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MiddleBrook Pharmaceuticals middlebrook 7h10 agar
Modified <t>Middlebrook</t> <t>7H10</t> medium containing 250 mg/L MG with mycobacteria primarily isolated from soil. a Sample collected at campus, b sample collected at community, and c collected at natural park. Black arrows indicate mycobacterial colonies, red arrows indicate dried soils
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1) Product Images from "Isolation of nontuberculous mycobacteria from soil using Middlebrook 7H10 agar with increased malachite green concentration"

Article Title: Isolation of nontuberculous mycobacteria from soil using Middlebrook 7H10 agar with increased malachite green concentration

Journal: AMB Express

doi: 10.1186/s13568-017-0373-6

Modified Middlebrook 7H10 medium containing 250 mg/L MG with mycobacteria primarily isolated from soil. a Sample collected at campus, b sample collected at community, and c collected at natural park. Black arrows indicate mycobacterial colonies, red arrows indicate dried soils
Figure Legend Snippet: Modified Middlebrook 7H10 medium containing 250 mg/L MG with mycobacteria primarily isolated from soil. a Sample collected at campus, b sample collected at community, and c collected at natural park. Black arrows indicate mycobacterial colonies, red arrows indicate dried soils

Techniques Used: Modification, Isolation

2) Product Images from "EPSP Synthase-Depleted Cells Are Aromatic Amino Acid Auxotrophs in Mycobacterium smegmatis"

Article Title: EPSP Synthase-Depleted Cells Are Aromatic Amino Acid Auxotrophs in Mycobacterium smegmatis

Journal: Microbiology Spectrum

doi: 10.1128/Spectrum.00009-21

Growth impairment of AroA -knockdown cells depends on growth conditions. (A to H) M. smegmatis growth curves and dilution spots in the presence or absence of anhydrotetracycline (ATc) (100 ng/mL). (A to D) Growth in rich media: liquid LB (curves) and solid LB (dilution spots shown in figure insets). (E to H) Growth in defined media: liquid 7H9 (curves) and solid 7H10 (dilution spots in figure insets). (A and E) Control gene mmpL3 . (B to D and F to H) aroA gene knocked down using sgRNAs directed to locations adjacent to PAM1, PAM2, and PAM3 (B to D and F to H). Growth curves with and without ATc are different with P
Figure Legend Snippet: Growth impairment of AroA -knockdown cells depends on growth conditions. (A to H) M. smegmatis growth curves and dilution spots in the presence or absence of anhydrotetracycline (ATc) (100 ng/mL). (A to D) Growth in rich media: liquid LB (curves) and solid LB (dilution spots shown in figure insets). (E to H) Growth in defined media: liquid 7H9 (curves) and solid 7H10 (dilution spots in figure insets). (A and E) Control gene mmpL3 . (B to D and F to H) aroA gene knocked down using sgRNAs directed to locations adjacent to PAM1, PAM2, and PAM3 (B to D and F to H). Growth curves with and without ATc are different with P

Techniques Used:

The D61W substitution in Ms EPSPS does not impair mycobacterial survival and growth in vitro . (A) Sequence alignment of EPSPS enzymes from M. smegmatis mc 2 155 (Ms), M. tuberculosis H37Rv (Mt), and E. coli CVM N33429PS (Ec). A multiple alignment for these proteins was made using Clustal Omega and then visualized and colored in Jalview. The color code represents the level of conservation of each amino acid, where darker shades of purple represent a higher conservation level. The enzymes from E. coli and M. tuberculosis have 52% and 78% positives and 31% and 68% of identity when aligned to the EPSPS from M. smegmatis , respectively. Amino acids indicated by black arrows were the ones chosen for mutagenesis. (B and C) PCR confirmation of double crossover (DCO) events leading to deletion of the original aroA allele in the M. smegmatis genome from merodiploid strains carrying an extra copy of the WT or mutated aroA gene encoding D61W EPSPS mutant. Genomic DNA was extracted from selected white colonies and used as the templates for PCRs in the presence of a forward primer upstream the 5′ AES (allelic exchange sequence), outside the region of recombination, and an internal reverse primer (see Table 3 ). The PCRs were performed on white colonies selected on LB medium (B) or 7H10 medium without supplementation with AroAAs (C). An amplicon of 1,813 bp was expected for allelic exchange mutants. (B) Lane M: 1 kb plus DNA ladder (Invitrogen). Lane 1: M. smegmatis mc 2 155 genomic DNA (negative control). Lanes 2 to 8: aroA -knockout cells obtained from a merodiploid strain carrying an extra copy of WT aroA gene. Lanes 9 to 10: aroA -knockout cells obtained from a merodiploid strain carrying an extra copy of aroA mutated that encodes the D61W Ms EPSPS protein. (C) Lane 1: M. smegmatis mc 2 155 genomic DNA (negative control). Lanes 2 to 7: aroA -deleted cells from merodiploid strains carrying an extra copy of WT aroA gene ( 2 – 6 ) or a mutant aroA gene encoding D61W EPSPS protein ( 7 ). (D) Control strains and a strain carrying an altered aroA sequence that encodes D61W EPSPS mutant ( aroA _D61W) and that have had the original aroA allele deleted were grown for 12 h in LB medium, under aerobic conditions. Aliquots were taken every 3 h for optical density measurement at 600 nm (OD 600 ). pNIP40::Ø: strain carrying the original aroA allele and an empty copy of the pNIP40/b vector integrated into the mycobacteriophage Ms6 chromosomal integration site; aroA _WT: strain having the original aroA allele deleted but carrying another copy of the WT aroA gene integrated. Error bars are standard deviation (SD) of three biological replicates. Growth curves from control strains pNIP40::Ø and aroA _WT are not statistically different compared to aroA _D61W growth curve at any time point ( P > 0.99 for all comparisons at 0 to 9 h; P = 0.49 for aroA _WT compared to aroA _D61W at 12 h; P > 0.99 for pNIP40::Ø compared to aroA _D61W at 12 h).
Figure Legend Snippet: The D61W substitution in Ms EPSPS does not impair mycobacterial survival and growth in vitro . (A) Sequence alignment of EPSPS enzymes from M. smegmatis mc 2 155 (Ms), M. tuberculosis H37Rv (Mt), and E. coli CVM N33429PS (Ec). A multiple alignment for these proteins was made using Clustal Omega and then visualized and colored in Jalview. The color code represents the level of conservation of each amino acid, where darker shades of purple represent a higher conservation level. The enzymes from E. coli and M. tuberculosis have 52% and 78% positives and 31% and 68% of identity when aligned to the EPSPS from M. smegmatis , respectively. Amino acids indicated by black arrows were the ones chosen for mutagenesis. (B and C) PCR confirmation of double crossover (DCO) events leading to deletion of the original aroA allele in the M. smegmatis genome from merodiploid strains carrying an extra copy of the WT or mutated aroA gene encoding D61W EPSPS mutant. Genomic DNA was extracted from selected white colonies and used as the templates for PCRs in the presence of a forward primer upstream the 5′ AES (allelic exchange sequence), outside the region of recombination, and an internal reverse primer (see Table 3 ). The PCRs were performed on white colonies selected on LB medium (B) or 7H10 medium without supplementation with AroAAs (C). An amplicon of 1,813 bp was expected for allelic exchange mutants. (B) Lane M: 1 kb plus DNA ladder (Invitrogen). Lane 1: M. smegmatis mc 2 155 genomic DNA (negative control). Lanes 2 to 8: aroA -knockout cells obtained from a merodiploid strain carrying an extra copy of WT aroA gene. Lanes 9 to 10: aroA -knockout cells obtained from a merodiploid strain carrying an extra copy of aroA mutated that encodes the D61W Ms EPSPS protein. (C) Lane 1: M. smegmatis mc 2 155 genomic DNA (negative control). Lanes 2 to 7: aroA -deleted cells from merodiploid strains carrying an extra copy of WT aroA gene ( 2 – 6 ) or a mutant aroA gene encoding D61W EPSPS protein ( 7 ). (D) Control strains and a strain carrying an altered aroA sequence that encodes D61W EPSPS mutant ( aroA _D61W) and that have had the original aroA allele deleted were grown for 12 h in LB medium, under aerobic conditions. Aliquots were taken every 3 h for optical density measurement at 600 nm (OD 600 ). pNIP40::Ø: strain carrying the original aroA allele and an empty copy of the pNIP40/b vector integrated into the mycobacteriophage Ms6 chromosomal integration site; aroA _WT: strain having the original aroA allele deleted but carrying another copy of the WT aroA gene integrated. Error bars are standard deviation (SD) of three biological replicates. Growth curves from control strains pNIP40::Ø and aroA _WT are not statistically different compared to aroA _D61W growth curve at any time point ( P > 0.99 for all comparisons at 0 to 9 h; P = 0.49 for aroA _WT compared to aroA _D61W at 12 h; P > 0.99 for pNIP40::Ø compared to aroA _D61W at 12 h).

Techniques Used: In Vitro, Sequencing, Mutagenesis, Polymerase Chain Reaction, Amplification, Negative Control, Knock-Out, Plasmid Preparation, Standard Deviation

aroA -deleted M. smegmatis cells are auxotrophic for aromatic amino acids. (A) Schematic representation of the allelic exchange event in the aroA locus. Two putative genes (MSMEG_1891 and MSMEG_1889) flank the aroA gene (MSMEG_1890) of M. smegmatis . The allelic exchange sequences (AESs) were designed to maintain possible transcriptional and translation regulatory sequences of these two genes. Most of the aroA gene sequence was replaced by a kanamycin resistance cassette (1,252 bp), which was also used as a selective marker for homologous recombination. The positions of primers used in PCRs described in panel B are indicated by black arrows. (B) Confirmation of aroA gene knockout by allelic exchange mutagenesis in merodiploid WT or control strain of M. smegmatis grown on defined 7H10 medium with AroAA supplementation. Double crossover (DCO) events on the original aroA locus in the M. smegmatis genome were confirmed by PCR amplification. Genomic DNA was extracted from selected white colonies and used as the templates for PCRs in the presence of a forward primer upstream the 5′ AES (allelic exchange sequence), outside the region of recombination, and an internal reverse primer (see Fig. 2A and Table 3 ). An amplicon of 1,813 bp is expected for allelic exchange mutants. Lane M: 1 kb plus DNA ladder (Invitrogen). Lanes 1 and 13: M. smegmatis cells grown on 7H10 medium without supplementation: aroA -deleted cells (A) from merodiploid strain containing an extra copy of WT aroA gene (positive controls). Lanes 2 to 12: M. smegmatis cells grown on defined 7H10 medium with AroAA supplementation. Lane 2: M. smegmatis mc 2 155 genomic DNA (negative control). Lanes 3 to 4: aroA -deleted cells from merodiploid strain carrying an extra copy of WT aroA gene. Lanes 5 to 12: aroA -deleted cells from M. smegmatis strain containing an integrated copy of vector pNIP40/b (empty vector, pNIP40::Ø), without an extra copy of aroA gene.
Figure Legend Snippet: aroA -deleted M. smegmatis cells are auxotrophic for aromatic amino acids. (A) Schematic representation of the allelic exchange event in the aroA locus. Two putative genes (MSMEG_1891 and MSMEG_1889) flank the aroA gene (MSMEG_1890) of M. smegmatis . The allelic exchange sequences (AESs) were designed to maintain possible transcriptional and translation regulatory sequences of these two genes. Most of the aroA gene sequence was replaced by a kanamycin resistance cassette (1,252 bp), which was also used as a selective marker for homologous recombination. The positions of primers used in PCRs described in panel B are indicated by black arrows. (B) Confirmation of aroA gene knockout by allelic exchange mutagenesis in merodiploid WT or control strain of M. smegmatis grown on defined 7H10 medium with AroAA supplementation. Double crossover (DCO) events on the original aroA locus in the M. smegmatis genome were confirmed by PCR amplification. Genomic DNA was extracted from selected white colonies and used as the templates for PCRs in the presence of a forward primer upstream the 5′ AES (allelic exchange sequence), outside the region of recombination, and an internal reverse primer (see Fig. 2A and Table 3 ). An amplicon of 1,813 bp is expected for allelic exchange mutants. Lane M: 1 kb plus DNA ladder (Invitrogen). Lanes 1 and 13: M. smegmatis cells grown on 7H10 medium without supplementation: aroA -deleted cells (A) from merodiploid strain containing an extra copy of WT aroA gene (positive controls). Lanes 2 to 12: M. smegmatis cells grown on defined 7H10 medium with AroAA supplementation. Lane 2: M. smegmatis mc 2 155 genomic DNA (negative control). Lanes 3 to 4: aroA -deleted cells from merodiploid strain carrying an extra copy of WT aroA gene. Lanes 5 to 12: aroA -deleted cells from M. smegmatis strain containing an integrated copy of vector pNIP40/b (empty vector, pNIP40::Ø), without an extra copy of aroA gene.

Techniques Used: Sequencing, Marker, Homologous Recombination, Gene Knockout, Mutagenesis, Polymerase Chain Reaction, Amplification, Negative Control, Plasmid Preparation

3) Product Images from "Aspartate aminotransferase Rv3722c governs aspartate-dependent nitrogen metabolism in Mycobacterium tuberculosis"

Article Title: Aspartate aminotransferase Rv3722c governs aspartate-dependent nitrogen metabolism in Mycobacterium tuberculosis

Journal: Nature Communications

doi: 10.1038/s41467-020-15876-8

Rv3722c is conditionally esssential in vitro. a Growth curve of Rv3722c-proficient and -deficient Mtb in 7H9 culture media. Rv3722c-TetON and Rv3722c-control were cultured in Middlebrook 7H9 culture media, with or without 500 ng mL −1 anhydrotetracycline (ATC). Bacterial growth was monitored by optical density at 600 nm. b Western blot showing the depletion of Rv3722c 7H9 culture media. Rv3722c-TetON was cultured in 7H9 with or without ATC for 12 days (corresponding to a ). Protein lysates were analyzed by western blotting, using an α-FLAG antibody. The proteasome subunit β (PrcB) was used as loading control. c Growth curve of Rv3722c-proficient and -deficient Mtb in 7H9 supplemented with casein hydrolysate. As a , but using 7H9 supplemented with 1% casein hydrolysate. d Spot assay of Rv3722c-proficient and -deficient Mtb on solid growth media. A serially diluted Rv3722c-TetON culture (OD 0.1) was spotted onto Middlebrook 7H10 agar with or without 1% casein hydrolysate, in the presence or absence of ATC, and cultured for 2 weeks. e Western blot showing depletion of Rv3722c. As b , but in 7H9 supplemented with 1% casein hydrolysate. f Growth curve of Rv3722c-proficient and -pre-depleted Mtb in 7H9 growth media. Rv3722c-TetON in 7H9 with or without ATC, after predepletion of Rv3722c in 7H9 with 1% casein hydrolysate without ATC. For all growth curves, data are represented as mean −/+ SD of three experimental replicates ( n = 3) representative of at least two independent experiments. See also Supplementary Fig. 1 . Source data are provided as a Source Data file.
Figure Legend Snippet: Rv3722c is conditionally esssential in vitro. a Growth curve of Rv3722c-proficient and -deficient Mtb in 7H9 culture media. Rv3722c-TetON and Rv3722c-control were cultured in Middlebrook 7H9 culture media, with or without 500 ng mL −1 anhydrotetracycline (ATC). Bacterial growth was monitored by optical density at 600 nm. b Western blot showing the depletion of Rv3722c 7H9 culture media. Rv3722c-TetON was cultured in 7H9 with or without ATC for 12 days (corresponding to a ). Protein lysates were analyzed by western blotting, using an α-FLAG antibody. The proteasome subunit β (PrcB) was used as loading control. c Growth curve of Rv3722c-proficient and -deficient Mtb in 7H9 supplemented with casein hydrolysate. As a , but using 7H9 supplemented with 1% casein hydrolysate. d Spot assay of Rv3722c-proficient and -deficient Mtb on solid growth media. A serially diluted Rv3722c-TetON culture (OD 0.1) was spotted onto Middlebrook 7H10 agar with or without 1% casein hydrolysate, in the presence or absence of ATC, and cultured for 2 weeks. e Western blot showing depletion of Rv3722c. As b , but in 7H9 supplemented with 1% casein hydrolysate. f Growth curve of Rv3722c-proficient and -pre-depleted Mtb in 7H9 growth media. Rv3722c-TetON in 7H9 with or without ATC, after predepletion of Rv3722c in 7H9 with 1% casein hydrolysate without ATC. For all growth curves, data are represented as mean −/+ SD of three experimental replicates ( n = 3) representative of at least two independent experiments. See also Supplementary Fig. 1 . Source data are provided as a Source Data file.

Techniques Used: In Vitro, Cell Culture, Western Blot, Spot Test

4) Product Images from "Mycobacterial Cultures Contain Cell Size and Density Specific Sub-populations of Cells with Significant Differential Susceptibility to Antibiotics, Oxidative and Nitrite Stress"

Article Title: Mycobacterial Cultures Contain Cell Size and Density Specific Sub-populations of Cells with Significant Differential Susceptibility to Antibiotics, Oxidative and Nitrite Stress

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2017.00463

Panels from Video S1 on the live cell imaging of Msm short cells generating micro colony and population regeneration potential of the cells in the SCF and NCF . (A) An Msm cell elongates and divides symmetrically to generate two short daughter cells. One of the short daughter cells (green), grew and divided asymmetrically to generate two unequal-sized daughter cells (indicated in cyan and pink). The other short daughter cell (red), grew and divided symmetrically to generate two comparably-sized short daughter cells (indicated in white and yellow). The daughter cells from the earlier asymmetric division (cyan) further divided symmetrically to generate two daughter cells. The short daughter cell (yellow) further grew and divided symmetrically to generate two daughter cells. Arrows show the site of constriction. Cells of length ≤ 2.60 ± 0.25 μm were considered short cells (as per Figure 1B ). (B) Fraction enriched for Msm short cells. (C) Cells from mid-log phase population. (D) Population generated from the short-cells-enriched fraction after reinoculation into Middlebrook 7H9 liquid medium. (E) Population generated from plating of the cells in the short-cells-enriched fraction on Middlebrook 7H10 agar. The compositions of the cells in (D,E) are similar to that in (C) . Arrows indicate short cells. Length of Msm short cells are shown in (B) . (F–I) Acid-fast stained cells in the SCF and NCF and the respective population generated from them after reinoculation into Middlebrook 7H9 media and incubated till the cultures reached 0.6 OD 600nm .
Figure Legend Snippet: Panels from Video S1 on the live cell imaging of Msm short cells generating micro colony and population regeneration potential of the cells in the SCF and NCF . (A) An Msm cell elongates and divides symmetrically to generate two short daughter cells. One of the short daughter cells (green), grew and divided asymmetrically to generate two unequal-sized daughter cells (indicated in cyan and pink). The other short daughter cell (red), grew and divided symmetrically to generate two comparably-sized short daughter cells (indicated in white and yellow). The daughter cells from the earlier asymmetric division (cyan) further divided symmetrically to generate two daughter cells. The short daughter cell (yellow) further grew and divided symmetrically to generate two daughter cells. Arrows show the site of constriction. Cells of length ≤ 2.60 ± 0.25 μm were considered short cells (as per Figure 1B ). (B) Fraction enriched for Msm short cells. (C) Cells from mid-log phase population. (D) Population generated from the short-cells-enriched fraction after reinoculation into Middlebrook 7H9 liquid medium. (E) Population generated from plating of the cells in the short-cells-enriched fraction on Middlebrook 7H10 agar. The compositions of the cells in (D,E) are similar to that in (C) . Arrows indicate short cells. Length of Msm short cells are shown in (B) . (F–I) Acid-fast stained cells in the SCF and NCF and the respective population generated from them after reinoculation into Middlebrook 7H9 media and incubated till the cultures reached 0.6 OD 600nm .

