Structured Review

Difco 7h9 medium
Macroscopic morphology of M. smegmatis mc 2 155 strain spreading on the surface of a motility agar plate. mc 2 155 was grown in 7H10, and a single colony was transferred with a toothpick to the center of a 0.3% agar plate containing <t>7H9</t> basal medium without any added carbon source. The plate was sealed with parafilm and incubated at 37°C for 2 weeks.
7h9 Medium, supplied by Difco, used in various techniques. Bioz Stars score: 95/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Images

1) Product Images from "Sliding Motility in Mycobacteria"

Article Title: Sliding Motility in Mycobacteria

Journal: Journal of Bacteriology

doi:

Macroscopic morphology of M. smegmatis mc 2 155 strain spreading on the surface of a motility agar plate. mc 2 155 was grown in 7H10, and a single colony was transferred with a toothpick to the center of a 0.3% agar plate containing 7H9 basal medium without any added carbon source. The plate was sealed with parafilm and incubated at 37°C for 2 weeks.
Figure Legend Snippet: Macroscopic morphology of M. smegmatis mc 2 155 strain spreading on the surface of a motility agar plate. mc 2 155 was grown in 7H10, and a single colony was transferred with a toothpick to the center of a 0.3% agar plate containing 7H9 basal medium without any added carbon source. The plate was sealed with parafilm and incubated at 37°C for 2 weeks.

Techniques Used: Incubation

Growth accompanies mycobacterial spreading. A 1:100 mix of GFP-labeled (light) and unlabeled (dark) mc 2 155 cells grown as described in Materials and Methods were plated on the surface of 0.3% M63 salts– (A) and 7H9 (with no added carbon source)– (B) agarose plates. Photographs were taken after 2 days of incubation at 37°C. Phase-contrast images showing the continuous spreading halo are on the left, and fluorescent micrographs of the same fields showing the locations of GFP-labeled cells are on the right. Bars, 25 μm.
Figure Legend Snippet: Growth accompanies mycobacterial spreading. A 1:100 mix of GFP-labeled (light) and unlabeled (dark) mc 2 155 cells grown as described in Materials and Methods were plated on the surface of 0.3% M63 salts– (A) and 7H9 (with no added carbon source)– (B) agarose plates. Photographs were taken after 2 days of incubation at 37°C. Phase-contrast images showing the continuous spreading halo are on the left, and fluorescent micrographs of the same fields showing the locations of GFP-labeled cells are on the right. Bars, 25 μm.

Techniques Used: Labeling, Incubation

Spreading phenotype of M. avium colony morphology variants 2151-SmD, 2151-SmT, Rg-O, and Rg-4 on 7H9–ADC–0.3% agarose plates. Photographs were taken 3 weeks after inoculation.
Figure Legend Snippet: Spreading phenotype of M. avium colony morphology variants 2151-SmD, 2151-SmT, Rg-O, and Rg-4 on 7H9–ADC–0.3% agarose plates. Photographs were taken 3 weeks after inoculation.

Techniques Used:

Pattern formation in a spreading Sm-1 colony. A 25-μl aliquot of a saturated Sm-1 culture was plated onto a 7H9–ADC–0.3% agarose plate. (A) Pictures of the spreading colony taken 1, 2, and 3 days after inoculation. (B and C) Electron micrographs of cells taken at day 3 from the transparent periphery (B) and opaque interior (C) of a spreading colony. Cells were negatively stained with 2% phosphotungstic acid. Bar, 1 μm. Arrows mark the structures discussed in the text.
Figure Legend Snippet: Pattern formation in a spreading Sm-1 colony. A 25-μl aliquot of a saturated Sm-1 culture was plated onto a 7H9–ADC–0.3% agarose plate. (A) Pictures of the spreading colony taken 1, 2, and 3 days after inoculation. (B and C) Electron micrographs of cells taken at day 3 from the transparent periphery (B) and opaque interior (C) of a spreading colony. Cells were negatively stained with 2% phosphotungstic acid. Bar, 1 μm. Arrows mark the structures discussed in the text.

Techniques Used: Staining

2) Product Images from "Mycobacterium smegmatis PafBC is involved in regulation of DNA damage response"

Article Title: Mycobacterium smegmatis PafBC is involved in regulation of DNA damage response

Journal: Scientific Reports

doi: 10.1038/s41598-017-14410-z

PafBC affects cellular levels of RecA in Msm . Analysis of RecA levels in cell-free lysates of Msm wild type, Msm Δ pafBC and Msm Δ pafBC - pafBC . Cells were grown in 7H9 at 37 °C. Cell-free lysates were analysed by immunblot using an anti- E. coli RecA antibody. The full-length blot is shown in Supplementary Fig. S6 . Equal loading was controlled by an anti-RpoB immunoblot.
Figure Legend Snippet: PafBC affects cellular levels of RecA in Msm . Analysis of RecA levels in cell-free lysates of Msm wild type, Msm Δ pafBC and Msm Δ pafBC - pafBC . Cells were grown in 7H9 at 37 °C. Cell-free lysates were analysed by immunblot using an anti- E. coli RecA antibody. The full-length blot is shown in Supplementary Fig. S6 . Equal loading was controlled by an anti-RpoB immunoblot.

Techniques Used:

( A ) DNA-damage induced upregulation of RecA is impaired in Msm Δ pafBC . Msm wild type, the pafBC deletion mutant and a complemented strain were grown in 7H9 to an OD 600 between 0.8 to 1.0 at 37 °C. Cultures were split, supplemented with mitomycin C (80 ng/ml) as indicated and were incubated for another 4 h. RecA levels of cell-free lysates were analysed by an anti-RecA immunoblot. Full-length blots are shown in Supplementary Fig. S8 . To control for equal loading, an anti-RpoB immunoblot was performed. ( B ) Levels of PafBC do not alter under DNA damaging growth conditions. Samples of Msm wild type cells were prepared as described in ( A ) and were analysed by an anti-PafBC immunoblot.
Figure Legend Snippet: ( A ) DNA-damage induced upregulation of RecA is impaired in Msm Δ pafBC . Msm wild type, the pafBC deletion mutant and a complemented strain were grown in 7H9 to an OD 600 between 0.8 to 1.0 at 37 °C. Cultures were split, supplemented with mitomycin C (80 ng/ml) as indicated and were incubated for another 4 h. RecA levels of cell-free lysates were analysed by an anti-RecA immunoblot. Full-length blots are shown in Supplementary Fig. S8 . To control for equal loading, an anti-RpoB immunoblot was performed. ( B ) Levels of PafBC do not alter under DNA damaging growth conditions. Samples of Msm wild type cells were prepared as described in ( A ) and were analysed by an anti-PafBC immunoblot.

Techniques Used: Mutagenesis, Incubation

Transcriptional regulation of recA in Msm Δ pafBC is affected at the level of the P1 LexA/RecA-independent promoter. ( A ) Schematic representation of the Msm recA promoter region and the GFP reporter constructs used to measure recA P1, P2, and P1-P2 promoter activity, respectively. Hatched areas indicate mutated promoter regions. ( B ) Msm wild type and Msm Δ pafBC carrying the reporter plasmids were grown in 7H9 to an OD 600 of 0.7 at 37 °C. Cultures were then supplemented with mitomycin C (MMC; 80 ng/ml) and were grown for an additional 4 h. Controls (STD) received no mitomycin ( C ) GFP fluorescence in lysates was measured and normalized to protein content. RFU, relative fluorescence units.
Figure Legend Snippet: Transcriptional regulation of recA in Msm Δ pafBC is affected at the level of the P1 LexA/RecA-independent promoter. ( A ) Schematic representation of the Msm recA promoter region and the GFP reporter constructs used to measure recA P1, P2, and P1-P2 promoter activity, respectively. Hatched areas indicate mutated promoter regions. ( B ) Msm wild type and Msm Δ pafBC carrying the reporter plasmids were grown in 7H9 to an OD 600 of 0.7 at 37 °C. Cultures were then supplemented with mitomycin C (MMC; 80 ng/ml) and were grown for an additional 4 h. Controls (STD) received no mitomycin ( C ) GFP fluorescence in lysates was measured and normalized to protein content. RFU, relative fluorescence units.

Techniques Used: Construct, Activity Assay, Fluorescence

3) Product Images from "The PhoP-Dependent ncRNA Mcr7 Modulates the TAT Secretion System in Mycobacterium tuberculosis"

Article Title: The PhoP-Dependent ncRNA Mcr7 Modulates the TAT Secretion System in Mycobacterium tuberculosis

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1004183

Reintroduction of mcr7 in an M. tuberculosis phoP mutant restores secretion of Tat-dependent substrates to wild type levels. A. qRT-PCR analysis of tatC , mcr7 and other genes from the PhoP regulon ( lipF , pks2 , pks3 , narK1 and fadD21 ). Bars represent fold changes in the expression levels in the phoP mutant and mcr7 -complemented strains relative to M. tuberculosis wild type grown exponentially in 7H9 medium. Expression of the tatC mRNA is independent of phoP mutation and mcr7 expression. Note that complementation with mcr7 does not affect expression of the PhoP regulon. Results are representative of three independent experiments and error bars are the standard deviation of the mean. B. Western blot experiments of GroEL2, Ag85C, EspD and EsxA in the whole cell and secreted fractions of M. tuberculosis wild type, its phoP mutant and the mcr7 -complemented strains. Equal protein amounts were loaded per well. GroEL2 is used as a control of bacterial integrity in each sample. Signal intensity was quantified and plotted in the graphs below the Western blot images. Results are representative of three independent experiments. C. Measure of β-lactamase activity of the BlaC protein by nitrocefin chromogenic assay. Activity is calculated relative to the CFU/ml in each strain.
Figure Legend Snippet: Reintroduction of mcr7 in an M. tuberculosis phoP mutant restores secretion of Tat-dependent substrates to wild type levels. A. qRT-PCR analysis of tatC , mcr7 and other genes from the PhoP regulon ( lipF , pks2 , pks3 , narK1 and fadD21 ). Bars represent fold changes in the expression levels in the phoP mutant and mcr7 -complemented strains relative to M. tuberculosis wild type grown exponentially in 7H9 medium. Expression of the tatC mRNA is independent of phoP mutation and mcr7 expression. Note that complementation with mcr7 does not affect expression of the PhoP regulon. Results are representative of three independent experiments and error bars are the standard deviation of the mean. B. Western blot experiments of GroEL2, Ag85C, EspD and EsxA in the whole cell and secreted fractions of M. tuberculosis wild type, its phoP mutant and the mcr7 -complemented strains. Equal protein amounts were loaded per well. GroEL2 is used as a control of bacterial integrity in each sample. Signal intensity was quantified and plotted in the graphs below the Western blot images. Results are representative of three independent experiments. C. Measure of β-lactamase activity of the BlaC protein by nitrocefin chromogenic assay. Activity is calculated relative to the CFU/ml in each strain.

Techniques Used: Mutagenesis, Quantitative RT-PCR, Expressing, Standard Deviation, Western Blot, Activity Assay, Chromogenic Assay

4) Product Images from "Mycobacterium smegmatis PafBC is involved in regulation of DNA damage response"

Article Title: Mycobacterium smegmatis PafBC is involved in regulation of DNA damage response

Journal: Scientific Reports

doi: 10.1038/s41598-017-14410-z

PafBC affects cellular levels of RecA in Msm . Analysis of RecA levels in cell-free lysates of Msm wild type, Msm Δ pafBC and Msm Δ pafBC - pafBC . Cells were grown in 7H9 at 37 °C. Cell-free lysates were analysed by immunblot using an anti- E. coli RecA antibody. The full-length blot is shown in Supplementary Fig. S6 . Equal loading was controlled by an anti-RpoB immunoblot.
Figure Legend Snippet: PafBC affects cellular levels of RecA in Msm . Analysis of RecA levels in cell-free lysates of Msm wild type, Msm Δ pafBC and Msm Δ pafBC - pafBC . Cells were grown in 7H9 at 37 °C. Cell-free lysates were analysed by immunblot using an anti- E. coli RecA antibody. The full-length blot is shown in Supplementary Fig. S6 . Equal loading was controlled by an anti-RpoB immunoblot.

