Mycobacterium tuberculosis Is Required for Virulence and Production of Androstenedione and Androstadienedione from Cholesterol ▿ Is Required for Virulence and Production of Androstenedione and Androstadienedione from Cholesterol ▿ †"

Article Title: A Thiolase of Mycobacterium tuberculosis Is Required for Virulence and Production of Androstenedione and Androstadienedione from Cholesterol ▿ Is Required for Virulence and Production of Androstenedione and Androstadienedione from Cholesterol ▿ †

Journal: Infection and Immunity

doi: 10.1128/IAI.00893-09

Mutation of fadA5 does not cause cholesterol toxicity. Strains were grown to exponential phase in 7H9 medium supplemented with glycerol and dextrose. Cultures were diluted into 7H9 medium containing glycerol and dextrose with or without cholesterol (1 mg ml −1 in 20% Tween). Values are for the wild-type H37Rv strain without cholesterol, wild-type H37Rv with cholesterol, the fadA5 mutant without cholesterol, and fadA5 mutant with cholesterol, as indicated. Data represent the results of the experiment done in triplicate.
Figure Legend Snippet: Mutation of fadA5 does not cause cholesterol toxicity. Strains were grown to exponential phase in 7H9 medium supplemented with glycerol and dextrose. Cultures were diluted into 7H9 medium containing glycerol and dextrose with or without cholesterol (1 mg ml −1 in 20% Tween). Values are for the wild-type H37Rv strain without cholesterol, wild-type H37Rv with cholesterol, the fadA5 mutant without cholesterol, and fadA5 mutant with cholesterol, as indicated. Data represent the results of the experiment done in triplicate.

Techniques Used: Mutagenesis

fadA5 is required for growth on cholesterol as the sole carbon source. The strains were grown in 7H9 medium containing 1 mg ml −1 (2.6 mM) cholesterol in tyloxapol or 7H9 medium containing only tyloxapol at 37°C. The wild-type H37Rv (Rv), fadA5 mutant, and complemented fadA5 (fadA5comp) strains were grown with and without cholesterol as indicated on the graph. Data represent results of each experiment run in duplicate.
Figure Legend Snippet: fadA5 is required for growth on cholesterol as the sole carbon source. The strains were grown in 7H9 medium containing 1 mg ml −1 (2.6 mM) cholesterol in tyloxapol or 7H9 medium containing only tyloxapol at 37°C. The wild-type H37Rv (Rv), fadA5 mutant, and complemented fadA5 (fadA5comp) strains were grown with and without cholesterol as indicated on the graph. Data represent results of each experiment run in duplicate.

Techniques Used: Mutagenesis

18) Product Images from "Rapid detection of Mycobacterium tuberculosis in sputum with a solvatochromic trehalose probe"

Article Title: Rapid detection of Mycobacterium tuberculosis in sputum with a solvatochromic trehalose probe

Journal: Science translational medicine

doi: 10.1126/scitranslmed.aam6310

DMN-Tre detects Mtb in sputum samples from patients with tuberculosis, similar to the auramine stain ( A ) Illustration of sputum sample labeling protocol: 16 sputum samples were collected from diagnosed treatment-naïve patients with tuberculosis and were decontaminated with N -acetyl-L-cysteine (NalC)/NaOH mixture following recommended standards. Decontaminated sputum samples were split in two equal aliquots, each incubated with 1 mM DMN-Tre or smeared with Auramine O stain, followed by imaging. ( B ) Microscopy analysis of four decontaminated sputum samples incubated with 1 mM DMN-Tre in 7H9 liquid medium for 2 hours in a 37°C atmospheric incubator. ( C ) Microscopy analysis of decontaminated sputum samples either treated with 1 mM DMN-Tre for 30 min at 37°C (top) or directly fixed onto microscope slide for Auramine O staining following standard kit protocol (bottom, orange pseudocolor). ( D ) Bar graph depiction of the total Mtb cell number detected over eight fields of view per sample with either DMN-Tre or Auramine O in the 16 sputum samples treated as in (C). Images were collected in the DIC and FITC (for DMN and auramine fluorescence) channels of a Zeiss Observer Z1-inverted fluorescent microscope. Scale bars, 5 μm.
Figure Legend Snippet: DMN-Tre detects Mtb in sputum samples from patients with tuberculosis, similar to the auramine stain ( A ) Illustration of sputum sample labeling protocol: 16 sputum samples were collected from diagnosed treatment-naïve patients with tuberculosis and were decontaminated with N -acetyl-L-cysteine (NalC)/NaOH mixture following recommended standards. Decontaminated sputum samples were split in two equal aliquots, each incubated with 1 mM DMN-Tre or smeared with Auramine O stain, followed by imaging. ( B ) Microscopy analysis of four decontaminated sputum samples incubated with 1 mM DMN-Tre in 7H9 liquid medium for 2 hours in a 37°C atmospheric incubator. ( C ) Microscopy analysis of decontaminated sputum samples either treated with 1 mM DMN-Tre for 30 min at 37°C (top) or directly fixed onto microscope slide for Auramine O staining following standard kit protocol (bottom, orange pseudocolor). ( D ) Bar graph depiction of the total Mtb cell number detected over eight fields of view per sample with either DMN-Tre or Auramine O in the 16 sputum samples treated as in (C). Images were collected in the DIC and FITC (for DMN and auramine fluorescence) channels of a Zeiss Observer Z1-inverted fluorescent microscope. Scale bars, 5 μm.

Techniques Used: Staining, Labeling, Incubation, Imaging, Microscopy, Fluorescence

DMN-Tre labeling is inhibited by tuberculosis drugs ( A and B ) Flow cytometry MFI analysis (A) and no-wash imaging (B) of control and drug-treated (37°C for 3 hours) Msmeg cells labeled with 100 μM DMN-Tre for 30 min. Drug cocktail contents: ethambutol (1 μg/ml), rifampicin (0.2 μg/ml), SQ109 (20 μg/ml), and isoniazid (20 μg/ml) in 7H9 medium. ( C ) Flow cytometry MFI analysis of wild-type (WT) Msmeg or KatG mutant cells treated with isoniazid (INH) at the indicated final concentrations (0, 3, 6, or 10 μg/ml of isoniazid) for 3 hours, followed by incubation with DMN-Tre for 30 min. Images were collected in the DIC, FITC/GFP (for DMN fluorescence), or RFP (for mCherry fluorescence) channels of a Nikon A1R confocal microscope. Scale bars, 5 μm. In (A) and (C), data are means ± SEM from at least two independent experiments. Data were analyzed by one-way ANOVA test (**** P
Figure Legend Snippet: DMN-Tre labeling is inhibited by tuberculosis drugs ( A and B ) Flow cytometry MFI analysis (A) and no-wash imaging (B) of control and drug-treated (37°C for 3 hours) Msmeg cells labeled with 100 μM DMN-Tre for 30 min. Drug cocktail contents: ethambutol (1 μg/ml), rifampicin (0.2 μg/ml), SQ109 (20 μg/ml), and isoniazid (20 μg/ml) in 7H9 medium. ( C ) Flow cytometry MFI analysis of wild-type (WT) Msmeg or KatG mutant cells treated with isoniazid (INH) at the indicated final concentrations (0, 3, 6, or 10 μg/ml of isoniazid) for 3 hours, followed by incubation with DMN-Tre for 30 min. Images were collected in the DIC, FITC/GFP (for DMN fluorescence), or RFP (for mCherry fluorescence) channels of a Nikon A1R confocal microscope. Scale bars, 5 μm. In (A) and (C), data are means ± SEM from at least two independent experiments. Data were analyzed by one-way ANOVA test (**** P

Techniques Used: Labeling, Flow Cytometry, Cytometry, Imaging, Mutagenesis, Incubation, Fluorescence, Microscopy

DMN-Tre labeling of Mycobacterium tuberculosis is inhibited by tuberculosis drug cocktail, unlike auramine staining ( A ) Microscopy analysis of Mycobacterium tuberculosis (Mtb) cells incubated with 100 μM DMN-Tre for 2 hours. ( B ) Flow cytometry MFI analysis of Mtb cells incubated with DMN-Tre for the indicated times: 0.5, 1, 2, and 3 hours or overnight (~16 hours). Error bars denote three biological replicates. ( C and D ) Microscopy analysis of control and drug-treated Mtb cells labeled with 100 μM DMN-Tre overnight (C) or stained with the auramine-based Fluorescent Stain kit for mycobacteria (05151, Sigma-Aldrich) (D). Drug cocktail contents: ethambutol (1 μg/ml), rifampicin (0.2 μg/ml), SQ109 (10 μg/ml), and isoniazid (10 μg/ml) in 7H9 medium. Images were collected in the DIC or FITC/GFP (for DMN and Auramine fluorescence) channels of a Nikon A1R confocal microscope. Cells stained with auramine were given an orange pseudocolor. Scale bars, 5 μm. In (B), data are means ± SEM from at least two independent experiments. Data were analyzed by one-way ANOVA test (* P
Figure Legend Snippet: DMN-Tre labeling of Mycobacterium tuberculosis is inhibited by tuberculosis drug cocktail, unlike auramine staining ( A ) Microscopy analysis of Mycobacterium tuberculosis (Mtb) cells incubated with 100 μM DMN-Tre for 2 hours. ( B ) Flow cytometry MFI analysis of Mtb cells incubated with DMN-Tre for the indicated times: 0.5, 1, 2, and 3 hours or overnight (~16 hours). Error bars denote three biological replicates. ( C and D ) Microscopy analysis of control and drug-treated Mtb cells labeled with 100 μM DMN-Tre overnight (C) or stained with the auramine-based Fluorescent Stain kit for mycobacteria (05151, Sigma-Aldrich) (D). Drug cocktail contents: ethambutol (1 μg/ml), rifampicin (0.2 μg/ml), SQ109 (10 μg/ml), and isoniazid (10 μg/ml) in 7H9 medium. Images were collected in the DIC or FITC/GFP (for DMN and Auramine fluorescence) channels of a Nikon A1R confocal microscope. Cells stained with auramine were given an orange pseudocolor. Scale bars, 5 μm. In (B), data are means ± SEM from at least two independent experiments. Data were analyzed by one-way ANOVA test (* P

Techniques Used: Labeling, Staining, Microscopy, Incubation, Flow Cytometry, Cytometry, Fluorescence

19) Product Images from "Two Faces of CwlM, an Essential PknB Substrate, in Mycobacterium tuberculosis"

Article Title: Two Faces of CwlM, an Essential PknB Substrate, in Mycobacterium tuberculosis

Journal: Cell Reports

doi: 10.1016/j.celrep.2018.09.004

T382A Mutant Mimics Phenotype of PknB-Depleted M. tuberculosis (A–D) The cwlM conditional mutant of M. tuberculosis was transformed with pMV306 plasmids containing cwlM variants. The resultant strains were grown in 7H9 medium (A) or in SMM (B) without pristinamycin. All of the strains grew similarly when 7H9 or SMM were supplemented with pristinamycin (data not shown for clarity). pMV, cwlM -CM pmv306 (the empty plasmid control); CwlM, cwlM -CM WT ; T382A and T382D phosphoablative and phosphomimetic mutants, respectively. Data are represented as means ± SEMs (n = 6). (C) Growth of strains on 7H10 agar. (D) Western blot of CwlM variants detected with anti-CwlM antibody. .
Figure Legend Snippet: T382A Mutant Mimics Phenotype of PknB-Depleted M. tuberculosis (A–D) The cwlM conditional mutant of M. tuberculosis was transformed with pMV306 plasmids containing cwlM variants. The resultant strains were grown in 7H9 medium (A) or in SMM (B) without pristinamycin. All of the strains grew similarly when 7H9 or SMM were supplemented with pristinamycin (data not shown for clarity). pMV, cwlM -CM pmv306 (the empty plasmid control); CwlM, cwlM -CM WT ; T382A and T382D phosphoablative and phosphomimetic mutants, respectively. Data are represented as means ± SEMs (n = 6). (C) Growth of strains on 7H10 agar. (D) Western blot of CwlM variants detected with anti-CwlM antibody. .