Techniques Used: Live Cell Imaging, Generated, Staining, Incubation

5) Product Images from "Aspartate aminotransferase Rv3722c governs aspartate-dependent nitrogen metabolism in Mycobacterium tuberculosis"

Article Title: Aspartate aminotransferase Rv3722c governs aspartate-dependent nitrogen metabolism in Mycobacterium tuberculosis

Journal: Nature Communications

doi: 10.1038/s41467-020-15876-8

Rv3722c is conditionally esssential in vitro. a Growth curve of Rv3722c-proficient and -deficient Mtb in 7H9 culture media. Rv3722c-TetON and Rv3722c-control were cultured in Middlebrook 7H9 culture media, with or without 500 ng mL −1 anhydrotetracycline (ATC). Bacterial growth was monitored by optical density at 600 nm. b Western blot showing the depletion of Rv3722c 7H9 culture media. Rv3722c-TetON was cultured in 7H9 with or without ATC for 12 days (corresponding to a ). Protein lysates were analyzed by western blotting, using an α-FLAG antibody. The proteasome subunit β (PrcB) was used as loading control. c Growth curve of Rv3722c-proficient and -deficient Mtb in 7H9 supplemented with casein hydrolysate. As a , but using 7H9 supplemented with 1% casein hydrolysate. d Spot assay of Rv3722c-proficient and -deficient Mtb on solid growth media. A serially diluted Rv3722c-TetON culture (OD 0.1) was spotted onto Middlebrook 7H10 agar with or without 1% casein hydrolysate, in the presence or absence of ATC, and cultured for 2 weeks. e Western blot showing depletion of Rv3722c. As b , but in 7H9 supplemented with 1% casein hydrolysate. f Growth curve of Rv3722c-proficient and -pre-depleted Mtb in 7H9 growth media. Rv3722c-TetON in 7H9 with or without ATC, after predepletion of Rv3722c in 7H9 with 1% casein hydrolysate without ATC. For all growth curves, data are represented as mean −/+ SD of three experimental replicates ( n = 3) representative of at least two independent experiments. See also Supplementary Fig. 1 . Source data are provided as a Source Data file.
Figure Legend Snippet: Rv3722c is conditionally esssential in vitro. a Growth curve of Rv3722c-proficient and -deficient Mtb in 7H9 culture media. Rv3722c-TetON and Rv3722c-control were cultured in Middlebrook 7H9 culture media, with or without 500 ng mL −1 anhydrotetracycline (ATC). Bacterial growth was monitored by optical density at 600 nm. b Western blot showing the depletion of Rv3722c 7H9 culture media. Rv3722c-TetON was cultured in 7H9 with or without ATC for 12 days (corresponding to a ). Protein lysates were analyzed by western blotting, using an α-FLAG antibody. The proteasome subunit β (PrcB) was used as loading control. c Growth curve of Rv3722c-proficient and -deficient Mtb in 7H9 supplemented with casein hydrolysate. As a , but using 7H9 supplemented with 1% casein hydrolysate. d Spot assay of Rv3722c-proficient and -deficient Mtb on solid growth media. A serially diluted Rv3722c-TetON culture (OD 0.1) was spotted onto Middlebrook 7H10 agar with or without 1% casein hydrolysate, in the presence or absence of ATC, and cultured for 2 weeks. e Western blot showing depletion of Rv3722c. As b , but in 7H9 supplemented with 1% casein hydrolysate. f Growth curve of Rv3722c-proficient and -pre-depleted Mtb in 7H9 growth media. Rv3722c-TetON in 7H9 with or without ATC, after predepletion of Rv3722c in 7H9 with 1% casein hydrolysate without ATC. For all growth curves, data are represented as mean −/+ SD of three experimental replicates ( n = 3) representative of at least two independent experiments. See also Supplementary Fig. 1 . Source data are provided as a Source Data file.

Techniques Used: In Vitro, Cell Culture, Western Blot, Spot Test

Rv3722c is required for virulence in macrophages and mice. a Timecourse of macrophage infection. Primary murine bone marrow-derived macrophages were treated with control or interferon γ (IFNγ) for activation, followed by infection with Rv3722c-control and Rv3722c-TetON precultured in 7H9 + casein hydrolysate with or without ATC to predeplete Rv3722c (multiplicity of infection of one). The number of colony-forming units (CFUs) was assessed by plating serial dilutions on 7H10 solid media supplemented with casein hydrolysate and ATC. Data are presented as mean +/− SD of three experimental replicates ( n = 3) representative of two independent experiments. b Timecourse of mouse infection. Mice were infected with aerosolized wild-type Mtb and Rv3722c-TetON precultured in Sauton’s without ATC to generate Rv3722c-deficient Mtb . After infection, mice were fed chow without doxycycline. The numbers of CFUs in lung and spleen were assessed by plating serial dilutions on 7H10 solid media with and without ATC to determine the number of non-ATC-dependent mutants. No non-ATC-dependent Rv3722c-TetON mutants were detected at any timepoint. Data are represented as mean +/− SD of five mice ( n = 5), except for the 24 h timepoints ( n = 3) and Rv3722c-TetON 3 weeks in the lung ( n = 4). Open symbols indicate samples in which the bacterial burden was below the limit of detection (
Figure Legend Snippet: Rv3722c is required for virulence in macrophages and mice. a Timecourse of macrophage infection. Primary murine bone marrow-derived macrophages were treated with control or interferon γ (IFNγ) for activation, followed by infection with Rv3722c-control and Rv3722c-TetON precultured in 7H9 + casein hydrolysate with or without ATC to predeplete Rv3722c (multiplicity of infection of one). The number of colony-forming units (CFUs) was assessed by plating serial dilutions on 7H10 solid media supplemented with casein hydrolysate and ATC. Data are presented as mean +/− SD of three experimental replicates ( n = 3) representative of two independent experiments. b Timecourse of mouse infection. Mice were infected with aerosolized wild-type Mtb and Rv3722c-TetON precultured in Sauton’s without ATC to generate Rv3722c-deficient Mtb . After infection, mice were fed chow without doxycycline. The numbers of CFUs in lung and spleen were assessed by plating serial dilutions on 7H10 solid media with and without ATC to determine the number of non-ATC-dependent mutants. No non-ATC-dependent Rv3722c-TetON mutants were detected at any timepoint. Data are represented as mean +/− SD of five mice ( n = 5), except for the 24 h timepoints ( n = 3) and Rv3722c-TetON 3 weeks in the lung ( n = 4). Open symbols indicate samples in which the bacterial burden was below the limit of detection (

Techniques Used: Mouse Assay, Infection, Derivative Assay, Activation Assay

6) Product Images from "Glby, Encoded by MAB_3167c, Is Required for In Vivo Growth of Mycobacteroides abscessus and Exhibits Mild β-Lactamase Activity"

Article Title: Glby, Encoded by MAB_3167c, Is Required for In Vivo Growth of Mycobacteroides abscessus and Exhibits Mild β-Lactamase Activity

Journal: Journal of Bacteriology

doi: 10.1128/jb.00046-22

In vitro growth phenotypes of Δglby . (A) Growth of parent strain Mab ATCC 19977 (WT), Δglby and Complement (COMP) strains in Middlebrook 7H9 broth at 96 h, 37°C, constant shaking at 200 RPM. Culture media only as negative controls (NC) was also included. (B) Time course of optical densities of cultures of WT, Δglby , and COMP measured as absorbance at 600 nm. Each time point represents the average from four distinct replicates and no sample tube was used more than once. Error bars are standard deviation of each sample. (C) Time course of CFU of the three strains. Error bars are standard deviation of each sample. (D) Generation time (time required for a strain to double in CFU) of each strain determined from exponential phase(s) of growth. Two generation times reported for Δglby represent the biphasic exponential growth stages of this strain.
Figure Legend Snippet: In vitro growth phenotypes of Δglby . (A) Growth of parent strain Mab ATCC 19977 (WT), Δglby and Complement (COMP) strains in Middlebrook 7H9 broth at 96 h, 37°C, constant shaking at 200 RPM. Culture media only as negative controls (NC) was also included. (B) Time course of optical densities of cultures of WT, Δglby , and COMP measured as absorbance at 600 nm. Each time point represents the average from four distinct replicates and no sample tube was used more than once. Error bars are standard deviation of each sample. (C) Time course of CFU of the three strains. Error bars are standard deviation of each sample. (D) Generation time (time required for a strain to double in CFU) of each strain determined from exponential phase(s) of growth. Two generation times reported for Δglby represent the biphasic exponential growth stages of this strain.

Techniques Used: In Vitro, Standard Deviation

7) Product Images from "Mechanistic insights into the antimycobacterial action of unani formulation, Qurs Sartan Kafoori"

Article Title: Mechanistic insights into the antimycobacterial action of unani formulation, Qurs Sartan Kafoori

Journal: Journal of Traditional and Complementary Medicine

doi: 10.1016/j.jtcme.2021.07.009

Effect of QSK on cell surface integrity (A) ZN staining images of control and cells treated with QSK. Scale bar depicts 20 μm. (B) Colony morphology of control and cells treated with QSK observed on 7H10-based medium at 10x magnification. Scale bar depicts 20 μm. (C) Cell sedimentation. Left panel shows O.D 600 of control cells and treated with QSK depicted on y axis with respect to time (hours) on x-axis. (D) Nitrocefin hydrolysis of control and cells treated with QSK. Mean of O.D 485 ± SD of three independent sets are depicted on y axis.
Figure Legend Snippet: Effect of QSK on cell surface integrity (A) ZN staining images of control and cells treated with QSK. Scale bar depicts 20 μm. (B) Colony morphology of control and cells treated with QSK observed on 7H10-based medium at 10x magnification. Scale bar depicts 20 μm. (C) Cell sedimentation. Left panel shows O.D 600 of control cells and treated with QSK depicted on y axis with respect to time (hours) on x-axis. (D) Nitrocefin hydrolysis of control and cells treated with QSK. Mean of O.D 485 ± SD of three independent sets are depicted on y axis.

Techniques Used: Staining, Sedimentation

8) Product Images from "Inactivation of nuclear factor κB by MIP-based drug combinations augments cell death of breast cancer cells"

Article Title: Inactivation of nuclear factor κB by MIP-based drug combinations augments cell death of breast cancer cells

Journal: Drug Design, Development and Therapy

doi: 10.2147/DDDT.S141925

Disc diffusion assay of MIP against ACA and CDDP with Neo as control. Note: Middlebrook 7H10 agar plates of discs were impregnated with ACA (3.0, 10.0 and 20.0 mM), CDDP (30, 100 and 200 mM) and Neo (0.5, 1.0, 3.0, 5.0, 8.0 and 10.0 μg/mL). Abbreviations: ACA, acetoxychavicol acetate; CDDP, cisplatin; MIP, Mycobacterium indicus pranii ; Neo, neomycin.
Figure Legend Snippet: Disc diffusion assay of MIP against ACA and CDDP with Neo as control. Note: Middlebrook 7H10 agar plates of discs were impregnated with ACA (3.0, 10.0 and 20.0 mM), CDDP (30, 100 and 200 mM) and Neo (0.5, 1.0, 3.0, 5.0, 8.0 and 10.0 μg/mL). Abbreviations: ACA, acetoxychavicol acetate; CDDP, cisplatin; MIP, Mycobacterium indicus pranii ; Neo, neomycin.

Techniques Used: Diffusion-based Assay

9) Product Images from "Methionine Sulfoxide Reductase A (MsrA) Deficiency Affects the Survival of Mycobacterium smegmatis within Macrophages"

Article Title: Methionine Sulfoxide Reductase A (MsrA) Deficiency Affects the Survival of Mycobacterium smegmatis within Macrophages

Journal: Journal of Bacteriology

doi: 10.1128/JB.186.11.3590-3598.2004

Survival of M. smegmatis wild-type strain MSWt, msrA mutant 97 (= MSΔ msrA ), and a complemented msrA mutant (MSΔ msrA /c) in murine macrophage-like J774A.1 cells. Naïve or IFN-γ-stimulated macrophages were infected at an MOI of 1 for 4 h, washed, lysed at intervals, and plated onto Middlebrook 7H10 agar plates to determine the number of CFU. ○, strain MSWt; •, mutant MSΔ msrA ; ▾, complemented strain MSΔ msrA /c. There are no error bars for some data points because of the small standard errors.
Figure Legend Snippet: Survival of M. smegmatis wild-type strain MSWt, msrA mutant 97 (= MSΔ msrA ), and a complemented msrA mutant (MSΔ msrA /c) in murine macrophage-like J774A.1 cells. Naïve or IFN-γ-stimulated macrophages were infected at an MOI of 1 for 4 h, washed, lysed at intervals, and plated onto Middlebrook 7H10 agar plates to determine the number of CFU. ○, strain MSWt; •, mutant MSΔ msrA ; ▾, complemented strain MSΔ msrA /c. There are no error bars for some data points because of the small standard errors.

Techniques Used: Mutagenesis, Infection

10) Product Images from "Inactivation of a New Potassium Channel Increases Rifampicin Resistance and Induces Collateral Sensitivity to Hydrophilic Antibiotics in Mycobacterium smegmatis"

Article Title: Inactivation of a New Potassium Channel Increases Rifampicin Resistance and Induces Collateral Sensitivity to Hydrophilic Antibiotics in Mycobacterium smegmatis

Journal: Antibiotics

doi: 10.3390/antibiotics11040509

K + effect on the growth of mc 2 155 and its ∆ mchK derivative under different conditions. ( a ) Effect of K + addition (10–200 mM KCl) on the growth of mc 2 155 and ∆ mchK at neutral pH. The ability of high K + concentrations to restore the growth of the ∆ mchK mutant was measured in cultures at mid-exponential phase (12 h). ( b ) Effect of extracellular pH (5–8) on the growth of mc 2 155 and the ∆ mchK mutant in Middlebrook 7H9 liquid medium after 12 h of incubation (initial OD 600 0.05). ( c ) Effect of K + addition (10–100 mM KCl) on the growth of mc 2 155 and ∆ mchK at pH 5.5. The OD 600 was measured after 12 h of incubation at 37 °C. Error bars indicate 95% CI (confidence intervals); (*) p
Figure Legend Snippet: K + effect on the growth of mc 2 155 and its ∆ mchK derivative under different conditions. ( a ) Effect of K + addition (10–200 mM KCl) on the growth of mc 2 155 and ∆ mchK at neutral pH. The ability of high K + concentrations to restore the growth of the ∆ mchK mutant was measured in cultures at mid-exponential phase (12 h). ( b ) Effect of extracellular pH (5–8) on the growth of mc 2 155 and the ∆ mchK mutant in Middlebrook 7H9 liquid medium after 12 h of incubation (initial OD 600 0.05). ( c ) Effect of K + addition (10–100 mM KCl) on the growth of mc 2 155 and ∆ mchK at pH 5.5. The OD 600 was measured after 12 h of incubation at 37 °C. Error bars indicate 95% CI (confidence intervals); (*) p

Techniques Used: Mutagenesis, Incubation

11) Product Images from "Inactivation of a New Potassium Channel Increases Rifampicin Resistance and Induces Collateral Sensitivity to Hydrophilic Antibiotics in Mycobacterium smegmatis"

Article Title: Inactivation of a New Potassium Channel Increases Rifampicin Resistance and Induces Collateral Sensitivity to Hydrophilic Antibiotics in Mycobacterium smegmatis

Journal: Antibiotics

doi: 10.3390/antibiotics11040509

K + effect on the growth of mc 2 155 and its ∆ mchK derivative under different conditions. ( a ) Effect of K + addition (10–200 mM KCl) on the growth of mc 2 155 and ∆ mchK at neutral pH. The ability of high K + concentrations to restore the growth of the ∆ mchK mutant was measured in cultures at mid-exponential phase (12 h). ( b ) Effect of extracellular pH (5–8) on the growth of mc 2 155 and the ∆ mchK mutant in Middlebrook 7H9 liquid medium after 12 h of incubation (initial OD 600 0.05). ( c ) Effect of K + addition (10–100 mM KCl) on the growth of mc 2 155 and ∆ mchK at pH 5.5. The OD 600 was measured after 12 h of incubation at 37 °C. Error bars indicate 95% CI (confidence intervals); (*) p
Figure Legend Snippet: K + effect on the growth of mc 2 155 and its ∆ mchK derivative under different conditions. ( a ) Effect of K + addition (10–200 mM KCl) on the growth of mc 2 155 and ∆ mchK at neutral pH. The ability of high K + concentrations to restore the growth of the ∆ mchK mutant was measured in cultures at mid-exponential phase (12 h). ( b ) Effect of extracellular pH (5–8) on the growth of mc 2 155 and the ∆ mchK mutant in Middlebrook 7H9 liquid medium after 12 h of incubation (initial OD 600 0.05). ( c ) Effect of K + addition (10–100 mM KCl) on the growth of mc 2 155 and ∆ mchK at pH 5.5. The OD 600 was measured after 12 h of incubation at 37 °C. Error bars indicate 95% CI (confidence intervals); (*) p

Techniques Used: Mutagenesis, Incubation

12) Product Images from "Inactivation of a New Potassium Channel Increases Rifampicin Resistance and Induces Collateral Sensitivity to Hydrophilic Antibiotics in Mycobacterium smegmatis"

Article Title: Inactivation of a New Potassium Channel Increases Rifampicin Resistance and Induces Collateral Sensitivity to Hydrophilic Antibiotics in Mycobacterium smegmatis

Journal: Antibiotics

doi: 10.3390/antibiotics11040509

K + effect on the growth of mc 2 155 and its ∆ mchK derivative under different conditions. ( a ) Effect of K + addition (10–200 mM KCl) on the growth of mc 2 155 and ∆ mchK at neutral pH. The ability of high K + concentrations to restore the growth of the ∆ mchK mutant was measured in cultures at mid-exponential phase (12 h). ( b ) Effect of extracellular pH (5–8) on the growth of mc 2 155 and the ∆ mchK mutant in Middlebrook 7H9 liquid medium after 12 h of incubation (initial OD 600 0.05). ( c ) Effect of K + addition (10–100 mM KCl) on the growth of mc 2 155 and ∆ mchK at pH 5.5. The OD 600 was measured after 12 h of incubation at 37 °C. Error bars indicate 95% CI (confidence intervals); (*) p
Figure Legend Snippet: K + effect on the growth of mc 2 155 and its ∆ mchK derivative under different conditions. ( a ) Effect of K + addition (10–200 mM KCl) on the growth of mc 2 155 and ∆ mchK at neutral pH. The ability of high K + concentrations to restore the growth of the ∆ mchK mutant was measured in cultures at mid-exponential phase (12 h). ( b ) Effect of extracellular pH (5–8) on the growth of mc 2 155 and the ∆ mchK mutant in Middlebrook 7H9 liquid medium after 12 h of incubation (initial OD 600 0.05). ( c ) Effect of K + addition (10–100 mM KCl) on the growth of mc 2 155 and ∆ mchK at pH 5.5. The OD 600 was measured after 12 h of incubation at 37 °C. Error bars indicate 95% CI (confidence intervals); (*) p

Techniques Used: Mutagenesis, Incubation

13) Product Images from "Essential Role of the ESX-5 Secretion System in Outer Membrane Permeability of Pathogenic Mycobacteria"

Article Title: Essential Role of the ESX-5 Secretion System in Outer Membrane Permeability of Pathogenic Mycobacteria

Journal: PLoS Genetics

doi: 10.1371/journal.pgen.1005190

Expression of MycP5 is essential for growth of M . bovis BCG. A, B) The BCG-Pasteur c- mycP5 -tet-on (A) and c- mycP5 -tet-off (B) mutants were grown for 21 days on Middlebrook 7H10 agar plates containing the indicated ATc concentrations. Full growth of c- mycP5 -tet-on was only observed at 10 μg/ml ATc, whereas this concentration of ATc did not completely abolish colony growth of c- mycP5 -tet-off. C, D) Resazurin reduction is dependent on ATc-induced expression/repression of mycP5 . Cells of the BCG-Pasteur c- mycP5 -tet-on (C), or c- mycP5- tet-off (D) mutants were grown as liquid cultures in 96-well microtiter plates for 6 days at 37°C at the indicated ATc concentrations, after which 10% Alamar Blue was added and fluorescence (585 nm) was measured after 16 h incubation to determine metabolic activity as a correlate of growth. Values are means of triplicates; error bars represent the standard deviation.
Figure Legend Snippet: Expression of MycP5 is essential for growth of M . bovis BCG. A, B) The BCG-Pasteur c- mycP5 -tet-on (A) and c- mycP5 -tet-off (B) mutants were grown for 21 days on Middlebrook 7H10 agar plates containing the indicated ATc concentrations. Full growth of c- mycP5 -tet-on was only observed at 10 μg/ml ATc, whereas this concentration of ATc did not completely abolish colony growth of c- mycP5 -tet-off. C, D) Resazurin reduction is dependent on ATc-induced expression/repression of mycP5 . Cells of the BCG-Pasteur c- mycP5 -tet-on (C), or c- mycP5- tet-off (D) mutants were grown as liquid cultures in 96-well microtiter plates for 6 days at 37°C at the indicated ATc concentrations, after which 10% Alamar Blue was added and fluorescence (585 nm) was measured after 16 h incubation to determine metabolic activity as a correlate of growth. Values are means of triplicates; error bars represent the standard deviation.