Techniques Used:

( A ) DNA-damage induced upregulation of RecA is impaired in Msm Δ pafBC . Msm wild type, the pafBC deletion mutant and a complemented strain were grown in 7H9 to an OD 600 between 0.8 to 1.0 at 37 °C. Cultures were split, supplemented with mitomycin C (80 ng/ml) as indicated and were incubated for another 4 h. RecA levels of cell-free lysates were analysed by an anti-RecA immunoblot. Full-length blots are shown in Supplementary Fig. S8 . To control for equal loading, an anti-RpoB immunoblot was performed. ( B ) Levels of PafBC do not alter under DNA damaging growth conditions. Samples of Msm wild type cells were prepared as described in ( A ) and were analysed by an anti-PafBC immunoblot.
Figure Legend Snippet: ( A ) DNA-damage induced upregulation of RecA is impaired in Msm Δ pafBC . Msm wild type, the pafBC deletion mutant and a complemented strain were grown in 7H9 to an OD 600 between 0.8 to 1.0 at 37 °C. Cultures were split, supplemented with mitomycin C (80 ng/ml) as indicated and were incubated for another 4 h. RecA levels of cell-free lysates were analysed by an anti-RecA immunoblot. Full-length blots are shown in Supplementary Fig. S8 . To control for equal loading, an anti-RpoB immunoblot was performed. ( B ) Levels of PafBC do not alter under DNA damaging growth conditions. Samples of Msm wild type cells were prepared as described in ( A ) and were analysed by an anti-PafBC immunoblot.

Techniques Used: Mutagenesis, Incubation

Transcriptional regulation of recA in Msm Δ pafBC is affected at the level of the P1 LexA/RecA-independent promoter. ( A ) Schematic representation of the Msm recA promoter region and the GFP reporter constructs used to measure recA P1, P2, and P1-P2 promoter activity, respectively. Hatched areas indicate mutated promoter regions. ( B ) Msm wild type and Msm Δ pafBC carrying the reporter plasmids were grown in 7H9 to an OD 600 of 0.7 at 37 °C. Cultures were then supplemented with mitomycin C (MMC; 80 ng/ml) and were grown for an additional 4 h. Controls (STD) received no mitomycin ( C ) GFP fluorescence in lysates was measured and normalized to protein content. RFU, relative fluorescence units.
Figure Legend Snippet: Transcriptional regulation of recA in Msm Δ pafBC is affected at the level of the P1 LexA/RecA-independent promoter. ( A ) Schematic representation of the Msm recA promoter region and the GFP reporter constructs used to measure recA P1, P2, and P1-P2 promoter activity, respectively. Hatched areas indicate mutated promoter regions. ( B ) Msm wild type and Msm Δ pafBC carrying the reporter plasmids were grown in 7H9 to an OD 600 of 0.7 at 37 °C. Cultures were then supplemented with mitomycin C (MMC; 80 ng/ml) and were grown for an additional 4 h. Controls (STD) received no mitomycin ( C ) GFP fluorescence in lysates was measured and normalized to protein content. RFU, relative fluorescence units.

Techniques Used: Construct, Activity Assay, Fluorescence

5) Product Images from "The PhoP-Dependent ncRNA Mcr7 Modulates the TAT Secretion System in Mycobacterium tuberculosis"

Article Title: The PhoP-Dependent ncRNA Mcr7 Modulates the TAT Secretion System in Mycobacterium tuberculosis

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1004183

Reintroduction of mcr7 in an M. tuberculosis phoP mutant restores secretion of Tat-dependent substrates to wild type levels. A. qRT-PCR analysis of tatC , mcr7 and other genes from the PhoP regulon ( lipF , pks2 , pks3 , narK1 and fadD21 ). Bars represent fold changes in the expression levels in the phoP mutant and mcr7 -complemented strains relative to M. tuberculosis wild type grown exponentially in 7H9 medium. Expression of the tatC mRNA is independent of phoP mutation and mcr7 expression. Note that complementation with mcr7 does not affect expression of the PhoP regulon. Results are representative of three independent experiments and error bars are the standard deviation of the mean. B. Western blot experiments of GroEL2, Ag85C, EspD and EsxA in the whole cell and secreted fractions of M. tuberculosis wild type, its phoP mutant and the mcr7 -complemented strains. Equal protein amounts were loaded per well. GroEL2 is used as a control of bacterial integrity in each sample. Signal intensity was quantified and plotted in the graphs below the Western blot images. Results are representative of three independent experiments. C. Measure of β-lactamase activity of the BlaC protein by nitrocefin chromogenic assay. Activity is calculated relative to the CFU/ml in each strain.
Figure Legend Snippet: Reintroduction of mcr7 in an M. tuberculosis phoP mutant restores secretion of Tat-dependent substrates to wild type levels. A. qRT-PCR analysis of tatC , mcr7 and other genes from the PhoP regulon ( lipF , pks2 , pks3 , narK1 and fadD21 ). Bars represent fold changes in the expression levels in the phoP mutant and mcr7 -complemented strains relative to M. tuberculosis wild type grown exponentially in 7H9 medium. Expression of the tatC mRNA is independent of phoP mutation and mcr7 expression. Note that complementation with mcr7 does not affect expression of the PhoP regulon. Results are representative of three independent experiments and error bars are the standard deviation of the mean. B. Western blot experiments of GroEL2, Ag85C, EspD and EsxA in the whole cell and secreted fractions of M. tuberculosis wild type, its phoP mutant and the mcr7 -complemented strains. Equal protein amounts were loaded per well. GroEL2 is used as a control of bacterial integrity in each sample. Signal intensity was quantified and plotted in the graphs below the Western blot images. Results are representative of three independent experiments. C. Measure of β-lactamase activity of the BlaC protein by nitrocefin chromogenic assay. Activity is calculated relative to the CFU/ml in each strain.

Techniques Used: Mutagenesis, Quantitative RT-PCR, Expressing, Standard Deviation, Western Blot, Activity Assay, Chromogenic Assay

6) Product Images from "Unexpected Genomic and Phenotypic Diversity of Mycobacterium africanum Lineage 5 Affects Drug Resistance, Protein Secretion, and Immunogenicity"

Article Title: Unexpected Genomic and Phenotypic Diversity of Mycobacterium africanum Lineage 5 Affects Drug Resistance, Protein Secretion, and Immunogenicity

Journal: Genome Biology and Evolution

doi: 10.1093/gbe/evy145

—Sublineage-specific growth characteristics of M. africanum sublineages. Precultures of the indicated strains were grown until midlogarithmic phase and were diluted to 0.05 OD 600 /ml in glass tubes containing 5 ml of liquid 7H9 medium supplemented with 0.05% Tween 80, ADC supplement and 0.2% pyruvate. Optical density was measured at the indicated time points (symbols). Data points are averages of three individual experiments performed in technical duplicates. Colors of line indicate the specific (sub)lineage of M. tuberculosis ( Mtb ) or M. africanum ( Maf ) on the legend (top left). Individual isolates can be identified by combination of colors and symbols used (bottom right).
Figure Legend Snippet: —Sublineage-specific growth characteristics of M. africanum sublineages. Precultures of the indicated strains were grown until midlogarithmic phase and were diluted to 0.05 OD 600 /ml in glass tubes containing 5 ml of liquid 7H9 medium supplemented with 0.05% Tween 80, ADC supplement and 0.2% pyruvate. Optical density was measured at the indicated time points (symbols). Data points are averages of three individual experiments performed in technical duplicates. Colors of line indicate the specific (sub)lineage of M. tuberculosis ( Mtb ) or M. africanum ( Maf ) on the legend (top left). Individual isolates can be identified by combination of colors and symbols used (bottom right).

Techniques Used:

7) Product Images from "Inactivation of lsr2 Results in a Hypermotile Phenotype in Mycobacterium smegmatis "

Article Title: Inactivation of lsr2 Results in a Hypermotile Phenotype in Mycobacterium smegmatis

Journal: Journal of Bacteriology

doi: 10.1128/JB.00023-08

M. smegmatis strains grown on low-agarose plates. (A) The effect of the agarose concentration on colony morphology is shown. 7H9 plates with decreased concentrations of agarose (0.8% and 0.3% [wt/vol]) were inoculated with 2-μl aliquots of M. smegmatis cultures grown to an OD 600 of 1.0. After 7 days of growth on plates, the edges of colonies were compared. On 0.3% agarose plates, the DL2008 colony showed fragmentation with separate large patches of cells (A4). (B) DL2008 forms a layer of “slime” on M63 plus 0.3% agarose, as visualized with a dissection microscope. The light refractive edge of the slime is shown (arrowheads), with patches of cells floating in it (arrows). Phase-contrast light microscope images of mc 2 155 cells (C) and DL2008 cells (D) at the perimeter of colonies. Magnification, ×200.
Figure Legend Snippet: M. smegmatis strains grown on low-agarose plates. (A) The effect of the agarose concentration on colony morphology is shown. 7H9 plates with decreased concentrations of agarose (0.8% and 0.3% [wt/vol]) were inoculated with 2-μl aliquots of M. smegmatis cultures grown to an OD 600 of 1.0. After 7 days of growth on plates, the edges of colonies were compared. On 0.3% agarose plates, the DL2008 colony showed fragmentation with separate large patches of cells (A4). (B) DL2008 forms a layer of “slime” on M63 plus 0.3% agarose, as visualized with a dissection microscope. The light refractive edge of the slime is shown (arrowheads), with patches of cells floating in it (arrows). Phase-contrast light microscope images of mc 2 155 cells (C) and DL2008 cells (D) at the perimeter of colonies. Magnification, ×200.

Techniques Used: Concentration Assay, Dissection, Microscopy, Light Microscopy

8) Product Images from "Selective small molecule inhibitor of the Mycobacterium tuberculosis fumarate hydratase reveals an allosteric regulatory site"

Article Title: Selective small molecule inhibitor of the Mycobacterium tuberculosis fumarate hydratase reveals an allosteric regulatory site

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi: 10.1073/pnas.1600630113

Compound 7 shows inhibition of bacterial growth rate under aerobic conditions. Treatment of M. tuberculosis H37Rv with the fumarate hydratase inhibitor 7 under aerobic conditions results in a decrease in bacterial growth. The fluorescence of cells constitutively expressing GFP was measured 12 d after dosing. Cultures were grown in 7H9 medium supplemented with glucose and casitone. The control cultures were treated with DMSO (black column), and 7 was dosed at concentrations up to 250 μM (blue columns). The relative fluorescence units (RFU) are represented as an average of replicates ( n = 4), and error bars indicate SEMs.
Figure Legend Snippet: Compound 7 shows inhibition of bacterial growth rate under aerobic conditions. Treatment of M. tuberculosis H37Rv with the fumarate hydratase inhibitor 7 under aerobic conditions results in a decrease in bacterial growth. The fluorescence of cells constitutively expressing GFP was measured 12 d after dosing. Cultures were grown in 7H9 medium supplemented with glucose and casitone. The control cultures were treated with DMSO (black column), and 7 was dosed at concentrations up to 250 μM (blue columns). The relative fluorescence units (RFU) are represented as an average of replicates ( n = 4), and error bars indicate SEMs.