Techniques Used: Mutagenesis, Transformation Assay, Plasmid Preparation, Western Blot

PknB-Mediated Phosphorylation of T382 Determines Distribution of CwlM in Cytoplasmic and Membrane Fractions of M. tuberculosis (A–D) Lysates obtained from cwlM -CM grown in SMM without pristinamycin to prevent induction of genomic cwlM (A and C) or from pknB -CM grown in SMM or standard 7H9 medium with or without pristinamycin (B and D) were fractionated and probed with anti-CwlM antibodies (A and B) or with anti-T382-P and anti-T382 antibodies (C and D). Anti-GarA and Anti-GlnA antibodies were used to confirm the purity of mycobacterial fractions. .
Figure Legend Snippet: PknB-Mediated Phosphorylation of T382 Determines Distribution of CwlM in Cytoplasmic and Membrane Fractions of M. tuberculosis (A–D) Lysates obtained from cwlM -CM grown in SMM without pristinamycin to prevent induction of genomic cwlM (A and C) or from pknB -CM grown in SMM or standard 7H9 medium with or without pristinamycin (B and D) were fractionated and probed with anti-CwlM antibodies (A and B) or with anti-T382-P and anti-T382 antibodies (C and D). Anti-GarA and Anti-GlnA antibodies were used to confirm the purity of mycobacterial fractions. .

Techniques Used:

20) Product Images from "Metabolic Network for the Biosynthesis of Intra- and Extracellular α-Glucans Required for Virulence of Mycobacterium tuberculosis"

Article Title: Metabolic Network for the Biosynthesis of Intra- and Extracellular α-Glucans Required for Virulence of Mycobacterium tuberculosis

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1005768

Compensatory flux of ADP-glucose through GlgA and OtsA links the GlgC-GlgA and TreS-Pep2 routes for α-glucan production. (A) α-Glucan visualization in M . smegmatis mutant strains. Cells were cultivated on Middlebrook 7H10 agar plates for 3 days and exposed to iodine vapor for staining of α-glucans. Branched α-glucans give a pale red-brown color. In the absence of branching enzyme GlgB, long linear glucans are produced resulting in a dark blue color of cells with the intensity of staining correlating with the amount of total cellular α-glucans. (B) Analysis of nucleotide sugar diphosphates in cell extracts of M . smegmatis mutant strains. Cells were cultivated for 24 h in Middlebrook 7H9 liquid medium. Due to trehalose auxotrophy of the M . smegmatis Δ glgA (u) Δ otsA mutant, 50 μM trehalose was added to all cultures. Cell suspensions were normalized to OD 600 nm , washed with PBS, concentrated 50-fold and disrupted by bead beating. Cell-free extracts were heat inactivated for 15 min at 100°C, and 1 H NMR spectroscopy was used to detect the anomeric protons of ADP-glucose.
Figure Legend Snippet: Compensatory flux of ADP-glucose through GlgA and OtsA links the GlgC-GlgA and TreS-Pep2 routes for α-glucan production. (A) α-Glucan visualization in M . smegmatis mutant strains. Cells were cultivated on Middlebrook 7H10 agar plates for 3 days and exposed to iodine vapor for staining of α-glucans. Branched α-glucans give a pale red-brown color. In the absence of branching enzyme GlgB, long linear glucans are produced resulting in a dark blue color of cells with the intensity of staining correlating with the amount of total cellular α-glucans. (B) Analysis of nucleotide sugar diphosphates in cell extracts of M . smegmatis mutant strains. Cells were cultivated for 24 h in Middlebrook 7H9 liquid medium. Due to trehalose auxotrophy of the M . smegmatis Δ glgA (u) Δ otsA mutant, 50 μM trehalose was added to all cultures. Cell suspensions were normalized to OD 600 nm , washed with PBS, concentrated 50-fold and disrupted by bead beating. Cell-free extracts were heat inactivated for 15 min at 100°C, and 1 H NMR spectroscopy was used to detect the anomeric protons of ADP-glucose.

Techniques Used: Mutagenesis, Staining, Produced, Nuclear Magnetic Resonance, Spectroscopy

The central importance of the GlgE pathway in intracellular and capsular α-glucan synthesis in M . tuberculosis . Quantification of intracellular (A) or extracellular (i.e. capsular) (B) α-glucan in M . tuberculosis H37Rv mutant strains. Cells were grown in Middlebrook 7H9 liquid medium for 7 days with shaking. Intracellular glucans were measured in hot water extracts of washed cells. Capsular glucans were measured from cell-free culture supernatants. Intracellular and capsular glucans were assayed by sandwich ELISA employing an α-glucan specific monoclonal antibody. Similar results for intracellular glucan content were also obtained using an enzymatic assay with cells from independent biological replicates ( S3 Fig ). Values were normalized based on OD 600 nm of cultures. Values in (A) and (B) represent means of triplicates ± SEM. (C) Visualization of the α-glucan capsule in the M . tuberculosis WT and the Δ glgC (u) Δ treS mutant by immunogold labelling. Cells were grown in liquid medium without shaking, fixed, labelled with an α-glucan specific monoclonal antibody, and analyzed by electron microscopy (scale bar 0.5 μm). (D) Quantitative evaluation of α-glucan capsule visualization as shown in (C), plotted as anti-α-glucan specific gold particles per cell. Values represent means ± SEM (WT n = 27, Δ glgC (u) Δ treS n = 28). Negative controls were not treated with the primary anti-α-glucan antibody (n = 32).
Figure Legend Snippet: The central importance of the GlgE pathway in intracellular and capsular α-glucan synthesis in M . tuberculosis . Quantification of intracellular (A) or extracellular (i.e. capsular) (B) α-glucan in M . tuberculosis H37Rv mutant strains. Cells were grown in Middlebrook 7H9 liquid medium for 7 days with shaking. Intracellular glucans were measured in hot water extracts of washed cells. Capsular glucans were measured from cell-free culture supernatants. Intracellular and capsular glucans were assayed by sandwich ELISA employing an α-glucan specific monoclonal antibody. Similar results for intracellular glucan content were also obtained using an enzymatic assay with cells from independent biological replicates ( S3 Fig ). Values were normalized based on OD 600 nm of cultures. Values in (A) and (B) represent means of triplicates ± SEM. (C) Visualization of the α-glucan capsule in the M . tuberculosis WT and the Δ glgC (u) Δ treS mutant by immunogold labelling. Cells were grown in liquid medium without shaking, fixed, labelled with an α-glucan specific monoclonal antibody, and analyzed by electron microscopy (scale bar 0.5 μm). (D) Quantitative evaluation of α-glucan capsule visualization as shown in (C), plotted as anti-α-glucan specific gold particles per cell. Values represent means ± SEM (WT n = 27, Δ glgC (u) Δ treS n = 28). Negative controls were not treated with the primary anti-α-glucan antibody (n = 32).

Techniques Used: Mutagenesis, Sandwich ELISA, Enzymatic Assay, Electron Microscopy

21) Product Images from "Two Faces of CwlM, an Essential PknB Substrate, in Mycobacterium tuberculosis"

Article Title: Two Faces of CwlM, an Essential PknB Substrate, in Mycobacterium tuberculosis

Journal: Cell Reports

doi: 10.1016/j.celrep.2018.09.004

T382A Mutant Mimics Phenotype of PknB-Depleted M. tuberculosis (A–D) The cwlM conditional mutant of M. tuberculosis was transformed with pMV306 plasmids containing cwlM variants. The resultant strains were grown in 7H9 medium (A) or in SMM (B) without pristinamycin. All of the strains grew similarly when 7H9 or SMM were supplemented with pristinamycin (data not shown for clarity). pMV, cwlM -CM pmv306 (the empty plasmid control); CwlM, cwlM -CM WT ; T382A and T382D phosphoablative and phosphomimetic mutants, respectively. Data are represented as means ± SEMs (n = 6). (C) Growth of strains on 7H10 agar. (D) Western blot of CwlM variants detected with anti-CwlM antibody. See also Figures S2 and S3 and Table S3 .
Figure Legend Snippet: T382A Mutant Mimics Phenotype of PknB-Depleted M. tuberculosis (A–D) The cwlM conditional mutant of M. tuberculosis was transformed with pMV306 plasmids containing cwlM variants. The resultant strains were grown in 7H9 medium (A) or in SMM (B) without pristinamycin. All of the strains grew similarly when 7H9 or SMM were supplemented with pristinamycin (data not shown for clarity). pMV, cwlM -CM pmv306 (the empty plasmid control); CwlM, cwlM -CM WT ; T382A and T382D phosphoablative and phosphomimetic mutants, respectively. Data are represented as means ± SEMs (n = 6). (C) Growth of strains on 7H10 agar. (D) Western blot of CwlM variants detected with anti-CwlM antibody. See also Figures S2 and S3 and Table S3 .