Techniques Used: Expressing, Concentration Assay, Fluorescence, Incubation, Activity Assay, Standard Deviation

14) Product Images from "Aspartate aminotransferase Rv3722c governs aspartate-dependent nitrogen metabolism in Mycobacterium tuberculosis"

Article Title: Aspartate aminotransferase Rv3722c governs aspartate-dependent nitrogen metabolism in Mycobacterium tuberculosis

Journal: Nature Communications

doi: 10.1038/s41467-020-15876-8

Rv3722c is conditionally esssential in vitro. a Growth curve of Rv3722c-proficient and -deficient Mtb in 7H9 culture media. Rv3722c-TetON and Rv3722c-control were cultured in Middlebrook 7H9 culture media, with or without 500 ng mL −1 anhydrotetracycline (ATC). Bacterial growth was monitored by optical density at 600 nm. b Western blot showing the depletion of Rv3722c 7H9 culture media. Rv3722c-TetON was cultured in 7H9 with or without ATC for 12 days (corresponding to a ). Protein lysates were analyzed by western blotting, using an α-FLAG antibody. The proteasome subunit β (PrcB) was used as loading control. c Growth curve of Rv3722c-proficient and -deficient Mtb in 7H9 supplemented with casein hydrolysate. As a , but using 7H9 supplemented with 1% casein hydrolysate. d Spot assay of Rv3722c-proficient and -deficient Mtb on solid growth media. A serially diluted Rv3722c-TetON culture (OD 0.1) was spotted onto Middlebrook 7H10 agar with or without 1% casein hydrolysate, in the presence or absence of ATC, and cultured for 2 weeks. e Western blot showing depletion of Rv3722c. As b , but in 7H9 supplemented with 1% casein hydrolysate. f Growth curve of Rv3722c-proficient and -pre-depleted Mtb in 7H9 growth media. Rv3722c-TetON in 7H9 with or without ATC, after predepletion of Rv3722c in 7H9 with 1% casein hydrolysate without ATC. For all growth curves, data are represented as mean −/+ SD of three experimental replicates ( n = 3) representative of at least two independent experiments. See also Supplementary Fig. 1 . Source data are provided as a Source Data file.
Figure Legend Snippet: Rv3722c is conditionally esssential in vitro. a Growth curve of Rv3722c-proficient and -deficient Mtb in 7H9 culture media. Rv3722c-TetON and Rv3722c-control were cultured in Middlebrook 7H9 culture media, with or without 500 ng mL −1 anhydrotetracycline (ATC). Bacterial growth was monitored by optical density at 600 nm. b Western blot showing the depletion of Rv3722c 7H9 culture media. Rv3722c-TetON was cultured in 7H9 with or without ATC for 12 days (corresponding to a ). Protein lysates were analyzed by western blotting, using an α-FLAG antibody. The proteasome subunit β (PrcB) was used as loading control. c Growth curve of Rv3722c-proficient and -deficient Mtb in 7H9 supplemented with casein hydrolysate. As a , but using 7H9 supplemented with 1% casein hydrolysate. d Spot assay of Rv3722c-proficient and -deficient Mtb on solid growth media. A serially diluted Rv3722c-TetON culture (OD 0.1) was spotted onto Middlebrook 7H10 agar with or without 1% casein hydrolysate, in the presence or absence of ATC, and cultured for 2 weeks. e Western blot showing depletion of Rv3722c. As b , but in 7H9 supplemented with 1% casein hydrolysate. f Growth curve of Rv3722c-proficient and -pre-depleted Mtb in 7H9 growth media. Rv3722c-TetON in 7H9 with or without ATC, after predepletion of Rv3722c in 7H9 with 1% casein hydrolysate without ATC. For all growth curves, data are represented as mean −/+ SD of three experimental replicates ( n = 3) representative of at least two independent experiments. See also Supplementary Fig. 1 . Source data are provided as a Source Data file.

Techniques Used: In Vitro, Cell Culture, Western Blot, Spot Test

Rv3722c is required for virulence in macrophages and mice. a Timecourse of macrophage infection. Primary murine bone marrow-derived macrophages were treated with control or interferon γ (IFNγ) for activation, followed by infection with Rv3722c-control and Rv3722c-TetON precultured in 7H9 + casein hydrolysate with or without ATC to predeplete Rv3722c (multiplicity of infection of one). The number of colony-forming units (CFUs) was assessed by plating serial dilutions on 7H10 solid media supplemented with casein hydrolysate and ATC. Data are presented as mean +/− SD of three experimental replicates ( n = 3) representative of two independent experiments. b Timecourse of mouse infection. Mice were infected with aerosolized wild-type Mtb and Rv3722c-TetON precultured in Sauton’s without ATC to generate Rv3722c-deficient Mtb . After infection, mice were fed chow without doxycycline. The numbers of CFUs in lung and spleen were assessed by plating serial dilutions on 7H10 solid media with and without ATC to determine the number of non-ATC-dependent mutants. No non-ATC-dependent Rv3722c-TetON mutants were detected at any timepoint. Data are represented as mean +/− SD of five mice ( n = 5), except for the 24 h timepoints ( n = 3) and Rv3722c-TetON 3 weeks in the lung ( n = 4). Open symbols indicate samples in which the bacterial burden was below the limit of detection (
Figure Legend Snippet: Rv3722c is required for virulence in macrophages and mice. a Timecourse of macrophage infection. Primary murine bone marrow-derived macrophages were treated with control or interferon γ (IFNγ) for activation, followed by infection with Rv3722c-control and Rv3722c-TetON precultured in 7H9 + casein hydrolysate with or without ATC to predeplete Rv3722c (multiplicity of infection of one). The number of colony-forming units (CFUs) was assessed by plating serial dilutions on 7H10 solid media supplemented with casein hydrolysate and ATC. Data are presented as mean +/− SD of three experimental replicates ( n = 3) representative of two independent experiments. b Timecourse of mouse infection. Mice were infected with aerosolized wild-type Mtb and Rv3722c-TetON precultured in Sauton’s without ATC to generate Rv3722c-deficient Mtb . After infection, mice were fed chow without doxycycline. The numbers of CFUs in lung and spleen were assessed by plating serial dilutions on 7H10 solid media with and without ATC to determine the number of non-ATC-dependent mutants. No non-ATC-dependent Rv3722c-TetON mutants were detected at any timepoint. Data are represented as mean +/− SD of five mice ( n = 5), except for the 24 h timepoints ( n = 3) and Rv3722c-TetON 3 weeks in the lung ( n = 4). Open symbols indicate samples in which the bacterial burden was below the limit of detection (

Techniques Used: Mouse Assay, Infection, Derivative Assay, Activation Assay

15) Product Images from "Mutations in Rv2983 as a novel determinant of resistance to nitroimidazole drugs in Mycobacterium tuberculosis"

Article Title: Mutations in Rv2983 as a novel determinant of resistance to nitroimidazole drugs in Mycobacterium tuberculosis

Journal: bioRxiv

doi: 10.1101/457754

Mutation of Rv2983 causes growth inhibition on commercial 7H10 agar and LJ slants, but not on commercial 7H11 agar. Aliquots of M. tuberculosis cultures were spread on various solid media purchased commercially after serial 10-fold dilutions. A-B. Mean CFU counts on 7H10 (A) and 7H11 (B) agar plates; C. Colonies on 7H10 agar plates after 21 (a) and 35 (b) days of incubation, and on 7H11 agar plates after 21 (c) days of incubation; D. Colonies on LJ slants inoculated with serially diluted aliquots after 28 and 35 days of incubation. 1: H37Rv wild type; 2: B101 mutant (Δ Rv2983 , A198P); 3: B101 mutant complemented with Rv2983 behind the native promoter; 4: B101 mutant complemented with Rv2983 behind the hsp60 promoter; 5. K91 mutant (Δ ddn , IS6110 ins in D108).
Figure Legend Snippet: Mutation of Rv2983 causes growth inhibition on commercial 7H10 agar and LJ slants, but not on commercial 7H11 agar. Aliquots of M. tuberculosis cultures were spread on various solid media purchased commercially after serial 10-fold dilutions. A-B. Mean CFU counts on 7H10 (A) and 7H11 (B) agar plates; C. Colonies on 7H10 agar plates after 21 (a) and 35 (b) days of incubation, and on 7H11 agar plates after 21 (c) days of incubation; D. Colonies on LJ slants inoculated with serially diluted aliquots after 28 and 35 days of incubation. 1: H37Rv wild type; 2: B101 mutant (Δ Rv2983 , A198P); 3: B101 mutant complemented with Rv2983 behind the native promoter; 4: B101 mutant complemented with Rv2983 behind the hsp60 promoter; 5. K91 mutant (Δ ddn , IS6110 ins in D108).

Techniques Used: Mutagenesis, Inhibition, Incubation

16) Product Images from "Alginate-Capped Silver Nanoparticles as a Potent Anti-mycobacterial Agent Against Mycobacterium tuberculosis"

Article Title: Alginate-Capped Silver Nanoparticles as a Potent Anti-mycobacterial Agent Against Mycobacterium tuberculosis

Journal: Frontiers in Pharmacology

doi: 10.3389/fphar.2021.746496

ALG-AgNPs effectively suppressed the growth of dormant-like bacilli in vitro . (A–B) The Mtb strain H37Rv was cultured with or without hypoxia to induce non-replicating persistence (NRP) or not. Then, the gene expression levels associated with the NRP state were assessed by QPCR, specifically (A) hspX and (B) esat-6 . (C) The bacteria were treated with PBS, rifampicin (RIF) (1 μg/ml), isoniazid (INH) (0.2 μg/ml), or different amounts of ALG-AgNPs (25, 50, and 100 μg/ml) in the NRP state or not (O 2 -replete) for 5 days. The bacterial growth was determined by CFU analysis on Middlebrook 7H10 agar and expressed as the number of CFU × 10 4 /ml. NRP: non-replicating persistence. RIF: rifampicin. INH: isoniazid. Data represent mean ± SEM of 3 experiments. * p
Figure Legend Snippet: ALG-AgNPs effectively suppressed the growth of dormant-like bacilli in vitro . (A–B) The Mtb strain H37Rv was cultured with or without hypoxia to induce non-replicating persistence (NRP) or not. Then, the gene expression levels associated with the NRP state were assessed by QPCR, specifically (A) hspX and (B) esat-6 . (C) The bacteria were treated with PBS, rifampicin (RIF) (1 μg/ml), isoniazid (INH) (0.2 μg/ml), or different amounts of ALG-AgNPs (25, 50, and 100 μg/ml) in the NRP state or not (O 2 -replete) for 5 days. The bacterial growth was determined by CFU analysis on Middlebrook 7H10 agar and expressed as the number of CFU × 10 4 /ml. NRP: non-replicating persistence. RIF: rifampicin. INH: isoniazid. Data represent mean ± SEM of 3 experiments. * p

Techniques Used: In Vitro, Cell Culture, Expressing, Real-time Polymerase Chain Reaction

ALG-AgNPs significantly inhibit the growth of cytosolic Mycobacterium tuberculosis in a macrophage infection model. (A) Schematic diagram of the experimental procedures. (B–E) THP-1 cells were differentiated into macrophages by PMA stimulation for 3 days, then infected with different strains of Mtb at a MOI of 1 for 24 h. After treatment with PBS, or different amounts of ALG-AgNPs (25, 50, and 100 μg/ml), or rifampicin (RIF) (1 μg/ml) for 5 days, the cells were lysed and plated on 7H10 agar plates. The bacterial growth was determined by CFU of (B) H37Rv, (C) W6 (Beijing strain), (D) KVGH264 (MDR), and (E) TCHL017 (XDR). MDR: Multidrug-resistant TB; XDR: extensively drug-resistant TB. Data represent mean ± SEM of 3 experiments. *** p
Figure Legend Snippet: ALG-AgNPs significantly inhibit the growth of cytosolic Mycobacterium tuberculosis in a macrophage infection model. (A) Schematic diagram of the experimental procedures. (B–E) THP-1 cells were differentiated into macrophages by PMA stimulation for 3 days, then infected with different strains of Mtb at a MOI of 1 for 24 h. After treatment with PBS, or different amounts of ALG-AgNPs (25, 50, and 100 μg/ml), or rifampicin (RIF) (1 μg/ml) for 5 days, the cells were lysed and plated on 7H10 agar plates. The bacterial growth was determined by CFU of (B) H37Rv, (C) W6 (Beijing strain), (D) KVGH264 (MDR), and (E) TCHL017 (XDR). MDR: Multidrug-resistant TB; XDR: extensively drug-resistant TB. Data represent mean ± SEM of 3 experiments. *** p

Techniques Used: Infection

ALG-AgNPs are a safe and effective therapeutic drug in zebrafish and mouse TB animal models. (A–C) After microinjection of M. marinum -DsRed into the caudal vein of zebrafish larvae, the infected larvae (5 fish/group/experiment) were treated with PBS, 100 μg/ml rifampicin (RIF) or 200 μg/ml ALG-AgNPs for 5 days. All data for zebrafish were from two experiments. (A) At 5 days post infection (dpi), the red fluorescence signal from M. marinum -DsRed infection was observed by fluorescence microscopy. Scale bar represents 1 mm. (B) The fluorescence signals of M. marinum -DsRed from zebrafish larvae treated with PBS ( n = 10), rifampicin ( n = 10) or ALG-AgNPs ( n = 10) were quantified using ImageJ software and expressed as fluorescence pixels per larvae. (C) For determination of bacterial burdens, five fish/group/experiment were pooled together and homogenized in 1 ml PBS containing 1% SDS. The bacterial burdens of homogenates from infected larvae were diluted 10 × and 100 × in triplicates and enumerated by CFU analysis of two experiments. (D) BALB/c mice were intravenously infected with Mtb strain H37Ra for 14 days, then treated with PBS ( n = 5), 10 mg/kg ( n = 5) or 50 mg/kg ( n = 5) of ALG-AgNPs, or 10 mg/kg rifampicin ( n = 5) for another 14 days, then the mice were euthanized. The lungs of treated mice were collected and homogenized. The homogenates were plated on 7H10 agar plates. Bacterial growth in the lungs was determined by CFU analysis. Data in (B–D) represent mean ± SEM. * p
Figure Legend Snippet: ALG-AgNPs are a safe and effective therapeutic drug in zebrafish and mouse TB animal models. (A–C) After microinjection of M. marinum -DsRed into the caudal vein of zebrafish larvae, the infected larvae (5 fish/group/experiment) were treated with PBS, 100 μg/ml rifampicin (RIF) or 200 μg/ml ALG-AgNPs for 5 days. All data for zebrafish were from two experiments. (A) At 5 days post infection (dpi), the red fluorescence signal from M. marinum -DsRed infection was observed by fluorescence microscopy. Scale bar represents 1 mm. (B) The fluorescence signals of M. marinum -DsRed from zebrafish larvae treated with PBS ( n = 10), rifampicin ( n = 10) or ALG-AgNPs ( n = 10) were quantified using ImageJ software and expressed as fluorescence pixels per larvae. (C) For determination of bacterial burdens, five fish/group/experiment were pooled together and homogenized in 1 ml PBS containing 1% SDS. The bacterial burdens of homogenates from infected larvae were diluted 10 × and 100 × in triplicates and enumerated by CFU analysis of two experiments. (D) BALB/c mice were intravenously infected with Mtb strain H37Ra for 14 days, then treated with PBS ( n = 5), 10 mg/kg ( n = 5) or 50 mg/kg ( n = 5) of ALG-AgNPs, or 10 mg/kg rifampicin ( n = 5) for another 14 days, then the mice were euthanized. The lungs of treated mice were collected and homogenized. The homogenates were plated on 7H10 agar plates. Bacterial growth in the lungs was determined by CFU analysis. Data in (B–D) represent mean ± SEM. * p

Techniques Used: Infection, Fluorescence In Situ Hybridization, Fluorescence, Microscopy, Software, Mouse Assay

17) Product Images from "Transcription Repressor Protein ZBTB25 Associates with HDAC1-Sin3a Complex in Mycobacterium tuberculosis-Infected Macrophages, and Its Inhibition Clears Pathogen by Autophagy"

Article Title: Transcription Repressor Protein ZBTB25 Associates with HDAC1-Sin3a Complex in Mycobacterium tuberculosis-Infected Macrophages, and Its Inhibition Clears Pathogen by Autophagy

Journal: mSphere

doi: 10.1128/mSphere.00036-21

Treatment of infected macrophages with DP (ZBTB25 inhibitor) and CI994 (HDAC1 inhibitor) enhanced the intracellular clearance of M. tuberculosis . THP-1-derived macrophages were infected with M. tuberculosis H37Rv. After M. tuberculosis infection, cells were treated with DP (20 μM) and/or CI994 (15 μM) or rifampicin (1 μg/ml) for 24 h. (A) Intracellular bacterial viability was determined based on the number of CFU. Isolation of bacilli from the macrophages was carried out at 24 h. The number of viable bacilli in each of the plate was assayed by plating lysed macrophages on 7H10 agar plates and incubating the plates at 37°C for 3 weeks and counting the CFU. Values are shown from 3 independent experiments (mean ± SD). *, survival is significantly different from infected samples (IF); P ≤ 0.05. (B) qPCR for the expression of the IL-12B mRNA transcript (normalized to β-actin mRNA expression). *#, IL-12B expression is significantly different from uninfected (UIF) and infected samples (IF), respectively; P ≤ 0.05. (C) ELISA of IL-12p40 after inhibitor treatment. *#, IL-12p40 levels are significantly different from uninfected (UIF) and infected samples (IF), respectively; P ≤ 0.05. (D) DP treatment blocks the recruitment of the HDAC1 silencing complex to the IL-12B promoter. Status of (i) ZBTB25, (ii) HDAC1, and (iii) Sin3a on the IL-12B promoter by ChIP PCR. (E) Immuno-cytochemical imaging shows HDAC1 does not colocalize with ZBTB25 inside the macrophage nucleus after DP treatment. (F) Intracellular viability of M. tuberculosis in infected PBMC after treatment with DP, CI994, and rifampicin was determined by counting the number of CFU. *, survival is significantly different from infected sample (IF); P ≤ 0.05.
Figure Legend Snippet: Treatment of infected macrophages with DP (ZBTB25 inhibitor) and CI994 (HDAC1 inhibitor) enhanced the intracellular clearance of M. tuberculosis . THP-1-derived macrophages were infected with M. tuberculosis H37Rv. After M. tuberculosis infection, cells were treated with DP (20 μM) and/or CI994 (15 μM) or rifampicin (1 μg/ml) for 24 h. (A) Intracellular bacterial viability was determined based on the number of CFU. Isolation of bacilli from the macrophages was carried out at 24 h. The number of viable bacilli in each of the plate was assayed by plating lysed macrophages on 7H10 agar plates and incubating the plates at 37°C for 3 weeks and counting the CFU. Values are shown from 3 independent experiments (mean ± SD). *, survival is significantly different from infected samples (IF); P ≤ 0.05. (B) qPCR for the expression of the IL-12B mRNA transcript (normalized to β-actin mRNA expression). *#, IL-12B expression is significantly different from uninfected (UIF) and infected samples (IF), respectively; P ≤ 0.05. (C) ELISA of IL-12p40 after inhibitor treatment. *#, IL-12p40 levels are significantly different from uninfected (UIF) and infected samples (IF), respectively; P ≤ 0.05. (D) DP treatment blocks the recruitment of the HDAC1 silencing complex to the IL-12B promoter. Status of (i) ZBTB25, (ii) HDAC1, and (iii) Sin3a on the IL-12B promoter by ChIP PCR. (E) Immuno-cytochemical imaging shows HDAC1 does not colocalize with ZBTB25 inside the macrophage nucleus after DP treatment. (F) Intracellular viability of M. tuberculosis in infected PBMC after treatment with DP, CI994, and rifampicin was determined by counting the number of CFU. *, survival is significantly different from infected sample (IF); P ≤ 0.05.