Techniques Used: Inhibition, Fluorescence, Expressing

9) Product Images from "A Ferritin Mutant of Mycobacterium tuberculosis Is Highly Susceptible to Killing by Antibiotics and Is Unable To Establish a Chronic Infection in Mice"

Article Title: A Ferritin Mutant of Mycobacterium tuberculosis Is Highly Susceptible to Killing by Antibiotics and Is Unable To Establish a Chronic Infection in Mice

Journal: Infection and Immunity

doi: 10.1128/IAI.00229-12

Sensitivity of the wild type and the Δ bfrB mutant to antibiotics. The wild-type (▲) and Δ bfrB (●) strains were cultured for 2 days in 7H9 medium with no drug added or with increasing concentrations of antibiotics. Dilutions
Figure Legend Snippet: Sensitivity of the wild type and the Δ bfrB mutant to antibiotics. The wild-type (▲) and Δ bfrB (●) strains were cultured for 2 days in 7H9 medium with no drug added or with increasing concentrations of antibiotics. Dilutions

Techniques Used: Mutagenesis, Cell Culture

Growth of M. tuberculosis strains in 7H9 medium. Wild-type strain H37Rv (◆), the Δ bfrA mutant (▲), the Δ bfrB mutant (●), and the complemented Δ bfrB mutant (■) were grown in 7H9 medium with agitation
Figure Legend Snippet: Growth of M. tuberculosis strains in 7H9 medium. Wild-type strain H37Rv (◆), the Δ bfrA mutant (▲), the Δ bfrB mutant (●), and the complemented Δ bfrB mutant (■) were grown in 7H9 medium with agitation

Techniques Used: Mutagenesis

10) Product Images from "A Ferritin Mutant of Mycobacterium tuberculosis Is Highly Susceptible to Killing by Antibiotics and Is Unable To Establish a Chronic Infection in Mice"

Article Title: A Ferritin Mutant of Mycobacterium tuberculosis Is Highly Susceptible to Killing by Antibiotics and Is Unable To Establish a Chronic Infection in Mice

Journal: Infection and Immunity

doi: 10.1128/IAI.00229-12

Sensitivity of the wild type and the Δ bfrB mutant to antibiotics. The wild-type (▲) and Δ bfrB (●) strains were cultured for 2 days in 7H9 medium with no drug added or with increasing concentrations of antibiotics. Dilutions
Figure Legend Snippet: Sensitivity of the wild type and the Δ bfrB mutant to antibiotics. The wild-type (▲) and Δ bfrB (●) strains were cultured for 2 days in 7H9 medium with no drug added or with increasing concentrations of antibiotics. Dilutions

Techniques Used: Mutagenesis, Cell Culture

Growth of M. tuberculosis strains in 7H9 medium. Wild-type strain H37Rv (◆), the Δ bfrA mutant (▲), the Δ bfrB mutant (●), and the complemented Δ bfrB mutant (■) were grown in 7H9 medium with agitation
Figure Legend Snippet: Growth of M. tuberculosis strains in 7H9 medium. Wild-type strain H37Rv (◆), the Δ bfrA mutant (▲), the Δ bfrB mutant (●), and the complemented Δ bfrB mutant (■) were grown in 7H9 medium with agitation

Techniques Used: Mutagenesis

11) Product Images from "Distinct Properties of Hexameric but Functionally Conserved Mycobacterium tuberculosis Transcription-Repair Coupling Factor"

Article Title: Distinct Properties of Hexameric but Functionally Conserved Mycobacterium tuberculosis Transcription-Repair Coupling Factor

Journal: PLoS ONE

doi: 10.1371/journal.pone.0019131

Determination of oligomeric status of MtbMfd in vivo . M. tuberculosis Ra strain was grown in 7H9 medium containing 10% ADC supplements and 0.05% tween 80 up to 0.7 OD. Crude cell lysate was prepared in Tris buffer pH 8.0 by sonication followed by ultracentrifugation at 100,000 g (S100). The supernatant was subjected to further analysis. A . Native-PAGE (6%) Western profile of cell free lysate of M. tuberculosis H37Ra in Tris-Glycine buffer pH 8.3 at room temperature, the blot was probed with anti-MtbMfd antibodies. Lanes 1 2, 25 and 50 µg of crude cell lysate of M. tuberculosis H37Ra and lane 3, purified MtbMfd protein. B . Crude cell lysate of M. tuberculosis Ra was subjected to gel filtration chromatography on superose 6 column and fractions were collected and analyzed on SDS-PAGE followed by Western blot using anti MtbMfd antibody. Lane 1, V corresponds to void volume fraction (protein complex or aggregates); lanes 2–4, 10.5, 11 and 11.5 ml fractions (hexamer of MtbMfd); lanes 5–8, 13.5,14,14.5 and 15 ml fractions (monomer of MtbMfd) and lane 9, purified MtbMfd used as a marker. Fractions 10.5–11.5 ml and 13.5–15 ml represents the hexamer and monomer species of MtbMfd present in the crude cell lysate.
Figure Legend Snippet: Determination of oligomeric status of MtbMfd in vivo . M. tuberculosis Ra strain was grown in 7H9 medium containing 10% ADC supplements and 0.05% tween 80 up to 0.7 OD. Crude cell lysate was prepared in Tris buffer pH 8.0 by sonication followed by ultracentrifugation at 100,000 g (S100). The supernatant was subjected to further analysis. A . Native-PAGE (6%) Western profile of cell free lysate of M. tuberculosis H37Ra in Tris-Glycine buffer pH 8.3 at room temperature, the blot was probed with anti-MtbMfd antibodies. Lanes 1 2, 25 and 50 µg of crude cell lysate of M. tuberculosis H37Ra and lane 3, purified MtbMfd protein. B . Crude cell lysate of M. tuberculosis Ra was subjected to gel filtration chromatography on superose 6 column and fractions were collected and analyzed on SDS-PAGE followed by Western blot using anti MtbMfd antibody. Lane 1, V corresponds to void volume fraction (protein complex or aggregates); lanes 2–4, 10.5, 11 and 11.5 ml fractions (hexamer of MtbMfd); lanes 5–8, 13.5,14,14.5 and 15 ml fractions (monomer of MtbMfd) and lane 9, purified MtbMfd used as a marker. Fractions 10.5–11.5 ml and 13.5–15 ml represents the hexamer and monomer species of MtbMfd present in the crude cell lysate.

Techniques Used: In Vivo, Sonication, Clear Native PAGE, Western Blot, Purification, Filtration, Chromatography, SDS Page, Marker

12) Product Images from "Mycobacterium smegmatis PafBC is involved in regulation of DNA damage response"

Article Title: Mycobacterium smegmatis PafBC is involved in regulation of DNA damage response

Journal: Scientific Reports

doi: 10.1038/s41598-017-14410-z

PafBC affects cellular levels of RecA in Msm . Analysis of RecA levels in cell-free lysates of Msm wild type, Msm Δ pafBC and Msm Δ pafBC - pafBC . Cells were grown in 7H9 at 37 °C. Cell-free lysates were analysed by immunblot using an anti- E. coli RecA antibody. The full-length blot is shown in Supplementary Fig. S6 . Equal loading was controlled by an anti-RpoB immunoblot.
Figure Legend Snippet: PafBC affects cellular levels of RecA in Msm . Analysis of RecA levels in cell-free lysates of Msm wild type, Msm Δ pafBC and Msm Δ pafBC - pafBC . Cells were grown in 7H9 at 37 °C. Cell-free lysates were analysed by immunblot using an anti- E. coli RecA antibody. The full-length blot is shown in Supplementary Fig. S6 . Equal loading was controlled by an anti-RpoB immunoblot.

Techniques Used:

( A ) DNA-damage induced upregulation of RecA is impaired in Msm Δ pafBC . Msm wild type, the pafBC deletion mutant and a complemented strain were grown in 7H9 to an OD 600 between 0.8 to 1.0 at 37 °C. Cultures were split, supplemented with mitomycin C (80 ng/ml) as indicated and were incubated for another 4 h. RecA levels of cell-free lysates were analysed by an anti-RecA immunoblot. Full-length blots are shown in Supplementary Fig. S8 . To control for equal loading, an anti-RpoB immunoblot was performed. ( B ) Levels of PafBC do not alter under DNA damaging growth conditions. Samples of Msm wild type cells were prepared as described in ( A ) and were analysed by an anti-PafBC immunoblot.
Figure Legend Snippet: ( A ) DNA-damage induced upregulation of RecA is impaired in Msm Δ pafBC . Msm wild type, the pafBC deletion mutant and a complemented strain were grown in 7H9 to an OD 600 between 0.8 to 1.0 at 37 °C. Cultures were split, supplemented with mitomycin C (80 ng/ml) as indicated and were incubated for another 4 h. RecA levels of cell-free lysates were analysed by an anti-RecA immunoblot. Full-length blots are shown in Supplementary Fig. S8 . To control for equal loading, an anti-RpoB immunoblot was performed. ( B ) Levels of PafBC do not alter under DNA damaging growth conditions. Samples of Msm wild type cells were prepared as described in ( A ) and were analysed by an anti-PafBC immunoblot.

Techniques Used: Mutagenesis, Incubation

Transcriptional regulation of recA in Msm Δ pafBC is affected at the level of the P1 LexA/RecA-independent promoter. ( A ) Schematic representation of the Msm recA promoter region and the GFP reporter constructs used to measure recA P1, P2, and P1-P2 promoter activity, respectively. Hatched areas indicate mutated promoter regions. ( B ) Msm wild type and Msm Δ pafBC carrying the reporter plasmids were grown in 7H9 to an OD 600 of 0.7 at 37 °C. Cultures were then supplemented with mitomycin C (MMC; 80 ng/ml) and were grown for an additional 4 h. Controls (STD) received no mitomycin ( C ) GFP fluorescence in lysates was measured and normalized to protein content. RFU, relative fluorescence units.
Figure Legend Snippet: Transcriptional regulation of recA in Msm Δ pafBC is affected at the level of the P1 LexA/RecA-independent promoter. ( A ) Schematic representation of the Msm recA promoter region and the GFP reporter constructs used to measure recA P1, P2, and P1-P2 promoter activity, respectively. Hatched areas indicate mutated promoter regions. ( B ) Msm wild type and Msm Δ pafBC carrying the reporter plasmids were grown in 7H9 to an OD 600 of 0.7 at 37 °C. Cultures were then supplemented with mitomycin C (MMC; 80 ng/ml) and were grown for an additional 4 h. Controls (STD) received no mitomycin ( C ) GFP fluorescence in lysates was measured and normalized to protein content. RFU, relative fluorescence units.

Techniques Used: Construct, Activity Assay, Fluorescence

13) Product Images from "A Ferritin Mutant of Mycobacterium tuberculosis Is Highly Susceptible to Killing by Antibiotics and Is Unable To Establish a Chronic Infection in Mice"

Article Title: A Ferritin Mutant of Mycobacterium tuberculosis Is Highly Susceptible to Killing by Antibiotics and Is Unable To Establish a Chronic Infection in Mice

Journal: Infection and Immunity

doi: 10.1128/IAI.00229-12

Sensitivity of the wild type and the Δ bfrB mutant to antibiotics. The wild-type (▲) and Δ bfrB (●) strains were cultured for 2 days in 7H9 medium with no drug added or with increasing concentrations of antibiotics. Dilutions
Figure Legend Snippet: Sensitivity of the wild type and the Δ bfrB mutant to antibiotics. The wild-type (▲) and Δ bfrB (●) strains were cultured for 2 days in 7H9 medium with no drug added or with increasing concentrations of antibiotics. Dilutions

Techniques Used: Mutagenesis, Cell Culture

Growth of M. tuberculosis strains in 7H9 medium. Wild-type strain H37Rv (◆), the Δ bfrA mutant (▲), the Δ bfrB mutant (●), and the complemented Δ bfrB mutant (■) were grown in 7H9 medium with agitation
Figure Legend Snippet: Growth of M. tuberculosis strains in 7H9 medium. Wild-type strain H37Rv (◆), the Δ bfrA mutant (▲), the Δ bfrB mutant (●), and the complemented Δ bfrB mutant (■) were grown in 7H9 medium with agitation

Techniques Used: Mutagenesis

14) Product Images from "Detection of mRNA Transcripts and Active Transcription in Persistent Mycobacterium tuberculosis Induced by Exposure to Rifampin or Pyrazinamide"

Article Title: Detection of mRNA Transcripts and Active Transcription in Persistent Mycobacterium tuberculosis Induced by Exposure to Rifampin or Pyrazinamide

Journal: Journal of Bacteriology

doi:

Detection of mRNA in M. tuberculosis H37Rv by RT-PCR before and after treatment with antituberculosis agents in vitro and in vivo. (a) RNA was extracted from 100-day cultures before rifampin treatment (10 6 CFU per RT reaction). (b) After rifampin (100 μg/ml) treatment for 5 days (0 CFU; same volume of culture used for RT as in panel a). (c) RNA was extracted from infected mice before antituberculosis drug treatment; week 0 (10 6 CFU per RT). (d) After antituberculosis drug treatment in mice; week 14 (0 CFU; same volume of tissue used for RT as in panel c). (e) The 100-day cultures were treated with rifampin (100 μg/ml) for 5 days. The cells were washed three times to remove rifampin and then incubated with 7H9 broth for 12 h. RT-PCR was performed with sigA , sigB , rpoB , and 16K primers before (R) and after (7H9) resuscitation with 7H9 medium. (f) The 100-day cultures were treated with rifampin for 5 days, washed free of rifampin, and immediately heat shocked at 45°C for 30 min compared to incubation at 37°C for 30 min. Detected genes are shown above lanes; the PCR product sizes are as follows: sigA , 306 bp; sigB , 273 bp; rpoB , 307 bp; dnaK , 275 bp; 16K ( hspX ), 242 bp; 16S , 336 bp. 10 −1 , 5 × 10 −1 , 10 −2 , and 10 −3 indicate that 10-, 50-, 100-, and 1,000-fold-diluted cDNAs were used for PCR. N, DNase I-treated RNA but no RT enzyme. −, PCR control with each primer and 10 μl of water instead of template. The following limits of detection (gene copies) were estimated by DNA PCR: 16S , 10 5 (equivalent to 100 CFU of a log-phase growth culture); sigA , 10 7 ; sigB , 10 6 ; 16K , 10 6 ; rpoB , 10 6 ; dnaK , 10 6 (equivalent to ∼10 4 CFU).
Figure Legend Snippet: Detection of mRNA in M. tuberculosis H37Rv by RT-PCR before and after treatment with antituberculosis agents in vitro and in vivo. (a) RNA was extracted from 100-day cultures before rifampin treatment (10 6 CFU per RT reaction). (b) After rifampin (100 μg/ml) treatment for 5 days (0 CFU; same volume of culture used for RT as in panel a). (c) RNA was extracted from infected mice before antituberculosis drug treatment; week 0 (10 6 CFU per RT). (d) After antituberculosis drug treatment in mice; week 14 (0 CFU; same volume of tissue used for RT as in panel c). (e) The 100-day cultures were treated with rifampin (100 μg/ml) for 5 days. The cells were washed three times to remove rifampin and then incubated with 7H9 broth for 12 h. RT-PCR was performed with sigA , sigB , rpoB , and 16K primers before (R) and after (7H9) resuscitation with 7H9 medium. (f) The 100-day cultures were treated with rifampin for 5 days, washed free of rifampin, and immediately heat shocked at 45°C for 30 min compared to incubation at 37°C for 30 min. Detected genes are shown above lanes; the PCR product sizes are as follows: sigA , 306 bp; sigB , 273 bp; rpoB , 307 bp; dnaK , 275 bp; 16K ( hspX ), 242 bp; 16S , 336 bp. 10 −1 , 5 × 10 −1 , 10 −2 , and 10 −3 indicate that 10-, 50-, 100-, and 1,000-fold-diluted cDNAs were used for PCR. N, DNase I-treated RNA but no RT enzyme. −, PCR control with each primer and 10 μl of water instead of template. The following limits of detection (gene copies) were estimated by DNA PCR: 16S , 10 5 (equivalent to 100 CFU of a log-phase growth culture); sigA , 10 7 ; sigB , 10 6 ; 16K , 10 6 ; rpoB , 10 6 ; dnaK , 10 6 (equivalent to ∼10 4 CFU).

Techniques Used: Reverse Transcription Polymerase Chain Reaction, In Vitro, In Vivo, Infection, Mouse Assay, Incubation, Polymerase Chain Reaction

15) Product Images from "An NAD+ Phosphorylase Toxin Triggers Mycobacterium tuberculosis Cell Death"

Article Title: An NAD+ Phosphorylase Toxin Triggers Mycobacterium tuberculosis Cell Death

Journal: Molecular Cell

doi: 10.1016/j.molcel.2019.01.028

Rv1989c-Rv1990c Is a Bactericidal TA System in Mtb (A) WT Mtb or Mtb ΔTA transformed with pGMC derivatives carrying Rv1990c, Rv1989c, or Rv1989c-Rv1990c as indicated were grown in 7H9 ADC Tween medium. Growth on solid medium was tested by spotting serial 10-fold dilutions on 7H11 OADC agar (−ATc) or the same medium supplemented with anhydrotetracycline (+ATc) to induce transcription from the P1 promoter. Plates were observed after 20 days at 37°C. Shown data are representative of three independent experiments. (B) Growth of WT Mtb or Mtb ΔTA transformed with pGMC derivatives carrying Rv1990c, Rv1989c, or Rv1989c-Rv1990c in liquid medium as monitored by turbidity measurements (McFarland units). ATc was added at day 0 to induce ectopic gene expression when indicated. Data are represented as mean of three technical replicates ± SD. Shown data are representative of three independent experiments. (C) Survival of Mtb ΔTA strains carrying pGMC-TetR-P1-Rv1989c as measured by CFU scoring of liquid cultures. ATc was added at time 0 to induce expression of Rv1989c. Samples were collected at the seven different time points, cells were washed with growth medium to remove ATc and 10-fold serial dilutions were spotted on agar plates for CFU counting. Data are represented as mean of three independent replicates ± SD. (D) Mtb ΔTA strains carrying pGMC-TetR-P1-Rv1989c or an empty vector control were grown for 4 days in the presence of ATc (+ATc) or left untreated (−ATc). Cells were labeled with the LIVE/DEAD Bac Light stains (Syto 9; propidium iodide (PI)) and analyzed by fluorescence-activated cell sorting (FACS). The empty vector control was either left unlabeled (negative control) or heat killed by incubation for 1 h at 100°C (positive control). Quadrants were established using the negative (no stain) and positive (heat-killed Mtb) controls as references. Shown data are representative of two independent experiments. (E) Bar diagram showing the fraction of Mtb ΔTA cells permeable to PI and Sypro 9 as determined by FACS analysis (see D). Data are represented as mean of two independent replicates ± SD. (F) Visualization of Syto9 (green) or PI (red) incorporation in Mtb ΔTA cells following ATc-induced expression of Rv1989c from pGMC-TetR-P1-Rv1989c by spinning disk confocal microscopy (see D). Mtb ΔTA cells transformed with empty vector were included as a negative control. PI incorporation is indicative of membrane damage. Representative maximum intensity Z projection images are shown. Scale bar, 5 μm. See also Figures S1 and S2 .
Figure Legend Snippet: Rv1989c-Rv1990c Is a Bactericidal TA System in Mtb (A) WT Mtb or Mtb ΔTA transformed with pGMC derivatives carrying Rv1990c, Rv1989c, or Rv1989c-Rv1990c as indicated were grown in 7H9 ADC Tween medium. Growth on solid medium was tested by spotting serial 10-fold dilutions on 7H11 OADC agar (−ATc) or the same medium supplemented with anhydrotetracycline (+ATc) to induce transcription from the P1 promoter. Plates were observed after 20 days at 37°C. Shown data are representative of three independent experiments. (B) Growth of WT Mtb or Mtb ΔTA transformed with pGMC derivatives carrying Rv1990c, Rv1989c, or Rv1989c-Rv1990c in liquid medium as monitored by turbidity measurements (McFarland units). ATc was added at day 0 to induce ectopic gene expression when indicated. Data are represented as mean of three technical replicates ± SD. Shown data are representative of three independent experiments. (C) Survival of Mtb ΔTA strains carrying pGMC-TetR-P1-Rv1989c as measured by CFU scoring of liquid cultures. ATc was added at time 0 to induce expression of Rv1989c. Samples were collected at the seven different time points, cells were washed with growth medium to remove ATc and 10-fold serial dilutions were spotted on agar plates for CFU counting. Data are represented as mean of three independent replicates ± SD. (D) Mtb ΔTA strains carrying pGMC-TetR-P1-Rv1989c or an empty vector control were grown for 4 days in the presence of ATc (+ATc) or left untreated (−ATc). Cells were labeled with the LIVE/DEAD Bac Light stains (Syto 9; propidium iodide (PI)) and analyzed by fluorescence-activated cell sorting (FACS). The empty vector control was either left unlabeled (negative control) or heat killed by incubation for 1 h at 100°C (positive control). Quadrants were established using the negative (no stain) and positive (heat-killed Mtb) controls as references. Shown data are representative of two independent experiments. (E) Bar diagram showing the fraction of Mtb ΔTA cells permeable to PI and Sypro 9 as determined by FACS analysis (see D). Data are represented as mean of two independent replicates ± SD. (F) Visualization of Syto9 (green) or PI (red) incorporation in Mtb ΔTA cells following ATc-induced expression of Rv1989c from pGMC-TetR-P1-Rv1989c by spinning disk confocal microscopy (see D). Mtb ΔTA cells transformed with empty vector were included as a negative control. PI incorporation is indicative of membrane damage. Representative maximum intensity Z projection images are shown. Scale bar, 5 μm. See also Figures S1 and S2 .

Techniques Used: Transformation Assay, Expressing, Plasmid Preparation, Labeling, BAC Assay, Fluorescence, FACS, Negative Control, Incubation, Positive Control, Staining, Confocal Microscopy

16) Product Images from "Mycobacterium smegmatis PafBC is involved in regulation of DNA damage response"

Article Title: Mycobacterium smegmatis PafBC is involved in regulation of DNA damage response

Journal: Scientific Reports

doi: 10.1038/s41598-017-14410-z

PafBC affects cellular levels of RecA in Msm . Analysis of RecA levels in cell-free lysates of Msm wild type, Msm Δ pafBC and Msm Δ pafBC - pafBC . Cells were grown in 7H9 at 37 °C. Cell-free lysates were analysed by immunblot using an anti- E. coli RecA antibody. The full-length blot is shown in Supplementary Fig. S6 . Equal loading was controlled by an anti-RpoB immunoblot.
Figure Legend Snippet: PafBC affects cellular levels of RecA in Msm . Analysis of RecA levels in cell-free lysates of Msm wild type, Msm Δ pafBC and Msm Δ pafBC - pafBC . Cells were grown in 7H9 at 37 °C. Cell-free lysates were analysed by immunblot using an anti- E. coli RecA antibody. The full-length blot is shown in Supplementary Fig. S6 . Equal loading was controlled by an anti-RpoB immunoblot.

Techniques Used:

( A ) DNA-damage induced upregulation of RecA is impaired in Msm Δ pafBC . Msm wild type, the pafBC deletion mutant and a complemented strain were grown in 7H9 to an OD 600 between 0.8 to 1.0 at 37 °C. Cultures were split, supplemented with mitomycin C (80 ng/ml) as indicated and were incubated for another 4 h. RecA levels of cell-free lysates were analysed by an anti-RecA immunoblot. Full-length blots are shown in Supplementary Fig. S8 . To control for equal loading, an anti-RpoB immunoblot was performed. ( B ) Levels of PafBC do not alter under DNA damaging growth conditions. Samples of Msm wild type cells were prepared as described in ( A ) and were analysed by an anti-PafBC immunoblot.
Figure Legend Snippet: ( A ) DNA-damage induced upregulation of RecA is impaired in Msm Δ pafBC . Msm wild type, the pafBC deletion mutant and a complemented strain were grown in 7H9 to an OD 600 between 0.8 to 1.0 at 37 °C. Cultures were split, supplemented with mitomycin C (80 ng/ml) as indicated and were incubated for another 4 h. RecA levels of cell-free lysates were analysed by an anti-RecA immunoblot. Full-length blots are shown in Supplementary Fig. S8 . To control for equal loading, an anti-RpoB immunoblot was performed. ( B ) Levels of PafBC do not alter under DNA damaging growth conditions. Samples of Msm wild type cells were prepared as described in ( A ) and were analysed by an anti-PafBC immunoblot.

Techniques Used: Mutagenesis, Incubation

Transcriptional regulation of recA in Msm Δ pafBC is affected at the level of the P1 LexA/RecA-independent promoter. ( A ) Schematic representation of the Msm recA promoter region and the GFP reporter constructs used to measure recA P1, P2, and P1-P2 promoter activity, respectively. Hatched areas indicate mutated promoter regions. ( B ) Msm wild type and Msm Δ pafBC carrying the reporter plasmids were grown in 7H9 to an OD 600 of 0.7 at 37 °C. Cultures were then supplemented with mitomycin C (MMC; 80 ng/ml) and were grown for an additional 4 h. Controls (STD) received no mitomycin ( C ) GFP fluorescence in lysates was measured and normalized to protein content. RFU, relative fluorescence units.
Figure Legend Snippet: Transcriptional regulation of recA in Msm Δ pafBC is affected at the level of the P1 LexA/RecA-independent promoter. ( A ) Schematic representation of the Msm recA promoter region and the GFP reporter constructs used to measure recA P1, P2, and P1-P2 promoter activity, respectively. Hatched areas indicate mutated promoter regions. ( B ) Msm wild type and Msm Δ pafBC carrying the reporter plasmids were grown in 7H9 to an OD 600 of 0.7 at 37 °C. Cultures were then supplemented with mitomycin C (MMC; 80 ng/ml) and were grown for an additional 4 h. Controls (STD) received no mitomycin ( C ) GFP fluorescence in lysates was measured and normalized to protein content. RFU, relative fluorescence units.