Techniques Used: Mutagenesis, Transformation Assay, Plasmid Preparation, Western Blot

PknB-Mediated Phosphorylation of T382 Determines Distribution of CwlM in Cytoplasmic and Membrane Fractions of M. tuberculosis (A–D) Lysates obtained from cwlM -CM grown in SMM without pristinamycin to prevent induction of genomic cwlM (A and C) or from pknB -CM grown in SMM or standard 7H9 medium with or without pristinamycin (B and D) were fractionated and probed with anti-CwlM antibodies (A and B) or with anti-T382-P and anti-T382 antibodies (C and D). Anti-GarA and Anti-GlnA antibodies were used to confirm the purity of mycobacterial fractions. See also Figure S4 .
Figure Legend Snippet: PknB-Mediated Phosphorylation of T382 Determines Distribution of CwlM in Cytoplasmic and Membrane Fractions of M. tuberculosis (A–D) Lysates obtained from cwlM -CM grown in SMM without pristinamycin to prevent induction of genomic cwlM (A and C) or from pknB -CM grown in SMM or standard 7H9 medium with or without pristinamycin (B and D) were fractionated and probed with anti-CwlM antibodies (A and B) or with anti-T382-P and anti-T382 antibodies (C and D). Anti-GarA and Anti-GlnA antibodies were used to confirm the purity of mycobacterial fractions. See also Figure S4 .

Techniques Used:

Osmoprotective Medium Supports Growth of a Conditional pknB Mutant (A–E) M. tuberculosis mutant was grown in standard 7H9 medium with (7H9 +pri ) or without (7H9 −pri ) pristinamycin or in sucrose-magnesium medium with (SMM +pri ) or without (SMM -pri ) pristinamycin at 37°C with shaking. Growth was monitored by (A) measurement of optical density at 580 nm and by (B) assessment of colony-forming unit (CFU) counts on 7H10 agar. Data are represented as means ± SEMs (n = 6). (C) PknB was detected using anti-PknB antibody; relative intensity of PknB bands presented as means ± SEMs (n = 3). (D) Scanning electron micrographs of M. tuberculosis bacteria. (E) Detection of nascent peptidoglycan by Van-BODIPY labeling. Scale bars, 1 μm.
Figure Legend Snippet: Osmoprotective Medium Supports Growth of a Conditional pknB Mutant (A–E) M. tuberculosis mutant was grown in standard 7H9 medium with (7H9 +pri ) or without (7H9 −pri ) pristinamycin or in sucrose-magnesium medium with (SMM +pri ) or without (SMM -pri ) pristinamycin at 37°C with shaking. Growth was monitored by (A) measurement of optical density at 580 nm and by (B) assessment of colony-forming unit (CFU) counts on 7H10 agar. Data are represented as means ± SEMs (n = 6). (C) PknB was detected using anti-PknB antibody; relative intensity of PknB bands presented as means ± SEMs (n = 3). (D) Scanning electron micrographs of M. tuberculosis bacteria. (E) Detection of nascent peptidoglycan by Van-BODIPY labeling. Scale bars, 1 μm.

Techniques Used: Mutagenesis, Labeling

22) Product Images from "Loss of a Functionally and Structurally Distinct ld-Transpeptidase, LdtMt5, Compromises Cell Wall Integrity in Mycobacterium tuberculosis *"

Article Title: Loss of a Functionally and Structurally Distinct ld-Transpeptidase, LdtMt5, Compromises Cell Wall Integrity in Mycobacterium tuberculosis *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M115.660753

Loss of Ldt Mt5 sensitizes M. tuberculosis to crystal violet and osmotic stress. Wild-type ( closed circles ), ldt Mt5 ::Tn ( open circles ), or ldt Mt2 ::Tn ( triangles ) M. tuberculosis were grown in 7H9 complete medium ( A ) or 7H9 medium supplemented with crystal
Figure Legend Snippet: Loss of Ldt Mt5 sensitizes M. tuberculosis to crystal violet and osmotic stress. Wild-type ( closed circles ), ldt Mt5 ::Tn ( open circles ), or ldt Mt2 ::Tn ( triangles ) M. tuberculosis were grown in 7H9 complete medium ( A ) or 7H9 medium supplemented with crystal

Techniques Used:

23) Product Images from "Mycobacterial Gene cuvA Is Required for Optimal Nutrient Utilization and Virulence"

Article Title: Mycobacterial Gene cuvA Is Required for Optimal Nutrient Utilization and Virulence

Journal: Infection and Immunity

doi: 10.1128/IAI.02207-14

Growth of M. tuberculosis strains in liquid and solid media. (A) Wild-type M. tuberculosis H37Rv, Δ cuvA Mt , and complemented Δ cuvA (Δ cuvA Mt /C) strains were grown in 7H9-ADC-Tw liquid medium with 0.2% glucose, and the OD 600 was measured daily. (B) Growth of the same strains on agar plates containing MM plus 0.2% glucose or MM plus 0.01% cholesterol. Serial 10-fold dilutions were spotted and incubated at 37°C, and photographs were taken after 1 month of incubation. In both panels, data shown are from one experiment that was repeated at least twice with similar results. Glc, 0.2% glucose; Chol., 0.01% cholesterol.
Figure Legend Snippet: Growth of M. tuberculosis strains in liquid and solid media. (A) Wild-type M. tuberculosis H37Rv, Δ cuvA Mt , and complemented Δ cuvA (Δ cuvA Mt /C) strains were grown in 7H9-ADC-Tw liquid medium with 0.2% glucose, and the OD 600 was measured daily. (B) Growth of the same strains on agar plates containing MM plus 0.2% glucose or MM plus 0.01% cholesterol. Serial 10-fold dilutions were spotted and incubated at 37°C, and photographs were taken after 1 month of incubation. In both panels, data shown are from one experiment that was repeated at least twice with similar results. Glc, 0.2% glucose; Chol., 0.01% cholesterol.

Techniques Used: Incubation, Gas Chromatography

24) Product Images from "Antibacterial Activity of Rifamycins for M. Smegmatis with Comparison of Oxidation and Binding to Tear Lipocalin"

Article Title: Antibacterial Activity of Rifamycins for M. Smegmatis with Comparison of Oxidation and Binding to Tear Lipocalin

Journal: Biochimica et biophysica acta

doi: 10.1016/j.bbapap.2014.02.001

Influence of antibiotics. Reduced versus oxidized rifamycin drugs. M. smegmatis mc 2 155 was incubated at pH 7.4 in PBS-buffer with rifamycin drugs (10 μg/ml) for 30 min. Thereafter 150 μl 7H9-G-TW was added and incubated for 48 h. (A) rifampin,
Figure Legend Snippet: Influence of antibiotics. Reduced versus oxidized rifamycin drugs. M. smegmatis mc 2 155 was incubated at pH 7.4 in PBS-buffer with rifamycin drugs (10 μg/ml) for 30 min. Thereafter 150 μl 7H9-G-TW was added and incubated for 48 h. (A) rifampin,

Techniques Used: Incubation

25) Product Images from "A rheostat mechanism governs the bifurcation of carbon flux in mycobacteria"

Article Title: A rheostat mechanism governs the bifurcation of carbon flux in mycobacteria

Journal: Nature Communications

doi: 10.1038/ncomms12527

Loss of ICD2 results in glutamate auxotrophy and impaired viability. ( a ) Growth (OD 600 ) of wild-type, Δ icd and Δ icd attB ::P np icd -complemented strains of M. smegmatis in Middlebrook 7H9 medium. Solid lines, culture density (OD 600 ). Dashed lines, glutamate concentration in culture medium. ( b ) Growth (OD 600 ) of wild-type, Δ icd and Δ icd attB ::P np icd -complemented strains of M. smegmatis in minimal medium supplemented with glucose and devoid of glutamate. Data are means±s.d. ( n =3 independent experiments). ( c ) Intracellular metabolites in wild-type, Δ icd and Δ icd attB ::P np icd -complemented strains of M. smegmatis 24 h after transferring cells into minimal medium supplemented with glucose and devoid of glutamate. Data are means±s.d. ( n =3 independent experiments). CIT/ICT, citrate/isocitrate; α-KG, alpha-ketoglutarate; GLU, glutamate. ( d , e ) Growth (OD 600 ) of wild-type, Δ icd1 , Δ icd2 and Δ icd2 attB ::P np icd2 -complemented strains of M. bovis BCG in minimal medium supplemented with glucose and glutamate ( d ) or without glutamate ( e ). ( f ) Survival (CFU) of wild-type, Δ icd and Δ icd attB ::P np icd -complemented strains of M. smegmatis in minimal medium supplemented with glucose and devoid of glutamate. Data are means±s.d. ( n =3 independent experiments each performed in triplicate). ( g ) Survival (CFU) of wild-type, Δ icd2 and Δ icd2 attB ::P np icd2 -complemented strains of M. bovis BCG in minimal medium supplemented with glucose and devoid of glutamate. Data are means±s.d. ( n =3 independent experiments each performed in triplicate).
Figure Legend Snippet: Loss of ICD2 results in glutamate auxotrophy and impaired viability. ( a ) Growth (OD 600 ) of wild-type, Δ icd and Δ icd attB ::P np icd -complemented strains of M. smegmatis in Middlebrook 7H9 medium. Solid lines, culture density (OD 600 ). Dashed lines, glutamate concentration in culture medium. ( b ) Growth (OD 600 ) of wild-type, Δ icd and Δ icd attB ::P np icd -complemented strains of M. smegmatis in minimal medium supplemented with glucose and devoid of glutamate. Data are means±s.d. ( n =3 independent experiments). ( c ) Intracellular metabolites in wild-type, Δ icd and Δ icd attB ::P np icd -complemented strains of M. smegmatis 24 h after transferring cells into minimal medium supplemented with glucose and devoid of glutamate. Data are means±s.d. ( n =3 independent experiments). CIT/ICT, citrate/isocitrate; α-KG, alpha-ketoglutarate; GLU, glutamate. ( d , e ) Growth (OD 600 ) of wild-type, Δ icd1 , Δ icd2 and Δ icd2 attB ::P np icd2 -complemented strains of M. bovis BCG in minimal medium supplemented with glucose and glutamate ( d ) or without glutamate ( e ). ( f ) Survival (CFU) of wild-type, Δ icd and Δ icd attB ::P np icd -complemented strains of M. smegmatis in minimal medium supplemented with glucose and devoid of glutamate. Data are means±s.d. ( n =3 independent experiments each performed in triplicate). ( g ) Survival (CFU) of wild-type, Δ icd2 and Δ icd2 attB ::P np icd2 -complemented strains of M. bovis BCG in minimal medium supplemented with glucose and devoid of glutamate. Data are means±s.d. ( n =3 independent experiments each performed in triplicate).