Techniques Used: Infection, Derivative Assay, Isolation, IF-P, Real-time Polymerase Chain Reaction, Expressing, Enzyme-linked Immunosorbent Assay, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Imaging

IL-12p40-mediated activation of autophagy depends on the JAK-STAT pathway. (A) Status of pJAK2 and pSTAT4 in infected macrophages after treatment with DP and CI994. (B and C) Densitometric analysis of pJAK2 (B) and STAT4 (C) bands normalized with that of β-actin. Each value represents mean ± SD from triplicate measurements. *, band intensity is significantly different from infected (IF) samples individually treated with inhibitors, respectively; P ≤ 0.05. (D) Infected THP-1-derived macrophage cells were treated with JAK2 inhibitor NSC33994 along with DP and CI994. THP-1-derived macrophages deficient in STAT4 were treated with DP and CI994. Intracellular bacterial viability was determined based on the number of CFU. Isolation of bacilli from the macrophages was carried out at 24 h. The number of viable bacilli in each of the plate was assayed by plating lysed macrophages on 7H10 agar plates and incubating the plates at 37°C for 3 weeks and counting the CFU. Each value represents mean ± SD from triplicate measurements. *, survival is significantly different from infected (IF) sample; P ≤ 0.05.
Figure Legend Snippet: IL-12p40-mediated activation of autophagy depends on the JAK-STAT pathway. (A) Status of pJAK2 and pSTAT4 in infected macrophages after treatment with DP and CI994. (B and C) Densitometric analysis of pJAK2 (B) and STAT4 (C) bands normalized with that of β-actin. Each value represents mean ± SD from triplicate measurements. *, band intensity is significantly different from infected (IF) samples individually treated with inhibitors, respectively; P ≤ 0.05. (D) Infected THP-1-derived macrophage cells were treated with JAK2 inhibitor NSC33994 along with DP and CI994. THP-1-derived macrophages deficient in STAT4 were treated with DP and CI994. Intracellular bacterial viability was determined based on the number of CFU. Isolation of bacilli from the macrophages was carried out at 24 h. The number of viable bacilli in each of the plate was assayed by plating lysed macrophages on 7H10 agar plates and incubating the plates at 37°C for 3 weeks and counting the CFU. Each value represents mean ± SD from triplicate measurements. *, survival is significantly different from infected (IF) sample; P ≤ 0.05.

Techniques Used: Activation Assay, Infection, Derivative Assay, Isolation

18) Product Images from "Host MKRN1-Mediated Mycobacterial PPE Protein Ubiquitination Suppresses Innate Immune Response"

Article Title: Host MKRN1-Mediated Mycobacterial PPE Protein Ubiquitination Suppresses Innate Immune Response

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2022.880315

Mtb PPE68 inhibits the production of TNF-α, IL-6 and NO and promotes mycobacterial survival in mice. (A) Procedure for the mouse Mtb infection experiment. (B-D) RT-qPCR analysis of lung TNF-α (B) , IL-6 (C) and iNOS (D) mRNA expression of C57BL/6 mice infected ( i.n. ) with 5 × 10 4 CFUs of H37Rv or RvΔPPE68 (n = 5, each group). (E-I) Lung TNF-α (E) , IL-6 (F) , NO (G) concentrations were measured by ELISA, and bacterial burdens in the lungs (H) , liver (I) , and spleen (J) of infected mice were determined by CFU counting on 7H10 agar plates. (K, L) Lung tissue sections were analyzed with H E (K) and acid-fast staining (L) . The red arrows indicate acid-fast staining-positive bacteria. The data are expressed as the mean ± SD of three independent experiments for B-J, and two tailed unpaired t test was used to calculate statistical significance. *** p
Figure Legend Snippet: Mtb PPE68 inhibits the production of TNF-α, IL-6 and NO and promotes mycobacterial survival in mice. (A) Procedure for the mouse Mtb infection experiment. (B-D) RT-qPCR analysis of lung TNF-α (B) , IL-6 (C) and iNOS (D) mRNA expression of C57BL/6 mice infected ( i.n. ) with 5 × 10 4 CFUs of H37Rv or RvΔPPE68 (n = 5, each group). (E-I) Lung TNF-α (E) , IL-6 (F) , NO (G) concentrations were measured by ELISA, and bacterial burdens in the lungs (H) , liver (I) , and spleen (J) of infected mice were determined by CFU counting on 7H10 agar plates. (K, L) Lung tissue sections were analyzed with H E (K) and acid-fast staining (L) . The red arrows indicate acid-fast staining-positive bacteria. The data are expressed as the mean ± SD of three independent experiments for B-J, and two tailed unpaired t test was used to calculate statistical significance. *** p

Techniques Used: Mouse Assay, Infection, Quantitative RT-PCR, Expressing, Enzyme-linked Immunosorbent Assay, Staining, Two Tailed Test

Adoptive transfer of SHP1/MKRN1 knockdown macrophages attenuates the PPE68-mediated suppression of inflammatory cytokine expression and promotion of bacterial survival in mice. (A) Procedure for the macrophage adoptive transfer experiment. (B-D) Lung TNF-α (B) , IL-6 (C) and NO (D) levels were evaluated by ELISA in the mouse adoptive transfer experiments. (E-G) RT-qPCR analysis of lung TNF-α (E) , IL-6 (F) and iNOS (G) mRNA expression in the MS::vector- or MS::PPE68-infected mice after adoptive transfer. (H) Mouse lung bacterial burden was determined by CFU counting on 7H10 agar plates (n = 5, each group). The data are expressed as the mean ± SD of three independent experiments for B-H, and two tailed unpaired t test was used to calculate statistical significance. p > 0.05, not significant (ns); **** p
Figure Legend Snippet: Adoptive transfer of SHP1/MKRN1 knockdown macrophages attenuates the PPE68-mediated suppression of inflammatory cytokine expression and promotion of bacterial survival in mice. (A) Procedure for the macrophage adoptive transfer experiment. (B-D) Lung TNF-α (B) , IL-6 (C) and NO (D) levels were evaluated by ELISA in the mouse adoptive transfer experiments. (E-G) RT-qPCR analysis of lung TNF-α (E) , IL-6 (F) and iNOS (G) mRNA expression in the MS::vector- or MS::PPE68-infected mice after adoptive transfer. (H) Mouse lung bacterial burden was determined by CFU counting on 7H10 agar plates (n = 5, each group). The data are expressed as the mean ± SD of three independent experiments for B-H, and two tailed unpaired t test was used to calculate statistical significance. p > 0.05, not significant (ns); **** p

Techniques Used: Adoptive Transfer Assay, Expressing, Mouse Assay, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Plasmid Preparation, Infection, Two Tailed Test

19) Product Images from "The Orphan Response Regulator Rv3143 Modulates the Activity of the NADH Dehydrogenase Complex (Nuo) in Mycobacterium tuberculosis via Protein–Protein Interactions"

Article Title: The Orphan Response Regulator Rv3143 Modulates the Activity of the NADH Dehydrogenase Complex (Nuo) in Mycobacterium tuberculosis via Protein–Protein Interactions

Journal: Frontiers in Cellular and Infection Microbiology

doi: 10.3389/fcimb.2022.909507

Kinetics of growth and viability of Mycobacterium smegmatis strains treated with DETA NONOate or menadione. The mutant Δ msmeg_2064 , wild-type Ms-wt, and complementing Δ msmeg_2064 – 2064 strains were grown in 7H9 Middlebrook-rich medium in the presence of reactive nitrogen (DETA NONOate) (A, B) and oxygen species (menadione) (C, D) . The growth of strains was evaluated by measuring the OD 600 at the indicated time points (A, C) , and the numbers of viable cells were determined as bacterial colony-forming units (CFU) per ml on 7H10/OADC plates (B, D) . Means ± SDs are shown from three independent experiments.
Figure Legend Snippet: Kinetics of growth and viability of Mycobacterium smegmatis strains treated with DETA NONOate or menadione. The mutant Δ msmeg_2064 , wild-type Ms-wt, and complementing Δ msmeg_2064 – 2064 strains were grown in 7H9 Middlebrook-rich medium in the presence of reactive nitrogen (DETA NONOate) (A, B) and oxygen species (menadione) (C, D) . The growth of strains was evaluated by measuring the OD 600 at the indicated time points (A, C) , and the numbers of viable cells were determined as bacterial colony-forming units (CFU) per ml on 7H10/OADC plates (B, D) . Means ± SDs are shown from three independent experiments.

Techniques Used: Mutagenesis

Kinetics of growth and viability of Mycobacterium smegmatis strains expressing decreased levels of ndh gene. The mutant strains ndh CRISPRi/dCas9 (Ms-wt-NDH) and Δ msmeg_2064-ndh CRISPRi/dCas9 (Δ msmeg_2064 -NDH) and control strains CRISPRi/dCas9 (Ms-wt-cas9) and Δ msmeg_2064 CRISPRi/dCas9 (Δ msmeg_2064 -cas9) were grown in 7H9/AD Middlebrook medium for 16 h at 37°C with the addition of anhydrotetracycline to deplete ndh . Then, bacteria were cultured in limited oxygen access for 10 days (A, B) and subjected to reaeration for 39 h (C,D). The growth of strains was evaluated by measuring the OD 600 at the indicated time points (A,C), and the numbers of viable cells were determined as bacterial colony-forming units (CFU) per ml on 7H10/OADC plates (B, D). Mean ± SD from three independent experiments are shown. Statistical significance was determined using Student’s t-test (A) ( * p
Figure Legend Snippet: Kinetics of growth and viability of Mycobacterium smegmatis strains expressing decreased levels of ndh gene. The mutant strains ndh CRISPRi/dCas9 (Ms-wt-NDH) and Δ msmeg_2064-ndh CRISPRi/dCas9 (Δ msmeg_2064 -NDH) and control strains CRISPRi/dCas9 (Ms-wt-cas9) and Δ msmeg_2064 CRISPRi/dCas9 (Δ msmeg_2064 -cas9) were grown in 7H9/AD Middlebrook medium for 16 h at 37°C with the addition of anhydrotetracycline to deplete ndh . Then, bacteria were cultured in limited oxygen access for 10 days (A, B) and subjected to reaeration for 39 h (C,D). The growth of strains was evaluated by measuring the OD 600 at the indicated time points (A,C), and the numbers of viable cells were determined as bacterial colony-forming units (CFU) per ml on 7H10/OADC plates (B, D). Mean ± SD from three independent experiments are shown. Statistical significance was determined using Student’s t-test (A) ( * p

Techniques Used: Expressing, Mutagenesis, Cell Culture

20) Product Images from "senX3-independent contribution of regX3 to Mycobacterium tuberculosis virulence"

Article Title: senX3-independent contribution of regX3 to Mycobacterium tuberculosis virulence

Journal: BMC Microbiology

doi: 10.1186/s12866-014-0265-8

Both senX3 and regX3 are required for Mtb survival during nutrient starvation, although there appears to be a senX3 -independent contribution of regX3 . The wild-type CDC1551, senX3 ::Tn, senX3 ::Tn Comp, regX3 ::Tn, and regX3 ::Tn Comp were subcultured in 1xPBS with 0.05% Tween-80 and CFU were counted at different time points after plating the diluted cultures on Middlebrook 7H10 plates (mean ± SD) and incubating for 21 days. Triplicate samples were used in the experiment and the experiment was repeated twice under the same condition. * p
Figure Legend Snippet: Both senX3 and regX3 are required for Mtb survival during nutrient starvation, although there appears to be a senX3 -independent contribution of regX3 . The wild-type CDC1551, senX3 ::Tn, senX3 ::Tn Comp, regX3 ::Tn, and regX3 ::Tn Comp were subcultured in 1xPBS with 0.05% Tween-80 and CFU were counted at different time points after plating the diluted cultures on Middlebrook 7H10 plates (mean ± SD) and incubating for 21 days. Triplicate samples were used in the experiment and the experiment was repeated twice under the same condition. * p

Techniques Used:

21) Product Images from "Severe inhibition of lipooligosaccharide synthesis induces TLR2-dependent elimination of Mycobacterium marinum from THP1-derived macrophages"

Article Title: Severe inhibition of lipooligosaccharide synthesis induces TLR2-dependent elimination of Mycobacterium marinum from THP1-derived macrophages

Journal: Microbial Cell Factories

doi: 10.1186/s12934-017-0829-z

Involvement of TLR2 and CR3 in phagocytosis of the M. marinum strains by macrophages. Comparison of the M. marinum strains phagocytosis rates after blocking of macrophage TLR2 or CR3. Macrophages were pretreated with blocking antibodies (anti-TLR2 or anti-CR3) for 1 h and incubated with the M. marinum strains for 2 h at an MOI of 10. Non-ingested bacteria were extensively washed and killed by gentamycin. Macrophages were then lysed with Triton X-100 and the cell lysates were plated onto Middlebrook 7H10 agar supplemented with 10% OADC for CFU determination. The data are presented as the mean number of ingested bacteria ± SEM from six separate experiments. For statistical significance: *p ≤ 0.03, (Mann–Whitney U test) the following datasets were compared: mutant/wild-type versus mutant/wild-type + blocking antibodies
Figure Legend Snippet: Involvement of TLR2 and CR3 in phagocytosis of the M. marinum strains by macrophages. Comparison of the M. marinum strains phagocytosis rates after blocking of macrophage TLR2 or CR3. Macrophages were pretreated with blocking antibodies (anti-TLR2 or anti-CR3) for 1 h and incubated with the M. marinum strains for 2 h at an MOI of 10. Non-ingested bacteria were extensively washed and killed by gentamycin. Macrophages were then lysed with Triton X-100 and the cell lysates were plated onto Middlebrook 7H10 agar supplemented with 10% OADC for CFU determination. The data are presented as the mean number of ingested bacteria ± SEM from six separate experiments. For statistical significance: *p ≤ 0.03, (Mann–Whitney U test) the following datasets were compared: mutant/wild-type versus mutant/wild-type + blocking antibodies

Techniques Used: Blocking Assay, Incubation, MANN-WHITNEY, Mutagenesis

Involvement of TLR2 and CR3 in intracellular M. marinum survival. Macrophages were pretreated with blocking antibodies (anti-TLR2 or anti-CR3) for 1 h and incubated with M. marinum mutants for 2 h at an MOI of 10. Non-ingested bacteria were extensively washed and killed by gentamycin. Macrophages were lysed with Triton X-100 or maintained in culture for another 2 days and then lysed, plated on Middlebrook 7H10 agar supplemented with 10% OADC and cultured for CFU determination. The data are presented as fold increase in CFU/ml (number of bacteria 2 days post-infection divided by the number of bacteria after 2-h of phagocytosis), expressed as the mean ± SEM from six separate experiments. For statistical significance: *p ≤ 0.03, (Mann–Whitney U test) the following datasets were compared: mutant/wild-type versus mutant/wild-type + blocking antibodies
Figure Legend Snippet: Involvement of TLR2 and CR3 in intracellular M. marinum survival. Macrophages were pretreated with blocking antibodies (anti-TLR2 or anti-CR3) for 1 h and incubated with M. marinum mutants for 2 h at an MOI of 10. Non-ingested bacteria were extensively washed and killed by gentamycin. Macrophages were lysed with Triton X-100 or maintained in culture for another 2 days and then lysed, plated on Middlebrook 7H10 agar supplemented with 10% OADC and cultured for CFU determination. The data are presented as fold increase in CFU/ml (number of bacteria 2 days post-infection divided by the number of bacteria after 2-h of phagocytosis), expressed as the mean ± SEM from six separate experiments. For statistical significance: *p ≤ 0.03, (Mann–Whitney U test) the following datasets were compared: mutant/wild-type versus mutant/wild-type + blocking antibodies

Techniques Used: Blocking Assay, Incubation, Cell Culture, Infection, MANN-WHITNEY, Mutagenesis

Phagocytosis of the M. marinum mutants by macrophages. Macrophages were incubated with M. marinum strains for 2 h at an MOI of 10. Non-ingested bacteria were extensively washed and killed by gentamycin. Macrophages were lysed with Triton X-100, and then cell lysates were plated onto Middlebrook 7H10 agar supplemented with 10% OADC for CFU determination. The number of ingested bacteria are presented as the mean ± SEM from 6 to 7 separate experiments. Figure describes the difference in phagocytosis between mutants, their complemented counterparts and wild-type M. marinum . Statistical significance: *p ≤ 0.03 (Mann–Whitney U test)
Figure Legend Snippet: Phagocytosis of the M. marinum mutants by macrophages. Macrophages were incubated with M. marinum strains for 2 h at an MOI of 10. Non-ingested bacteria were extensively washed and killed by gentamycin. Macrophages were lysed with Triton X-100, and then cell lysates were plated onto Middlebrook 7H10 agar supplemented with 10% OADC for CFU determination. The number of ingested bacteria are presented as the mean ± SEM from 6 to 7 separate experiments. Figure describes the difference in phagocytosis between mutants, their complemented counterparts and wild-type M. marinum . Statistical significance: *p ≤ 0.03 (Mann–Whitney U test)