Techniques Used: Construct, Activity Assay, Fluorescence

17) Product Images from "A Ferritin Mutant of Mycobacterium tuberculosis Is Highly Susceptible to Killing by Antibiotics and Is Unable To Establish a Chronic Infection in Mice"

Article Title: A Ferritin Mutant of Mycobacterium tuberculosis Is Highly Susceptible to Killing by Antibiotics and Is Unable To Establish a Chronic Infection in Mice

Journal: Infection and Immunity

doi: 10.1128/IAI.00229-12

Sensitivity of the wild type and the Δ bfrB mutant to antibiotics. The wild-type (▲) and Δ bfrB (●) strains were cultured for 2 days in 7H9 medium with no drug added or with increasing concentrations of antibiotics. Dilutions
Figure Legend Snippet: Sensitivity of the wild type and the Δ bfrB mutant to antibiotics. The wild-type (▲) and Δ bfrB (●) strains were cultured for 2 days in 7H9 medium with no drug added or with increasing concentrations of antibiotics. Dilutions

Techniques Used: Mutagenesis, Cell Culture

Growth of M. tuberculosis strains in 7H9 medium. Wild-type strain H37Rv (◆), the Δ bfrA mutant (▲), the Δ bfrB mutant (●), and the complemented Δ bfrB mutant (■) were grown in 7H9 medium with agitation
Figure Legend Snippet: Growth of M. tuberculosis strains in 7H9 medium. Wild-type strain H37Rv (◆), the Δ bfrA mutant (▲), the Δ bfrB mutant (●), and the complemented Δ bfrB mutant (■) were grown in 7H9 medium with agitation

Techniques Used: Mutagenesis

18) Product Images from "A Ferritin Mutant of Mycobacterium tuberculosis Is Highly Susceptible to Killing by Antibiotics and Is Unable To Establish a Chronic Infection in Mice"

Article Title: A Ferritin Mutant of Mycobacterium tuberculosis Is Highly Susceptible to Killing by Antibiotics and Is Unable To Establish a Chronic Infection in Mice

Journal: Infection and Immunity

doi: 10.1128/IAI.00229-12

Sensitivity of the wild type and the Δ bfrB mutant to antibiotics. The wild-type (▲) and Δ bfrB (●) strains were cultured for 2 days in 7H9 medium with no drug added or with increasing concentrations of antibiotics. Dilutions
Figure Legend Snippet: Sensitivity of the wild type and the Δ bfrB mutant to antibiotics. The wild-type (▲) and Δ bfrB (●) strains were cultured for 2 days in 7H9 medium with no drug added or with increasing concentrations of antibiotics. Dilutions

Techniques Used: Mutagenesis, Cell Culture

Growth of M. tuberculosis strains in 7H9 medium. Wild-type strain H37Rv (◆), the Δ bfrA mutant (▲), the Δ bfrB mutant (●), and the complemented Δ bfrB mutant (■) were grown in 7H9 medium with agitation
Figure Legend Snippet: Growth of M. tuberculosis strains in 7H9 medium. Wild-type strain H37Rv (◆), the Δ bfrA mutant (▲), the Δ bfrB mutant (●), and the complemented Δ bfrB mutant (■) were grown in 7H9 medium with agitation

Techniques Used: Mutagenesis

19) Product Images from "A Ferritin Mutant of Mycobacterium tuberculosis Is Highly Susceptible to Killing by Antibiotics and Is Unable To Establish a Chronic Infection in Mice"

Article Title: A Ferritin Mutant of Mycobacterium tuberculosis Is Highly Susceptible to Killing by Antibiotics and Is Unable To Establish a Chronic Infection in Mice

Journal: Infection and Immunity

doi: 10.1128/IAI.00229-12

Sensitivity of the wild type and the Δ bfrB mutant to antibiotics. The wild-type (▲) and Δ bfrB (●) strains were cultured for 2 days in 7H9 medium with no drug added or with increasing concentrations of antibiotics. Dilutions
Figure Legend Snippet: Sensitivity of the wild type and the Δ bfrB mutant to antibiotics. The wild-type (▲) and Δ bfrB (●) strains were cultured for 2 days in 7H9 medium with no drug added or with increasing concentrations of antibiotics. Dilutions

Techniques Used: Mutagenesis, Cell Culture

Growth of M. tuberculosis strains in 7H9 medium. Wild-type strain H37Rv (◆), the Δ bfrA mutant (▲), the Δ bfrB mutant (●), and the complemented Δ bfrB mutant (■) were grown in 7H9 medium with agitation
Figure Legend Snippet: Growth of M. tuberculosis strains in 7H9 medium. Wild-type strain H37Rv (◆), the Δ bfrA mutant (▲), the Δ bfrB mutant (●), and the complemented Δ bfrB mutant (■) were grown in 7H9 medium with agitation

Techniques Used: Mutagenesis

20) Product Images from "Unexpected Genomic and Phenotypic Diversity of Mycobacterium africanum Lineage 5 Affects Drug Resistance, Protein Secretion, and Immunogenicity"

Article Title: Unexpected Genomic and Phenotypic Diversity of Mycobacterium africanum Lineage 5 Affects Drug Resistance, Protein Secretion, and Immunogenicity

Journal: Genome Biology and Evolution

doi: 10.1093/gbe/evy145

—Sublineage-specific growth characteristics of M. africanum sublineages. Precultures of the indicated strains were grown until midlogarithmic phase and were diluted to 0.05 OD 600 /ml in glass tubes containing 5 ml of liquid 7H9 medium supple mented with 0.05% Tween 80, ADC supplement and 0.2% pyruvate. Optical density was measured at the indicated time points (symbols). Data points are averages of three individual experiments performed in technical duplicates. Colors of line indicate the specific (sub)lineage of M. tuberculosis ( Mtb ) or M. africanum ( Maf ) on the legend (top left). Individual isolates can be identified by combination of colors and symbols used (bottom right).
Figure Legend Snippet: —Sublineage-specific growth characteristics of M. africanum sublineages. Precultures of the indicated strains were grown until midlogarithmic phase and were diluted to 0.05 OD 600 /ml in glass tubes containing 5 ml of liquid 7H9 medium supple mented with 0.05% Tween 80, ADC supplement and 0.2% pyruvate. Optical density was measured at the indicated time points (symbols). Data points are averages of three individual experiments performed in technical duplicates. Colors of line indicate the specific (sub)lineage of M. tuberculosis ( Mtb ) or M. africanum ( Maf ) on the legend (top left). Individual isolates can be identified by combination of colors and symbols used (bottom right).

Techniques Used:

21) Product Images from "Treatment of Mycobacterium tuberculosis with antisense oligonucleotides to glutamine synthetase mRNA inhibits glutamine synthetase activity, formation of the poly-l-glutamate/glutamine cell wall structure, and bacterial replication"

Article Title: Treatment of Mycobacterium tuberculosis with antisense oligonucleotides to glutamine synthetase mRNA inhibits glutamine synthetase activity, formation of the poly-l-glutamate/glutamine cell wall structure, and bacterial replication

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi:

Inhibition of cell proliferation of M. tuberculosis Erdman and M. smegmatis 1–2c wild type and recombinant strain by antisense PS-ODNs. Duplicate bacterial cultures were grown for 6 weeks ( M. tuberculosis ) or 6 days ( M. smegmatis ) in 1–2 ml of 7H9 medium in the presence of the antisense PS-ODNs, individually or combined, at concentrations of 10 μM. At each time point, cultures were harvested, washed, serially diluted, and plated on 7H11 agar medium. Viable bacteria were enumerated after an incubation period of 2 weeks ( M. tuberculosis ) or 3 days ( M. smegmatis ). SDs varied between 15 and 20%.
Figure Legend Snippet: Inhibition of cell proliferation of M. tuberculosis Erdman and M. smegmatis 1–2c wild type and recombinant strain by antisense PS-ODNs. Duplicate bacterial cultures were grown for 6 weeks ( M. tuberculosis ) or 6 days ( M. smegmatis ) in 1–2 ml of 7H9 medium in the presence of the antisense PS-ODNs, individually or combined, at concentrations of 10 μM. At each time point, cultures were harvested, washed, serially diluted, and plated on 7H11 agar medium. Viable bacteria were enumerated after an incubation period of 2 weeks ( M. tuberculosis ) or 3 days ( M. smegmatis ). SDs varied between 15 and 20%.

Techniques Used: Inhibition, Recombinant, Incubation

Inhibition of cell proliferation of M. tuberculosis by antisense PS-ODNs in the presence of various concentrations of ethambutol or polymyxin B nonapeptide. Duplicate bacterial cultures were grown for 6 weeks in 1–2 ml of 7H9 medium in the presence of the antisense PS-ODNs, individually or combined, at concentrations of 10 μM and 0, 0.1, 0.25, or 0.5 μg/ml ethambutol (EMB) and/or polymyxin B nonapeptide (PMBN) to “soften” the cell wall. At each time point, cultures were harvested, washed, serially diluted, plated on 7H11 agar medium, and cfu enumerated after 2 weeks. SDs varied between 15 and 20%. Values for single PS-ODNs were combined to yield one dashed line; the range of values for single PS-ODNs is indicated by the broad, solid, vertical bar.
Figure Legend Snippet: Inhibition of cell proliferation of M. tuberculosis by antisense PS-ODNs in the presence of various concentrations of ethambutol or polymyxin B nonapeptide. Duplicate bacterial cultures were grown for 6 weeks in 1–2 ml of 7H9 medium in the presence of the antisense PS-ODNs, individually or combined, at concentrations of 10 μM and 0, 0.1, 0.25, or 0.5 μg/ml ethambutol (EMB) and/or polymyxin B nonapeptide (PMBN) to “soften” the cell wall. At each time point, cultures were harvested, washed, serially diluted, plated on 7H11 agar medium, and cfu enumerated after 2 weeks. SDs varied between 15 and 20%. Values for single PS-ODNs were combined to yield one dashed line; the range of values for single PS-ODNs is indicated by the broad, solid, vertical bar.

Techniques Used: Inhibition

22) Product Images from "Treatment of Mycobacterium tuberculosis with antisense oligonucleotides to glutamine synthetase mRNA inhibits glutamine synthetase activity, formation of the poly-l-glutamate/glutamine cell wall structure, and bacterial replication"

Article Title: Treatment of Mycobacterium tuberculosis with antisense oligonucleotides to glutamine synthetase mRNA inhibits glutamine synthetase activity, formation of the poly-l-glutamate/glutamine cell wall structure, and bacterial replication

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi:

Inhibition of cell proliferation of M. tuberculosis Erdman and M. smegmatis 1–2c wild type and recombinant strain by antisense PS-ODNs. Duplicate bacterial cultures were grown for 6 weeks ( M. tuberculosis ) or 6 days ( M. smegmatis ) in 1–2 ml of 7H9 medium in the presence of the antisense PS-ODNs, individually or combined, at concentrations of 10 μM. At each time point, cultures were harvested, washed, serially diluted, and plated on 7H11 agar medium. Viable bacteria were enumerated after an incubation period of 2 weeks ( M. tuberculosis ) or 3 days ( M. smegmatis ). SDs varied between 15 and 20%.
Figure Legend Snippet: Inhibition of cell proliferation of M. tuberculosis Erdman and M. smegmatis 1–2c wild type and recombinant strain by antisense PS-ODNs. Duplicate bacterial cultures were grown for 6 weeks ( M. tuberculosis ) or 6 days ( M. smegmatis ) in 1–2 ml of 7H9 medium in the presence of the antisense PS-ODNs, individually or combined, at concentrations of 10 μM. At each time point, cultures were harvested, washed, serially diluted, and plated on 7H11 agar medium. Viable bacteria were enumerated after an incubation period of 2 weeks ( M. tuberculosis ) or 3 days ( M. smegmatis ). SDs varied between 15 and 20%.