Techniques Used: Concentration Assay, Transferring

26) Product Images from "Methionine Antagonizes para-Aminosalicylic Acid Activity via Affecting Folate Precursor Biosynthesis in Mycobacterium tuberculosis"

Article Title: Methionine Antagonizes para-Aminosalicylic Acid Activity via Affecting Folate Precursor Biosynthesis in Mycobacterium tuberculosis

Journal: Frontiers in Cellular and Infection Microbiology

doi: 10.3389/fcimb.2018.00399

Disruption of bioB is growth inhibitory and potentiates drug action. (A,B) M. bovis BCG bioB :: himar1 was grown to an OD 600 of ~0.5, washed three times to remove residual biotin with biotin-free 7H9 medium, and resuspended in biotin-free 7H9 medium to a starting OD 600 of 0.01. Cultures were then supplemented with biotin and incubated for 14 days with OD 600 readings taken at the given time points. Error bars denote standard deviation and are representative of 2 separate experiments. (B) M. bovis BCG and bioB :: himar1 were grown to an OD 600 of ~0.5, washed three times to remove residual biotin, and resuspended in biotin-free 7H9 medium to a starting OD 600 of 0.01. Cultures were then supplemented with biotin (0.05 and 5) and incubated for 14 days with OD 600 readings taken at the given time points. MIC 90 is defined as the minimum concentration of inhibitor required to restrict at least 90% of growth relative to growth seen in the no-drug control cultures. PAS, para -aminosalicylic acid; SMX, sulfamethoxazole; RIF, rifampin; INH, isoniazid. Results shown are representative of 3 separate experiments.
Figure Legend Snippet: Disruption of bioB is growth inhibitory and potentiates drug action. (A,B) M. bovis BCG bioB :: himar1 was grown to an OD 600 of ~0.5, washed three times to remove residual biotin with biotin-free 7H9 medium, and resuspended in biotin-free 7H9 medium to a starting OD 600 of 0.01. Cultures were then supplemented with biotin and incubated for 14 days with OD 600 readings taken at the given time points. Error bars denote standard deviation and are representative of 2 separate experiments. (B) M. bovis BCG and bioB :: himar1 were grown to an OD 600 of ~0.5, washed three times to remove residual biotin, and resuspended in biotin-free 7H9 medium to a starting OD 600 of 0.01. Cultures were then supplemented with biotin (0.05 and 5) and incubated for 14 days with OD 600 readings taken at the given time points. MIC 90 is defined as the minimum concentration of inhibitor required to restrict at least 90% of growth relative to growth seen in the no-drug control cultures. PAS, para -aminosalicylic acid; SMX, sulfamethoxazole; RIF, rifampin; INH, isoniazid. Results shown are representative of 3 separate experiments.

Techniques Used: Incubation, Standard Deviation, Concentration Assay

Methionine can affect but not bypass essentiality of upstream folate biosynthetic pathways in M. tuberculosis . (A) New working model of methionine-mediated PAS antagonism. (B) M. tuberculosis Δ pabB was grown to an OD 600 of ~0.5, washed three times with PABA-free 7H9 medium to remove residual PABA and resuspended in PABA-free 7H9 medium to a starting OD 600 of 0.01. Cultures were then supplemented with the indicated metabolites and incubated for 14 days with OD 600 readings taken at the given time points.
Figure Legend Snippet: Methionine can affect but not bypass essentiality of upstream folate biosynthetic pathways in M. tuberculosis . (A) New working model of methionine-mediated PAS antagonism. (B) M. tuberculosis Δ pabB was grown to an OD 600 of ~0.5, washed three times with PABA-free 7H9 medium to remove residual PABA and resuspended in PABA-free 7H9 medium to a starting OD 600 of 0.01. Cultures were then supplemented with the indicated metabolites and incubated for 14 days with OD 600 readings taken at the given time points.

Techniques Used: Incubation

27) Product Images from "Fumarase deficiency causes protein and metabolite succination and intoxicates Mycobacterium tuberculosis"

Article Title: Fumarase deficiency causes protein and metabolite succination and intoxicates Mycobacterium tuberculosis

Journal: Cell chemical biology

doi: 10.1016/j.chembiol.2017.01.005

Metabolic consequences of Fum deficiency Intrabacterial pool sizes of selected metabolites in the indicated Mtb strains after 24 hours cultivation on filters atop 7H9 medium with 0.2% glucose with or without atc. Pool sizes are expressed as area under the curve normalized to protein content. Data are mean values +/− SD of three biological replicates and are representative of two independent experiments. Statistically significant differences between atc-treated and untreated cultures that were observed in two biologically independent experiments are reported. *p≤0.05, **p≤0.005 by Student’s t-test. The differences between pool sizes in atc treated and untreated WT were not reproducibly statistically significant. Enzymatic reactions are depicted as arrows; the dashed arrow indicates that the respective enzyme has not been identified in Mtb .
Figure Legend Snippet: Metabolic consequences of Fum deficiency Intrabacterial pool sizes of selected metabolites in the indicated Mtb strains after 24 hours cultivation on filters atop 7H9 medium with 0.2% glucose with or without atc. Pool sizes are expressed as area under the curve normalized to protein content. Data are mean values +/− SD of three biological replicates and are representative of two independent experiments. Statistically significant differences between atc-treated and untreated cultures that were observed in two biologically independent experiments are reported. *p≤0.05, **p≤0.005 by Student’s t-test. The differences between pool sizes in atc treated and untreated WT were not reproducibly statistically significant. Enzymatic reactions are depicted as arrows; the dashed arrow indicates that the respective enzyme has not been identified in Mtb .

Techniques Used:

28) Product Images from "Viability, biofilm formation, and MazEF expression in drug-sensitive and drug-resistant Mycobacterium tuberculosis strains circulating in Xinjiang, China"

Article Title: Viability, biofilm formation, and MazEF expression in drug-sensitive and drug-resistant Mycobacterium tuberculosis strains circulating in Xinjiang, China

Journal: Infection and Drug Resistance

doi: 10.2147/IDR.S148648

Growth profiles of MTB in standard, hypoxic and nutrient starvation conditions at different culture time points. Notes: ( A ) MTB incubated under standard conditions in 7H9 medium. Difference in growth ability became apparent on the eighth day, and resistant strains grew more rapidly than H37Rv ( P
Figure Legend Snippet: Growth profiles of MTB in standard, hypoxic and nutrient starvation conditions at different culture time points. Notes: ( A ) MTB incubated under standard conditions in 7H9 medium. Difference in growth ability became apparent on the eighth day, and resistant strains grew more rapidly than H37Rv ( P

Techniques Used: Incubation

29) Product Images from "Functional analysis of an intergenic non-coding sequence within mce1 operon of M.tuberculosis"

Article Title: Functional analysis of an intergenic non-coding sequence within mce1 operon of M.tuberculosis

Journal: BMC Microbiology

doi: 10.1186/1471-2180-10-128

Regulation of heterologous promoter by IGPr . dps promoter activity under induced conditions in different constructs in terms of β -gal activity units expressed as nmol ONPG converted to o-nitrophenol per min per milligram of protein. The transformants were grown in Middlebrook 7H9 medium supplemented with 0.02% glucose (Induced). Each experiment was carried out in triplicates and S.D is indicated as error bars.
Figure Legend Snippet: Regulation of heterologous promoter by IGPr . dps promoter activity under induced conditions in different constructs in terms of β -gal activity units expressed as nmol ONPG converted to o-nitrophenol per min per milligram of protein. The transformants were grown in Middlebrook 7H9 medium supplemented with 0.02% glucose (Induced). Each experiment was carried out in triplicates and S.D is indicated as error bars.

Techniques Used: Activity Assay, Construct

30) Product Images from "Rapid detection of Mycobacterium tuberculosis in sputum with a solvatochromic trehalose probe"

Article Title: Rapid detection of Mycobacterium tuberculosis in sputum with a solvatochromic trehalose probe

Journal: Science translational medicine

doi: 10.1126/scitranslmed.aam6310

DMN-Tre detects Mtb in sputum samples from patients with tuberculosis, similar to the auramine stain ( A ) Illustration of sputum sample labeling protocol: 16 sputum samples were collected from diagnosed treatment-naïve patients with tuberculosis and were decontaminated with N -acetyl-L-cysteine (NalC)/NaOH mixture following recommended standards. Decontaminated sputum samples were split in two equal aliquots, each incubated with 1 mM DMN-Tre or smeared with Auramine O stain, followed by imaging. ( B ) Microscopy analysis of four decontaminated sputum samples incubated with 1 mM DMN-Tre in 7H9 liquid medium for 2 hours in a 37°C atmospheric incubator. ( C ) Microscopy analysis of decontaminated sputum samples either treated with 1 mM DMN-Tre for 30 min at 37°C (top) or directly fixed onto microscope slide for Auramine O staining following standard kit protocol (bottom, orange pseudocolor). ( D ) Bar graph depiction of the total Mtb cell number detected over eight fields of view per sample with either DMN-Tre or Auramine O in the 16 sputum samples treated as in (C). Images were collected in the DIC and FITC (for DMN and auramine fluorescence) channels of a Zeiss Observer Z1-inverted fluorescent microscope. Scale bars, 5 μm.
Figure Legend Snippet: DMN-Tre detects Mtb in sputum samples from patients with tuberculosis, similar to the auramine stain ( A ) Illustration of sputum sample labeling protocol: 16 sputum samples were collected from diagnosed treatment-naïve patients with tuberculosis and were decontaminated with N -acetyl-L-cysteine (NalC)/NaOH mixture following recommended standards. Decontaminated sputum samples were split in two equal aliquots, each incubated with 1 mM DMN-Tre or smeared with Auramine O stain, followed by imaging. ( B ) Microscopy analysis of four decontaminated sputum samples incubated with 1 mM DMN-Tre in 7H9 liquid medium for 2 hours in a 37°C atmospheric incubator. ( C ) Microscopy analysis of decontaminated sputum samples either treated with 1 mM DMN-Tre for 30 min at 37°C (top) or directly fixed onto microscope slide for Auramine O staining following standard kit protocol (bottom, orange pseudocolor). ( D ) Bar graph depiction of the total Mtb cell number detected over eight fields of view per sample with either DMN-Tre or Auramine O in the 16 sputum samples treated as in (C). Images were collected in the DIC and FITC (for DMN and auramine fluorescence) channels of a Zeiss Observer Z1-inverted fluorescent microscope. Scale bars, 5 μm.