Techniques Used: Incubation, MANN-WHITNEY

Intracellular survival of the LOS mutants in macrophages. Macrophages were incubated with M. marinum strains for 2 h at an MOI of 10. Non-ingested bacteria were extensively washed and killed by gentamycin. Macrophages were either lysed with Triton X-100 or maintained in culture for 2 additional days prior to lysis. Cell lysates were plated onto Middlebrook 7H10 agar supplemented with 10% OADC for CFU determination. The data are presented as fold increase in CFU/ml (number of bacteria 2 days post-infection divided by the number of bacteria after 2-h of phagocytosis), expressed as the mean ± SEM from 6 to 7 separate experiments. Figure describes the difference in intracellular survival between mutants, their complemented counterparts and wild-type M. marinum . Statistical significance: *p ≤ 0.05 (Mann–Whitney U test)
Figure Legend Snippet: Intracellular survival of the LOS mutants in macrophages. Macrophages were incubated with M. marinum strains for 2 h at an MOI of 10. Non-ingested bacteria were extensively washed and killed by gentamycin. Macrophages were either lysed with Triton X-100 or maintained in culture for 2 additional days prior to lysis. Cell lysates were plated onto Middlebrook 7H10 agar supplemented with 10% OADC for CFU determination. The data are presented as fold increase in CFU/ml (number of bacteria 2 days post-infection divided by the number of bacteria after 2-h of phagocytosis), expressed as the mean ± SEM from 6 to 7 separate experiments. Figure describes the difference in intracellular survival between mutants, their complemented counterparts and wild-type M. marinum . Statistical significance: *p ≤ 0.05 (Mann–Whitney U test)

Techniques Used: Incubation, Lysis, Infection, MANN-WHITNEY

22) Product Images from "Isolation of nontuberculous mycobacteria from soil using Middlebrook 7H10 agar with increased malachite green concentration"

Article Title: Isolation of nontuberculous mycobacteria from soil using Middlebrook 7H10 agar with increased malachite green concentration

Journal: AMB Express

doi: 10.1186/s13568-017-0373-6

Modified Middlebrook 7H10 medium containing 250 mg/L MG with mycobacteria primarily isolated from soil. a Sample collected at campus, b sample collected at community, and c collected at natural park. Black arrows indicate mycobacterial colonies, red arrows indicate dried soils
Figure Legend Snippet: Modified Middlebrook 7H10 medium containing 250 mg/L MG with mycobacteria primarily isolated from soil. a Sample collected at campus, b sample collected at community, and c collected at natural park. Black arrows indicate mycobacterial colonies, red arrows indicate dried soils

Techniques Used: Modification, Isolation

23) Product Images from "Proteasome Accessory Factor C (pafC) Is a novel gene Involved in Mycobacterium Intrinsic Resistance to broad-spectrum antibiotics - Fluoroquinolones"

Article Title: Proteasome Accessory Factor C (pafC) Is a novel gene Involved in Mycobacterium Intrinsic Resistance to broad-spectrum antibiotics - Fluoroquinolones

Journal: Scientific Reports

doi: 10.1038/srep11910

Growth of M. smegmatis mc 2 155 and M371 under fluoroquinolones exposure. Ten-fold serial dilutions of wild-type, M371, M371-pALACE- pafC and M371-pALACE were spotted on Middlebrook 7H10 containing indicated concentration of moxifloxacin, norfloxacin, ofloxacin and ciprofloxacin. Then the result was recorded when incubated at 37 °C for 3 days.
Figure Legend Snippet: Growth of M. smegmatis mc 2 155 and M371 under fluoroquinolones exposure. Ten-fold serial dilutions of wild-type, M371, M371-pALACE- pafC and M371-pALACE were spotted on Middlebrook 7H10 containing indicated concentration of moxifloxacin, norfloxacin, ofloxacin and ciprofloxacin. Then the result was recorded when incubated at 37 °C for 3 days.

Techniques Used: Concentration Assay, Incubation

24) Product Images from "A ?-Lactamase Based Reporter System for ESX Dependent Protein Translocation in Mycobacteria"

Article Title: A ?-Lactamase Based Reporter System for ESX Dependent Protein Translocation in Mycobacteria

Journal: PLoS ONE

doi: 10.1371/journal.pone.0035453

Growth of M. smegmatis strains on an agar plate containing ampicillin. Freshly grown cultures were diluted to an OD 600 of 0.015. 1 µl of each strain was streaked in a separate sector on a Middlebrook 7H10 plate containing ampicillin [120 µg/ml]. The plate was incubated for 3 days at 37°C. The parental strain in the sectors 1–5 is M. smegmatis Δ blaS . The parental strain in sectors 6–8 is M. smegmatis Δ blaS Δ eccD 3 . Additional roman numbers correspond to the reporter vector in each strain (cf. tables 1 + 2 ). Reporter constructs: (−): none; 0: BlaTEM ; II: Sec-BlaTEM-‘FbpB; IV: BlaTEM-EsxG; and VI: BlaTEM-EsxB. Note: The reporter construct BlaTEM-EsxG from pIV- blaTEM (sector 7) is no longer able to confer ampicillin resistance when a key component of ESX-3 (EccD 3 ) is deleted. In contrast, constructs specific for Sec (sector 6) and ESX-1 (sector 8) dependent translocation still confer an ampicillin resistance phenotype.
Figure Legend Snippet: Growth of M. smegmatis strains on an agar plate containing ampicillin. Freshly grown cultures were diluted to an OD 600 of 0.015. 1 µl of each strain was streaked in a separate sector on a Middlebrook 7H10 plate containing ampicillin [120 µg/ml]. The plate was incubated for 3 days at 37°C. The parental strain in the sectors 1–5 is M. smegmatis Δ blaS . The parental strain in sectors 6–8 is M. smegmatis Δ blaS Δ eccD 3 . Additional roman numbers correspond to the reporter vector in each strain (cf. tables 1 + 2 ). Reporter constructs: (−): none; 0: BlaTEM ; II: Sec-BlaTEM-‘FbpB; IV: BlaTEM-EsxG; and VI: BlaTEM-EsxB. Note: The reporter construct BlaTEM-EsxG from pIV- blaTEM (sector 7) is no longer able to confer ampicillin resistance when a key component of ESX-3 (EccD 3 ) is deleted. In contrast, constructs specific for Sec (sector 6) and ESX-1 (sector 8) dependent translocation still confer an ampicillin resistance phenotype.

Techniques Used: Incubation, Plasmid Preparation, Construct, Size-exclusion Chromatography, Translocation Assay

25) Product Images from "Genetic models of latent tuberculosis in mice reveal differential control by adaptive immunity"

Article Title: Genetic models of latent tuberculosis in mice reveal differential control by adaptive immunity

Journal: bioRxiv

doi: 10.1101/2021.02.08.430271

BCG vaccination fails to prevent TrxB2-DUC reactivation. (A) CFU from lungs and ( B ) spleens of mice infected with TrxB2-DUC and vaccinated with BCG or injected with PBS on day 140 post infection (arrow). Groups of 21 mice were analyzed on day 280 post infection, the proportion of mice with CFU is indicated in brackets. Twenty-five days post vaccination immune responses in lungs ( C , F ) and in spleens ( D , G ) were analyzed. Cytokine expressing CD4 T cells were measured by intracellular cytokine staining in CD4 + , CD44 + T lymphocytes following stimulation of lung leukocytes ( C ) and splenocytes ( D ) with purified protein derivative (PPD). (E) Quantification of BCG CFU in lungs and spleens. On day 1, 25 and 40 post vaccination CFU in lungs and spleens were determined by culturing organ homogenates on 7H10 agar plates. ( F , G ) Effector memory CD4 and CD8 T cells were quantified by flow cytometry in lungs ( F ) and spleens ( G ) in groups of 3-4 PBS treated and 5 BCG vaccinated mice. The differences between PBS treated and BCG vaccinated mice were analyzed by unpaired, two tailed t-test. * P
Figure Legend Snippet: BCG vaccination fails to prevent TrxB2-DUC reactivation. (A) CFU from lungs and ( B ) spleens of mice infected with TrxB2-DUC and vaccinated with BCG or injected with PBS on day 140 post infection (arrow). Groups of 21 mice were analyzed on day 280 post infection, the proportion of mice with CFU is indicated in brackets. Twenty-five days post vaccination immune responses in lungs ( C , F ) and in spleens ( D , G ) were analyzed. Cytokine expressing CD4 T cells were measured by intracellular cytokine staining in CD4 + , CD44 + T lymphocytes following stimulation of lung leukocytes ( C ) and splenocytes ( D ) with purified protein derivative (PPD). (E) Quantification of BCG CFU in lungs and spleens. On day 1, 25 and 40 post vaccination CFU in lungs and spleens were determined by culturing organ homogenates on 7H10 agar plates. ( F , G ) Effector memory CD4 and CD8 T cells were quantified by flow cytometry in lungs ( F ) and spleens ( G ) in groups of 3-4 PBS treated and 5 BCG vaccinated mice. The differences between PBS treated and BCG vaccinated mice were analyzed by unpaired, two tailed t-test. * P

Techniques Used: Mouse Assay, Infection, Injection, Expressing, Staining, Purification, Flow Cytometry, Two Tailed Test

26) Product Images from "Glby, Encoded by MAB_3167c, Is Required for In Vivo Growth of Mycobacteroides abscessus and Exhibits Mild β-Lactamase Activity"

Article Title: Glby, Encoded by MAB_3167c, Is Required for In Vivo Growth of Mycobacteroides abscessus and Exhibits Mild β-Lactamase Activity

Journal: Journal of Bacteriology

doi: 10.1128/jb.00046-22

In vitro growth phenotypes of Δglby . (A) Growth of parent strain Mab ATCC 19977 (WT), Δglby and Complement (COMP) strains in Middlebrook 7H9 broth at 96 h, 37°C, constant shaking at 200 RPM. Culture media only as negative controls (NC) was also included. (B) Time course of optical densities of cultures of WT, Δglby , and COMP measured as absorbance at 600 nm. Each time point represents the average from four distinct replicates and no sample tube was used more than once. Error bars are standard deviation of each sample. (C) Time course of CFU of the three strains. Error bars are standard deviation of each sample. (D) Generation time (time required for a strain to double in CFU) of each strain determined from exponential phase(s) of growth. Two generation times reported for Δglby represent the biphasic exponential growth stages of this strain.
Figure Legend Snippet: In vitro growth phenotypes of Δglby . (A) Growth of parent strain Mab ATCC 19977 (WT), Δglby and Complement (COMP) strains in Middlebrook 7H9 broth at 96 h, 37°C, constant shaking at 200 RPM. Culture media only as negative controls (NC) was also included. (B) Time course of optical densities of cultures of WT, Δglby , and COMP measured as absorbance at 600 nm. Each time point represents the average from four distinct replicates and no sample tube was used more than once. Error bars are standard deviation of each sample. (C) Time course of CFU of the three strains. Error bars are standard deviation of each sample. (D) Generation time (time required for a strain to double in CFU) of each strain determined from exponential phase(s) of growth. Two generation times reported for Δglby represent the biphasic exponential growth stages of this strain.

Techniques Used: In Vitro, Standard Deviation

27) Product Images from "Phosphorylation of KasB Regulates Virulence and Acid-Fastness in Mycobacterium tuberculosis"

Article Title: Phosphorylation of KasB Regulates Virulence and Acid-Fastness in Mycobacterium tuberculosis

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1004115

Infection of immunocompromised SCID mice with M. tuberculosis kasB isogenic mutants. (A) Survival curves in infected SCID mice. Low-dose aerosol infection of SCID mice (as in B ) was performed with the following Mtb strains: parental, KasB_T334A/T336A, KasB_T334D/T336D and Δ kasB . (B) Growth of Mtb kasB strains in the lungs of SCID mice. At 1, 7, 21 and 56 days post-infection, one lung from each infected SCID mouse was harvested, homogenized and serial dilutions were plated on Middlebrook 7H10 supplemented with 10% OADC and 0.2% glycerol. (C) Pathology of lungs from infected SCID mice. One lung from each infected SCID mouse was harvested and fixed in 10% paraformaldehyde for a month prior to photography. (D) CFU plots in the liver and spleen three weeks (black bars) and eight weeks (grey bars) post-infection.
Figure Legend Snippet: Infection of immunocompromised SCID mice with M. tuberculosis kasB isogenic mutants. (A) Survival curves in infected SCID mice. Low-dose aerosol infection of SCID mice (as in B ) was performed with the following Mtb strains: parental, KasB_T334A/T336A, KasB_T334D/T336D and Δ kasB . (B) Growth of Mtb kasB strains in the lungs of SCID mice. At 1, 7, 21 and 56 days post-infection, one lung from each infected SCID mouse was harvested, homogenized and serial dilutions were plated on Middlebrook 7H10 supplemented with 10% OADC and 0.2% glycerol. (C) Pathology of lungs from infected SCID mice. One lung from each infected SCID mouse was harvested and fixed in 10% paraformaldehyde for a month prior to photography. (D) CFU plots in the liver and spleen three weeks (black bars) and eight weeks (grey bars) post-infection.

Techniques Used: Infection, Mouse Assay

Infection of immunocompetent C57Bl/6 mice with M. tuberculosis kasB isogenic strains. (A) Growth of Mtb kasB strains in the lungs. Low-dose aerosol infection was performed with the following Mtb strains: parental, KasB_T334A/T336A, KasB_T334D/T336D, and Δ kasB . Lungs from infected mice were harvested at 1, 7, 21 and 57 days post-infection, homogenized and serial dilutions were plated onto Middlebrook 7H10 plates supplemented with 10% OADC and 0.2% glycerol. (B) Pathology slides of lungs from infected mice at 21 days post-infection. Lung tissue sections from mice infected with the Mtb CDC1551 parental, KasB_T334A/T336A, KasB_T334D/T336D, and Δ kasB were stained with hematoxylin/eosin and observed. Magnification = ×20. (C) CFU plots in the liver and spleen three weeks (black bars) and eight weeks (grey bars) post-infection.
Figure Legend Snippet: Infection of immunocompetent C57Bl/6 mice with M. tuberculosis kasB isogenic strains. (A) Growth of Mtb kasB strains in the lungs. Low-dose aerosol infection was performed with the following Mtb strains: parental, KasB_T334A/T336A, KasB_T334D/T336D, and Δ kasB . Lungs from infected mice were harvested at 1, 7, 21 and 57 days post-infection, homogenized and serial dilutions were plated onto Middlebrook 7H10 plates supplemented with 10% OADC and 0.2% glycerol. (B) Pathology slides of lungs from infected mice at 21 days post-infection. Lung tissue sections from mice infected with the Mtb CDC1551 parental, KasB_T334A/T336A, KasB_T334D/T336D, and Δ kasB were stained with hematoxylin/eosin and observed. Magnification = ×20. (C) CFU plots in the liver and spleen three weeks (black bars) and eight weeks (grey bars) post-infection.

Techniques Used: Infection, Mouse Assay, Staining

28) Product Images from "Pasakbumin A controls the growth of Mycobacterium tuberculosis by enhancing the autophagy and production of antibacterial mediators in mouse macrophages"

Article Title: Pasakbumin A controls the growth of Mycobacterium tuberculosis by enhancing the autophagy and production of antibacterial mediators in mouse macrophages

Journal: bioRxiv

doi: 10.1101/348144

Pasakbumin A controls intracellular Mtb growth by increasing the production of NO and pro-inflammatory cytokine in H37Rv-infected macrophages. Raw264.7 macrophages were stimulated with pasakbumin A (Pas A, 10 μM) for 48 h after infection with H37Rv (at MOIs of 1 or 5). (A) Intracellular bacterial survival was determined by counting the number of CFUs at 3-weeks after inoculation. (B) TNF-α and IL-10 production in culture supernatants from non-infected (NI) or H37Rv-infected cells was measured by ELISA. (C) Bar graph in the left panel represents NO production (as indicated by the nitrite level) with the diazotization (Griess method) assay. Right panel represents NOS2 protein by western blot analysis. Actin served as a loading control. A complete western blot images is provided in S1 Data File . (D) Cell viability was assessed using a trypan-blue exclusion assay. (E) The cultures were grown in 7H9 medium supplemented with 10% ADC containing 0.2% glycerol at 37°C for 72 h with or without pasakbumin A (Pas A, 10 μM). Left, bacterial growth was measured as OD 600 at 72 h after Mtb inoculation. Right, colony-forming units (CFUs) were measured by plating bacterial dilutions onto 7H10 agar supplemented with 10% OADC containing 0.5% glycerol at 72 h. Error bars represents the standard deviation of the mean. Statistical significance is indicated as **, p
Figure Legend Snippet: Pasakbumin A controls intracellular Mtb growth by increasing the production of NO and pro-inflammatory cytokine in H37Rv-infected macrophages. Raw264.7 macrophages were stimulated with pasakbumin A (Pas A, 10 μM) for 48 h after infection with H37Rv (at MOIs of 1 or 5). (A) Intracellular bacterial survival was determined by counting the number of CFUs at 3-weeks after inoculation. (B) TNF-α and IL-10 production in culture supernatants from non-infected (NI) or H37Rv-infected cells was measured by ELISA. (C) Bar graph in the left panel represents NO production (as indicated by the nitrite level) with the diazotization (Griess method) assay. Right panel represents NOS2 protein by western blot analysis. Actin served as a loading control. A complete western blot images is provided in S1 Data File . (D) Cell viability was assessed using a trypan-blue exclusion assay. (E) The cultures were grown in 7H9 medium supplemented with 10% ADC containing 0.2% glycerol at 37°C for 72 h with or without pasakbumin A (Pas A, 10 μM). Left, bacterial growth was measured as OD 600 at 72 h after Mtb inoculation. Right, colony-forming units (CFUs) were measured by plating bacterial dilutions onto 7H10 agar supplemented with 10% OADC containing 0.5% glycerol at 72 h. Error bars represents the standard deviation of the mean. Statistical significance is indicated as **, p

Techniques Used: Infection, Enzyme-linked Immunosorbent Assay, Western Blot, Trypan Blue Exclusion Assay, Standard Deviation

29) Product Images from "Glby, Encoded by MAB_3167c, Is Required for In Vivo Growth of Mycobacteroides abscessus and Exhibits Mild β-Lactamase Activity"

Article Title: Glby, Encoded by MAB_3167c, Is Required for In Vivo Growth of Mycobacteroides abscessus and Exhibits Mild β-Lactamase Activity

Journal: Journal of Bacteriology

doi: 10.1128/jb.00046-22

In vitro growth phenotypes of Δglby . (A) Growth of parent strain Mab ATCC 19977 (WT), Δglby and Complement (COMP) strains in Middlebrook 7H9 broth at 96 h, 37°C, constant shaking at 200 RPM. Culture media only as negative controls (NC) was also included. (B) Time course of optical densities of cultures of WT, Δglby , and COMP measured as absorbance at 600 nm. Each time point represents the average from four distinct replicates and no sample tube was used more than once. Error bars are standard deviation of each sample. (C) Time course of CFU of the three strains. Error bars are standard deviation of each sample. (D) Generation time (time required for a strain to double in CFU) of each strain determined from exponential phase(s) of growth. Two generation times reported for Δglby represent the biphasic exponential growth stages of this strain.
Figure Legend Snippet: In vitro growth phenotypes of Δglby . (A) Growth of parent strain Mab ATCC 19977 (WT), Δglby and Complement (COMP) strains in Middlebrook 7H9 broth at 96 h, 37°C, constant shaking at 200 RPM. Culture media only as negative controls (NC) was also included. (B) Time course of optical densities of cultures of WT, Δglby , and COMP measured as absorbance at 600 nm. Each time point represents the average from four distinct replicates and no sample tube was used more than once. Error bars are standard deviation of each sample. (C) Time course of CFU of the three strains. Error bars are standard deviation of each sample. (D) Generation time (time required for a strain to double in CFU) of each strain determined from exponential phase(s) of growth. Two generation times reported for Δglby represent the biphasic exponential growth stages of this strain.