Techniques Used: Inhibition, Recombinant, Incubation

Inhibition of cell proliferation of M. tuberculosis by antisense PS-ODNs in the presence of various concentrations of ethambutol or polymyxin B nonapeptide. Duplicate bacterial cultures were grown for 6 weeks in 1–2 ml of 7H9 medium in the presence of the antisense PS-ODNs, individually or combined, at concentrations of 10 μM and 0, 0.1, 0.25, or 0.5 μg/ml ethambutol (EMB) and/or polymyxin B nonapeptide (PMBN) to “soften” the cell wall. At each time point, cultures were harvested, washed, serially diluted, plated on 7H11 agar medium, and cfu enumerated after 2 weeks. SDs varied between 15 and 20%. Values for single PS-ODNs were combined to yield one dashed line; the range of values for single PS-ODNs is indicated by the broad, solid, vertical bar.
Figure Legend Snippet: Inhibition of cell proliferation of M. tuberculosis by antisense PS-ODNs in the presence of various concentrations of ethambutol or polymyxin B nonapeptide. Duplicate bacterial cultures were grown for 6 weeks in 1–2 ml of 7H9 medium in the presence of the antisense PS-ODNs, individually or combined, at concentrations of 10 μM and 0, 0.1, 0.25, or 0.5 μg/ml ethambutol (EMB) and/or polymyxin B nonapeptide (PMBN) to “soften” the cell wall. At each time point, cultures were harvested, washed, serially diluted, plated on 7H11 agar medium, and cfu enumerated after 2 weeks. SDs varied between 15 and 20%. Values for single PS-ODNs were combined to yield one dashed line; the range of values for single PS-ODNs is indicated by the broad, solid, vertical bar.

Techniques Used: Inhibition

23) Product Images from "A Ferritin Mutant of Mycobacterium tuberculosis Is Highly Susceptible to Killing by Antibiotics and Is Unable To Establish a Chronic Infection in Mice"

Article Title: A Ferritin Mutant of Mycobacterium tuberculosis Is Highly Susceptible to Killing by Antibiotics and Is Unable To Establish a Chronic Infection in Mice

Journal: Infection and Immunity

doi: 10.1128/IAI.00229-12

Sensitivity of the wild type and the Δ bfrB mutant to antibiotics. The wild-type (▲) and Δ bfrB (●) strains were cultured for 2 days in 7H9 medium with no drug added or with increasing concentrations of antibiotics. Dilutions
Figure Legend Snippet: Sensitivity of the wild type and the Δ bfrB mutant to antibiotics. The wild-type (▲) and Δ bfrB (●) strains were cultured for 2 days in 7H9 medium with no drug added or with increasing concentrations of antibiotics. Dilutions

Techniques Used: Mutagenesis, Cell Culture

Growth of M. tuberculosis strains in 7H9 medium. Wild-type strain H37Rv (◆), the Δ bfrA mutant (▲), the Δ bfrB mutant (●), and the complemented Δ bfrB mutant (■) were grown in 7H9 medium with agitation
Figure Legend Snippet: Growth of M. tuberculosis strains in 7H9 medium. Wild-type strain H37Rv (◆), the Δ bfrA mutant (▲), the Δ bfrB mutant (●), and the complemented Δ bfrB mutant (■) were grown in 7H9 medium with agitation

Techniques Used: Mutagenesis

24) Product Images from "A Ferritin Mutant of Mycobacterium tuberculosis Is Highly Susceptible to Killing by Antibiotics and Is Unable To Establish a Chronic Infection in Mice"

Article Title: A Ferritin Mutant of Mycobacterium tuberculosis Is Highly Susceptible to Killing by Antibiotics and Is Unable To Establish a Chronic Infection in Mice

Journal: Infection and Immunity

doi: 10.1128/IAI.00229-12

Sensitivity of the wild type and the Δ bfrB mutant to antibiotics. The wild-type (▲) and Δ bfrB (●) strains were cultured for 2 days in 7H9 medium with no drug added or with increasing concentrations of antibiotics. Dilutions
Figure Legend Snippet: Sensitivity of the wild type and the Δ bfrB mutant to antibiotics. The wild-type (▲) and Δ bfrB (●) strains were cultured for 2 days in 7H9 medium with no drug added or with increasing concentrations of antibiotics. Dilutions

Techniques Used: Mutagenesis, Cell Culture

Growth of M. tuberculosis strains in 7H9 medium. Wild-type strain H37Rv (◆), the Δ bfrA mutant (▲), the Δ bfrB mutant (●), and the complemented Δ bfrB mutant (■) were grown in 7H9 medium with agitation
Figure Legend Snippet: Growth of M. tuberculosis strains in 7H9 medium. Wild-type strain H37Rv (◆), the Δ bfrA mutant (▲), the Δ bfrB mutant (●), and the complemented Δ bfrB mutant (■) were grown in 7H9 medium with agitation

Techniques Used: Mutagenesis

25) Product Images from "A Ferritin Mutant of Mycobacterium tuberculosis Is Highly Susceptible to Killing by Antibiotics and Is Unable To Establish a Chronic Infection in Mice"

Article Title: A Ferritin Mutant of Mycobacterium tuberculosis Is Highly Susceptible to Killing by Antibiotics and Is Unable To Establish a Chronic Infection in Mice

Journal: Infection and Immunity

doi: 10.1128/IAI.00229-12

Sensitivity of the wild type and the Δ bfrB mutant to antibiotics. The wild-type (▲) and Δ bfrB (●) strains were cultured for 2 days in 7H9 medium with no drug added or with increasing concentrations of antibiotics. Dilutions
Figure Legend Snippet: Sensitivity of the wild type and the Δ bfrB mutant to antibiotics. The wild-type (▲) and Δ bfrB (●) strains were cultured for 2 days in 7H9 medium with no drug added or with increasing concentrations of antibiotics. Dilutions

Techniques Used: Mutagenesis, Cell Culture

Growth of M. tuberculosis strains in 7H9 medium. Wild-type strain H37Rv (◆), the Δ bfrA mutant (▲), the Δ bfrB mutant (●), and the complemented Δ bfrB mutant (■) were grown in 7H9 medium with agitation
Figure Legend Snippet: Growth of M. tuberculosis strains in 7H9 medium. Wild-type strain H37Rv (◆), the Δ bfrA mutant (▲), the Δ bfrB mutant (●), and the complemented Δ bfrB mutant (■) were grown in 7H9 medium with agitation

Techniques Used: Mutagenesis

26) Product Images from "Targeting the Mycobacterium tuberculosis 30/32-kDa mycolyl transferase complex as a therapeutic strategy against tuberculosis: Proof of principle by using antisense technology"

Article Title: Targeting the Mycobacterium tuberculosis 30/32-kDa mycolyl transferase complex as a therapeutic strategy against tuberculosis: Proof of principle by using antisense technology

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi: 10.1073/pnas.242612299

Inhibition of M. tuberculosis growth by antisense PS-ODNs specific for the 30/32-kDa protein gene complex. Duplicate bacterial cultures were grown for 6 wk in 2 ml of 7H9 medium in the presence of antisense (PS-ODN) or mismatched PS-ODN (MM PS-ODN) at concentrations of 10 μM. For cultures with multiple PS-ODNs, each of the PS-ODNs or MM PS-ODNs was added at a concentration of 10 μM. All PS-ODNs were added once at the start of the observation period. At each time point, culture aliquots were harvested, washed, and plated on 7H11 agar medium. The cfu were enumerated after incubating plates for 2 wk. The maximal standard deviation for all of the data points was ≤6.7%. ( a ) PS-ODNs specific for various internal regions of the transcript of the 30-kDa protein gene. ( b ) PS-ODNs specific for an internal domain of each gene transcript of the mycolic acid transferase complex (30/32A/32B/24-kDa proteins). ( c ) PS-ODNs specific for the 5′ end of each gene transcript of the mycolic acid transferase complex (30/32A/32B/24-kDa proteins).
Figure Legend Snippet: Inhibition of M. tuberculosis growth by antisense PS-ODNs specific for the 30/32-kDa protein gene complex. Duplicate bacterial cultures were grown for 6 wk in 2 ml of 7H9 medium in the presence of antisense (PS-ODN) or mismatched PS-ODN (MM PS-ODN) at concentrations of 10 μM. For cultures with multiple PS-ODNs, each of the PS-ODNs or MM PS-ODNs was added at a concentration of 10 μM. All PS-ODNs were added once at the start of the observation period. At each time point, culture aliquots were harvested, washed, and plated on 7H11 agar medium. The cfu were enumerated after incubating plates for 2 wk. The maximal standard deviation for all of the data points was ≤6.7%. ( a ) PS-ODNs specific for various internal regions of the transcript of the 30-kDa protein gene. ( b ) PS-ODNs specific for an internal domain of each gene transcript of the mycolic acid transferase complex (30/32A/32B/24-kDa proteins). ( c ) PS-ODNs specific for the 5′ end of each gene transcript of the mycolic acid transferase complex (30/32A/32B/24-kDa proteins).

Techniques Used: Inhibition, Concentration Assay, Standard Deviation

27) Product Images from "Mycobacterium smegmatis PafBC is involved in regulation of DNA damage response"

Article Title: Mycobacterium smegmatis PafBC is involved in regulation of DNA damage response

Journal: Scientific Reports

doi: 10.1038/s41598-017-14410-z

PafBC affects cellular levels of RecA in Msm . Analysis of RecA levels in cell-free lysates of Msm wild type, Msm Δ pafBC and Msm Δ pafBC - pafBC . Cells were grown in 7H9 at 37 °C. Cell-free lysates were analysed by immunblot using an anti- E. coli RecA antibody. The full-length blot is shown in Supplementary Fig. S6 . Equal loading was controlled by an anti-RpoB immunoblot.
Figure Legend Snippet: PafBC affects cellular levels of RecA in Msm . Analysis of RecA levels in cell-free lysates of Msm wild type, Msm Δ pafBC and Msm Δ pafBC - pafBC . Cells were grown in 7H9 at 37 °C. Cell-free lysates were analysed by immunblot using an anti- E. coli RecA antibody. The full-length blot is shown in Supplementary Fig. S6 . Equal loading was controlled by an anti-RpoB immunoblot.

Techniques Used:

( A ) DNA-damage induced upregulation of RecA is impaired in Msm Δ pafBC . Msm wild type, the pafBC deletion mutant and a complemented strain were grown in 7H9 to an OD 600 between 0.8 to 1.0 at 37 °C. Cultures were split, supplemented with mitomycin C (80 ng/ml) as indicated and were incubated for another 4 h. RecA levels of cell-free lysates were analysed by an anti-RecA immunoblot. Full-length blots are shown in Supplementary Fig. S8 . To control for equal loading, an anti-RpoB immunoblot was performed. ( B ) Levels of PafBC do not alter under DNA damaging growth conditions. Samples of Msm wild type cells were prepared as described in ( A ) and were analysed by an anti-PafBC immunoblot.
Figure Legend Snippet: ( A ) DNA-damage induced upregulation of RecA is impaired in Msm Δ pafBC . Msm wild type, the pafBC deletion mutant and a complemented strain were grown in 7H9 to an OD 600 between 0.8 to 1.0 at 37 °C. Cultures were split, supplemented with mitomycin C (80 ng/ml) as indicated and were incubated for another 4 h. RecA levels of cell-free lysates were analysed by an anti-RecA immunoblot. Full-length blots are shown in Supplementary Fig. S8 . To control for equal loading, an anti-RpoB immunoblot was performed. ( B ) Levels of PafBC do not alter under DNA damaging growth conditions. Samples of Msm wild type cells were prepared as described in ( A ) and were analysed by an anti-PafBC immunoblot.

Techniques Used: Mutagenesis, Incubation

Transcriptional regulation of recA in Msm Δ pafBC is affected at the level of the P1 LexA/RecA-independent promoter. ( A ) Schematic representation of the Msm recA promoter region and the GFP reporter constructs used to measure recA P1, P2, and P1-P2 promoter activity, respectively. Hatched areas indicate mutated promoter regions. ( B ) Msm wild type and Msm Δ pafBC carrying the reporter plasmids were grown in 7H9 to an OD 600 of 0.7 at 37 °C. Cultures were then supplemented with mitomycin C (MMC; 80 ng/ml) and were grown for an additional 4 h. Controls (STD) received no mitomycin ( C ) GFP fluorescence in lysates was measured and normalized to protein content. RFU, relative fluorescence units.
Figure Legend Snippet: Transcriptional regulation of recA in Msm Δ pafBC is affected at the level of the P1 LexA/RecA-independent promoter. ( A ) Schematic representation of the Msm recA promoter region and the GFP reporter constructs used to measure recA P1, P2, and P1-P2 promoter activity, respectively. Hatched areas indicate mutated promoter regions. ( B ) Msm wild type and Msm Δ pafBC carrying the reporter plasmids were grown in 7H9 to an OD 600 of 0.7 at 37 °C. Cultures were then supplemented with mitomycin C (MMC; 80 ng/ml) and were grown for an additional 4 h. Controls (STD) received no mitomycin ( C ) GFP fluorescence in lysates was measured and normalized to protein content. RFU, relative fluorescence units.