Techniques Used: Staining, Labeling, Incubation, Imaging, Microscopy, Fluorescence

DMN-Tre labeling is inhibited by tuberculosis drugs ( A and B ) Flow cytometry MFI analysis (A) and no-wash imaging (B) of control and drug-treated (37°C for 3 hours) Msmeg cells labeled with 100 μM DMN-Tre for 30 min. Drug cocktail contents: ethambutol (1 μg/ml), rifampicin (0.2 μg/ml), SQ109 (20 μg/ml), and isoniazid (20 μg/ml) in 7H9 medium. ( C ) Flow cytometry MFI analysis of wild-type (WT) Msmeg or KatG mutant cells treated with isoniazid (INH) at the indicated final concentrations (0, 3, 6, or 10 μg/ml of isoniazid) for 3 hours, followed by incubation with DMN-Tre for 30 min. Images were collected in the DIC, FITC/GFP (for DMN fluorescence), or RFP (for mCherry fluorescence) channels of a Nikon A1R confocal microscope. Scale bars, 5 μm. In (A) and (C), data are means ± SEM from at least two independent experiments. Data were analyzed by one-way ANOVA test (**** P
Figure Legend Snippet: DMN-Tre labeling is inhibited by tuberculosis drugs ( A and B ) Flow cytometry MFI analysis (A) and no-wash imaging (B) of control and drug-treated (37°C for 3 hours) Msmeg cells labeled with 100 μM DMN-Tre for 30 min. Drug cocktail contents: ethambutol (1 μg/ml), rifampicin (0.2 μg/ml), SQ109 (20 μg/ml), and isoniazid (20 μg/ml) in 7H9 medium. ( C ) Flow cytometry MFI analysis of wild-type (WT) Msmeg or KatG mutant cells treated with isoniazid (INH) at the indicated final concentrations (0, 3, 6, or 10 μg/ml of isoniazid) for 3 hours, followed by incubation with DMN-Tre for 30 min. Images were collected in the DIC, FITC/GFP (for DMN fluorescence), or RFP (for mCherry fluorescence) channels of a Nikon A1R confocal microscope. Scale bars, 5 μm. In (A) and (C), data are means ± SEM from at least two independent experiments. Data were analyzed by one-way ANOVA test (**** P

Techniques Used: Labeling, Flow Cytometry, Cytometry, Imaging, Mutagenesis, Incubation, Fluorescence, Microscopy

DMN-Tre labeling of Mycobacterium tuberculosis is inhibited by tuberculosis drug cocktail, unlike auramine staining ( A ) Microscopy analysis of Mycobacterium tuberculosis (Mtb) cells incubated with 100 μM DMN-Tre for 2 hours. ( B ) Flow cytometry MFI analysis of Mtb cells incubated with DMN-Tre for the indicated times: 0.5, 1, 2, and 3 hours or overnight (~16 hours). Error bars denote three biological replicates. ( C and D ) Microscopy analysis of control and drug-treated Mtb cells labeled with 100 μM DMN-Tre overnight (C) or stained with the auramine-based Fluorescent Stain kit for mycobacteria (05151, Sigma-Aldrich) (D). Drug cocktail contents: ethambutol (1 μg/ml), rifampicin (0.2 μg/ml), SQ109 (10 μg/ml), and isoniazid (10 μg/ml) in 7H9 medium. Images were collected in the DIC or FITC/GFP (for DMN and Auramine fluorescence) channels of a Nikon A1R confocal microscope. Cells stained with auramine were given an orange pseudocolor. Scale bars, 5 μm. In (B), data are means ± SEM from at least two independent experiments. Data were analyzed by one-way ANOVA test (* P
Figure Legend Snippet: DMN-Tre labeling of Mycobacterium tuberculosis is inhibited by tuberculosis drug cocktail, unlike auramine staining ( A ) Microscopy analysis of Mycobacterium tuberculosis (Mtb) cells incubated with 100 μM DMN-Tre for 2 hours. ( B ) Flow cytometry MFI analysis of Mtb cells incubated with DMN-Tre for the indicated times: 0.5, 1, 2, and 3 hours or overnight (~16 hours). Error bars denote three biological replicates. ( C and D ) Microscopy analysis of control and drug-treated Mtb cells labeled with 100 μM DMN-Tre overnight (C) or stained with the auramine-based Fluorescent Stain kit for mycobacteria (05151, Sigma-Aldrich) (D). Drug cocktail contents: ethambutol (1 μg/ml), rifampicin (0.2 μg/ml), SQ109 (10 μg/ml), and isoniazid (10 μg/ml) in 7H9 medium. Images were collected in the DIC or FITC/GFP (for DMN and Auramine fluorescence) channels of a Nikon A1R confocal microscope. Cells stained with auramine were given an orange pseudocolor. Scale bars, 5 μm. In (B), data are means ± SEM from at least two independent experiments. Data were analyzed by one-way ANOVA test (* P

Techniques Used: Labeling, Staining, Microscopy, Incubation, Flow Cytometry, Cytometry, Fluorescence

31) Product Images from "A fluorescence-based reporter for monitoring expression of mycobacterial cytochrome bd in response to antibacterials and during infection"

Article Title: A fluorescence-based reporter for monitoring expression of mycobacterial cytochrome bd in response to antibacterials and during infection

Journal: Scientific Reports

doi: 10.1038/s41598-017-10944-4

Construction and basic characterization of the cytochrome bd expression reporter. ( A ) Genetic construction of the reporter plasmid with the mCherry gene under control of the cydA promoter. ( B ) Growth of Mycobacterium marinum carrying the cydA reporter in 7H9 medium. Three independent biological replicates were monitored for growth. Error bars indicate the standard deviation (s.d.) value. ( C ) mCherry fluorescence emitted by the cydA reporter during in vitro culture under aerated (normoxia) conditions and hypoxic conditions. Three independent biological replicates were monitored. The fluorescence signal was corrected for autofluorescence of WT bacteria without a plasmid. Error bars indicate the s.d. ( D ) cydA induction in response to the nitric oxide donor DETA-NO (1x MIC and 10x MIC) on day 3 after induction. Three independent biological replicates were monitored. Error bars indicate the s.d. value.
Figure Legend Snippet: Construction and basic characterization of the cytochrome bd expression reporter. ( A ) Genetic construction of the reporter plasmid with the mCherry gene under control of the cydA promoter. ( B ) Growth of Mycobacterium marinum carrying the cydA reporter in 7H9 medium. Three independent biological replicates were monitored for growth. Error bars indicate the standard deviation (s.d.) value. ( C ) mCherry fluorescence emitted by the cydA reporter during in vitro culture under aerated (normoxia) conditions and hypoxic conditions. Three independent biological replicates were monitored. The fluorescence signal was corrected for autofluorescence of WT bacteria without a plasmid. Error bars indicate the s.d. ( D ) cydA induction in response to the nitric oxide donor DETA-NO (1x MIC and 10x MIC) on day 3 after induction. Three independent biological replicates were monitored. Error bars indicate the s.d. value.

Techniques Used: Expressing, Plasmid Preparation, Standard Deviation, Fluorescence, In Vitro

32) Product Images from "The prrAB Two-Component System Is Essential for Mycobacterium tuberculosis Viability and Is Induced under Nitrogen-Limiting Conditions"

Article Title: The prrAB Two-Component System Is Essential for Mycobacterium tuberculosis Viability and Is Induced under Nitrogen-Limiting Conditions

Journal: Journal of Bacteriology

doi: 10.1128/JB.06258-11

prrA and prrB transcription and PrrA levels during in vitro growth. (A) Quantitation of prrA and prrB transcripts during M. tuberculosis H37Rv in vitro exponential growth in supplemented Middlebrook 7H9 broth with glycerol. The optical density at 600
Figure Legend Snippet: prrA and prrB transcription and PrrA levels during in vitro growth. (A) Quantitation of prrA and prrB transcripts during M. tuberculosis H37Rv in vitro exponential growth in supplemented Middlebrook 7H9 broth with glycerol. The optical density at 600

Techniques Used: In Vitro, Quantitation Assay

33) Product Images from "Inorganic Phosphate Limitation Modulates Capsular Polysaccharide Composition in Mycobacteria *"

Article Title: Inorganic Phosphate Limitation Modulates Capsular Polysaccharide Composition in Mycobacteria *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M116.722454

Chemical activation of the stringent response by SHX up-regulates the capsule polysaccharides α-glucan and AM in M. smegmatis and α-glucan in M. tuberculosis . M. smegmatis (mc 2 155) and M. tuberculosis (mc 2 6020) strains were grown in 7H9-based liquid P i medium without ADC, and SHX was added at 6.5 h and 4 days, respectively. Bacterial cells were collected at different time points and shaved with a detergent. The obtained capsular extracts were subsequently assayed by immunodot-blotting to analyze the capsular polysaccharides α-glucan or AM using specific anti-glucan (IV56B6) or anti-AM (F30.5) mAb. A , M. smegmatis challenged with 100 μg/ml SHX showed strong reduction in growth in comparison with bacteria grown without SHX. Error bars represent S.D. The cells grown in A were used to determine α-glucan ( B ) and AM ( C ) levels. A strong induction of α-glucan and AM is observed after 3 and 17 h of SHX addition. D , direct visualization of capsular polysaccharides α-glucan ( upper panel ) and AM ( lower panel ) in M. smegmatis WT by immunogold labeling. Cells were cultured in liquid, fixed 2 days after SHX addition, and labeled with IV56B6 ( upper panel ) or F30.5 ( lower panel ) mAb. Quantitative evaluation of the EM analysis of α-glucan ( E ) and AM ( F ) is shown. Values represent means of three experiments ±S.E. G , M. tuberculosis (mc 2 6020) strain treated with SHX (50,100, 150, and 200 μg/ml) showed dose-dependent up-regulation of capsular α-glucan after 48 h. Data are representative for three independent experiments. Dil , diluted/dilutes or dilutions.
Figure Legend Snippet: Chemical activation of the stringent response by SHX up-regulates the capsule polysaccharides α-glucan and AM in M. smegmatis and α-glucan in M. tuberculosis . M. smegmatis (mc 2 155) and M. tuberculosis (mc 2 6020) strains were grown in 7H9-based liquid P i medium without ADC, and SHX was added at 6.5 h and 4 days, respectively. Bacterial cells were collected at different time points and shaved with a detergent. The obtained capsular extracts were subsequently assayed by immunodot-blotting to analyze the capsular polysaccharides α-glucan or AM using specific anti-glucan (IV56B6) or anti-AM (F30.5) mAb. A , M. smegmatis challenged with 100 μg/ml SHX showed strong reduction in growth in comparison with bacteria grown without SHX. Error bars represent S.D. The cells grown in A were used to determine α-glucan ( B ) and AM ( C ) levels. A strong induction of α-glucan and AM is observed after 3 and 17 h of SHX addition. D , direct visualization of capsular polysaccharides α-glucan ( upper panel ) and AM ( lower panel ) in M. smegmatis WT by immunogold labeling. Cells were cultured in liquid, fixed 2 days after SHX addition, and labeled with IV56B6 ( upper panel ) or F30.5 ( lower panel ) mAb. Quantitative evaluation of the EM analysis of α-glucan ( E ) and AM ( F ) is shown. Values represent means of three experiments ±S.E. G , M. tuberculosis (mc 2 6020) strain treated with SHX (50,100, 150, and 200 μg/ml) showed dose-dependent up-regulation of capsular α-glucan after 48 h. Data are representative for three independent experiments. Dil , diluted/dilutes or dilutions.