Techniques Used: In Vitro, Standard Deviation

30) Product Images from "Inactivation of a New Potassium Channel Increases Rifampicin Resistance and Induces Collateral Sensitivity to Hydrophilic Antibiotics in Mycobacterium smegmatis"

Article Title: Inactivation of a New Potassium Channel Increases Rifampicin Resistance and Induces Collateral Sensitivity to Hydrophilic Antibiotics in Mycobacterium smegmatis

Journal: Antibiotics

doi: 10.3390/antibiotics11040509

K + effect on the growth of mc 2 155 and its ∆ mchK derivative under different conditions. ( a ) Effect of K + addition (10–200 mM KCl) on the growth of mc 2 155 and ∆ mchK at neutral pH. The ability of high K + concentrations to restore the growth of the ∆ mchK mutant was measured in cultures at mid-exponential phase (12 h). ( b ) Effect of extracellular pH (5–8) on the growth of mc 2 155 and the ∆ mchK mutant in Middlebrook 7H9 liquid medium after 12 h of incubation (initial OD 600 0.05). ( c ) Effect of K + addition (10–100 mM KCl) on the growth of mc 2 155 and ∆ mchK at pH 5.5. The OD 600 was measured after 12 h of incubation at 37 °C. Error bars indicate 95% CI (confidence intervals); (*) p
Figure Legend Snippet: K + effect on the growth of mc 2 155 and its ∆ mchK derivative under different conditions. ( a ) Effect of K + addition (10–200 mM KCl) on the growth of mc 2 155 and ∆ mchK at neutral pH. The ability of high K + concentrations to restore the growth of the ∆ mchK mutant was measured in cultures at mid-exponential phase (12 h). ( b ) Effect of extracellular pH (5–8) on the growth of mc 2 155 and the ∆ mchK mutant in Middlebrook 7H9 liquid medium after 12 h of incubation (initial OD 600 0.05). ( c ) Effect of K + addition (10–100 mM KCl) on the growth of mc 2 155 and ∆ mchK at pH 5.5. The OD 600 was measured after 12 h of incubation at 37 °C. Error bars indicate 95% CI (confidence intervals); (*) p

Techniques Used: Mutagenesis, Incubation

31) Product Images from "Either Non-Homologous Ends Joining or Homologous Recombination Is Required to Repair Double-Strand Breaks in the Genome of Macrophage-Internalized Mycobacterium tuberculosis"

Article Title: Either Non-Homologous Ends Joining or Homologous Recombination Is Required to Repair Double-Strand Breaks in the Genome of Macrophage-Internalized Mycobacterium tuberculosis

Journal: PLoS ONE

doi: 10.1371/journal.pone.0092799

Effect of inhibition of ROS and NO production on the survival of Mtb strains inside MØs. MØs were incubated with apocynin or L-NIL for 30 minutes, then infected with Mtb wild-type or Δ( ku,ligD,recA ) for 2 hours (MOI = 10) and washed with HBSS. On the day of infection and after 6 days of culture, MØs were lysed with Triton X-100 and cell lysates were plated onto Middlebrook 7H10 agar supplemented with 10% OADC. After 21 days of culture, CFUs were counted. Data are presented as fold increase in CFUs, expressed as means ± SEM (n = 5; #p≤0.04, Δ( ku,ligD,recA ) vs. Δ( ku,ligD,recA ) + apocynin or L-NIL; Mann-Whitney U test).
Figure Legend Snippet: Effect of inhibition of ROS and NO production on the survival of Mtb strains inside MØs. MØs were incubated with apocynin or L-NIL for 30 minutes, then infected with Mtb wild-type or Δ( ku,ligD,recA ) for 2 hours (MOI = 10) and washed with HBSS. On the day of infection and after 6 days of culture, MØs were lysed with Triton X-100 and cell lysates were plated onto Middlebrook 7H10 agar supplemented with 10% OADC. After 21 days of culture, CFUs were counted. Data are presented as fold increase in CFUs, expressed as means ± SEM (n = 5; #p≤0.04, Δ( ku,ligD,recA ) vs. Δ( ku,ligD,recA ) + apocynin or L-NIL; Mann-Whitney U test).

Techniques Used: Inhibition, Incubation, Infection, MANN-WHITNEY

Survival of Mtb strains inside MØs. (A) MØs were infected with Mtb wild-type or its mutants, Δ( ku,ligD,recA ), Δ( ku,ligD ) or Δ recA , for 2 hours (MOI = 10), washed with HBSS, and cultured for 2, 4, and 6 days. (B and C) MØs were infected with Mtb wild-type or its mutants (B) or complemented strains Δ( ku,ligD,recA )- P recA recA – complemented strain I or Δ( ku,ligD,recA )- P kuligD ku-ligD – complemented strain II (C) for 6 days. On the day of infection and after 6 days of culture, MØs were lysed with Triton X-100 and cell lysates were plated onto Middlebrook 7H10 agar supplemented with 10% OADC. After 21 days of culture, CFUs were counted. Data are presented as fold increase in CFUs, expressed as means ± SEM (n = 5; *p≤0.05, Δ( ku,ligD,recA ) vs. wild-type; #p≤0.05, complemented strain I and II vs. Δ( ku,ligD,recA ) Mann-Whitney U test).
Figure Legend Snippet: Survival of Mtb strains inside MØs. (A) MØs were infected with Mtb wild-type or its mutants, Δ( ku,ligD,recA ), Δ( ku,ligD ) or Δ recA , for 2 hours (MOI = 10), washed with HBSS, and cultured for 2, 4, and 6 days. (B and C) MØs were infected with Mtb wild-type or its mutants (B) or complemented strains Δ( ku,ligD,recA )- P recA recA – complemented strain I or Δ( ku,ligD,recA )- P kuligD ku-ligD – complemented strain II (C) for 6 days. On the day of infection and after 6 days of culture, MØs were lysed with Triton X-100 and cell lysates were plated onto Middlebrook 7H10 agar supplemented with 10% OADC. After 21 days of culture, CFUs were counted. Data are presented as fold increase in CFUs, expressed as means ± SEM (n = 5; *p≤0.05, Δ( ku,ligD,recA ) vs. wild-type; #p≤0.05, complemented strain I and II vs. Δ( ku,ligD,recA ) Mann-Whitney U test).

Techniques Used: Infection, Cell Culture, MANN-WHITNEY

32) Product Images from "Inactivation of a New Potassium Channel Increases Rifampicin Resistance and Induces Collateral Sensitivity to Hydrophilic Antibiotics in Mycobacterium smegmatis"

Article Title: Inactivation of a New Potassium Channel Increases Rifampicin Resistance and Induces Collateral Sensitivity to Hydrophilic Antibiotics in Mycobacterium smegmatis

Journal: Antibiotics

doi: 10.3390/antibiotics11040509

K + effect on the growth of mc 2 155 and its ∆ mchK derivative under different conditions. ( a ) Effect of K + addition (10–200 mM KCl) on the growth of mc 2 155 and ∆ mchK at neutral pH. The ability of high K + concentrations to restore the growth of the ∆ mchK mutant was measured in cultures at mid-exponential phase (12 h). ( b ) Effect of extracellular pH (5–8) on the growth of mc 2 155 and the ∆ mchK mutant in Middlebrook 7H9 liquid medium after 12 h of incubation (initial OD 600 0.05). ( c ) Effect of K + addition (10–100 mM KCl) on the growth of mc 2 155 and ∆ mchK at pH 5.5. The OD 600 was measured after 12 h of incubation at 37 °C. Error bars indicate 95% CI (confidence intervals); (*) p
Figure Legend Snippet: K + effect on the growth of mc 2 155 and its ∆ mchK derivative under different conditions. ( a ) Effect of K + addition (10–200 mM KCl) on the growth of mc 2 155 and ∆ mchK at neutral pH. The ability of high K + concentrations to restore the growth of the ∆ mchK mutant was measured in cultures at mid-exponential phase (12 h). ( b ) Effect of extracellular pH (5–8) on the growth of mc 2 155 and the ∆ mchK mutant in Middlebrook 7H9 liquid medium after 12 h of incubation (initial OD 600 0.05). ( c ) Effect of K + addition (10–100 mM KCl) on the growth of mc 2 155 and ∆ mchK at pH 5.5. The OD 600 was measured after 12 h of incubation at 37 °C. Error bars indicate 95% CI (confidence intervals); (*) p

Techniques Used: Mutagenesis, Incubation

33) Product Images from "Either Non-Homologous Ends Joining or Homologous Recombination Is Required to Repair Double-Strand Breaks in the Genome of Macrophage-Internalized Mycobacterium tuberculosis"

Article Title: Either Non-Homologous Ends Joining or Homologous Recombination Is Required to Repair Double-Strand Breaks in the Genome of Macrophage-Internalized Mycobacterium tuberculosis

Journal: PLoS ONE

doi: 10.1371/journal.pone.0092799

Effect of inhibition of ROS and NO production on the survival of Mtb strains inside MØs. MØs were incubated with apocynin or L-NIL for 30 minutes, then infected with Mtb wild-type or Δ( ku,ligD,recA ) for 2 hours (MOI = 10) and washed with HBSS. On the day of infection and after 6 days of culture, MØs were lysed with Triton X-100 and cell lysates were plated onto Middlebrook 7H10 agar supplemented with 10% OADC. After 21 days of culture, CFUs were counted. Data are presented as fold increase in CFUs, expressed as means ± SEM (n = 5; #p≤0.04, Δ( ku,ligD,recA ) vs. Δ( ku,ligD,recA ) + apocynin or L-NIL; Mann-Whitney U test).
Figure Legend Snippet: Effect of inhibition of ROS and NO production on the survival of Mtb strains inside MØs. MØs were incubated with apocynin or L-NIL for 30 minutes, then infected with Mtb wild-type or Δ( ku,ligD,recA ) for 2 hours (MOI = 10) and washed with HBSS. On the day of infection and after 6 days of culture, MØs were lysed with Triton X-100 and cell lysates were plated onto Middlebrook 7H10 agar supplemented with 10% OADC. After 21 days of culture, CFUs were counted. Data are presented as fold increase in CFUs, expressed as means ± SEM (n = 5; #p≤0.04, Δ( ku,ligD,recA ) vs. Δ( ku,ligD,recA ) + apocynin or L-NIL; Mann-Whitney U test).

Techniques Used: Inhibition, Incubation, Infection, MANN-WHITNEY

Survival of Mtb strains inside MØs. (A) MØs were infected with Mtb wild-type or its mutants, Δ( ku,ligD,recA ), Δ( ku,ligD ) or Δ recA , for 2 hours (MOI = 10), washed with HBSS, and cultured for 2, 4, and 6 days. (B and C) MØs were infected with Mtb wild-type or its mutants (B) or complemented strains Δ( ku,ligD,recA )- P recA recA – complemented strain I or Δ( ku,ligD,recA )- P kuligD ku-ligD – complemented strain II (C) for 6 days. On the day of infection and after 6 days of culture, MØs were lysed with Triton X-100 and cell lysates were plated onto Middlebrook 7H10 agar supplemented with 10% OADC. After 21 days of culture, CFUs were counted. Data are presented as fold increase in CFUs, expressed as means ± SEM (n = 5; *p≤0.05, Δ( ku,ligD,recA ) vs. wild-type; #p≤0.05, complemented strain I and II vs. Δ( ku,ligD,recA ) Mann-Whitney U test).
Figure Legend Snippet: Survival of Mtb strains inside MØs. (A) MØs were infected with Mtb wild-type or its mutants, Δ( ku,ligD,recA ), Δ( ku,ligD ) or Δ recA , for 2 hours (MOI = 10), washed with HBSS, and cultured for 2, 4, and 6 days. (B and C) MØs were infected with Mtb wild-type or its mutants (B) or complemented strains Δ( ku,ligD,recA )- P recA recA – complemented strain I or Δ( ku,ligD,recA )- P kuligD ku-ligD – complemented strain II (C) for 6 days. On the day of infection and after 6 days of culture, MØs were lysed with Triton X-100 and cell lysates were plated onto Middlebrook 7H10 agar supplemented with 10% OADC. After 21 days of culture, CFUs were counted. Data are presented as fold increase in CFUs, expressed as means ± SEM (n = 5; *p≤0.05, Δ( ku,ligD,recA ) vs. wild-type; #p≤0.05, complemented strain I and II vs. Δ( ku,ligD,recA ) Mann-Whitney U test).

Techniques Used: Infection, Cell Culture, MANN-WHITNEY

34) Product Images from "Aspartate aminotransferase Rv3722c governs aspartate-dependent nitrogen metabolism in Mycobacterium tuberculosis"

Article Title: Aspartate aminotransferase Rv3722c governs aspartate-dependent nitrogen metabolism in Mycobacterium tuberculosis

Journal: Nature Communications

doi: 10.1038/s41467-020-15876-8

Rv3722c is conditionally esssential in vitro. a Growth curve of Rv3722c-proficient and -deficient Mtb in 7H9 culture media. Rv3722c-TetON and Rv3722c-control were cultured in Middlebrook 7H9 culture media, with or without 500 ng mL −1 anhydrotetracycline (ATC). Bacterial growth was monitored by optical density at 600 nm. b Western blot showing the depletion of Rv3722c 7H9 culture media. Rv3722c-TetON was cultured in 7H9 with or without ATC for 12 days (corresponding to a ). Protein lysates were analyzed by western blotting, using an α-FLAG antibody. The proteasome subunit β (PrcB) was used as loading control. c Growth curve of Rv3722c-proficient and -deficient Mtb in 7H9 supplemented with casein hydrolysate. As a , but using 7H9 supplemented with 1% casein hydrolysate. d Spot assay of Rv3722c-proficient and -deficient Mtb on solid growth media. A serially diluted Rv3722c-TetON culture (OD 0.1) was spotted onto Middlebrook 7H10 agar with or without 1% casein hydrolysate, in the presence or absence of ATC, and cultured for 2 weeks. e Western blot showing depletion of Rv3722c. As b , but in 7H9 supplemented with 1% casein hydrolysate. f Growth curve of Rv3722c-proficient and -pre-depleted Mtb in 7H9 growth media. Rv3722c-TetON in 7H9 with or without ATC, after predepletion of Rv3722c in 7H9 with 1% casein hydrolysate without ATC. For all growth curves, data are represented as mean −/+ SD of three experimental replicates ( n = 3) representative of at least two independent experiments. See also Supplementary Fig. 1 . Source data are provided as a Source Data file.
Figure Legend Snippet: Rv3722c is conditionally esssential in vitro. a Growth curve of Rv3722c-proficient and -deficient Mtb in 7H9 culture media. Rv3722c-TetON and Rv3722c-control were cultured in Middlebrook 7H9 culture media, with or without 500 ng mL −1 anhydrotetracycline (ATC). Bacterial growth was monitored by optical density at 600 nm. b Western blot showing the depletion of Rv3722c 7H9 culture media. Rv3722c-TetON was cultured in 7H9 with or without ATC for 12 days (corresponding to a ). Protein lysates were analyzed by western blotting, using an α-FLAG antibody. The proteasome subunit β (PrcB) was used as loading control. c Growth curve of Rv3722c-proficient and -deficient Mtb in 7H9 supplemented with casein hydrolysate. As a , but using 7H9 supplemented with 1% casein hydrolysate. d Spot assay of Rv3722c-proficient and -deficient Mtb on solid growth media. A serially diluted Rv3722c-TetON culture (OD 0.1) was spotted onto Middlebrook 7H10 agar with or without 1% casein hydrolysate, in the presence or absence of ATC, and cultured for 2 weeks. e Western blot showing depletion of Rv3722c. As b , but in 7H9 supplemented with 1% casein hydrolysate. f Growth curve of Rv3722c-proficient and -pre-depleted Mtb in 7H9 growth media. Rv3722c-TetON in 7H9 with or without ATC, after predepletion of Rv3722c in 7H9 with 1% casein hydrolysate without ATC. For all growth curves, data are represented as mean −/+ SD of three experimental replicates ( n = 3) representative of at least two independent experiments. See also Supplementary Fig. 1 . Source data are provided as a Source Data file.

Techniques Used: In Vitro, Cell Culture, Western Blot, Spot Test

35) Product Images from "Design of novel peptide inhibitors against the conserved bacterial transcription terminator, Rho"

Article Title: Design of novel peptide inhibitors against the conserved bacterial transcription terminator, Rho

Journal: The Journal of Biological Chemistry

doi: 10.1016/j.jbc.2021.100653

In vitro and in vivo effects of the peptides on the Rho proteins from some pathogens. A , inhibition of the ATPase activities of Rho proteins from different pathogenic bacteria by the peptides as indicated. The autoradiograms showing the fractions of ATP hydrolyzed by Rho in the presence or absence of 50 μM peptide 16 and peptide 33 and 5 μM Psu. Poly (rC) was used as a substrate in cases of Mycobacteria and Xanthomonas Rho, whereas RNA with the λtR1 terminator was used for Salmonella and Vibrio Rho. SDs were obtained from two to three measurements. B , Rho-dependent RNA release assays from the roadblock complex (the same as described for Fig. 3 E ) in the absence and presence of 35 μM peptide 33. Fifty nanomolar of each of the Rho proteins from indicated pathogenic bacteria were used in these assays. The supernatant fraction (S) contains half of the released RNA, whereas the P fraction contains the rest of the sample (half of the supernatant + pellet). All the other experimental conditions were the same as in Figure 3 E . The fraction of released RNA ([2S]/([S] + [P])) in each case is shown as bar diagrams. The SDs were calculated from two to three measurements. C , Mycobacterium smegmatis strain mc 2 155 was transformed with either a pSTKT empty vector or pSTKT plasmid expressing either peptide 33 or WT Psu. Transformants were subsequently streaked onto 7H10 plates. Mycobacterium bovis BCG strain was transformed with the same plasmids as above. Transformants of the M. bovis strain were obtained after 3 weeks, and colonies were further streaked to show the growth differences in the M. bovis strains upon expression of either peptide 33 or the WT Psu. The spliced part of the images in Figure 9 , A and C are indicated by vertical lines .
Figure Legend Snippet: In vitro and in vivo effects of the peptides on the Rho proteins from some pathogens. A , inhibition of the ATPase activities of Rho proteins from different pathogenic bacteria by the peptides as indicated. The autoradiograms showing the fractions of ATP hydrolyzed by Rho in the presence or absence of 50 μM peptide 16 and peptide 33 and 5 μM Psu. Poly (rC) was used as a substrate in cases of Mycobacteria and Xanthomonas Rho, whereas RNA with the λtR1 terminator was used for Salmonella and Vibrio Rho. SDs were obtained from two to three measurements. B , Rho-dependent RNA release assays from the roadblock complex (the same as described for Fig. 3 E ) in the absence and presence of 35 μM peptide 33. Fifty nanomolar of each of the Rho proteins from indicated pathogenic bacteria were used in these assays. The supernatant fraction (S) contains half of the released RNA, whereas the P fraction contains the rest of the sample (half of the supernatant + pellet). All the other experimental conditions were the same as in Figure 3 E . The fraction of released RNA ([2S]/([S] + [P])) in each case is shown as bar diagrams. The SDs were calculated from two to three measurements. C , Mycobacterium smegmatis strain mc 2 155 was transformed with either a pSTKT empty vector or pSTKT plasmid expressing either peptide 33 or WT Psu. Transformants were subsequently streaked onto 7H10 plates. Mycobacterium bovis BCG strain was transformed with the same plasmids as above. Transformants of the M. bovis strain were obtained after 3 weeks, and colonies were further streaked to show the growth differences in the M. bovis strains upon expression of either peptide 33 or the WT Psu. The spliced part of the images in Figure 9 , A and C are indicated by vertical lines .

Techniques Used: In Vitro, In Vivo, Inhibition, Transformation Assay, Plasmid Preparation, Expressing

36) Product Images from "A Murine Model of Mycobacterium kansasii Infection Reproducing Necrotic Lung Pathology Reveals Considerable Heterogeneity in Virulence of Clinical Isolates"

Article Title: A Murine Model of Mycobacterium kansasii Infection Reproducing Necrotic Lung Pathology Reveals Considerable Heterogeneity in Virulence of Clinical Isolates

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2021.718477

Colony morphotype variants of Mycobacterium kansasii strains. Mycobacterial strains were grown on Middlebrook 7H10 agar for 21 days ( M. kansasii ) and 28 days ( Mycobacterium tuberculosis ), and the macrocolony images were captured. (A) M. kansasii clinical isolates (images are demonstrated for strains 8835, 4404, and 10953) exhibited a rough colony shape (R) similar to that of the reference M. kansasii strain 12478 or the M. tuberculosis strain H37Rv, with exception of the isolate 6849, which showed smooth morphotype (S). (B) All M. kansasii strains grown on the agar exhibited yellow colonies under exposure to light.
Figure Legend Snippet: Colony morphotype variants of Mycobacterium kansasii strains. Mycobacterial strains were grown on Middlebrook 7H10 agar for 21 days ( M. kansasii ) and 28 days ( Mycobacterium tuberculosis ), and the macrocolony images were captured. (A) M. kansasii clinical isolates (images are demonstrated for strains 8835, 4404, and 10953) exhibited a rough colony shape (R) similar to that of the reference M. kansasii strain 12478 or the M. tuberculosis strain H37Rv, with exception of the isolate 6849, which showed smooth morphotype (S). (B) All M. kansasii strains grown on the agar exhibited yellow colonies under exposure to light.

Techniques Used:

37) Product Images from "Rv3722c governs aspartate-dependent nitrogen metabolism in Mycobacterium tuberculosis"

Article Title: Rv3722c governs aspartate-dependent nitrogen metabolism in Mycobacterium tuberculosis

Journal: bioRxiv

doi: 10.1101/784462

Rv3722c is conditionally esssential in vitro . A) Growth curve of Rv3722c-proficient and – deficient Mtb in 7H9 culture media. Rv3722c-TetOn and Rv3722c-control were cultured in Middlebrook 7H9 culture media with or without 500 ng/mL anhydrotetracycline (ATC). Bacterial growth was monitored by optical density at 600 nm. B) Western blot showing the depletion of Rv3722c 7H9 culture media. Rv3722c-TetON was cultured in 7H9 with or without ATC for 12 days (corresponding to A). Protein lysates were analyzed by Western blotting, using an α-FLAG antibody. The proteasome subunit β (PrcB) was used as loading control. C) Growth curve of Rv3722c-proficient and –deficient Mtb in 7H9 supplemented with casein hydrolysate. As A, but using 7H9 supplemented with 1% casein hydrolysate. D) Spot assay of Rv3722c-proficient and –deficient Mtb on solid growth media. A serially diluted Rv3722c-TetON culture (OD 0.1) was spotted onto Middlebrook 7H10 agar with or without 1% casein hydrolysate, in the presence or absence of ATC, and cultures for 2 weeks. E) Western blot showing depletion of Rv3722c. As B, but in 7H9 supplemented with 1% casein hydrolysate. F) Growth curve of of Rv3722c-proficient and –pre-depleted Mtb in 7H9 growth media. Rv3722c-TetOn in 7H9 with or without ATC, after pre-depletion of Rv3722c in 7H9 with 1% casein hydrolysate without ATC. For all growth curves, data are represented as mean -/+ SD of three experimental replicates (n=3) representative of at least two independent experiments. See also Fig S1 .
Figure Legend Snippet: Rv3722c is conditionally esssential in vitro . A) Growth curve of Rv3722c-proficient and – deficient Mtb in 7H9 culture media. Rv3722c-TetOn and Rv3722c-control were cultured in Middlebrook 7H9 culture media with or without 500 ng/mL anhydrotetracycline (ATC). Bacterial growth was monitored by optical density at 600 nm. B) Western blot showing the depletion of Rv3722c 7H9 culture media. Rv3722c-TetON was cultured in 7H9 with or without ATC for 12 days (corresponding to A). Protein lysates were analyzed by Western blotting, using an α-FLAG antibody. The proteasome subunit β (PrcB) was used as loading control. C) Growth curve of Rv3722c-proficient and –deficient Mtb in 7H9 supplemented with casein hydrolysate. As A, but using 7H9 supplemented with 1% casein hydrolysate. D) Spot assay of Rv3722c-proficient and –deficient Mtb on solid growth media. A serially diluted Rv3722c-TetON culture (OD 0.1) was spotted onto Middlebrook 7H10 agar with or without 1% casein hydrolysate, in the presence or absence of ATC, and cultures for 2 weeks. E) Western blot showing depletion of Rv3722c. As B, but in 7H9 supplemented with 1% casein hydrolysate. F) Growth curve of of Rv3722c-proficient and –pre-depleted Mtb in 7H9 growth media. Rv3722c-TetOn in 7H9 with or without ATC, after pre-depletion of Rv3722c in 7H9 with 1% casein hydrolysate without ATC. For all growth curves, data are represented as mean -/+ SD of three experimental replicates (n=3) representative of at least two independent experiments. See also Fig S1 .

Techniques Used: In Vitro, Cell Culture, Western Blot, Spot Test

Rv3722c is required for virulence in macrophages and mice. A) Timecourse of macrophage infection. Primary murine bone marrow-derived macrophages were treated with control or interferon γ (IFNγ) for activation, followed by infection with Rv3722c-control and Rv3722c-TetON precultured in 7H9+casein hydrolysate with or without ATC to pre-deplete Rv3722c (multiplicity of infection of 1). The number of colony-forming units (CFU’s) were assessed by plating serial dilutions on 7H10 solid media supplemented with casein hydrolysate and ATC. Data are presented as mean +/- SD of three experimental replicates (n=3) representative of two independent experiments; **: p
Figure Legend Snippet: Rv3722c is required for virulence in macrophages and mice. A) Timecourse of macrophage infection. Primary murine bone marrow-derived macrophages were treated with control or interferon γ (IFNγ) for activation, followed by infection with Rv3722c-control and Rv3722c-TetON precultured in 7H9+casein hydrolysate with or without ATC to pre-deplete Rv3722c (multiplicity of infection of 1). The number of colony-forming units (CFU’s) were assessed by plating serial dilutions on 7H10 solid media supplemented with casein hydrolysate and ATC. Data are presented as mean +/- SD of three experimental replicates (n=3) representative of two independent experiments; **: p

Techniques Used: Mouse Assay, Infection, Derivative Assay, Activation Assay

Rv3722c is dispensible in defined Sauton’s minimal media A) Growth curve of Rv3722c-proficient and –deficient Mtb in defined Sauton’ s minimal media. Rv3722c-TetOn and Rv3722c-control cultured in Middlebrook 7H9 culture media with 500 ng/mL anhydrotetracycline (ATC) were used to inoculate Sauton’s minimal media with and without 500 ng/mL ATC. Bacterial growth was monitored for 12 days, by optical density at 600 nm. Data are represented as mean -/+ SD of three experimental replicates (n=3), representative of at least two independent experiments. B) Western blot showing depletion of Rv3722c after 12 days of culturing in Sauton’s without ATC (A). Protein lysates were analyzed by Western blotting, using an α-FLAG antibody. The proteasome subunit β (PrcB) was used as loading control.
Figure Legend Snippet: Rv3722c is dispensible in defined Sauton’s minimal media A) Growth curve of Rv3722c-proficient and –deficient Mtb in defined Sauton’ s minimal media. Rv3722c-TetOn and Rv3722c-control cultured in Middlebrook 7H9 culture media with 500 ng/mL anhydrotetracycline (ATC) were used to inoculate Sauton’s minimal media with and without 500 ng/mL ATC. Bacterial growth was monitored for 12 days, by optical density at 600 nm. Data are represented as mean -/+ SD of three experimental replicates (n=3), representative of at least two independent experiments. B) Western blot showing depletion of Rv3722c after 12 days of culturing in Sauton’s without ATC (A). Protein lysates were analyzed by Western blotting, using an α-FLAG antibody. The proteasome subunit β (PrcB) was used as loading control.

Techniques Used: Cell Culture, Western Blot

Rv3722c is required for virulence in mice. Mice were infected with aerosolized Rv3722c-TetON pre-cultured in Sauton’s with and without ATC to generate Rv3722c-proficient and -deficient Mtb , respectively. After infection, mice were fed chow with (Rv3722c-proficient Mtb ) or without (Rv3722-deficient Mtb ) doxycycline. The number of CFU’s were assessed by plating serial dilutions on 7H10 solid media with ATC. The number of non ATC-responsive mutants were determined by plating on 7H10 solid media with and without ATC (n=2). Data are represented as mean +/- SD (n=3-5). Within 3 weeks after aerosol infection of mice, Rv3722-deficient Mtb were almost completely replaced by escape mutants that were no longer ATC-responsive.
Figure Legend Snippet: Rv3722c is required for virulence in mice. Mice were infected with aerosolized Rv3722c-TetON pre-cultured in Sauton’s with and without ATC to generate Rv3722c-proficient and -deficient Mtb , respectively. After infection, mice were fed chow with (Rv3722c-proficient Mtb ) or without (Rv3722-deficient Mtb ) doxycycline. The number of CFU’s were assessed by plating serial dilutions on 7H10 solid media with ATC. The number of non ATC-responsive mutants were determined by plating on 7H10 solid media with and without ATC (n=2). Data are represented as mean +/- SD (n=3-5). Within 3 weeks after aerosol infection of mice, Rv3722-deficient Mtb were almost completely replaced by escape mutants that were no longer ATC-responsive.

Techniques Used: Mouse Assay, Infection, Cell Culture

38) Product Images from "Mycobacterial Cultures Contain Cell Size and Density Specific Sub-populations of Cells with Significant Differential Susceptibility to Antibiotics, Oxidative and Nitrite Stress"

Article Title: Mycobacterial Cultures Contain Cell Size and Density Specific Sub-populations of Cells with Significant Differential Susceptibility to Antibiotics, Oxidative and Nitrite Stress

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2017.00463

Panels from Video S1 on the live cell imaging of Msm short cells generating micro colony and population regeneration potential of the cells in the SCF and NCF . (A) An Msm cell elongates and divides symmetrically to generate two short daughter cells. One of the short daughter cells (green), grew and divided asymmetrically to generate two unequal-sized daughter cells (indicated in cyan and pink). The other short daughter cell (red), grew and divided symmetrically to generate two comparably-sized short daughter cells (indicated in white and yellow). The daughter cells from the earlier asymmetric division (cyan) further divided symmetrically to generate two daughter cells. The short daughter cell (yellow) further grew and divided symmetrically to generate two daughter cells. Arrows show the site of constriction. Cells of length ≤ 2.60 ± 0.25 μm were considered short cells (as per Figure 1B ). (B) Fraction enriched for Msm short cells. (C) Cells from mid-log phase population. (D) Population generated from the short-cells-enriched fraction after reinoculation into Middlebrook 7H9 liquid medium. (E) Population generated from plating of the cells in the short-cells-enriched fraction on Middlebrook 7H10 agar. The compositions of the cells in (D,E) are similar to that in (C) . Arrows indicate short cells. Length of Msm short cells are shown in (B) . (F–I) Acid-fast stained cells in the SCF and NCF and the respective population generated from them after reinoculation into Middlebrook 7H9 media and incubated till the cultures reached 0.6 OD 600nm .
Figure Legend Snippet: Panels from Video S1 on the live cell imaging of Msm short cells generating micro colony and population regeneration potential of the cells in the SCF and NCF . (A) An Msm cell elongates and divides symmetrically to generate two short daughter cells. One of the short daughter cells (green), grew and divided asymmetrically to generate two unequal-sized daughter cells (indicated in cyan and pink). The other short daughter cell (red), grew and divided symmetrically to generate two comparably-sized short daughter cells (indicated in white and yellow). The daughter cells from the earlier asymmetric division (cyan) further divided symmetrically to generate two daughter cells. The short daughter cell (yellow) further grew and divided symmetrically to generate two daughter cells. Arrows show the site of constriction. Cells of length ≤ 2.60 ± 0.25 μm were considered short cells (as per Figure 1B ). (B) Fraction enriched for Msm short cells. (C) Cells from mid-log phase population. (D) Population generated from the short-cells-enriched fraction after reinoculation into Middlebrook 7H9 liquid medium. (E) Population generated from plating of the cells in the short-cells-enriched fraction on Middlebrook 7H10 agar. The compositions of the cells in (D,E) are similar to that in (C) . Arrows indicate short cells. Length of Msm short cells are shown in (B) . (F–I) Acid-fast stained cells in the SCF and NCF and the respective population generated from them after reinoculation into Middlebrook 7H9 media and incubated till the cultures reached 0.6 OD 600nm .

Techniques Used: Live Cell Imaging, Generated, Staining, Incubation

39) Product Images from "Isolation of nontuberculous mycobacteria from soil using Middlebrook 7H10 agar with increased malachite green concentration"

Article Title: Isolation of nontuberculous mycobacteria from soil using Middlebrook 7H10 agar with increased malachite green concentration

Journal: AMB Express

doi: 10.1186/s13568-017-0373-6

Modified Middlebrook 7H10 medium containing 250 mg/L MG with mycobacteria primarily isolated from soil. a Sample collected at campus, b sample collected at community, and c collected at natural park. Black arrows indicate mycobacterial colonies, red arrows indicate dried soils
Figure Legend Snippet: Modified Middlebrook 7H10 medium containing 250 mg/L MG with mycobacteria primarily isolated from soil. a Sample collected at campus, b sample collected at community, and c collected at natural park. Black arrows indicate mycobacterial colonies, red arrows indicate dried soils

Techniques Used: Modification, Isolation

40) Product Images from "Isolation of nontuberculous mycobacteria from soil using Middlebrook 7H10 agar with increased malachite green concentration"

Article Title: Isolation of nontuberculous mycobacteria from soil using Middlebrook 7H10 agar with increased malachite green concentration

Journal: AMB Express

doi: 10.1186/s13568-017-0373-6

Modified Middlebrook 7H10 medium containing 250 mg/L MG with mycobacteria primarily isolated from soil. a Sample collected at campus, b sample collected at community, and c collected at natural park. Black arrows indicate mycobacterial colonies, red arrows indicate dried soils
Figure Legend Snippet: Modified Middlebrook 7H10 medium containing 250 mg/L MG with mycobacteria primarily isolated from soil. a Sample collected at campus, b sample collected at community, and c collected at natural park. Black arrows indicate mycobacterial colonies, red arrows indicate dried soils

Techniques Used: Modification, Isolation

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    MiddleBrook Pharmaceuticals middlebrook 7h10 agar
    Modified <t>Middlebrook</t> <t>7H10</t> medium containing 250 mg/L MG with mycobacteria primarily isolated from soil. a Sample collected at campus, b sample collected at community, and c collected at natural park. Black arrows indicate mycobacterial colonies, red arrows indicate dried soils
    Middlebrook 7h10 Agar, supplied by MiddleBrook Pharmaceuticals, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    MiddleBrook Pharmaceuticals agar 7h10 medium
    Phenotypic characterization of BCG.HIVA 2auxo.int strain. We assessed the phenotype of lysine auxotrophy, lysine complementation and kanamycin resistance of BCG.HIVA 2auxo.int strain (A) BCG lysine auxotroph strain plated on non-lysine supplemented <t>7H10;</t> (B) BCG lysine auxotroph strain plated on lysine supplemented 7H10; (C) BCG.HIVA 2auxo.int plated on 7H10 without lysine and kanamycin supplementation; (D) BCG.HIVA 2auxo.int plated on 7H10 without lysine supplementation and with kanamycin.
    Agar 7h10 Medium, supplied by MiddleBrook Pharmaceuticals, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    MiddleBrook Pharmaceuticals 7h10 agar plates
    Bactericidal activity of HAMLET (HL) against M. tuberculosis . (A) M. tuberculosis H37Rv was grown in HdB minimal medium to an OD 600 of 1.3, and then 5 ml of culture was seeded into 12-well microplates. M. tuberculosis was treated with 300 μg/ml HAMLET on days 0, 1, and 2 as indicated by the arrows. The bactericidal activity of HAMLET was determined by measuring the optical density at 600 nm. Error bars represent standard errors of mean values of biological triplicates. (B) Images of the wells were taken on the second day of HAMLET treatment. (C) Tenfold serial dilutions of cultures of untreated and HAMLET-treated M. tuberculosis strains were plated on Middlebrook <t>7H10</t> agar after the first treatment with HAMLET (day 0). Agar plates were imaged after incubation at 37°C for 15 days.
    7h10 Agar Plates, supplied by MiddleBrook Pharmaceuticals, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Modified Middlebrook 7H10 medium containing 250 mg/L MG with mycobacteria primarily isolated from soil. a Sample collected at campus, b sample collected at community, and c collected at natural park. Black arrows indicate mycobacterial colonies, red arrows indicate dried soils

    Journal: AMB Express

    Article Title: Isolation of nontuberculous mycobacteria from soil using Middlebrook 7H10 agar with increased malachite green concentration

    doi: 10.1186/s13568-017-0373-6

    Figure Lengend Snippet: Modified Middlebrook 7H10 medium containing 250 mg/L MG with mycobacteria primarily isolated from soil. a Sample collected at campus, b sample collected at community, and c collected at natural park. Black arrows indicate mycobacterial colonies, red arrows indicate dried soils

    Article Snippet: Modified Middlebrook 7H10 agar with 25 mg/L MG or lower was insufficient to inhibit nontarget bacteria whereas MG at the concentration of 2500 in the medium can also suppress mycobacteria.

    Techniques: Modification, Isolation

    Growth impairment of AroA -knockdown cells depends on growth conditions. (A to H) M. smegmatis growth curves and dilution spots in the presence or absence of anhydrotetracycline (ATc) (100 ng/mL). (A to D) Growth in rich media: liquid LB (curves) and solid LB (dilution spots shown in figure insets). (E to H) Growth in defined media: liquid 7H9 (curves) and solid 7H10 (dilution spots in figure insets). (A and E) Control gene mmpL3 . (B to D and F to H) aroA gene knocked down using sgRNAs directed to locations adjacent to PAM1, PAM2, and PAM3 (B to D and F to H). Growth curves with and without ATc are different with P

    Journal: Microbiology Spectrum

    Article Title: EPSP Synthase-Depleted Cells Are Aromatic Amino Acid Auxotrophs in Mycobacterium smegmatis

    doi: 10.1128/Spectrum.00009-21

    Figure Lengend Snippet: Growth impairment of AroA -knockdown cells depends on growth conditions. (A to H) M. smegmatis growth curves and dilution spots in the presence or absence of anhydrotetracycline (ATc) (100 ng/mL). (A to D) Growth in rich media: liquid LB (curves) and solid LB (dilution spots shown in figure insets). (E to H) Growth in defined media: liquid 7H9 (curves) and solid 7H10 (dilution spots in figure insets). (A and E) Control gene mmpL3 . (B to D and F to H) aroA gene knocked down using sgRNAs directed to locations adjacent to PAM1, PAM2, and PAM3 (B to D and F to H). Growth curves with and without ATc are different with P

    Article Snippet: M. smegmatis was used for gene disruption and knockdown experiments, grown in LB (tryptone, yeast extract, NaCl, and agar) medium, Difco Middlebrook 7H9 broth (Becton, Dickinson - BD), supplemented with 0.05% (vol/vol) Tween 80 (Sigma-Aldrich), and 0.2% (vol/vol) glycerol (Merck), or Difco Middlebrook 7H10 agar (BD), supplemented with 0.5% (vol/vol) glycerol.

    Techniques:

    The D61W substitution in Ms EPSPS does not impair mycobacterial survival and growth in vitro . (A) Sequence alignment of EPSPS enzymes from M. smegmatis mc 2 155 (Ms), M. tuberculosis H37Rv (Mt), and E. coli CVM N33429PS (Ec). A multiple alignment for these proteins was made using Clustal Omega and then visualized and colored in Jalview. The color code represents the level of conservation of each amino acid, where darker shades of purple represent a higher conservation level. The enzymes from E. coli and M. tuberculosis have 52% and 78% positives and 31% and 68% of identity when aligned to the EPSPS from M. smegmatis , respectively. Amino acids indicated by black arrows were the ones chosen for mutagenesis. (B and C) PCR confirmation of double crossover (DCO) events leading to deletion of the original aroA allele in the M. smegmatis genome from merodiploid strains carrying an extra copy of the WT or mutated aroA gene encoding D61W EPSPS mutant. Genomic DNA was extracted from selected white colonies and used as the templates for PCRs in the presence of a forward primer upstream the 5′ AES (allelic exchange sequence), outside the region of recombination, and an internal reverse primer (see Table 3 ). The PCRs were performed on white colonies selected on LB medium (B) or 7H10 medium without supplementation with AroAAs (C). An amplicon of 1,813 bp was expected for allelic exchange mutants. (B) Lane M: 1 kb plus DNA ladder (Invitrogen). Lane 1: M. smegmatis mc 2 155 genomic DNA (negative control). Lanes 2 to 8: aroA -knockout cells obtained from a merodiploid strain carrying an extra copy of WT aroA gene. Lanes 9 to 10: aroA -knockout cells obtained from a merodiploid strain carrying an extra copy of aroA mutated that encodes the D61W Ms EPSPS protein. (C) Lane 1: M. smegmatis mc 2 155 genomic DNA (negative control). Lanes 2 to 7: aroA -deleted cells from merodiploid strains carrying an extra copy of WT aroA gene ( 2 – 6 ) or a mutant aroA gene encoding D61W EPSPS protein ( 7 ). (D) Control strains and a strain carrying an altered aroA sequence that encodes D61W EPSPS mutant ( aroA _D61W) and that have had the original aroA allele deleted were grown for 12 h in LB medium, under aerobic conditions. Aliquots were taken every 3 h for optical density measurement at 600 nm (OD 600 ). pNIP40::Ø: strain carrying the original aroA allele and an empty copy of the pNIP40/b vector integrated into the mycobacteriophage Ms6 chromosomal integration site; aroA _WT: strain having the original aroA allele deleted but carrying another copy of the WT aroA gene integrated. Error bars are standard deviation (SD) of three biological replicates. Growth curves from control strains pNIP40::Ø and aroA _WT are not statistically different compared to aroA _D61W growth curve at any time point ( P > 0.99 for all comparisons at 0 to 9 h; P = 0.49 for aroA _WT compared to aroA _D61W at 12 h; P > 0.99 for pNIP40::Ø compared to aroA _D61W at 12 h).

    Journal: Microbiology Spectrum

    Article Title: EPSP Synthase-Depleted Cells Are Aromatic Amino Acid Auxotrophs in Mycobacterium smegmatis

    doi: 10.1128/Spectrum.00009-21

    Figure Lengend Snippet: The D61W substitution in Ms EPSPS does not impair mycobacterial survival and growth in vitro . (A) Sequence alignment of EPSPS enzymes from M. smegmatis mc 2 155 (Ms), M. tuberculosis H37Rv (Mt), and E. coli CVM N33429PS (Ec). A multiple alignment for these proteins was made using Clustal Omega and then visualized and colored in Jalview. The color code represents the level of conservation of each amino acid, where darker shades of purple represent a higher conservation level. The enzymes from E. coli and M. tuberculosis have 52% and 78% positives and 31% and 68% of identity when aligned to the EPSPS from M. smegmatis , respectively. Amino acids indicated by black arrows were the ones chosen for mutagenesis. (B and C) PCR confirmation of double crossover (DCO) events leading to deletion of the original aroA allele in the M. smegmatis genome from merodiploid strains carrying an extra copy of the WT or mutated aroA gene encoding D61W EPSPS mutant. Genomic DNA was extracted from selected white colonies and used as the templates for PCRs in the presence of a forward primer upstream the 5′ AES (allelic exchange sequence), outside the region of recombination, and an internal reverse primer (see Table 3 ). The PCRs were performed on white colonies selected on LB medium (B) or 7H10 medium without supplementation with AroAAs (C). An amplicon of 1,813 bp was expected for allelic exchange mutants. (B) Lane M: 1 kb plus DNA ladder (Invitrogen). Lane 1: M. smegmatis mc 2 155 genomic DNA (negative control). Lanes 2 to 8: aroA -knockout cells obtained from a merodiploid strain carrying an extra copy of WT aroA gene. Lanes 9 to 10: aroA -knockout cells obtained from a merodiploid strain carrying an extra copy of aroA mutated that encodes the D61W Ms EPSPS protein. (C) Lane 1: M. smegmatis mc 2 155 genomic DNA (negative control). Lanes 2 to 7: aroA -deleted cells from merodiploid strains carrying an extra copy of WT aroA gene ( 2 – 6 ) or a mutant aroA gene encoding D61W EPSPS protein ( 7 ). (D) Control strains and a strain carrying an altered aroA sequence that encodes D61W EPSPS mutant ( aroA _D61W) and that have had the original aroA allele deleted were grown for 12 h in LB medium, under aerobic conditions. Aliquots were taken every 3 h for optical density measurement at 600 nm (OD 600 ). pNIP40::Ø: strain carrying the original aroA allele and an empty copy of the pNIP40/b vector integrated into the mycobacteriophage Ms6 chromosomal integration site; aroA _WT: strain having the original aroA allele deleted but carrying another copy of the WT aroA gene integrated. Error bars are standard deviation (SD) of three biological replicates. Growth curves from control strains pNIP40::Ø and aroA _WT are not statistically different compared to aroA _D61W growth curve at any time point ( P > 0.99 for all comparisons at 0 to 9 h; P = 0.49 for aroA _WT compared to aroA _D61W at 12 h; P > 0.99 for pNIP40::Ø compared to aroA _D61W at 12 h).

    Article Snippet: M. smegmatis was used for gene disruption and knockdown experiments, grown in LB (tryptone, yeast extract, NaCl, and agar) medium, Difco Middlebrook 7H9 broth (Becton, Dickinson - BD), supplemented with 0.05% (vol/vol) Tween 80 (Sigma-Aldrich), and 0.2% (vol/vol) glycerol (Merck), or Difco Middlebrook 7H10 agar (BD), supplemented with 0.5% (vol/vol) glycerol.

    Techniques: In Vitro, Sequencing, Mutagenesis, Polymerase Chain Reaction, Amplification, Negative Control, Knock-Out, Plasmid Preparation, Standard Deviation

    aroA -deleted M. smegmatis cells are auxotrophic for aromatic amino acids. (A) Schematic representation of the allelic exchange event in the aroA locus. Two putative genes (MSMEG_1891 and MSMEG_1889) flank the aroA gene (MSMEG_1890) of M. smegmatis . The allelic exchange sequences (AESs) were designed to maintain possible transcriptional and translation regulatory sequences of these two genes. Most of the aroA gene sequence was replaced by a kanamycin resistance cassette (1,252 bp), which was also used as a selective marker for homologous recombination. The positions of primers used in PCRs described in panel B are indicated by black arrows. (B) Confirmation of aroA gene knockout by allelic exchange mutagenesis in merodiploid WT or control strain of M. smegmatis grown on defined 7H10 medium with AroAA supplementation. Double crossover (DCO) events on the original aroA locus in the M. smegmatis genome were confirmed by PCR amplification. Genomic DNA was extracted from selected white colonies and used as the templates for PCRs in the presence of a forward primer upstream the 5′ AES (allelic exchange sequence), outside the region of recombination, and an internal reverse primer (see Fig. 2A and Table 3 ). An amplicon of 1,813 bp is expected for allelic exchange mutants. Lane M: 1 kb plus DNA ladder (Invitrogen). Lanes 1 and 13: M. smegmatis cells grown on 7H10 medium without supplementation: aroA -deleted cells (A) from merodiploid strain containing an extra copy of WT aroA gene (positive controls). Lanes 2 to 12: M. smegmatis cells grown on defined 7H10 medium with AroAA supplementation. Lane 2: M. smegmatis mc 2 155 genomic DNA (negative control). Lanes 3 to 4: aroA -deleted cells from merodiploid strain carrying an extra copy of WT aroA gene. Lanes 5 to 12: aroA -deleted cells from M. smegmatis strain containing an integrated copy of vector pNIP40/b (empty vector, pNIP40::Ø), without an extra copy of aroA gene.

    Journal: Microbiology Spectrum

    Article Title: EPSP Synthase-Depleted Cells Are Aromatic Amino Acid Auxotrophs in Mycobacterium smegmatis

    doi: 10.1128/Spectrum.00009-21

    Figure Lengend Snippet: aroA -deleted M. smegmatis cells are auxotrophic for aromatic amino acids. (A) Schematic representation of the allelic exchange event in the aroA locus. Two putative genes (MSMEG_1891 and MSMEG_1889) flank the aroA gene (MSMEG_1890) of M. smegmatis . The allelic exchange sequences (AESs) were designed to maintain possible transcriptional and translation regulatory sequences of these two genes. Most of the aroA gene sequence was replaced by a kanamycin resistance cassette (1,252 bp), which was also used as a selective marker for homologous recombination. The positions of primers used in PCRs described in panel B are indicated by black arrows. (B) Confirmation of aroA gene knockout by allelic exchange mutagenesis in merodiploid WT or control strain of M. smegmatis grown on defined 7H10 medium with AroAA supplementation. Double crossover (DCO) events on the original aroA locus in the M. smegmatis genome were confirmed by PCR amplification. Genomic DNA was extracted from selected white colonies and used as the templates for PCRs in the presence of a forward primer upstream the 5′ AES (allelic exchange sequence), outside the region of recombination, and an internal reverse primer (see Fig. 2A and Table 3 ). An amplicon of 1,813 bp is expected for allelic exchange mutants. Lane M: 1 kb plus DNA ladder (Invitrogen). Lanes 1 and 13: M. smegmatis cells grown on 7H10 medium without supplementation: aroA -deleted cells (A) from merodiploid strain containing an extra copy of WT aroA gene (positive controls). Lanes 2 to 12: M. smegmatis cells grown on defined 7H10 medium with AroAA supplementation. Lane 2: M. smegmatis mc 2 155 genomic DNA (negative control). Lanes 3 to 4: aroA -deleted cells from merodiploid strain carrying an extra copy of WT aroA gene. Lanes 5 to 12: aroA -deleted cells from M. smegmatis strain containing an integrated copy of vector pNIP40/b (empty vector, pNIP40::Ø), without an extra copy of aroA gene.

    Article Snippet: M. smegmatis was used for gene disruption and knockdown experiments, grown in LB (tryptone, yeast extract, NaCl, and agar) medium, Difco Middlebrook 7H9 broth (Becton, Dickinson - BD), supplemented with 0.05% (vol/vol) Tween 80 (Sigma-Aldrich), and 0.2% (vol/vol) glycerol (Merck), or Difco Middlebrook 7H10 agar (BD), supplemented with 0.5% (vol/vol) glycerol.

    Techniques: Sequencing, Marker, Homologous Recombination, Gene Knockout, Mutagenesis, Polymerase Chain Reaction, Amplification, Negative Control, Plasmid Preparation

    Phenotypic characterization of BCG.HIVA 2auxo.int strain. We assessed the phenotype of lysine auxotrophy, lysine complementation and kanamycin resistance of BCG.HIVA 2auxo.int strain (A) BCG lysine auxotroph strain plated on non-lysine supplemented 7H10; (B) BCG lysine auxotroph strain plated on lysine supplemented 7H10; (C) BCG.HIVA 2auxo.int plated on 7H10 without lysine and kanamycin supplementation; (D) BCG.HIVA 2auxo.int plated on 7H10 without lysine supplementation and with kanamycin.

    Journal: Human Vaccines & Immunotherapeutics

    Article Title: Preclinical development of BCG.HIVA2auxo.int, harboring an integrative expression vector, for a HIV-TB Pediatric vaccine. Enhancement of stability and specific HIV-1 T-cell immunity

    doi: 10.1080/21645515.2017.1316911

    Figure Lengend Snippet: Phenotypic characterization of BCG.HIVA 2auxo.int strain. We assessed the phenotype of lysine auxotrophy, lysine complementation and kanamycin resistance of BCG.HIVA 2auxo.int strain (A) BCG lysine auxotroph strain plated on non-lysine supplemented 7H10; (B) BCG lysine auxotroph strain plated on lysine supplemented 7H10; (C) BCG.HIVA 2auxo.int plated on 7H10 without lysine and kanamycin supplementation; (D) BCG.HIVA 2auxo.int plated on 7H10 without lysine supplementation and with kanamycin.

    Article Snippet: Bloom, and T. Hsu was transformed with the p2auxo.HIVAint plasmid DNA without the kanamycin resistance gene by electroporation.. Mycobacterial cultures were grown in Middlebrook 7H9 broth medium or on Middlebrook agar 7H10 medium supplemented with albumin-dextrose-catalase (ADC; Difco) containing 0.05% Tween 80.

    Techniques:

    Bactericidal activity of HAMLET (HL) against M. tuberculosis . (A) M. tuberculosis H37Rv was grown in HdB minimal medium to an OD 600 of 1.3, and then 5 ml of culture was seeded into 12-well microplates. M. tuberculosis was treated with 300 μg/ml HAMLET on days 0, 1, and 2 as indicated by the arrows. The bactericidal activity of HAMLET was determined by measuring the optical density at 600 nm. Error bars represent standard errors of mean values of biological triplicates. (B) Images of the wells were taken on the second day of HAMLET treatment. (C) Tenfold serial dilutions of cultures of untreated and HAMLET-treated M. tuberculosis strains were plated on Middlebrook 7H10 agar after the first treatment with HAMLET (day 0). Agar plates were imaged after incubation at 37°C for 15 days.

    Journal: Antimicrobial Agents and Chemotherapy

    Article Title: A Protein Complex from Human Milk Enhances the Activity of Antibiotics and Drugs against Mycobacterium tuberculosis

    doi: 10.1128/AAC.01846-18

    Figure Lengend Snippet: Bactericidal activity of HAMLET (HL) against M. tuberculosis . (A) M. tuberculosis H37Rv was grown in HdB minimal medium to an OD 600 of 1.3, and then 5 ml of culture was seeded into 12-well microplates. M. tuberculosis was treated with 300 μg/ml HAMLET on days 0, 1, and 2 as indicated by the arrows. The bactericidal activity of HAMLET was determined by measuring the optical density at 600 nm. Error bars represent standard errors of mean values of biological triplicates. (B) Images of the wells were taken on the second day of HAMLET treatment. (C) Tenfold serial dilutions of cultures of untreated and HAMLET-treated M. tuberculosis strains were plated on Middlebrook 7H10 agar after the first treatment with HAMLET (day 0). Agar plates were imaged after incubation at 37°C for 15 days.

    Article Snippet: Each day, the OD600 was measured, and 10 μl of each sample of serial 10-fold dilutions was dropped on Middlebrook 7H10 agar plates.

    Techniques: Activity Assay, Incubation

    Activity of recombinant HAMLET against M. tuberculosis in macrophages. The viability of differentiated THP-1 macrophages in the presence of increasing concentrations of recombinant α-lactalbumin (A), oleic acid (B), and recombinant HAMLET (C) was determined using the microplate alamarBlue assay. Error bars represent standard errors of mean values of biological triplicates. The MIC 90 is represented by dotted lines. (D) Differentiated THP-1 macrophages were infected with M. tuberculosis H37Rv at an MOI of 10 for 4 h. Then, recombinant HAMLET was added at the indicated amounts, and the viability of M. tuberculosis was determined for two days using a drop assay on Middlebrook 7H10 agar plates. The uninfected macrophages are shown as a negative control.

    Journal: Antimicrobial Agents and Chemotherapy

    Article Title: A Protein Complex from Human Milk Enhances the Activity of Antibiotics and Drugs against Mycobacterium tuberculosis

    doi: 10.1128/AAC.01846-18

    Figure Lengend Snippet: Activity of recombinant HAMLET against M. tuberculosis in macrophages. The viability of differentiated THP-1 macrophages in the presence of increasing concentrations of recombinant α-lactalbumin (A), oleic acid (B), and recombinant HAMLET (C) was determined using the microplate alamarBlue assay. Error bars represent standard errors of mean values of biological triplicates. The MIC 90 is represented by dotted lines. (D) Differentiated THP-1 macrophages were infected with M. tuberculosis H37Rv at an MOI of 10 for 4 h. Then, recombinant HAMLET was added at the indicated amounts, and the viability of M. tuberculosis was determined for two days using a drop assay on Middlebrook 7H10 agar plates. The uninfected macrophages are shown as a negative control.

    Article Snippet: Each day, the OD600 was measured, and 10 μl of each sample of serial 10-fold dilutions was dropped on Middlebrook 7H10 agar plates.

    Techniques: Activity Assay, Recombinant, Alamar Blue Assay, Infection, Negative Control

    Recombinant HAMLET increases the efficacy of bedaquiline, delamanid, and clarithromycin against M. tuberculosis in infected macrophages. Differentiated THP-1 cells were infected with M. tuberculosis H37Rv at an MOI of 10, washed, and treated with serial dilutions of bedaquiline (A), delamanid (B), and clarithromycin (C) in the presence and absence of 200 μg/ml recombinant HAMLET for two days. Ten-microliter drops of lysed macrophages were then plated on Middlebrook 7H10 agar plates which were incubated for 13 days at 37°C.

    Journal: Antimicrobial Agents and Chemotherapy

    Article Title: A Protein Complex from Human Milk Enhances the Activity of Antibiotics and Drugs against Mycobacterium tuberculosis

    doi: 10.1128/AAC.01846-18

    Figure Lengend Snippet: Recombinant HAMLET increases the efficacy of bedaquiline, delamanid, and clarithromycin against M. tuberculosis in infected macrophages. Differentiated THP-1 cells were infected with M. tuberculosis H37Rv at an MOI of 10, washed, and treated with serial dilutions of bedaquiline (A), delamanid (B), and clarithromycin (C) in the presence and absence of 200 μg/ml recombinant HAMLET for two days. Ten-microliter drops of lysed macrophages were then plated on Middlebrook 7H10 agar plates which were incubated for 13 days at 37°C.

    Article Snippet: Each day, the OD600 was measured, and 10 μl of each sample of serial 10-fold dilutions was dropped on Middlebrook 7H10 agar plates.

    Techniques: Recombinant, Infection, Incubation