Techniques Used: Construct, Activity Assay, Fluorescence

28) Product Images from "A Ferritin Mutant of Mycobacterium tuberculosis Is Highly Susceptible to Killing by Antibiotics and Is Unable To Establish a Chronic Infection in Mice"

Article Title: A Ferritin Mutant of Mycobacterium tuberculosis Is Highly Susceptible to Killing by Antibiotics and Is Unable To Establish a Chronic Infection in Mice

Journal: Infection and Immunity

doi: 10.1128/IAI.00229-12

Sensitivity of the wild type and the Δ bfrB mutant to antibiotics. The wild-type (▲) and Δ bfrB (●) strains were cultured for 2 days in 7H9 medium with no drug added or with increasing concentrations of antibiotics. Dilutions
Figure Legend Snippet: Sensitivity of the wild type and the Δ bfrB mutant to antibiotics. The wild-type (▲) and Δ bfrB (●) strains were cultured for 2 days in 7H9 medium with no drug added or with increasing concentrations of antibiotics. Dilutions

Techniques Used: Mutagenesis, Cell Culture

Growth of M. tuberculosis strains in 7H9 medium. Wild-type strain H37Rv (◆), the Δ bfrA mutant (▲), the Δ bfrB mutant (●), and the complemented Δ bfrB mutant (■) were grown in 7H9 medium with agitation
Figure Legend Snippet: Growth of M. tuberculosis strains in 7H9 medium. Wild-type strain H37Rv (◆), the Δ bfrA mutant (▲), the Δ bfrB mutant (●), and the complemented Δ bfrB mutant (■) were grown in 7H9 medium with agitation

Techniques Used: Mutagenesis

29) Product Images from "A Ferritin Mutant of Mycobacterium tuberculosis Is Highly Susceptible to Killing by Antibiotics and Is Unable To Establish a Chronic Infection in Mice"

Article Title: A Ferritin Mutant of Mycobacterium tuberculosis Is Highly Susceptible to Killing by Antibiotics and Is Unable To Establish a Chronic Infection in Mice

Journal: Infection and Immunity

doi: 10.1128/IAI.00229-12

Sensitivity of the wild type and the Δ bfrB mutant to antibiotics. The wild-type (▲) and Δ bfrB (●) strains were cultured for 2 days in 7H9 medium with no drug added or with increasing concentrations of antibiotics. Dilutions
Figure Legend Snippet: Sensitivity of the wild type and the Δ bfrB mutant to antibiotics. The wild-type (▲) and Δ bfrB (●) strains were cultured for 2 days in 7H9 medium with no drug added or with increasing concentrations of antibiotics. Dilutions

Techniques Used: Mutagenesis, Cell Culture

Growth of M. tuberculosis strains in 7H9 medium. Wild-type strain H37Rv (◆), the Δ bfrA mutant (▲), the Δ bfrB mutant (●), and the complemented Δ bfrB mutant (■) were grown in 7H9 medium with agitation
Figure Legend Snippet: Growth of M. tuberculosis strains in 7H9 medium. Wild-type strain H37Rv (◆), the Δ bfrA mutant (▲), the Δ bfrB mutant (●), and the complemented Δ bfrB mutant (■) were grown in 7H9 medium with agitation

Techniques Used: Mutagenesis

30) Product Images from "Treatment of Mycobacterium tuberculosis with antisense oligonucleotides to glutamine synthetase mRNA inhibits glutamine synthetase activity, formation of the poly-l-glutamate/glutamine cell wall structure, and bacterial replication"

Article Title: Treatment of Mycobacterium tuberculosis with antisense oligonucleotides to glutamine synthetase mRNA inhibits glutamine synthetase activity, formation of the poly-l-glutamate/glutamine cell wall structure, and bacterial replication

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi:

Inhibition of cell proliferation of M. tuberculosis Erdman and M. smegmatis 1–2c wild type and recombinant strain by antisense PS-ODNs. Duplicate bacterial cultures were grown for 6 weeks ( M. tuberculosis ) or 6 days ( M. smegmatis ) in 1–2 ml of 7H9 medium in the presence of the antisense PS-ODNs, individually or combined, at concentrations of 10 μM. At each time point, cultures were harvested, washed, serially diluted, and plated on 7H11 agar medium. Viable bacteria were enumerated after an incubation period of 2 weeks ( M. tuberculosis ) or 3 days ( M. smegmatis ). SDs varied between 15 and 20%.
Figure Legend Snippet: Inhibition of cell proliferation of M. tuberculosis Erdman and M. smegmatis 1–2c wild type and recombinant strain by antisense PS-ODNs. Duplicate bacterial cultures were grown for 6 weeks ( M. tuberculosis ) or 6 days ( M. smegmatis ) in 1–2 ml of 7H9 medium in the presence of the antisense PS-ODNs, individually or combined, at concentrations of 10 μM. At each time point, cultures were harvested, washed, serially diluted, and plated on 7H11 agar medium. Viable bacteria were enumerated after an incubation period of 2 weeks ( M. tuberculosis ) or 3 days ( M. smegmatis ). SDs varied between 15 and 20%.

Techniques Used: Inhibition, Recombinant, Incubation

Inhibition of cell proliferation of M. tuberculosis by antisense PS-ODNs in the presence of various concentrations of ethambutol or polymyxin B nonapeptide. Duplicate bacterial cultures were grown for 6 weeks in 1–2 ml of 7H9 medium in the presence of the antisense PS-ODNs, individually or combined, at concentrations of 10 μM and 0, 0.1, 0.25, or 0.5 μg/ml ethambutol (EMB) and/or polymyxin B nonapeptide (PMBN) to “soften” the cell wall. At each time point, cultures were harvested, washed, serially diluted, plated on 7H11 agar medium, and cfu enumerated after 2 weeks. SDs varied between 15 and 20%. Values for single PS-ODNs were combined to yield one dashed line; the range of values for single PS-ODNs is indicated by the broad, solid, vertical bar.
Figure Legend Snippet: Inhibition of cell proliferation of M. tuberculosis by antisense PS-ODNs in the presence of various concentrations of ethambutol or polymyxin B nonapeptide. Duplicate bacterial cultures were grown for 6 weeks in 1–2 ml of 7H9 medium in the presence of the antisense PS-ODNs, individually or combined, at concentrations of 10 μM and 0, 0.1, 0.25, or 0.5 μg/ml ethambutol (EMB) and/or polymyxin B nonapeptide (PMBN) to “soften” the cell wall. At each time point, cultures were harvested, washed, serially diluted, plated on 7H11 agar medium, and cfu enumerated after 2 weeks. SDs varied between 15 and 20%. Values for single PS-ODNs were combined to yield one dashed line; the range of values for single PS-ODNs is indicated by the broad, solid, vertical bar.

Techniques Used: Inhibition

Related Articles

Clone Assay:

Article Title: The PhoP-Dependent ncRNA Mcr7 Modulates the TAT Secretion System in Mycobacterium tuberculosis
Article Snippet: Mycobacterial strains were grown at 37°C in 7H9 medium (Difco) supplemented with 0.05% Tween 80 and 10% albumin-dextrose-catalase (ADC, Middlebrook) or on 7H10 plates supplemented with 10% ADC. .. Escherichia coli DH5α used for cloning procedures was grown at 37°C in LB broth or on LB agar plates.

Filtration:

Article Title: Distinct Properties of Hexameric but Functionally Conserved Mycobacterium tuberculosis Transcription-Repair Coupling Factor
Article Snippet: Growth and preparation of M. tuberculosis H37Ra cell extract M. tuberculosis H37Ra strain was grown in 7H9 medium (Difco, BD, USA) containing 10% ADC supplements (for 1 liter; 8.5 g NaCl, 50 g BSA, 20 g glucose and 0.03 g Catalase) and 0.05% Tween 80 to 0.7 OD. .. The supernatant was used for native-PAGE Western and gel filtration analysis for .

Mutagenesis:

Article Title: A Ferritin Mutant of Mycobacterium tuberculosis Is Highly Susceptible to Killing by Antibiotics and Is Unable To Establish a Chronic Infection in Mice
Article Snippet: .. As shown in , the growth of the Δ bfrB mutant was stimulated by the addition of up to 40 to 50 μM FeCl3 to LIMM, but higher concentrations, such as those in 7H9 medium, inhibited growth compared to supplementation with 40 μM FeCl3 . .. Moreover, the addition of 2′,2-dypiridyl (DPI), a permeable ferrous iron chelator, to 7H9 medium improved the growth of the Δ bfrB mutant, indicating that the reduced growth in 7H9 medium was iron dependent ( ).

Isolation:

Article Title: Unexpected Genomic and Phenotypic Diversity of Mycobacterium africanum Lineage 5 Affects Drug Resistance, Protein Secretion, and Immunogenicity
Article Snippet: Growth Conditions All newly analyzed isolates of M. africanum ( , online) were isolated in different locations in France and subsequently sent to the national reference laboratory. .. Cultivation of all isolates ( , online) was performed on 7H11 solid medium with OADC supplement (Difco), or in liquid 7H9 medium supplemented with ADC (Difco), 0.05% Tween 80, and 0.2% w/v pyruvate ( ).

Cell Culture:

Article Title: Inactivation of lsr2 Results in a Hypermotile Phenotype in Mycobacterium smegmatis
Article Snippet: .. Briefly, cells were cultured in 7H9 medium to mid-logarithmic phase (optical density at 600 nm [OD600 ], 0.8 to 1.0) before spotting 2-μl aliquots onto motility medium consisting of M63 salts liquid medium (Difco) supplemented with 0.5% Casamino Acids, 0.2% glycerol, 1 mM MgCl2 , and 10 μM FeCl2 , solidified with agarose. ..

Concentration Assay:

Article Title: Mycobacterium smegmatis PafBC is involved in regulation of DNA damage response
Article Snippet: Bacteria were grown in LB medium supplemented with 0.05% (v/v) TWEEN-80 (LB-T) or 7H9 medium (Difco Laboratories) without OADC supplement. .. When appropriate, hygromycin B was added to a final concentration of 100 µg ml−1 .

Incubation:

Article Title: Inactivation of lsr2 Results in a Hypermotile Phenotype in Mycobacterium smegmatis
Article Snippet: Briefly, cells were cultured in 7H9 medium to mid-logarithmic phase (optical density at 600 nm [OD600 ], 0.8 to 1.0) before spotting 2-μl aliquots onto motility medium consisting of M63 salts liquid medium (Difco) supplemented with 0.5% Casamino Acids, 0.2% glycerol, 1 mM MgCl2 , and 10 μM FeCl2 , solidified with agarose. .. The inoculated plates were incubated for 24 h at 37°C in plastic bags containing moistened paper towels to ensure bacteria grew under humidified conditions.

Article Title: Mycobacterium smegmatis PafBC is involved in regulation of DNA damage response
Article Snippet: .. Then cells were resuspended in 7H9 medium without acetamide and incubation of cultures continued for 9 h. Samples were withdrawn at the given time points. ..

Article Title: A Ferritin Mutant of Mycobacterium tuberculosis Is Highly Susceptible to Killing by Antibiotics and Is Unable To Establish a Chronic Infection in Mice
Article Snippet: .. Briefly, 2 × 105 bacteria in microtiter plates were incubated at 37°C in 7H9 medium containing increasing concentrations of streptonigrin. .. Twenty microliters of alamarBlue was added, and 24 h later, the fluorescence emitted by cell-reduced alamarBlue was measured with a GloMax-Multi detection system (Promega).

Article Title: Sliding Motility in Mycobacteria
Article Snippet: M63 or 7H9 medium supplemented as indicated were solidified with 0.3% agar (Difco) or 0.1 to 0.8% ultrapure SeaKem LE agarose (FMC Bioproducts). .. Spreading was evaluated visually after incubation of parafilm-sealed plates at 37°C in a humidified incubator (50% relative humidity) for the indicated period of time.

other:

Article Title: Mycobacterium smegmatis PafBC is involved in regulation of DNA damage response
Article Snippet: Msm SMR5, Msm ΔpafBC and Msm ΔprcB carrying pMyC-His6 -recA were grown in 7H9 medium to an OD600 of 1.0.