Techniques Used: Activation Assay, Labeling, Cell Culture

Mycobacterial growth medium 7H9 supplement ADC inactivates activity of stringent response activator SHX in M. smegmatis . M. smegmatis WT strain was grown in liquid 7H9 or in 7H9-based P i medium. The 7H9 and P i media were supplemented with or without ADC, and P i medium was supplemented with albumin. The bacterial growth of M. smegmatis was monitored by A 600 over time; at 4 h after inoculation, the bacterial cultures where supplemented with or without 100 μg/ml SHX. A , bacterial growth is arrested in P i medium upon addition with SHX in comparison without SHX, indicative for SHX-dependent activation of the stringent response. In 7H9 medium supplemented with SHX, bacterial growth arrest is not observed, a clear sign that this compound is not active. B , removal of ADC from 7H9 medium shows SHX-mediated growth arrest in M. smegmatis , a strong indication that ADC supplement is responsible for inactivation of compound SHX. C , ADC addition to P i medium shows no activity of SHX on bacterial growth in M. smegmatis , and P i medium supplemented with or without albumin ( A ) shows SHX-mediated bacterial growth arrest. This indicates that dextrose and/or catalase is responsible for inactivation of compound SHX. Data are representative for six independent experiments.
Figure Legend Snippet: Mycobacterial growth medium 7H9 supplement ADC inactivates activity of stringent response activator SHX in M. smegmatis . M. smegmatis WT strain was grown in liquid 7H9 or in 7H9-based P i medium. The 7H9 and P i media were supplemented with or without ADC, and P i medium was supplemented with albumin. The bacterial growth of M. smegmatis was monitored by A 600 over time; at 4 h after inoculation, the bacterial cultures where supplemented with or without 100 μg/ml SHX. A , bacterial growth is arrested in P i medium upon addition with SHX in comparison without SHX, indicative for SHX-dependent activation of the stringent response. In 7H9 medium supplemented with SHX, bacterial growth arrest is not observed, a clear sign that this compound is not active. B , removal of ADC from 7H9 medium shows SHX-mediated growth arrest in M. smegmatis , a strong indication that ADC supplement is responsible for inactivation of compound SHX. C , ADC addition to P i medium shows no activity of SHX on bacterial growth in M. smegmatis , and P i medium supplemented with or without albumin ( A ) shows SHX-mediated bacterial growth arrest. This indicates that dextrose and/or catalase is responsible for inactivation of compound SHX. Data are representative for six independent experiments.

Techniques Used: Activity Assay, Activation Assay

Capsular α-glucan production of P i -stressed M. smegmatis , M. marinum , and auxotrophic M. tuberculosis . The mycobacterial strains M. smegmatis mc 2 155, M. marinum E11, and auxotrophic M. tuberculosis mc 2 6020 were grown in 7H9-based liquid medium supplemented with different concentrations of P i , and cells were collected at various time points (see below) The bacterial cells were “shaved” with a detergent, and the obtained capsular extracts were subsequently assayed by immunodot-blotting to analyze the capsular α-glucan level using anti-glucan mAb (IV56B6). A , growth curves of M. smegmatis grown in different concentrations of P i show a P i concentration-dependent growth arrest. B , immunodot-blotting analysis of capsular α-glucan of A at 48 h revealed P i stress-mediated induction of α-glucan in M. smegmatis. C , immunodot-blotting analysis of capsular α-glucan in M. marinum ( left panel ) and M. tuberculosis ( right panel ) grown under low and high P i at days 5 and 6, respectively. In both mycobacterial strains, the capsular α-glucan levels are elevated under low P i . The data presented here are representative for four independent experiments. Dil , diluted/dilutes.
Figure Legend Snippet: Capsular α-glucan production of P i -stressed M. smegmatis , M. marinum , and auxotrophic M. tuberculosis . The mycobacterial strains M. smegmatis mc 2 155, M. marinum E11, and auxotrophic M. tuberculosis mc 2 6020 were grown in 7H9-based liquid medium supplemented with different concentrations of P i , and cells were collected at various time points (see below) The bacterial cells were “shaved” with a detergent, and the obtained capsular extracts were subsequently assayed by immunodot-blotting to analyze the capsular α-glucan level using anti-glucan mAb (IV56B6). A , growth curves of M. smegmatis grown in different concentrations of P i show a P i concentration-dependent growth arrest. B , immunodot-blotting analysis of capsular α-glucan of A at 48 h revealed P i stress-mediated induction of α-glucan in M. smegmatis. C , immunodot-blotting analysis of capsular α-glucan in M. marinum ( left panel ) and M. tuberculosis ( right panel ) grown under low and high P i at days 5 and 6, respectively. In both mycobacterial strains, the capsular α-glucan levels are elevated under low P i . The data presented here are representative for four independent experiments. Dil , diluted/dilutes.

Techniques Used: Concentration Assay

Related Articles

Infection:

Article Title: Mycobacterial Gene cuvA Is Required for Optimal Nutrient Utilization and Virulence
Article Snippet: A complemented M. tuberculosis Δ cuvA strain was constructed by expressing cuvA under the control of the hsp70 promoter in the vector pJEB402 ( ) to allow stable expression during prolonged incubation in vitro and during infection experiments. .. When grown in Middlebrook 7H9 liquid medium containing 0.2% glucose, the M. tuberculosis Δ cuvA ( ) and M. smegmatis Δ cuvA (see Fig. S4A in the supplemental material) strains grew at similar rates and to the same final densities as the wild-type parental strains.

Article Title: Viability, biofilm formation, and MazEF expression in drug-sensitive and drug-resistant Mycobacterium tuberculosis strains circulating in Xinjiang, China
Article Snippet: H37Rv, H37RvΔmazEF 3, H37RvΔmazEF 6, and H37RvΔmazEF 9 were grown at 37°C in Middlebrook 7H9 liquid medium supplemented with 10% oleic albumin dextrose catalase or on Löwenstein–Jensen slants. .. To determine what role the mazEF 3,6,9 TASs play in MTB growth and survival, we compared cell growth between the parent H37Rv strain and H37RvΔmazEF 3,6,9-deletion mutants under both normal and stressful culture conditions, including hypoxia and nutrient starvation or after in vitro intracellular infection of host macrophages.

Clone Assay:

Article Title: Mycobacterial Gene cuvA Is Required for Optimal Nutrient Utilization and Virulence
Article Snippet: Escherichia coli TOP10 (Invitrogen) was used for cloning and was grown in LB broth. .. For measurements of growth on solid medium, cells grown in Middlebrook 7H9 liquid medium to an optical density at 600 nm (OD600 ) of 0.5 were washed in phosphate-buffered saline containing 0.05% tyloxapol (PBS-Tx), and 10-fold serial dilutions in PBS were spotted on agar plates containing MM as described above (without tyloxapol) plus a 0.01% concentration of the carbon source.

Centrifugation:

Article Title: A rheostat mechanism governs the bifurcation of carbon flux in mycobacteria
Article Snippet: .. Metabolite measurements Bacteria were grown in Middlebrook 7H9 liquid medium at 37 °C with aeration (180 r.p.m.) to mid-exponential phase, harvested by centrifugation (3,000g , 5 min) washed three times with PBS containing 0.05% Tween-80 (Sigma), inoculated into Middlebrook 7H9 liquid medium supplemented with 2 g l−1 glucose or acetate (initial OD600 0.05), and grown at 37 °C to mid-log phase (OD600 ∼0.5). .. Samples equivalent to a biomass of 1 ml culture at OD600 2 were collected for metabolome analysis by fast filtration and metabolism was quenched at –20 °C in a mixture of acetonitrile:methanol:water (2:2:1), as described .

Article Title: Unexpected Link between Lipooligosaccharide Biosynthesis and Surface Protein Release in Mycobacterium marinum *
Article Snippet: Subsequently, the bacteria were washed twice in Middlebrook 7H9 medium without supplement to remove all traces of bovine serum albumin (BSA) and grown overnight in Middlebrook 7H9 liquid medium supplemented with 0.2% dextrose and optionally 0.05% Tween 80. .. Secreted proteins present in cell-free supernatants were obtained by centrifugation and subsequent passage through a 0.45-μm filter and concentrated by precipitation with 10% trichloroacetic acid in 10% acetone.

Amplification:

Article Title: Mycobacterial Gene cuvA Is Required for Optimal Nutrient Utilization and Virulence
Article Snippet: For measurements of growth on solid medium, cells grown in Middlebrook 7H9 liquid medium to an optical density at 600 nm (OD600 ) of 0.5 were washed in phosphate-buffered saline containing 0.05% tyloxapol (PBS-Tx), and 10-fold serial dilutions in PBS were spotted on agar plates containing MM as described above (without tyloxapol) plus a 0.01% concentration of the carbon source. .. For subcellular localization, CuvA was expressed as a carboxy-terminal GFP fusion. cuvA was PCR amplified from genomic DNA of M. tuberculosis H37RV by use of primers Rv1422AE and Rv1422rPac1. gfp was PCR amplified from pTracerCMV (Invitrogen) by use of primers GFP3PacI and GFP4XK.

Filtration:

Article Title: A rheostat mechanism governs the bifurcation of carbon flux in mycobacteria
Article Snippet: Metabolite measurements Bacteria were grown in Middlebrook 7H9 liquid medium at 37 °C with aeration (180 r.p.m.) to mid-exponential phase, harvested by centrifugation (3,000g , 5 min) washed three times with PBS containing 0.05% Tween-80 (Sigma), inoculated into Middlebrook 7H9 liquid medium supplemented with 2 g l−1 glucose or acetate (initial OD600 0.05), and grown at 37 °C to mid-log phase (OD600 ∼0.5). .. Samples equivalent to a biomass of 1 ml culture at OD600 2 were collected for metabolome analysis by fast filtration and metabolism was quenched at –20 °C in a mixture of acetonitrile:methanol:water (2:2:1), as described .

In Vitro:

Article Title: Mycobacterial Gene cuvA Is Required for Optimal Nutrient Utilization and Virulence
Article Snippet: A complemented M. tuberculosis Δ cuvA strain was constructed by expressing cuvA under the control of the hsp70 promoter in the vector pJEB402 ( ) to allow stable expression during prolonged incubation in vitro and during infection experiments. .. When grown in Middlebrook 7H9 liquid medium containing 0.2% glucose, the M. tuberculosis Δ cuvA ( ) and M. smegmatis Δ cuvA (see Fig. S4A in the supplemental material) strains grew at similar rates and to the same final densities as the wild-type parental strains.

Article Title: Viability, biofilm formation, and MazEF expression in drug-sensitive and drug-resistant Mycobacterium tuberculosis strains circulating in Xinjiang, China
Article Snippet: H37Rv, H37RvΔmazEF 3, H37RvΔmazEF 6, and H37RvΔmazEF 9 were grown at 37°C in Middlebrook 7H9 liquid medium supplemented with 10% oleic albumin dextrose catalase or on Löwenstein–Jensen slants. .. To determine what role the mazEF 3,6,9 TASs play in MTB growth and survival, we compared cell growth between the parent H37Rv strain and H37RvΔmazEF 3,6,9-deletion mutants under both normal and stressful culture conditions, including hypoxia and nutrient starvation or after in vitro intracellular infection of host macrophages.

Southern Blot:

Article Title: The Twin-Arginine Translocation Pathway of Mycobacterium smegmatis Is Functional and Required for the Export of Mycobacterial ?-Lactamases
Article Snippet: These small colonies were subsequently proven by PCR (data not shown) and Southern blot analysis (Fig. ) to be tatA or tatC deletion mutants. .. We compared growth of the Δ tat mutants to that of wild-type M. smegmatis in Middlebrook 7H9 liquid medium.

Electron Microscopy:

Article Title: Metabolic Network for the Biosynthesis of Intra- and Extracellular α-Glucans Required for Virulence of Mycobacterium tuberculosis
Article Snippet: .. Immunogold labeling and electron microscopy for visualization of capsular α-glucans M . tuberculosis WT and the ΔglgC (u) ΔtreS double mutant were grown in Middlebrook 7H9 liquid medium supplemented with 10% (v/v) ADC enrichment (BD, Breda, The Netherlands) and 0.05% (v/v) Tween 80 without shaking for 17 days at 37°C. ..

Mutagenesis:

Article Title: Mycobacterial Gene cuvA Is Required for Optimal Nutrient Utilization and Virulence
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Article Title: Metabolic Network for the Biosynthesis of Intra- and Extracellular α-Glucans Required for Virulence of Mycobacterium tuberculosis
Article Snippet: .. Immunogold labeling and electron microscopy for visualization of capsular α-glucans M . tuberculosis WT and the ΔglgC (u) ΔtreS double mutant were grown in Middlebrook 7H9 liquid medium supplemented with 10% (v/v) ADC enrichment (BD, Breda, The Netherlands) and 0.05% (v/v) Tween 80 without shaking for 17 days at 37°C. ..

Article Title: The Twin-Arginine Translocation Pathway of Mycobacterium smegmatis Is Functional and Required for the Export of Mycobacterial ?-Lactamases
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Subcloning:

Article Title: Role of Mycobacterium tuberculosis Ser/Thr Kinase PknF: Implications in Glucose Transport and Cell Division
Article Snippet: M. tuberculosis H37 Rv and M. smegmatis LR222 were grown in Middlebrook 7H9 liquid medium and on Middlebrook 7H10 agar. .. E. coli DH5α was used for all subcloning procedures and was grown in LB medium/agar.

Protein Concentration:

Article Title: Unexpected Link between Lipooligosaccharide Biosynthesis and Surface Protein Release in Mycobacterium marinum *
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Construct:

Article Title: Mycobacterial Gene cuvA Is Required for Optimal Nutrient Utilization and Virulence
Article Snippet: The same construct was used to complement the M. smegmatis mutant with M. tuberculosis cuvA . .. When grown in Middlebrook 7H9 liquid medium containing 0.2% glucose, the M. tuberculosis Δ cuvA ( ) and M. smegmatis Δ cuvA (see Fig. S4A in the supplemental material) strains grew at similar rates and to the same final densities as the wild-type parental strains.

Concentration Assay:

Article Title: Mycobacterial Gene cuvA Is Required for Optimal Nutrient Utilization and Virulence
Article Snippet: .. For measurements of growth on solid medium, cells grown in Middlebrook 7H9 liquid medium to an optical density at 600 nm (OD600 ) of 0.5 were washed in phosphate-buffered saline containing 0.05% tyloxapol (PBS-Tx), and 10-fold serial dilutions in PBS were spotted on agar plates containing MM as described above (without tyloxapol) plus a 0.01% concentration of the carbon source. ..

Incubation:

Article Title: Mycobacterial Gene cuvA Is Required for Optimal Nutrient Utilization and Virulence
Article Snippet: For measurements of growth on solid medium, cells grown in Middlebrook 7H9 liquid medium to an optical density at 600 nm (OD600 ) of 0.5 were washed in phosphate-buffered saline containing 0.05% tyloxapol (PBS-Tx), and 10-fold serial dilutions in PBS were spotted on agar plates containing MM as described above (without tyloxapol) plus a 0.01% concentration of the carbon source. .. Plates were incubated at 37°C and photographed after 1 month for M. tuberculosis and after 2 days for M. smegmatis .

Article Title: Mycobacterial Gene cuvA Is Required for Optimal Nutrient Utilization and Virulence
Article Snippet: A complemented M. tuberculosis Δ cuvA strain was constructed by expressing cuvA under the control of the hsp70 promoter in the vector pJEB402 ( ) to allow stable expression during prolonged incubation in vitro and during infection experiments. .. When grown in Middlebrook 7H9 liquid medium containing 0.2% glucose, the M. tuberculosis Δ cuvA ( ) and M. smegmatis Δ cuvA (see Fig. S4A in the supplemental material) strains grew at similar rates and to the same final densities as the wild-type parental strains.

Article Title: Assessing the role of Rv1222 (RseA) as an anti-sigma factor of the Mycobacterium tuberculosis extracytoplasmic sigma factor SigE
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Article Title: The Twin-Arginine Translocation Pathway of Mycobacterium smegmatis Is Functional and Required for the Export of Mycobacterial ?-Lactamases
Article Snippet: However, further incubation for 2 additional days at 37°C yielded a population of slow-growing secondary recombinants. .. We compared growth of the Δ tat mutants to that of wild-type M. smegmatis in Middlebrook 7H9 liquid medium.

other:

Article Title: Proteomic Analysis of Drug-Resistant Mycobacteria: Co-Evolution of Copper and INH Resistance
Article Snippet: Bacterial strains and growth conditions M . smegmatis strains were grown in Middlebrook 7H9 liquid medium supplemented with 10% ADS (50 g/L albumin, 20 g/L D-glucose and 8.1 g/L sodium chloride), 0.05% Tween 80 and 0.2% glycerol or on Middlebrook 7H10 agar supplemented with 0.5% glycerol and 10% ADS.

Article Title: The Mycobacterium tuberculosis relBE toxin:antitoxin genes are stress-responsive modules that regulate growth through translation inhibition
Article Snippet: M. tuberculosis H37Rv was grown at 37°C in Middlebrook 7H9 liquid medium or on Middlebrook 7H10 agar (Difco) supplemented with ADS (0.5% bovine serum albumin-frac- tion V, 0.2% dextrose, 0.85% saline) enrichment and 0.05% Tween 80 (Tw).

Article Title: Chlorine Disinfection of Atypical Mycobacteria Isolated from a Water Distribution System
Article Snippet: To test the effect of the medium composition on inactivation rates, M. gordonae was grown in tap water previously filtered through a 0.45-μm-pore-size filter and supplemented with 10% Middlebrook 7H9 liquid medium.

Labeling:

Article Title: Metabolic Network for the Biosynthesis of Intra- and Extracellular α-Glucans Required for Virulence of Mycobacterium tuberculosis
Article Snippet: .. Immunogold labeling and electron microscopy for visualization of capsular α-glucans M . tuberculosis WT and the ΔglgC (u) ΔtreS double mutant were grown in Middlebrook 7H9 liquid medium supplemented with 10% (v/v) ADC enrichment (BD, Breda, The Netherlands) and 0.05% (v/v) Tween 80 without shaking for 17 days at 37°C. ..

Expressing:

Article Title: Mycobacterial Gene cuvA Is Required for Optimal Nutrient Utilization and Virulence
Article Snippet: To complement the M. smegmatis mutant with the M. smegmatis cuvA homologue MSMEG_3080 , this gene was expressed under the control of a Tet repressor-regulated promoter to allow inducible expression of this gene ( ). .. When grown in Middlebrook 7H9 liquid medium containing 0.2% glucose, the M. tuberculosis Δ cuvA ( ) and M. smegmatis Δ cuvA (see Fig. S4A in the supplemental material) strains grew at similar rates and to the same final densities as the wild-type parental strains.

BIA-KA:

Article Title: Unexpected Link between Lipooligosaccharide Biosynthesis and Surface Protein Release in Mycobacterium marinum *
Article Snippet: Subsequently, the bacteria were washed twice in Middlebrook 7H9 medium without supplement to remove all traces of bovine serum albumin (BSA) and grown overnight in Middlebrook 7H9 liquid medium supplemented with 0.2% dextrose and optionally 0.05% Tween 80. .. Subsequently, the bacteria were washed twice in Middlebrook 7H9 medium without supplement to remove all traces of bovine serum albumin (BSA) and grown overnight in Middlebrook 7H9 liquid medium supplemented with 0.2% dextrose and optionally 0.05% Tween 80.

Polymerase Chain Reaction:

Article Title: Mycobacterial Gene cuvA Is Required for Optimal Nutrient Utilization and Virulence
Article Snippet: For measurements of growth on solid medium, cells grown in Middlebrook 7H9 liquid medium to an optical density at 600 nm (OD600 ) of 0.5 were washed in phosphate-buffered saline containing 0.05% tyloxapol (PBS-Tx), and 10-fold serial dilutions in PBS were spotted on agar plates containing MM as described above (without tyloxapol) plus a 0.01% concentration of the carbon source. .. For subcellular localization, CuvA was expressed as a carboxy-terminal GFP fusion. cuvA was PCR amplified from genomic DNA of M. tuberculosis H37RV by use of primers Rv1422AE and Rv1422rPac1. gfp was PCR amplified from pTracerCMV (Invitrogen) by use of primers GFP3PacI and GFP4XK.

Article Title: Viability, biofilm formation, and MazEF expression in drug-sensitive and drug-resistant Mycobacterium tuberculosis strains circulating in Xinjiang, China
Article Snippet: Construction and identification of mazEF 3,6,9-deletion mutants from H37Rv Construction and identification of deletion mutants of TAS mazEF 3,6,9 of MTB H37Rv was conducted by splicing-overlap extension polymerase chain reaction. .. H37Rv, H37RvΔmazEF 3, H37RvΔmazEF 6, and H37RvΔmazEF 9 were grown at 37°C in Middlebrook 7H9 liquid medium supplemented with 10% oleic albumin dextrose catalase or on Löwenstein–Jensen slants.

Article Title: The Twin-Arginine Translocation Pathway of Mycobacterium smegmatis Is Functional and Required for the Export of Mycobacterial ?-Lactamases
Article Snippet: These small colonies were subsequently proven by PCR (data not shown) and Southern blot analysis (Fig. ) to be tatA or tatC deletion mutants. .. We compared growth of the Δ tat mutants to that of wild-type M. smegmatis in Middlebrook 7H9 liquid medium.

SDS Page:

Article Title: Unexpected Link between Lipooligosaccharide Biosynthesis and Surface Protein Release in Mycobacterium marinum *
Article Snippet: Paragraph title: SDS-PAGE and Immunoblotting ... Subsequently, the bacteria were washed twice in Middlebrook 7H9 medium without supplement to remove all traces of bovine serum albumin (BSA) and grown overnight in Middlebrook 7H9 liquid medium supplemented with 0.2% dextrose and optionally 0.05% Tween 80.

Plasmid Preparation:

Article Title: Mycobacterial Gene cuvA Is Required for Optimal Nutrient Utilization and Virulence
Article Snippet: A complemented M. tuberculosis Δ cuvA strain was constructed by expressing cuvA under the control of the hsp70 promoter in the vector pJEB402 ( ) to allow stable expression during prolonged incubation in vitro and during infection experiments. .. When grown in Middlebrook 7H9 liquid medium containing 0.2% glucose, the M. tuberculosis Δ cuvA ( ) and M. smegmatis Δ cuvA (see Fig. S4A in the supplemental material) strains grew at similar rates and to the same final densities as the wild-type parental strains.

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    MiddleBrook Pharmaceuticals middlebrook 7h9
    (A) Growth curves of TB101 in <t>Middlebrook</t> <t>7H9</t> containing different concentrations of ATc. The optical density at 540 nm was recorded at different time points and used to compile the growth curves. (B) Killing curves of the pimA conditional mutant TB101 in the presence of ATc. The conditional mutant was grown in Middlebrook 7H9 containing 200 ng/ml ATc. Starting from day 1, samples were collected at different time points and plated on Middlebrook 7H10 to determine the viable counts. Each experiment was repeated at least twice, giving similar results. Filled squares, optical density; open squares, viable counts.
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    MiddleBrook Pharmaceuticals 7h9 liquid medium
    Growth of M. tuberculosis strains in liquid and solid media. (A) Wild-type M. tuberculosis H37Rv, Δ cuvA Mt , and complemented Δ cuvA (Δ cuvA Mt /C) strains were grown in <t>7H9-ADC-Tw</t> liquid medium with 0.2% glucose, and the OD 600 was measured daily. (B) Growth of the same strains on agar plates containing MM plus 0.2% glucose or MM plus 0.01% cholesterol. Serial 10-fold dilutions were spotted and incubated at 37°C, and photographs were taken after 1 month of incubation. In both panels, data shown are from one experiment that was repeated at least twice with similar results. Glc, 0.2% glucose; Chol., 0.01% cholesterol.
    7h9 Liquid Medium, supplied by MiddleBrook Pharmaceuticals, used in various techniques. Bioz Stars score: 99/100, based on 23 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    (A) Growth curves of TB101 in Middlebrook 7H9 containing different concentrations of ATc. The optical density at 540 nm was recorded at different time points and used to compile the growth curves. (B) Killing curves of the pimA conditional mutant TB101 in the presence of ATc. The conditional mutant was grown in Middlebrook 7H9 containing 200 ng/ml ATc. Starting from day 1, samples were collected at different time points and plated on Middlebrook 7H10 to determine the viable counts. Each experiment was repeated at least twice, giving similar results. Filled squares, optical density; open squares, viable counts.

    Journal: Journal of Bacteriology

    Article Title: The Phosphatidyl-myo-Inositol Mannosyltransferase PimA Is Essential for Mycobacterium tuberculosis Growth In Vitro and In Vivo

    doi: 10.1128/JB.01346-13

    Figure Lengend Snippet: (A) Growth curves of TB101 in Middlebrook 7H9 containing different concentrations of ATc. The optical density at 540 nm was recorded at different time points and used to compile the growth curves. (B) Killing curves of the pimA conditional mutant TB101 in the presence of ATc. The conditional mutant was grown in Middlebrook 7H9 containing 200 ng/ml ATc. Starting from day 1, samples were collected at different time points and plated on Middlebrook 7H10 to determine the viable counts. Each experiment was repeated at least twice, giving similar results. Filled squares, optical density; open squares, viable counts.

    Article Snippet: M. tuberculosis H37Rv and its derivative, TB38, were grown at 37°C in Middlebrook 7H9 (liquid medium) or 7H10 (solid medium) (Difco) supplemented with 0.05% (vol/vol) Tween 80 (Sigma-Aldrich), 0.2% (vol/vol) glycerol (Sigma-Aldrich), and 10% ADN (2% glucose, 5% bovine serum albumin, 0.85% NaCl).

    Techniques: Mutagenesis

    Growth of M. tuberculosis strains in liquid and solid media. (A) Wild-type M. tuberculosis H37Rv, Δ cuvA Mt , and complemented Δ cuvA (Δ cuvA Mt /C) strains were grown in 7H9-ADC-Tw liquid medium with 0.2% glucose, and the OD 600 was measured daily. (B) Growth of the same strains on agar plates containing MM plus 0.2% glucose or MM plus 0.01% cholesterol. Serial 10-fold dilutions were spotted and incubated at 37°C, and photographs were taken after 1 month of incubation. In both panels, data shown are from one experiment that was repeated at least twice with similar results. Glc, 0.2% glucose; Chol., 0.01% cholesterol.

    Journal: Infection and Immunity

    Article Title: Mycobacterial Gene cuvA Is Required for Optimal Nutrient Utilization and Virulence

    doi: 10.1128/IAI.02207-14

    Figure Lengend Snippet: Growth of M. tuberculosis strains in liquid and solid media. (A) Wild-type M. tuberculosis H37Rv, Δ cuvA Mt , and complemented Δ cuvA (Δ cuvA Mt /C) strains were grown in 7H9-ADC-Tw liquid medium with 0.2% glucose, and the OD 600 was measured daily. (B) Growth of the same strains on agar plates containing MM plus 0.2% glucose or MM plus 0.01% cholesterol. Serial 10-fold dilutions were spotted and incubated at 37°C, and photographs were taken after 1 month of incubation. In both panels, data shown are from one experiment that was repeated at least twice with similar results. Glc, 0.2% glucose; Chol., 0.01% cholesterol.

    Article Snippet: For measurements of growth on solid medium, cells grown in Middlebrook 7H9 liquid medium to an optical density at 600 nm (OD600 ) of 0.5 were washed in phosphate-buffered saline containing 0.05% tyloxapol (PBS-Tx), and 10-fold serial dilutions in PBS were spotted on agar plates containing MM as described above (without tyloxapol) plus a 0.01% concentration of the carbon source.

    Techniques: Incubation, Gas Chromatography

    Malachite green interferes with postantibiotic recovery of M. smegmatis on solid culture medium. (A and B) M. smegmatis was grown to mid-exponential phase in Middlebrook 7H9 broth and incubation was continued after the addition of 50 μg/ml isoniazid

    Journal: Antimicrobial Agents and Chemotherapy

    Article Title: Malachite Green Interferes with Postantibiotic Recovery of Mycobacteria

    doi: 10.1128/AAC.00406-12

    Figure Lengend Snippet: Malachite green interferes with postantibiotic recovery of M. smegmatis on solid culture medium. (A and B) M. smegmatis was grown to mid-exponential phase in Middlebrook 7H9 broth and incubation was continued after the addition of 50 μg/ml isoniazid

    Article Snippet: Cells were grown at 37°C to mid-exponential phase in Middlebrook 7H9 liquid culture medium ( ) and diluted 10-fold in fresh 7H9 broth.

    Techniques: Incubation

    Inhibition of postantibiotic recovery by malachite green is specific to antibiotics that target cell wall biogenesis. (A to F) M. smegmatis was grown to mid-exponential phase in Middlebrook 7H9 broth, and incubation was continued for another 24 h after

    Journal: Antimicrobial Agents and Chemotherapy

    Article Title: Malachite Green Interferes with Postantibiotic Recovery of Mycobacteria

    doi: 10.1128/AAC.00406-12

    Figure Lengend Snippet: Inhibition of postantibiotic recovery by malachite green is specific to antibiotics that target cell wall biogenesis. (A to F) M. smegmatis was grown to mid-exponential phase in Middlebrook 7H9 broth, and incubation was continued for another 24 h after

    Article Snippet: Cells were grown at 37°C to mid-exponential phase in Middlebrook 7H9 liquid culture medium ( ) and diluted 10-fold in fresh 7H9 broth.

    Techniques: Inhibition, Incubation

    Effect of albumin or ROS scavengers on inhibition of postantibiotic recovery by malachite green. (A to C) M. smegmatis was grown to mid-exponential phase in Middlebrook 7H9 broth (A and C) or in 7H9 broth containing 10% OADC (B) and incubation was continued

    Journal: Antimicrobial Agents and Chemotherapy

    Article Title: Malachite Green Interferes with Postantibiotic Recovery of Mycobacteria

    doi: 10.1128/AAC.00406-12

    Figure Lengend Snippet: Effect of albumin or ROS scavengers on inhibition of postantibiotic recovery by malachite green. (A to C) M. smegmatis was grown to mid-exponential phase in Middlebrook 7H9 broth (A and C) or in 7H9 broth containing 10% OADC (B) and incubation was continued

    Article Snippet: Cells were grown at 37°C to mid-exponential phase in Middlebrook 7H9 liquid culture medium ( ) and diluted 10-fold in fresh 7H9 broth.

    Techniques: Inhibition, Incubation

    Malachite green interferes with postantibiotic recovery of M. tuberculosis on solid culture medium. (A to F) M. tuberculosis was grown to mid-exponential phase in Middlebrook 7H9 broth, and incubation was continued for another 48 h after the addition

    Journal: Antimicrobial Agents and Chemotherapy

    Article Title: Malachite Green Interferes with Postantibiotic Recovery of Mycobacteria

    doi: 10.1128/AAC.00406-12

    Figure Lengend Snippet: Malachite green interferes with postantibiotic recovery of M. tuberculosis on solid culture medium. (A to F) M. tuberculosis was grown to mid-exponential phase in Middlebrook 7H9 broth, and incubation was continued for another 48 h after the addition

    Article Snippet: Cells were grown at 37°C to mid-exponential phase in Middlebrook 7H9 liquid culture medium ( ) and diluted 10-fold in fresh 7H9 broth.

    Techniques: Incubation