Expressing:

Article Title: The PhoP-Dependent ncRNA Mcr7 Modulates the TAT Secretion System in Mycobacterium tuberculosis
Article Snippet: .. Figure shows fold change in gene expression in bacteria grown in 7H9 medium. ..

Sonication:

Article Title: Distinct Properties of Hexameric but Functionally Conserved Mycobacterium tuberculosis Transcription-Repair Coupling Factor
Article Snippet: Growth and preparation of M. tuberculosis H37Ra cell extract M. tuberculosis H37Ra strain was grown in 7H9 medium (Difco, BD, USA) containing 10% ADC supplements (for 1 liter; 8.5 g NaCl, 50 g BSA, 20 g glucose and 0.03 g Catalase) and 0.05% Tween 80 to 0.7 OD. .. Crude cell lysate was prepared in Tris buffer pH 8.0 (20 mM Tris-Cl, 100 mM NaCl, 10% glycerol, 0.1 mM EDTA and 5 mM β-mercaptoethanol) by sonication followed by ultracentrifugation at 100,000 g (S100) for 3 hrs at 4°C.

Marker:

Article Title: Selective small molecule inhibitor of the Mycobacterium tuberculosis fumarate hydratase reveals an allosteric regulatory site
Article Snippet: .. Because the plasmid contains a selectable kanamycin marker, cultures were maintained in 25 μg/mL kanamycin in 7H9 medium (Difco) supplemented with 4 g/L glucose and 0.3 g/L casitone (Difco). .. Experiments were conducted in black, clear-bottomed 96-well plates (Greiner).

Western Blot:

Article Title: Distinct Properties of Hexameric but Functionally Conserved Mycobacterium tuberculosis Transcription-Repair Coupling Factor
Article Snippet: Growth and preparation of M. tuberculosis H37Ra cell extract M. tuberculosis H37Ra strain was grown in 7H9 medium (Difco, BD, USA) containing 10% ADC supplements (for 1 liter; 8.5 g NaCl, 50 g BSA, 20 g glucose and 0.03 g Catalase) and 0.05% Tween 80 to 0.7 OD. .. The supernatant was used for native-PAGE Western and gel filtration analysis for .

Clear Native PAGE:

Article Title: Distinct Properties of Hexameric but Functionally Conserved Mycobacterium tuberculosis Transcription-Repair Coupling Factor
Article Snippet: Growth and preparation of M. tuberculosis H37Ra cell extract M. tuberculosis H37Ra strain was grown in 7H9 medium (Difco, BD, USA) containing 10% ADC supplements (for 1 liter; 8.5 g NaCl, 50 g BSA, 20 g glucose and 0.03 g Catalase) and 0.05% Tween 80 to 0.7 OD. .. The supernatant was used for native-PAGE Western and gel filtration analysis for .

Plasmid Preparation:

Article Title: Selective small molecule inhibitor of the Mycobacterium tuberculosis fumarate hydratase reveals an allosteric regulatory site
Article Snippet: .. Because the plasmid contains a selectable kanamycin marker, cultures were maintained in 25 μg/mL kanamycin in 7H9 medium (Difco) supplemented with 4 g/L glucose and 0.3 g/L casitone (Difco). .. Experiments were conducted in black, clear-bottomed 96-well plates (Greiner).

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  • 94
    Difco 7h9 minimal medium
    Increased qsdA and qsdC mRNA levels in a Δ qsdR mutant strain. qRT-PCR analysis shows the qsdA and qsdC mRNA levels in the Δ qsdR mutant and in the complementing strain (Δ qsdR-qsdR ) grown in <t>7H9</t> medium at the mid-exponential (10 h) and stationary (19 h) phases. These mRNA levels are relative to the wild type strain. Data shown are mean values obtained from three independent experiments. Statistical analysis was performed by the DataAssist TM software (v3.01) used for calculating relative quantitation of gene expression, based on the comparative C T (2 -ΔΔCT ) method. ∗ p -value
    7h9 Minimal Medium, supplied by Difco, used in various techniques. Bioz Stars score: 94/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Difco 7h9 liquid medium
    Growth of M. smegmatis in various nitrogen sources. Wild-type SMR5 and glnR deletion strain MH1 were grown in the presence of the indicated substances (10 mM final concentration) as sole nitrogen source. Standard <t>7H9</t> medium was used as positive control; 7H9 lacking any nitrogen source as negative control (7H9-N). (A) Putative nitrogen sources deduced from microarray results, (B) putative nitrogen sources used by closely related species.
    7h9 Liquid Medium, supplied by Difco, used in various techniques. Bioz Stars score: 94/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Difco middlebrook 7h9 medium
    Kinetics and inducibility of fructose, glucose, and glycerol uptake by M. smegmatis . (A) M. smegmatis mc 2 155 was grown in <t>Middlebrook</t> <t>7H9</t> medium in the presence of 2% glycerol (open circles) or 2% fructose (closed circles). Accumulation
    Middlebrook 7h9 Medium, supplied by Difco, used in various techniques. Bioz Stars score: 95/100, based on 36 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Difco 7h9 broth
    Serial monitoring outcomes of RIF stability in L-J medium and <t>7H9</t> broth when kept at 37°C and 4°C. (a) Stability of RIF in 7H9 and L-J media when kept at 37°C. (b) Stability of RIF in 7H9 and L-J media when kept at 4°C.
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    Increased qsdA and qsdC mRNA levels in a Δ qsdR mutant strain. qRT-PCR analysis shows the qsdA and qsdC mRNA levels in the Δ qsdR mutant and in the complementing strain (Δ qsdR-qsdR ) grown in 7H9 medium at the mid-exponential (10 h) and stationary (19 h) phases. These mRNA levels are relative to the wild type strain. Data shown are mean values obtained from three independent experiments. Statistical analysis was performed by the DataAssist TM software (v3.01) used for calculating relative quantitation of gene expression, based on the comparative C T (2 -ΔΔCT ) method. ∗ p -value

    Journal: Frontiers in Microbiology

    Article Title: A Rhodococcal Transcriptional Regulatory Mechanism Detects the Common Lactone Ring of AHL Quorum-Sensing Signals and Triggers the Quorum-Quenching Response

    doi: 10.3389/fmicb.2018.02800

    Figure Lengend Snippet: Increased qsdA and qsdC mRNA levels in a Δ qsdR mutant strain. qRT-PCR analysis shows the qsdA and qsdC mRNA levels in the Δ qsdR mutant and in the complementing strain (Δ qsdR-qsdR ) grown in 7H9 medium at the mid-exponential (10 h) and stationary (19 h) phases. These mRNA levels are relative to the wild type strain. Data shown are mean values obtained from three independent experiments. Statistical analysis was performed by the DataAssist TM software (v3.01) used for calculating relative quantitation of gene expression, based on the comparative C T (2 -ΔΔCT ) method. ∗ p -value

    Article Snippet: R. erythropolis strains were cultivated in LBP ( ) for conjugative transfer of DNA and in 7H9 minimal medium (Difco) supplemented with hexanoate at 6 mM (Sigma-Aldrich) as carbon source for induction assays.

    Techniques: Mutagenesis, Quantitative RT-PCR, Software, Quantitation Assay, Expressing

    Induction of the qsd operon expression by 3-oxo-C 8 -HSL. qRT-PCR analysis of the qsdA , qsdC , and qsdR transcription was conducted in the R. erythropolis R138 wild-type strain grown in 7H9 medium in which 3-oxo-C 8 -HSL was added at mid-exponential phase. Expression levels of qsdA , qsdC , and qsdR are relative to those obtained in the same medium without the inducer. The dotted line indicates the same mRNA expression between the induced condition and without the inducers. Data shown are mean values obtained from three independent experiments. Statistical analysis was performed by the DataAssist TM software (v3.01) used for calculating relative quantitation of gene expression, based on the comparative C T (2 -ΔΔCT ) method. ∗ p -value

    Journal: Frontiers in Microbiology

    Article Title: A Rhodococcal Transcriptional Regulatory Mechanism Detects the Common Lactone Ring of AHL Quorum-Sensing Signals and Triggers the Quorum-Quenching Response

    doi: 10.3389/fmicb.2018.02800

    Figure Lengend Snippet: Induction of the qsd operon expression by 3-oxo-C 8 -HSL. qRT-PCR analysis of the qsdA , qsdC , and qsdR transcription was conducted in the R. erythropolis R138 wild-type strain grown in 7H9 medium in which 3-oxo-C 8 -HSL was added at mid-exponential phase. Expression levels of qsdA , qsdC , and qsdR are relative to those obtained in the same medium without the inducer. The dotted line indicates the same mRNA expression between the induced condition and without the inducers. Data shown are mean values obtained from three independent experiments. Statistical analysis was performed by the DataAssist TM software (v3.01) used for calculating relative quantitation of gene expression, based on the comparative C T (2 -ΔΔCT ) method. ∗ p -value

    Article Snippet: R. erythropolis strains were cultivated in LBP ( ) for conjugative transfer of DNA and in 7H9 minimal medium (Difco) supplemented with hexanoate at 6 mM (Sigma-Aldrich) as carbon source for induction assays.

    Techniques: Expressing, Quantitative RT-PCR, Software, Quantitation Assay

    Growth of M. smegmatis in various nitrogen sources. Wild-type SMR5 and glnR deletion strain MH1 were grown in the presence of the indicated substances (10 mM final concentration) as sole nitrogen source. Standard 7H9 medium was used as positive control; 7H9 lacking any nitrogen source as negative control (7H9-N). (A) Putative nitrogen sources deduced from microarray results, (B) putative nitrogen sources used by closely related species.

    Journal: BMC Research Notes

    Article Title: Nitrogen starvation-induced transcriptome alterations and influence of transcription regulator mutants in Mycobacterium smegmatis

    doi: 10.1186/1756-0500-6-482

    Figure Lengend Snippet: Growth of M. smegmatis in various nitrogen sources. Wild-type SMR5 and glnR deletion strain MH1 were grown in the presence of the indicated substances (10 mM final concentration) as sole nitrogen source. Standard 7H9 medium was used as positive control; 7H9 lacking any nitrogen source as negative control (7H9-N). (A) Putative nitrogen sources deduced from microarray results, (B) putative nitrogen sources used by closely related species.

    Article Snippet: Mycobacterial strains were grown in Middlebrook 7H9 liquid medium (Difco Laboratories; per 900 ml approx.

    Techniques: Concentration Assay, Positive Control, Negative Control, Microarray

    Kinetics and inducibility of fructose, glucose, and glycerol uptake by M. smegmatis . (A) M. smegmatis mc 2 155 was grown in Middlebrook 7H9 medium in the presence of 2% glycerol (open circles) or 2% fructose (closed circles). Accumulation

    Journal:

    Article Title: A Genomic View of Sugar Transport in Mycobacterium smegmatis and Mycobacterium tuberculosis ▿

    doi: 10.1128/JB.00257-07

    Figure Lengend Snippet: Kinetics and inducibility of fructose, glucose, and glycerol uptake by M. smegmatis . (A) M. smegmatis mc 2 155 was grown in Middlebrook 7H9 medium in the presence of 2% glycerol (open circles) or 2% fructose (closed circles). Accumulation

    Article Snippet: M. smegmatis mc2 155 was grown in liquid cultures using Middlebrook 7H9 medium (Difco) supplemented with 0.2% glycerol and 0.05% Tween 80 or minimal Hartmans-de Bont (HB) medium ( ) at 37°C.

    Techniques:

    Serial monitoring outcomes of RIF stability in L-J medium and 7H9 broth when kept at 37°C and 4°C. (a) Stability of RIF in 7H9 and L-J media when kept at 37°C. (b) Stability of RIF in 7H9 and L-J media when kept at 4°C.

    Journal: Journal of Clinical Microbiology

    Article Title: Rifampin Stability in 7H9 Broth and L?wenstein-Jensen Medium ▿

    doi: 10.1128/JCM.01951-10

    Figure Lengend Snippet: Serial monitoring outcomes of RIF stability in L-J medium and 7H9 broth when kept at 37°C and 4°C. (a) Stability of RIF in 7H9 and L-J media when kept at 37°C. (b) Stability of RIF in 7H9 and L-J media when kept at 4°C.

    Article Snippet: Rifampin was not stable in either L-J medium or 7H9 broth.

    Techniques: