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Rel Mtb -mediated (p)ppGpp hydrolysis is required for growth in vitro and maintenance of ATP and GTP levels when (p)ppGpp is being produced. (A and B) An M. tuberculosis Δ rel Mtb strain containing an episomal vector expressing the WT TetR that induces expression of rel Mtb H80A from a cassette integrated at the attB site in the genome in the presence of ATc (Tet-Rel Mtb H80A strain, designated H80A here) and a WT M. tuberculosis strain containing an empty vector in the attB site and transformed with the same TetR-expressing plasmid (control strain). (A) Transcript levels in exponential-growth-phase cultures in liquid 7H9 media. rel Mtb H80A transcripts from the cassette at the attB site were detected in the Tet-Rel Mtb H80A strain, and transcripts from the endogenous rel Mtb gene were detected in the control strain. The first comparison shows the ratio of transcript levels in the presence compared to the absence of ATc in each strain (+ATc/−ATc), and it illustrates the induction of rel Mtb H80A in the Tet-Rel Mtb H80A strain when exposed to ATc and the unresponsiveness of the rel Mtb gene to ATc in the control strain. The second comparison shows the ratio of transcript levels in the Tet-Rel Mtb H80A strain compared to the control strain (H80A/control) under each condition to illustrate the low level of transcription of rel Mtb H80A in the absence of ATc compared to that of induced cultures. (B) M. tuberculosis Tet-Rel Mtb H80A and control strains were diluted and plated on <t>7H10</t> plates in the absence or presence of ATc. (C to G) M. smegmatis Δ rel Msm strains containing an episomal vector expressing the WT TetR that turns on expression of rel Mtb WT (Tet-Rel Mtb WT strain; designated the control) or rel Mtb H80A (Tet-Rel Mtb H80A strain; designated H80A here) from cassettes integrated at the attB site in the genome in the presence of ATc. (C and D) M. smegmatis strains were diluted and plated on 7H10 (C) and LB (D) plates in the absence or presence of ATc. (E) Graphic representation of the number of M. smegmatis CFU that grew in the presence compared to the absence of ATc when grown on LB, where all Tet-Rel Mtb H80A strain bacteria that grew in the presence of ATc on LB were suppressors and were no longer responsive to ATc treatment. Data are means ± SEM from 13 replicates. (F) ATP and GTP levels in M. smegmatis strains grown in the absence or presence of ATc for 6 h to an ODλ 600 of ∼0.6 in LB liquid media were measured by LC-MS/MS. The ratio of levels in the Tet-Rel Mtb H80A strain (H80A) to those in the Tet-Rel Mtb WT -expressing strain (control) under the same conditions is graphed. Data are the means ± SEM from 4 to 6 replicates. (E and F) The significance of differences was determined by calculating P values by Student's t tests; one asterisk indicates significance with a P value of
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1) Product Images from "Essential Roles for Mycobacterium tuberculosis Rel beyond the Production of (p)ppGpp"

Article Title: Essential Roles for Mycobacterium tuberculosis Rel beyond the Production of (p)ppGpp

Journal: Journal of Bacteriology

doi: 10.1128/JB.00759-13

Rel Mtb -mediated (p)ppGpp hydrolysis is required for growth in vitro and maintenance of ATP and GTP levels when (p)ppGpp is being produced. (A and B) An M. tuberculosis Δ rel Mtb strain containing an episomal vector expressing the WT TetR that induces expression of rel Mtb H80A from a cassette integrated at the attB site in the genome in the presence of ATc (Tet-Rel Mtb H80A strain, designated H80A here) and a WT M. tuberculosis strain containing an empty vector in the attB site and transformed with the same TetR-expressing plasmid (control strain). (A) Transcript levels in exponential-growth-phase cultures in liquid 7H9 media. rel Mtb H80A transcripts from the cassette at the attB site were detected in the Tet-Rel Mtb H80A strain, and transcripts from the endogenous rel Mtb gene were detected in the control strain. The first comparison shows the ratio of transcript levels in the presence compared to the absence of ATc in each strain (+ATc/−ATc), and it illustrates the induction of rel Mtb H80A in the Tet-Rel Mtb H80A strain when exposed to ATc and the unresponsiveness of the rel Mtb gene to ATc in the control strain. The second comparison shows the ratio of transcript levels in the Tet-Rel Mtb H80A strain compared to the control strain (H80A/control) under each condition to illustrate the low level of transcription of rel Mtb H80A in the absence of ATc compared to that of induced cultures. (B) M. tuberculosis Tet-Rel Mtb H80A and control strains were diluted and plated on 7H10 plates in the absence or presence of ATc. (C to G) M. smegmatis Δ rel Msm strains containing an episomal vector expressing the WT TetR that turns on expression of rel Mtb WT (Tet-Rel Mtb WT strain; designated the control) or rel Mtb H80A (Tet-Rel Mtb H80A strain; designated H80A here) from cassettes integrated at the attB site in the genome in the presence of ATc. (C and D) M. smegmatis strains were diluted and plated on 7H10 (C) and LB (D) plates in the absence or presence of ATc. (E) Graphic representation of the number of M. smegmatis CFU that grew in the presence compared to the absence of ATc when grown on LB, where all Tet-Rel Mtb H80A strain bacteria that grew in the presence of ATc on LB were suppressors and were no longer responsive to ATc treatment. Data are means ± SEM from 13 replicates. (F) ATP and GTP levels in M. smegmatis strains grown in the absence or presence of ATc for 6 h to an ODλ 600 of ∼0.6 in LB liquid media were measured by LC-MS/MS. The ratio of levels in the Tet-Rel Mtb H80A strain (H80A) to those in the Tet-Rel Mtb WT -expressing strain (control) under the same conditions is graphed. Data are the means ± SEM from 4 to 6 replicates. (E and F) The significance of differences was determined by calculating P values by Student's t tests; one asterisk indicates significance with a P value of
Figure Legend Snippet: Rel Mtb -mediated (p)ppGpp hydrolysis is required for growth in vitro and maintenance of ATP and GTP levels when (p)ppGpp is being produced. (A and B) An M. tuberculosis Δ rel Mtb strain containing an episomal vector expressing the WT TetR that induces expression of rel Mtb H80A from a cassette integrated at the attB site in the genome in the presence of ATc (Tet-Rel Mtb H80A strain, designated H80A here) and a WT M. tuberculosis strain containing an empty vector in the attB site and transformed with the same TetR-expressing plasmid (control strain). (A) Transcript levels in exponential-growth-phase cultures in liquid 7H9 media. rel Mtb H80A transcripts from the cassette at the attB site were detected in the Tet-Rel Mtb H80A strain, and transcripts from the endogenous rel Mtb gene were detected in the control strain. The first comparison shows the ratio of transcript levels in the presence compared to the absence of ATc in each strain (+ATc/−ATc), and it illustrates the induction of rel Mtb H80A in the Tet-Rel Mtb H80A strain when exposed to ATc and the unresponsiveness of the rel Mtb gene to ATc in the control strain. The second comparison shows the ratio of transcript levels in the Tet-Rel Mtb H80A strain compared to the control strain (H80A/control) under each condition to illustrate the low level of transcription of rel Mtb H80A in the absence of ATc compared to that of induced cultures. (B) M. tuberculosis Tet-Rel Mtb H80A and control strains were diluted and plated on 7H10 plates in the absence or presence of ATc. (C to G) M. smegmatis Δ rel Msm strains containing an episomal vector expressing the WT TetR that turns on expression of rel Mtb WT (Tet-Rel Mtb WT strain; designated the control) or rel Mtb H80A (Tet-Rel Mtb H80A strain; designated H80A here) from cassettes integrated at the attB site in the genome in the presence of ATc. (C and D) M. smegmatis strains were diluted and plated on 7H10 (C) and LB (D) plates in the absence or presence of ATc. (E) Graphic representation of the number of M. smegmatis CFU that grew in the presence compared to the absence of ATc when grown on LB, where all Tet-Rel Mtb H80A strain bacteria that grew in the presence of ATc on LB were suppressors and were no longer responsive to ATc treatment. Data are means ± SEM from 13 replicates. (F) ATP and GTP levels in M. smegmatis strains grown in the absence or presence of ATc for 6 h to an ODλ 600 of ∼0.6 in LB liquid media were measured by LC-MS/MS. The ratio of levels in the Tet-Rel Mtb H80A strain (H80A) to those in the Tet-Rel Mtb WT -expressing strain (control) under the same conditions is graphed. Data are the means ± SEM from 4 to 6 replicates. (E and F) The significance of differences was determined by calculating P values by Student's t tests; one asterisk indicates significance with a P value of

Techniques Used: In Vitro, Produced, Plasmid Preparation, Expressing, Transformation Assay, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry

Rv1366 is not necessary for M. tuberculosis growth or biofilm formation in vitro . (A) Southern blot analysis of WT M. tuberculosis Erdman (lane 1) and the Δ rv1366 strain (lanes 2 to 4) using EcoRI-digested genomic DNA and a probe that spans nucleotides 1537706 to 1538406 of the M. tuberculosis genome. EcoRI digestion of WT M. tuberculosis yields a band at 5,083 bp. EcoRI digestion of Δ rv1366 results in a 2,226-bp band due to the replacement of the rv1366 gene with a hygromycin resistance cassette. (B) Southern blot analysis of WT M. tuberculosis Erdman (lane 1) and the Δ rel Mtb Δ rv1366 strain (lanes 2 to 6) using SmaI-digested genomic DNA and a probe that spans nucleotides 2910191 to 2910844 of the M. tuberculosis genome. SmaI digestion of WT M. tuberculosis yields a band at 2,257 bp. SmaI digestion of the Δ rel Mtb Δ rv1366 strain results in a 4,769-bp band due to the replacement of the rel Mtb gene with a hygromycin resistance cassette. (C and D) M. tuberculosis Δ rel Mtb , Δ rv1366 , and Δ rel Mtb Δ rv1366 strains containing a vector that constitutively expresses Rel Mtb WT or Rv1366 or containing an empty vector (−) integrated at the attB site in the genome were normalized to the same ODλ 600 and either diluted and plated on 7H10 plates (C) or grown in Sauton's media in static, biofilm-forming conditions in a 24-well dish (D). (C) 7H10 plates were incubated for 3 weeks at 37°C. (D) Biofilms were incubated for 4 weeks at 37°C. The parent strain used is labeled above each photo, and the protein expressed from the attB site is labeled below the photo.
Figure Legend Snippet: Rv1366 is not necessary for M. tuberculosis growth or biofilm formation in vitro . (A) Southern blot analysis of WT M. tuberculosis Erdman (lane 1) and the Δ rv1366 strain (lanes 2 to 4) using EcoRI-digested genomic DNA and a probe that spans nucleotides 1537706 to 1538406 of the M. tuberculosis genome. EcoRI digestion of WT M. tuberculosis yields a band at 5,083 bp. EcoRI digestion of Δ rv1366 results in a 2,226-bp band due to the replacement of the rv1366 gene with a hygromycin resistance cassette. (B) Southern blot analysis of WT M. tuberculosis Erdman (lane 1) and the Δ rel Mtb Δ rv1366 strain (lanes 2 to 6) using SmaI-digested genomic DNA and a probe that spans nucleotides 2910191 to 2910844 of the M. tuberculosis genome. SmaI digestion of WT M. tuberculosis yields a band at 2,257 bp. SmaI digestion of the Δ rel Mtb Δ rv1366 strain results in a 4,769-bp band due to the replacement of the rel Mtb gene with a hygromycin resistance cassette. (C and D) M. tuberculosis Δ rel Mtb , Δ rv1366 , and Δ rel Mtb Δ rv1366 strains containing a vector that constitutively expresses Rel Mtb WT or Rv1366 or containing an empty vector (−) integrated at the attB site in the genome were normalized to the same ODλ 600 and either diluted and plated on 7H10 plates (C) or grown in Sauton's media in static, biofilm-forming conditions in a 24-well dish (D). (C) 7H10 plates were incubated for 3 weeks at 37°C. (D) Biofilms were incubated for 4 weeks at 37°C. The parent strain used is labeled above each photo, and the protein expressed from the attB site is labeled below the photo.

Techniques Used: In Vitro, Southern Blot, Plasmid Preparation, Incubation, Labeling

Rel Mtb -mediated (p)ppGpp synthesis is necessary for optimal M. tuberculosis growth and biofilm formation in vitro. M. tuberculosis Δ rel Mtb strains containing a vector that constitutively expresses Rel Mtb WT (WT) or Rel Mtb H344Y (H344Y) or containing an empty vector (−) integrated at the attB site in the genome were normalized to the same ODλ 600 and either diluted and plated on 7H10 plates (A) or grown in Sauton's medium in static, biofilm-forming conditions in a 24-well dish (B). (A) 7H10 plates were incubated for 3 weeks at 37°C. (B) Biofilms were incubated for 4 weeks at 37°C. The parent strain used is labeled above each photo, and the protein expressed from the attB site is labeled below the photo.
Figure Legend Snippet: Rel Mtb -mediated (p)ppGpp synthesis is necessary for optimal M. tuberculosis growth and biofilm formation in vitro. M. tuberculosis Δ rel Mtb strains containing a vector that constitutively expresses Rel Mtb WT (WT) or Rel Mtb H344Y (H344Y) or containing an empty vector (−) integrated at the attB site in the genome were normalized to the same ODλ 600 and either diluted and plated on 7H10 plates (A) or grown in Sauton's medium in static, biofilm-forming conditions in a 24-well dish (B). (A) 7H10 plates were incubated for 3 weeks at 37°C. (B) Biofilms were incubated for 4 weeks at 37°C. The parent strain used is labeled above each photo, and the protein expressed from the attB site is labeled below the photo.

Techniques Used: In Vitro, Plasmid Preparation, Incubation, Labeling

Rel Mtb (p)ppGpp hydrolase activity is essential for acute and chronic M. tuberculosis infection of mice. Bacterial titers in the lungs (A, E, and G) and spleens (B and H) of C57BL/6 mice infected with either control or Tet-Rel Mtb H80A (designated H80A in the figure) M. tuberculosis strains. The parent strain for the control is WT M. tuberculosis Erdman, and the parent strain for Tet-Rel Mtb H80A is M. tuberculosis Δ rel Mtb . Both strains express TetR from a plasmid that confers streptomycin resistance and contain a kanamycin resistance cassette integrated into the attB site that, in the case of Tet-Rel Mtb H80A , carries the rel Mtb H80A allele. The strain symbols in panel A are the same for all panels. (A to C) Mice were given normal mouse chow throughout infection. (E and F) Mice were administered doxycycline-containing mouse chow starting at day 1 postinfection (designated by the arrow). (G to I) Mice were administered doxycycline-containing mouse chow starting at day 63 postinfection (designated by the arrow). (C, F, and I) The ratio of CFU from the lungs or spleens grown on 7H10 plates containing streptomycin and kanamycin compared to 7H10 containing no antibiotics (No AB). ND denotes when no colonies were recovered after plating 5% of the lung homogenate (limit of detection, 20 CFU). Data are means ± SEM of 6 Tet-Rel Mtb H80A strain-infected mice and 3 control strain-infected mice per time point from two replicate experiments. (E, G, and H) The significance of differences were determined by calculating P values by Student's t tests; two asterisks indicate significance with a P value of
Figure Legend Snippet: Rel Mtb (p)ppGpp hydrolase activity is essential for acute and chronic M. tuberculosis infection of mice. Bacterial titers in the lungs (A, E, and G) and spleens (B and H) of C57BL/6 mice infected with either control or Tet-Rel Mtb H80A (designated H80A in the figure) M. tuberculosis strains. The parent strain for the control is WT M. tuberculosis Erdman, and the parent strain for Tet-Rel Mtb H80A is M. tuberculosis Δ rel Mtb . Both strains express TetR from a plasmid that confers streptomycin resistance and contain a kanamycin resistance cassette integrated into the attB site that, in the case of Tet-Rel Mtb H80A , carries the rel Mtb H80A allele. The strain symbols in panel A are the same for all panels. (A to C) Mice were given normal mouse chow throughout infection. (E and F) Mice were administered doxycycline-containing mouse chow starting at day 1 postinfection (designated by the arrow). (G to I) Mice were administered doxycycline-containing mouse chow starting at day 63 postinfection (designated by the arrow). (C, F, and I) The ratio of CFU from the lungs or spleens grown on 7H10 plates containing streptomycin and kanamycin compared to 7H10 containing no antibiotics (No AB). ND denotes when no colonies were recovered after plating 5% of the lung homogenate (limit of detection, 20 CFU). Data are means ± SEM of 6 Tet-Rel Mtb H80A strain-infected mice and 3 control strain-infected mice per time point from two replicate experiments. (E, G, and H) The significance of differences were determined by calculating P values by Student's t tests; two asterisks indicate significance with a P value of

Techniques Used: Activity Assay, Infection, Mouse Assay, Plasmid Preparation

2) Product Images from "The EXIT Strategy: an Approach for Identifying Bacterial Proteins Exported during Host Infection"

Article Title: The EXIT Strategy: an Approach for Identifying Bacterial Proteins Exported during Host Infection

Journal: mBio

doi: 10.1128/mBio.00333-17

Strategy for identification of in vivo induced exported proteins. (a) Identification of in vivo induced exported proteins. Spleens from β-lactam-treated mice infected with the EXIT library were harvested after 2 weeks of infection. Spleen homogenates were plated in parallel on 7H10 agar without β-lactam to recover all clones (red Venn diagram) and on 7H10 agar containing β-lactam to recover clones exporting ‘BlaTEM fusion proteins during in vivo growth and in vitro growth (purple Venn diagram). The population of clones identified only or in significantly greater abundance on media lacking β-lactams represents proteins whose export was induced during infection (blue). (b) Sequenced read count values recovered from agar with or without β-lactam for the 593 EXIT proteins were plotted to compare abundances after β-lactam treatment in vivo , with the abundance after dual β-lactam treatment in vivo and in vitro indicated. The majority of proteins identified as exported in vivo remained highly abundant after additional β-lactam treatment in vitro (black). A total of 38 genes (highlighted in red) were identified as statistically less abundant after in vitro β-lactam selection, representing proteins exported significantly more in vivo than in vitro (see Materials and Methods for details on statistical analysis). (c) In vivo induced exported proteins with roles promoting growth in macrophages ( rv1508 :: tn , rv3707c:tn , rv0559c :: tn , and rv2536 :: tn ). Murine bone marrow-derived macrophages were infected with M. tuberculosis CDC1551 transposon mutants lacking individual in vivo induced exported proteins. At specific times postinfection, macrophage lysates were plated to measure intracellular CFU. The fold change in CFU over the course of the infection is plotted relative to the bacterial burden at day 0 postinfection. Statistical significance was determined by one-way analysis of variance (ANOVA) with multiple comparisons performed by the use of the Holm-Sidak (normal by Shapiro-Wilk) or Student-Newman-Keuls (nonnormal) test (*, P
Figure Legend Snippet: Strategy for identification of in vivo induced exported proteins. (a) Identification of in vivo induced exported proteins. Spleens from β-lactam-treated mice infected with the EXIT library were harvested after 2 weeks of infection. Spleen homogenates were plated in parallel on 7H10 agar without β-lactam to recover all clones (red Venn diagram) and on 7H10 agar containing β-lactam to recover clones exporting ‘BlaTEM fusion proteins during in vivo growth and in vitro growth (purple Venn diagram). The population of clones identified only or in significantly greater abundance on media lacking β-lactams represents proteins whose export was induced during infection (blue). (b) Sequenced read count values recovered from agar with or without β-lactam for the 593 EXIT proteins were plotted to compare abundances after β-lactam treatment in vivo , with the abundance after dual β-lactam treatment in vivo and in vitro indicated. The majority of proteins identified as exported in vivo remained highly abundant after additional β-lactam treatment in vitro (black). A total of 38 genes (highlighted in red) were identified as statistically less abundant after in vitro β-lactam selection, representing proteins exported significantly more in vivo than in vitro (see Materials and Methods for details on statistical analysis). (c) In vivo induced exported proteins with roles promoting growth in macrophages ( rv1508 :: tn , rv3707c:tn , rv0559c :: tn , and rv2536 :: tn ). Murine bone marrow-derived macrophages were infected with M. tuberculosis CDC1551 transposon mutants lacking individual in vivo induced exported proteins. At specific times postinfection, macrophage lysates were plated to measure intracellular CFU. The fold change in CFU over the course of the infection is plotted relative to the bacterial burden at day 0 postinfection. Statistical significance was determined by one-way analysis of variance (ANOVA) with multiple comparisons performed by the use of the Holm-Sidak (normal by Shapiro-Wilk) or Student-Newman-Keuls (nonnormal) test (*, P

Techniques Used: In Vivo, Mouse Assay, Infection, Clone Assay, In Vitro, Selection, Derivative Assay

(a) The ‘BlaTEM reporter. The ‘BlaTEM reporter is compatible with proteins localized to the bacterial cytoplasmic membrane or cell wall or secreted from the bacterial cell. The right panel indicates in-frame fusions to categories of exported proteins that confer β-lactam resistance (red). In-frame fusions to cytoplasmic proteins or the cytoplasmic domain of integral membrane proteins (purple) do not confer β-lactam resistance. (b) EXIT strategy. In step 1, a comprehensive library of 5 × 10 6 plasmids containing fragments of M. tuberculosis (Mtb) genomic DNA fused to the ‘ bla TEM reporter was constructed. The plasmid library was transformed into the ΔblaC β-lactamase-sensitive mutant of M. tuberculosis , and 5 × 10 6 transformants were pooled to generate the EXIT library. In step 2, mice were infected by intravenous injection with the EXIT library and treated with β-lactam antibiotics (oral gavage twice daily) to select for EXIT clones exporting ‘BlaTEM fusion proteins. β-lactam treatment began 1 day after infection and continued to 2 weeks after infection. Mice were sacrificed, and spleens and lungs were harvested and homogenized. In step 3, organ homogenates were plated on 7H10 agar and grown to recover M. tuberculosis clones that survived β-lactam treatment during infection. Plates were scraped, and colonies were pooled separately for lungs and spleens. In step 4, plasmids from the recovered bacteria and the input samples were isolated and the fusion junction was sequenced using next-generation sequencing. Sequencing primers were designed to read out of the ‘ bla TEM reporter and sequence the immediately adjacent M. tuberculosis DNA. Sequences were aligned to the M. tuberculosis genome. Unique sequences were counted to identify the abundance of each fusion junction site within the population. The genes that were most highly abundant after in vivo β-lactam treatment were identified, and the results corresponded to plasmids producing in-frame exported ‘BlaTEM fusion proteins.
Figure Legend Snippet: (a) The ‘BlaTEM reporter. The ‘BlaTEM reporter is compatible with proteins localized to the bacterial cytoplasmic membrane or cell wall or secreted from the bacterial cell. The right panel indicates in-frame fusions to categories of exported proteins that confer β-lactam resistance (red). In-frame fusions to cytoplasmic proteins or the cytoplasmic domain of integral membrane proteins (purple) do not confer β-lactam resistance. (b) EXIT strategy. In step 1, a comprehensive library of 5 × 10 6 plasmids containing fragments of M. tuberculosis (Mtb) genomic DNA fused to the ‘ bla TEM reporter was constructed. The plasmid library was transformed into the ΔblaC β-lactamase-sensitive mutant of M. tuberculosis , and 5 × 10 6 transformants were pooled to generate the EXIT library. In step 2, mice were infected by intravenous injection with the EXIT library and treated with β-lactam antibiotics (oral gavage twice daily) to select for EXIT clones exporting ‘BlaTEM fusion proteins. β-lactam treatment began 1 day after infection and continued to 2 weeks after infection. Mice were sacrificed, and spleens and lungs were harvested and homogenized. In step 3, organ homogenates were plated on 7H10 agar and grown to recover M. tuberculosis clones that survived β-lactam treatment during infection. Plates were scraped, and colonies were pooled separately for lungs and spleens. In step 4, plasmids from the recovered bacteria and the input samples were isolated and the fusion junction was sequenced using next-generation sequencing. Sequencing primers were designed to read out of the ‘ bla TEM reporter and sequence the immediately adjacent M. tuberculosis DNA. Sequences were aligned to the M. tuberculosis genome. Unique sequences were counted to identify the abundance of each fusion junction site within the population. The genes that were most highly abundant after in vivo β-lactam treatment were identified, and the results corresponded to plasmids producing in-frame exported ‘BlaTEM fusion proteins.

Techniques Used: Transmission Electron Microscopy, Construct, Plasmid Preparation, Transformation Assay, Mutagenesis, Mouse Assay, Infection, Injection, Clone Assay, Isolation, Next-Generation Sequencing, Sequencing, In Vivo

3) Product Images from "ideR, an Essential Gene in Mycobacterium tuberculosis: Role of IdeR in Iron-Dependent Gene Expression, Iron Metabolism, and Oxidative Stress Response †"

Article Title: ideR, an Essential Gene in Mycobacterium tuberculosis: Role of IdeR in Iron-Dependent Gene Expression, Iron Metabolism, and Oxidative Stress Response †

Journal: Infection and Immunity

doi: 10.1128/IAI.70.7.3371-3381.2002

Sensitivity to oxidative stress. Shown are the diameters of the zones of growth inhibition produced in the presence of 600 mM hydrogen peroxide (A) and 5 mM plumbagin (B). Wild-type H37Rv, ST22 ( ideR :: aph ), and ST52 ( ideR complemented) were grown in 7H9 medium and plated in 7H10 medium. The values represent means plus standard deviations (the experiments were performed in triplicate).
Figure Legend Snippet: Sensitivity to oxidative stress. Shown are the diameters of the zones of growth inhibition produced in the presence of 600 mM hydrogen peroxide (A) and 5 mM plumbagin (B). Wild-type H37Rv, ST22 ( ideR :: aph ), and ST52 ( ideR complemented) were grown in 7H9 medium and plated in 7H10 medium. The values represent means plus standard deviations (the experiments were performed in triplicate).

Techniques Used: Inhibition, Produced

4) Product Images from "Real-Time Investigation of Tuberculosis Transmission: Developing the Respiratory Aerosol Sampling Chamber (RASC)"

Article Title: Real-Time Investigation of Tuberculosis Transmission: Developing the Respiratory Aerosol Sampling Chamber (RASC)

Journal: PLoS ONE

doi: 10.1371/journal.pone.0146658

Isolation and visualization of viable mycobacteria in the RASC. (A) M . smegmatis :: gfp growth on solid 7H10 agar plates from the Six-Stage Viable Andersen Cascade Impactor after wet release of 200 μl diluted culture into the RASC (30 000, 3000 and 300 colony forming units—CFU). The columns indicate the particle sizes captured on each plate across the 6 stages of the impactor, and the rows indicate the estimated total number of CFU passing through the impactor. Each release was repeated three times and the mean and SD for each plate are presented below the typical growth pattern distribution seen in the particle release. In all the releases the sampling was run for 5 minutes at 28 l/min resulting in the potential total capture of 3000, 300 and 30 CFU respectively. (B) SEM (left) and fluorescent microscopy (right) of M . smegmatis :: gfp isolated on a PM10 impactor following experimental release.
Figure Legend Snippet: Isolation and visualization of viable mycobacteria in the RASC. (A) M . smegmatis :: gfp growth on solid 7H10 agar plates from the Six-Stage Viable Andersen Cascade Impactor after wet release of 200 μl diluted culture into the RASC (30 000, 3000 and 300 colony forming units—CFU). The columns indicate the particle sizes captured on each plate across the 6 stages of the impactor, and the rows indicate the estimated total number of CFU passing through the impactor. Each release was repeated three times and the mean and SD for each plate are presented below the typical growth pattern distribution seen in the particle release. In all the releases the sampling was run for 5 minutes at 28 l/min resulting in the potential total capture of 3000, 300 and 30 CFU respectively. (B) SEM (left) and fluorescent microscopy (right) of M . smegmatis :: gfp isolated on a PM10 impactor following experimental release.

Techniques Used: Isolation, Sampling, Microscopy

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

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

Journal: Infection and Immunity

doi: 10.1128/IAI.00229-12

Sensitivity of M. tuberculosis strains to oxidative stress. (A and B) Diameters of the zones of inhibition produced in the presence of 500 mM H 2 O 2 (A) or 40 mM menadione (B). KO, knockout. (C) Diameters of the zone of inhibition on 7H10 agar (white columns)
Figure Legend Snippet: Sensitivity of M. tuberculosis strains to oxidative stress. (A and B) Diameters of the zones of inhibition produced in the presence of 500 mM H 2 O 2 (A) or 40 mM menadione (B). KO, knockout. (C) Diameters of the zone of inhibition on 7H10 agar (white columns)

Techniques Used: Inhibition, Produced, Knock-Out

6) Product Images from "Essential Roles for Mycobacterium tuberculosis Rel beyond the Production of (p)ppGpp"

Article Title: Essential Roles for Mycobacterium tuberculosis Rel beyond the Production of (p)ppGpp

Journal: Journal of Bacteriology

doi: 10.1128/JB.00759-13

Rel Mtb -mediated (p)ppGpp hydrolysis is required for growth in vitro and maintenance of ATP and GTP levels when (p)ppGpp is being produced. (A and B) An M. tuberculosis Δ rel Mtb strain containing an episomal vector expressing the WT TetR that induces expression of rel Mtb H80A from a cassette integrated at the attB site in the genome in the presence of ATc (Tet-Rel Mtb H80A strain, designated H80A here) and a WT M. tuberculosis strain containing an empty vector in the attB site and transformed with the same TetR-expressing plasmid (control strain). (A) Transcript levels in exponential-growth-phase cultures in liquid 7H9 media. rel Mtb H80A transcripts from the cassette at the attB site were detected in the Tet-Rel Mtb H80A strain, and transcripts from the endogenous rel Mtb gene were detected in the control strain. The first comparison shows the ratio of transcript levels in the presence compared to the absence of ATc in each strain (+ATc/−ATc), and it illustrates the induction of rel Mtb H80A in the Tet-Rel Mtb H80A strain when exposed to ATc and the unresponsiveness of the rel Mtb gene to ATc in the control strain. The second comparison shows the ratio of transcript levels in the Tet-Rel Mtb H80A strain compared to the control strain (H80A/control) under each condition to illustrate the low level of transcription of rel Mtb H80A in the absence of ATc compared to that of induced cultures. (B) M. tuberculosis Tet-Rel Mtb H80A and control strains were diluted and plated on 7H10 plates in the absence or presence of ATc. (C to G) M. smegmatis Δ rel Msm strains containing an episomal vector expressing the WT TetR that turns on expression of rel Mtb WT (Tet-Rel Mtb WT strain; designated the control) or rel Mtb H80A (Tet-Rel Mtb H80A strain; designated H80A here) from cassettes integrated at the attB site in the genome in the presence of ATc. (C and D) M. smegmatis strains were diluted and plated on 7H10 (C) and LB (D) plates in the absence or presence of ATc. (E) Graphic representation of the number of M. smegmatis CFU that grew in the presence compared to the absence of ATc when grown on LB, where all Tet-Rel Mtb H80A strain bacteria that grew in the presence of ATc on LB were suppressors and were no longer responsive to ATc treatment. Data are means ± SEM from 13 replicates. (F) ATP and GTP levels in M. smegmatis strains grown in the absence or presence of ATc for 6 h to an ODλ 600 of ∼0.6 in LB liquid media were measured by LC-MS/MS. The ratio of levels in the Tet-Rel Mtb H80A strain (H80A) to those in the Tet-Rel Mtb WT -expressing strain (control) under the same conditions is graphed. Data are the means ± SEM from 4 to 6 replicates. (E and F) The significance of differences was determined by calculating P values by Student's t tests; one asterisk indicates significance with a P value of
Figure Legend Snippet: Rel Mtb -mediated (p)ppGpp hydrolysis is required for growth in vitro and maintenance of ATP and GTP levels when (p)ppGpp is being produced. (A and B) An M. tuberculosis Δ rel Mtb strain containing an episomal vector expressing the WT TetR that induces expression of rel Mtb H80A from a cassette integrated at the attB site in the genome in the presence of ATc (Tet-Rel Mtb H80A strain, designated H80A here) and a WT M. tuberculosis strain containing an empty vector in the attB site and transformed with the same TetR-expressing plasmid (control strain). (A) Transcript levels in exponential-growth-phase cultures in liquid 7H9 media. rel Mtb H80A transcripts from the cassette at the attB site were detected in the Tet-Rel Mtb H80A strain, and transcripts from the endogenous rel Mtb gene were detected in the control strain. The first comparison shows the ratio of transcript levels in the presence compared to the absence of ATc in each strain (+ATc/−ATc), and it illustrates the induction of rel Mtb H80A in the Tet-Rel Mtb H80A strain when exposed to ATc and the unresponsiveness of the rel Mtb gene to ATc in the control strain. The second comparison shows the ratio of transcript levels in the Tet-Rel Mtb H80A strain compared to the control strain (H80A/control) under each condition to illustrate the low level of transcription of rel Mtb H80A in the absence of ATc compared to that of induced cultures. (B) M. tuberculosis Tet-Rel Mtb H80A and control strains were diluted and plated on 7H10 plates in the absence or presence of ATc. (C to G) M. smegmatis Δ rel Msm strains containing an episomal vector expressing the WT TetR that turns on expression of rel Mtb WT (Tet-Rel Mtb WT strain; designated the control) or rel Mtb H80A (Tet-Rel Mtb H80A strain; designated H80A here) from cassettes integrated at the attB site in the genome in the presence of ATc. (C and D) M. smegmatis strains were diluted and plated on 7H10 (C) and LB (D) plates in the absence or presence of ATc. (E) Graphic representation of the number of M. smegmatis CFU that grew in the presence compared to the absence of ATc when grown on LB, where all Tet-Rel Mtb H80A strain bacteria that grew in the presence of ATc on LB were suppressors and were no longer responsive to ATc treatment. Data are means ± SEM from 13 replicates. (F) ATP and GTP levels in M. smegmatis strains grown in the absence or presence of ATc for 6 h to an ODλ 600 of ∼0.6 in LB liquid media were measured by LC-MS/MS. The ratio of levels in the Tet-Rel Mtb H80A strain (H80A) to those in the Tet-Rel Mtb WT -expressing strain (control) under the same conditions is graphed. Data are the means ± SEM from 4 to 6 replicates. (E and F) The significance of differences was determined by calculating P values by Student's t tests; one asterisk indicates significance with a P value of

Techniques Used: In Vitro, Produced, Plasmid Preparation, Expressing, Transformation Assay, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry

Rv1366 is not necessary for M. tuberculosis growth or biofilm formation in vitro . (A) Southern blot analysis of WT M. tuberculosis Erdman (lane 1) and the Δ rv1366 strain (lanes 2 to 4) using EcoRI-digested genomic DNA and a probe that spans nucleotides 1537706 to 1538406 of the M. tuberculosis genome. EcoRI digestion of WT M. tuberculosis yields a band at 5,083 bp. EcoRI digestion of Δ rv1366 results in a 2,226-bp band due to the replacement of the rv1366 gene with a hygromycin resistance cassette. (B) Southern blot analysis of WT M. tuberculosis Erdman (lane 1) and the Δ rel Mtb Δ rv1366 strain (lanes 2 to 6) using SmaI-digested genomic DNA and a probe that spans nucleotides 2910191 to 2910844 of the M. tuberculosis genome. SmaI digestion of WT M. tuberculosis yields a band at 2,257 bp. SmaI digestion of the Δ rel Mtb Δ rv1366 strain results in a 4,769-bp band due to the replacement of the rel Mtb gene with a hygromycin resistance cassette. (C and D) M. tuberculosis Δ rel Mtb , Δ rv1366 , and Δ rel Mtb Δ rv1366 strains containing a vector that constitutively expresses Rel Mtb WT or Rv1366 or containing an empty vector (−) integrated at the attB site in the genome were normalized to the same ODλ 600 and either diluted and plated on 7H10 plates (C) or grown in Sauton's media in static, biofilm-forming conditions in a 24-well dish (D). (C) 7H10 plates were incubated for 3 weeks at 37°C. (D) Biofilms were incubated for 4 weeks at 37°C. The parent strain used is labeled above each photo, and the protein expressed from the attB site is labeled below the photo.
Figure Legend Snippet: Rv1366 is not necessary for M. tuberculosis growth or biofilm formation in vitro . (A) Southern blot analysis of WT M. tuberculosis Erdman (lane 1) and the Δ rv1366 strain (lanes 2 to 4) using EcoRI-digested genomic DNA and a probe that spans nucleotides 1537706 to 1538406 of the M. tuberculosis genome. EcoRI digestion of WT M. tuberculosis yields a band at 5,083 bp. EcoRI digestion of Δ rv1366 results in a 2,226-bp band due to the replacement of the rv1366 gene with a hygromycin resistance cassette. (B) Southern blot analysis of WT M. tuberculosis Erdman (lane 1) and the Δ rel Mtb Δ rv1366 strain (lanes 2 to 6) using SmaI-digested genomic DNA and a probe that spans nucleotides 2910191 to 2910844 of the M. tuberculosis genome. SmaI digestion of WT M. tuberculosis yields a band at 2,257 bp. SmaI digestion of the Δ rel Mtb Δ rv1366 strain results in a 4,769-bp band due to the replacement of the rel Mtb gene with a hygromycin resistance cassette. (C and D) M. tuberculosis Δ rel Mtb , Δ rv1366 , and Δ rel Mtb Δ rv1366 strains containing a vector that constitutively expresses Rel Mtb WT or Rv1366 or containing an empty vector (−) integrated at the attB site in the genome were normalized to the same ODλ 600 and either diluted and plated on 7H10 plates (C) or grown in Sauton's media in static, biofilm-forming conditions in a 24-well dish (D). (C) 7H10 plates were incubated for 3 weeks at 37°C. (D) Biofilms were incubated for 4 weeks at 37°C. The parent strain used is labeled above each photo, and the protein expressed from the attB site is labeled below the photo.

Techniques Used: In Vitro, Southern Blot, Plasmid Preparation, Incubation, Labeling

Rel Mtb -mediated (p)ppGpp synthesis is necessary for optimal M. tuberculosis growth and biofilm formation in vitro. M. tuberculosis Δ rel Mtb strains containing a vector that constitutively expresses Rel Mtb WT (WT) or Rel Mtb H344Y (H344Y) or containing an empty vector (−) integrated at the attB site in the genome were normalized to the same ODλ 600 and either diluted and plated on 7H10 plates (A) or grown in Sauton's medium in static, biofilm-forming conditions in a 24-well dish (B). (A) 7H10 plates were incubated for 3 weeks at 37°C. (B) Biofilms were incubated for 4 weeks at 37°C. The parent strain used is labeled above each photo, and the protein expressed from the attB site is labeled below the photo.
Figure Legend Snippet: Rel Mtb -mediated (p)ppGpp synthesis is necessary for optimal M. tuberculosis growth and biofilm formation in vitro. M. tuberculosis Δ rel Mtb strains containing a vector that constitutively expresses Rel Mtb WT (WT) or Rel Mtb H344Y (H344Y) or containing an empty vector (−) integrated at the attB site in the genome were normalized to the same ODλ 600 and either diluted and plated on 7H10 plates (A) or grown in Sauton's medium in static, biofilm-forming conditions in a 24-well dish (B). (A) 7H10 plates were incubated for 3 weeks at 37°C. (B) Biofilms were incubated for 4 weeks at 37°C. The parent strain used is labeled above each photo, and the protein expressed from the attB site is labeled below the photo.

Techniques Used: In Vitro, Plasmid Preparation, Incubation, Labeling

Rel Mtb (p)ppGpp hydrolase activity is essential for acute and chronic M. tuberculosis infection of mice. Bacterial titers in the lungs (A, E, and G) and spleens (B and H) of C57BL/6 mice infected with either control or Tet-Rel Mtb H80A (designated H80A in the figure) M. tuberculosis strains. The parent strain for the control is WT M. tuberculosis Erdman, and the parent strain for Tet-Rel Mtb H80A is M. tuberculosis Δ rel Mtb . Both strains express TetR from a plasmid that confers streptomycin resistance and contain a kanamycin resistance cassette integrated into the attB site that, in the case of Tet-Rel Mtb H80A , carries the rel Mtb H80A allele. The strain symbols in panel A are the same for all panels. (A to C) Mice were given normal mouse chow throughout infection. (E and F) Mice were administered doxycycline-containing mouse chow starting at day 1 postinfection (designated by the arrow). (G to I) Mice were administered doxycycline-containing mouse chow starting at day 63 postinfection (designated by the arrow). (C, F, and I) The ratio of CFU from the lungs or spleens grown on 7H10 plates containing streptomycin and kanamycin compared to 7H10 containing no antibiotics (No AB). ND denotes when no colonies were recovered after plating 5% of the lung homogenate (limit of detection, 20 CFU). Data are means ± SEM of 6 Tet-Rel Mtb H80A strain-infected mice and 3 control strain-infected mice per time point from two replicate experiments. (E, G, and H) The significance of differences were determined by calculating P values by Student's t tests; two asterisks indicate significance with a P value of
Figure Legend Snippet: Rel Mtb (p)ppGpp hydrolase activity is essential for acute and chronic M. tuberculosis infection of mice. Bacterial titers in the lungs (A, E, and G) and spleens (B and H) of C57BL/6 mice infected with either control or Tet-Rel Mtb H80A (designated H80A in the figure) M. tuberculosis strains. The parent strain for the control is WT M. tuberculosis Erdman, and the parent strain for Tet-Rel Mtb H80A is M. tuberculosis Δ rel Mtb . Both strains express TetR from a plasmid that confers streptomycin resistance and contain a kanamycin resistance cassette integrated into the attB site that, in the case of Tet-Rel Mtb H80A , carries the rel Mtb H80A allele. The strain symbols in panel A are the same for all panels. (A to C) Mice were given normal mouse chow throughout infection. (E and F) Mice were administered doxycycline-containing mouse chow starting at day 1 postinfection (designated by the arrow). (G to I) Mice were administered doxycycline-containing mouse chow starting at day 63 postinfection (designated by the arrow). (C, F, and I) The ratio of CFU from the lungs or spleens grown on 7H10 plates containing streptomycin and kanamycin compared to 7H10 containing no antibiotics (No AB). ND denotes when no colonies were recovered after plating 5% of the lung homogenate (limit of detection, 20 CFU). Data are means ± SEM of 6 Tet-Rel Mtb H80A strain-infected mice and 3 control strain-infected mice per time point from two replicate experiments. (E, G, and H) The significance of differences were determined by calculating P values by Student's t tests; two asterisks indicate significance with a P value of

Techniques Used: Activity Assay, Infection, Mouse Assay, Plasmid Preparation

7) Product Images from "CD8+ T Cells Participate in the Memory Immune Response to Mycobacterium tuberculosis"

Article Title: CD8+ T Cells Participate in the Memory Immune Response to Mycobacterium tuberculosis

Journal: Infection and Immunity

doi: 10.1128/IAI.69.7.4320-4328.2001

Course of M. tuberculosis aerosol challenge in the lungs of memory and naive mice. Memory immune (●) and naive (□) mice were infected with ∼60 M. tuberculosis bacilli via aerosol. The numbers of viable bacilli were determined by plating serial dilutions of lung homogenates onto 7H10 plates and counting the colonies after 3 weeks at 37°C. Each time point represents four mice, and the experiment was repeated once. The standard error bars are too small to see on this graph. ∗, P ≤ 0.001, comparing naive and memory mice at each time point.
Figure Legend Snippet: Course of M. tuberculosis aerosol challenge in the lungs of memory and naive mice. Memory immune (●) and naive (□) mice were infected with ∼60 M. tuberculosis bacilli via aerosol. The numbers of viable bacilli were determined by plating serial dilutions of lung homogenates onto 7H10 plates and counting the colonies after 3 weeks at 37°C. Each time point represents four mice, and the experiment was repeated once. The standard error bars are too small to see on this graph. ∗, P ≤ 0.001, comparing naive and memory mice at each time point.

Techniques Used: Mouse Assay, Infection

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

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

Journal: Infection and Immunity

doi: 10.1128/IAI.00229-12

Sensitivity of M. tuberculosis strains to oxidative stress. (A and B) Diameters of the zones of inhibition produced in the presence of 500 mM H 2 O 2 (A) or 40 mM menadione (B). KO, knockout. (C) Diameters of the zone of inhibition on 7H10 agar (white columns)
Figure Legend Snippet: Sensitivity of M. tuberculosis strains to oxidative stress. (A and B) Diameters of the zones of inhibition produced in the presence of 500 mM H 2 O 2 (A) or 40 mM menadione (B). KO, knockout. (C) Diameters of the zone of inhibition on 7H10 agar (white columns)

Techniques Used: Inhibition, Produced, Knock-Out

9) Product Images from "A Promoter Mutation Causes Differential Nitrate Reductase Activity of Mycobacterium tuberculosis and Mycobacterium bovis"

Article Title: A Promoter Mutation Causes Differential Nitrate Reductase Activity of Mycobacterium tuberculosis and Mycobacterium bovis

Journal: Journal of Bacteriology

doi: 10.1128/JB.186.9.2856-2861.2004

Diagnostic nitrate reductase activity of M. tuberculosis , M. bovis , M. bovis BCG, and various T215C mutants of M. tuberculosis. All strains were subcultured on 7H10 agar plates for 3 weeks. One loop of bacilli was used for inoculation of phosphate buffer supplemented with nitrate. Following incubation for 2 h at 37°C, production of nitrite was tested. The presence of nitrite was visualized by adding naphthylamide and sulfanilic acid reagents, which form a red diazonium dye when reacting with nitrite.
Figure Legend Snippet: Diagnostic nitrate reductase activity of M. tuberculosis , M. bovis , M. bovis BCG, and various T215C mutants of M. tuberculosis. All strains were subcultured on 7H10 agar plates for 3 weeks. One loop of bacilli was used for inoculation of phosphate buffer supplemented with nitrate. Following incubation for 2 h at 37°C, production of nitrite was tested. The presence of nitrite was visualized by adding naphthylamide and sulfanilic acid reagents, which form a red diazonium dye when reacting with nitrite.

Techniques Used: Diagnostic Assay, Activity Assay, Incubation

10) Product Images from "Interaction of CarD with RNA Polymerase Mediates Mycobacterium tuberculosis Viability, Rifampin Resistance, and Pathogenesis"

Article Title: Interaction of CarD with RNA Polymerase Mediates Mycobacterium tuberculosis Viability, Rifampin Resistance, and Pathogenesis

Journal: Journal of Bacteriology

doi: 10.1128/JB.00879-12

Weakening the interaction between CarD and the RNAP compromises the survival of mycobacteria during oxidative stress. (A and B) Survival of M. smegmatis strains during oxidative stress. The log-phase M. smegmatis Δ carD attB ∷ tet-carD strains expressing CarD WT , CarD R25E , or CarD R47E in LB were treated for 1 h with either 10 mM or 25 mM H 2 O 2 . After treatment, dilutions were plated on LB. Panel A shows one such experiment, and B graphically represents survival as a ratio of CFU in treated cultures to that in untreated cultures. (C) Survival of the M. tuberculosis strains during oxidative stress. The log-phase M. tuberculosis Δ carD attB ∷ tet-carD strains expressing CarD WT or CarD R47E growing in 7H9 broth were treated for 75 h with 25 mM H 2 O 2 . After treatment, dilutions were plated on 7H10 agar, and survival is graphically represented as the ratio of CFU in treated cultures to that in untreated cultures. The graphs in panels B and C show the mean± standard error of the mean (SEM), and each sample is represented by a black circle. The significance levels in panels B and C were determined by calculating P values by Student's t test; an asterisk indicates significance with a P value of
Figure Legend Snippet: Weakening the interaction between CarD and the RNAP compromises the survival of mycobacteria during oxidative stress. (A and B) Survival of M. smegmatis strains during oxidative stress. The log-phase M. smegmatis Δ carD attB ∷ tet-carD strains expressing CarD WT , CarD R25E , or CarD R47E in LB were treated for 1 h with either 10 mM or 25 mM H 2 O 2 . After treatment, dilutions were plated on LB. Panel A shows one such experiment, and B graphically represents survival as a ratio of CFU in treated cultures to that in untreated cultures. (C) Survival of the M. tuberculosis strains during oxidative stress. The log-phase M. tuberculosis Δ carD attB ∷ tet-carD strains expressing CarD WT or CarD R47E growing in 7H9 broth were treated for 75 h with 25 mM H 2 O 2 . After treatment, dilutions were plated on 7H10 agar, and survival is graphically represented as the ratio of CFU in treated cultures to that in untreated cultures. The graphs in panels B and C show the mean± standard error of the mean (SEM), and each sample is represented by a black circle. The significance levels in panels B and C were determined by calculating P values by Student's t test; an asterisk indicates significance with a P value of

Techniques Used: Expressing

11) Product Images from "Polymorphic Nucleotide within the Promoter of Nitrate Reductase (NarGHJI) Is Specific for Mycobacterium tuberculosis"

Article Title: Polymorphic Nucleotide within the Promoter of Nitrate Reductase (NarGHJI) Is Specific for Mycobacterium tuberculosis

Journal: Journal of Clinical Microbiology

doi: 10.1128/JCM.41.7.3252-3259.2003

Diagnostic nitrate reductase activity of M. tuberculosis and the narG mutant of M. tuberculosis . Three-week-old cultures from M. tuberculosis wild-type and the narG mutant of M. tuberculosis on 7H10 agar plates were used to inoculate three loops (tube a), one loop (tube b), and one-third loop (tube c) of bacilli into phosphate buffer containing 10 mM nitrate and tested for the accumulation of nitrite after 2 h at 37°C.
Figure Legend Snippet: Diagnostic nitrate reductase activity of M. tuberculosis and the narG mutant of M. tuberculosis . Three-week-old cultures from M. tuberculosis wild-type and the narG mutant of M. tuberculosis on 7H10 agar plates were used to inoculate three loops (tube a), one loop (tube b), and one-third loop (tube c) of bacilli into phosphate buffer containing 10 mM nitrate and tested for the accumulation of nitrite after 2 h at 37°C.

Techniques Used: Diagnostic Assay, Activity Assay, Mutagenesis

12) Product Images from "Interaction of CarD with RNA Polymerase Mediates Mycobacterium tuberculosis Viability, Rifampin Resistance, and Pathogenesis"

Article Title: Interaction of CarD with RNA Polymerase Mediates Mycobacterium tuberculosis Viability, Rifampin Resistance, and Pathogenesis

Journal: Journal of Bacteriology

doi: 10.1128/JB.00879-12

Weakening the interaction between CarD and the RNAP compromises the survival of mycobacteria during oxidative stress. (A and B) Survival of M. smegmatis strains during oxidative stress. The log-phase M. smegmatis Δ carD attB ∷ tet-carD strains expressing CarD WT , CarD R25E , or CarD R47E in LB were treated for 1 h with either 10 mM or 25 mM H 2 O 2 . After treatment, dilutions were plated on LB. Panel A shows one such experiment, and B graphically represents survival as a ratio of CFU in treated cultures to that in untreated cultures. (C) Survival of the M. tuberculosis strains during oxidative stress. The log-phase M. tuberculosis Δ carD attB ∷ tet-carD strains expressing CarD WT or CarD R47E growing in 7H9 broth were treated for 75 h with 25 mM H 2 O 2 . After treatment, dilutions were plated on 7H10 agar, and survival is graphically represented as the ratio of CFU in treated cultures to that in untreated cultures. The graphs in panels B and C show the mean± standard error of the mean (SEM), and each sample is represented by a black circle. The significance levels in panels B and C were determined by calculating P values by Student's t test; an asterisk indicates significance with a P value of
Figure Legend Snippet: Weakening the interaction between CarD and the RNAP compromises the survival of mycobacteria during oxidative stress. (A and B) Survival of M. smegmatis strains during oxidative stress. The log-phase M. smegmatis Δ carD attB ∷ tet-carD strains expressing CarD WT , CarD R25E , or CarD R47E in LB were treated for 1 h with either 10 mM or 25 mM H 2 O 2 . After treatment, dilutions were plated on LB. Panel A shows one such experiment, and B graphically represents survival as a ratio of CFU in treated cultures to that in untreated cultures. (C) Survival of the M. tuberculosis strains during oxidative stress. The log-phase M. tuberculosis Δ carD attB ∷ tet-carD strains expressing CarD WT or CarD R47E growing in 7H9 broth were treated for 75 h with 25 mM H 2 O 2 . After treatment, dilutions were plated on 7H10 agar, and survival is graphically represented as the ratio of CFU in treated cultures to that in untreated cultures. The graphs in panels B and C show the mean± standard error of the mean (SEM), and each sample is represented by a black circle. The significance levels in panels B and C were determined by calculating P values by Student's t test; an asterisk indicates significance with a P value of

Techniques Used: Expressing

13) Product Images from "Essential Roles for Mycobacterium tuberculosis Rel beyond the Production of (p)ppGpp"

Article Title: Essential Roles for Mycobacterium tuberculosis Rel beyond the Production of (p)ppGpp

Journal: Journal of Bacteriology

doi: 10.1128/JB.00759-13

Rel Mtb -mediated (p)ppGpp hydrolysis is required for growth in vitro and maintenance of ATP and GTP levels when (p)ppGpp is being produced. (A and B) An M. tuberculosis Δ rel Mtb strain containing an episomal vector expressing the WT TetR that induces expression of rel Mtb H80A from a cassette integrated at the attB site in the genome in the presence of ATc (Tet-Rel Mtb H80A strain, designated H80A here) and a WT M. tuberculosis strain containing an empty vector in the attB site and transformed with the same TetR-expressing plasmid (control strain). (A) Transcript levels in exponential-growth-phase cultures in liquid 7H9 media. rel Mtb H80A transcripts from the cassette at the attB site were detected in the Tet-Rel Mtb H80A strain, and transcripts from the endogenous rel Mtb gene were detected in the control strain. The first comparison shows the ratio of transcript levels in the presence compared to the absence of ATc in each strain (+ATc/−ATc), and it illustrates the induction of rel Mtb H80A in the Tet-Rel Mtb H80A strain when exposed to ATc and the unresponsiveness of the rel Mtb gene to ATc in the control strain. The second comparison shows the ratio of transcript levels in the Tet-Rel Mtb H80A strain compared to the control strain (H80A/control) under each condition to illustrate the low level of transcription of rel Mtb H80A in the absence of ATc compared to that of induced cultures. (B) M. tuberculosis Tet-Rel Mtb H80A and control strains were diluted and plated on 7H10 plates in the absence or presence of ATc. (C to G) M. smegmatis Δ rel Msm strains containing an episomal vector expressing the WT TetR that turns on expression of rel Mtb WT (Tet-Rel Mtb WT strain; designated the control) or rel Mtb H80A (Tet-Rel Mtb H80A strain; designated H80A here) from cassettes integrated at the attB site in the genome in the presence of ATc. (C and D) M. smegmatis strains were diluted and plated on 7H10 (C) and LB (D) plates in the absence or presence of ATc. (E) Graphic representation of the number of M. smegmatis CFU that grew in the presence compared to the absence of ATc when grown on LB, where all Tet-Rel Mtb H80A strain bacteria that grew in the presence of ATc on LB were suppressors and were no longer responsive to ATc treatment. Data are means ± SEM from 13 replicates. (F) ATP and GTP levels in M. smegmatis strains grown in the absence or presence of ATc for 6 h to an ODλ 600 of ∼0.6 in LB liquid media were measured by LC-MS/MS. The ratio of levels in the Tet-Rel Mtb H80A strain (H80A) to those in the Tet-Rel Mtb WT -expressing strain (control) under the same conditions is graphed. Data are the means ± SEM from 4 to 6 replicates. (E and F) The significance of differences was determined by calculating P values by Student's t tests; one asterisk indicates significance with a P value of
Figure Legend Snippet: Rel Mtb -mediated (p)ppGpp hydrolysis is required for growth in vitro and maintenance of ATP and GTP levels when (p)ppGpp is being produced. (A and B) An M. tuberculosis Δ rel Mtb strain containing an episomal vector expressing the WT TetR that induces expression of rel Mtb H80A from a cassette integrated at the attB site in the genome in the presence of ATc (Tet-Rel Mtb H80A strain, designated H80A here) and a WT M. tuberculosis strain containing an empty vector in the attB site and transformed with the same TetR-expressing plasmid (control strain). (A) Transcript levels in exponential-growth-phase cultures in liquid 7H9 media. rel Mtb H80A transcripts from the cassette at the attB site were detected in the Tet-Rel Mtb H80A strain, and transcripts from the endogenous rel Mtb gene were detected in the control strain. The first comparison shows the ratio of transcript levels in the presence compared to the absence of ATc in each strain (+ATc/−ATc), and it illustrates the induction of rel Mtb H80A in the Tet-Rel Mtb H80A strain when exposed to ATc and the unresponsiveness of the rel Mtb gene to ATc in the control strain. The second comparison shows the ratio of transcript levels in the Tet-Rel Mtb H80A strain compared to the control strain (H80A/control) under each condition to illustrate the low level of transcription of rel Mtb H80A in the absence of ATc compared to that of induced cultures. (B) M. tuberculosis Tet-Rel Mtb H80A and control strains were diluted and plated on 7H10 plates in the absence or presence of ATc. (C to G) M. smegmatis Δ rel Msm strains containing an episomal vector expressing the WT TetR that turns on expression of rel Mtb WT (Tet-Rel Mtb WT strain; designated the control) or rel Mtb H80A (Tet-Rel Mtb H80A strain; designated H80A here) from cassettes integrated at the attB site in the genome in the presence of ATc. (C and D) M. smegmatis strains were diluted and plated on 7H10 (C) and LB (D) plates in the absence or presence of ATc. (E) Graphic representation of the number of M. smegmatis CFU that grew in the presence compared to the absence of ATc when grown on LB, where all Tet-Rel Mtb H80A strain bacteria that grew in the presence of ATc on LB were suppressors and were no longer responsive to ATc treatment. Data are means ± SEM from 13 replicates. (F) ATP and GTP levels in M. smegmatis strains grown in the absence or presence of ATc for 6 h to an ODλ 600 of ∼0.6 in LB liquid media were measured by LC-MS/MS. The ratio of levels in the Tet-Rel Mtb H80A strain (H80A) to those in the Tet-Rel Mtb WT -expressing strain (control) under the same conditions is graphed. Data are the means ± SEM from 4 to 6 replicates. (E and F) The significance of differences was determined by calculating P values by Student's t tests; one asterisk indicates significance with a P value of

Techniques Used: In Vitro, Produced, Plasmid Preparation, Expressing, Transformation Assay, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry

Rv1366 is not necessary for M. tuberculosis growth or biofilm formation in vitro . (A) Southern blot analysis of WT M. tuberculosis Erdman (lane 1) and the Δ rv1366 strain (lanes 2 to 4) using EcoRI-digested genomic DNA and a probe that spans nucleotides 1537706 to 1538406 of the M. tuberculosis genome. EcoRI digestion of WT M. tuberculosis yields a band at 5,083 bp. EcoRI digestion of Δ rv1366 results in a 2,226-bp band due to the replacement of the rv1366 gene with a hygromycin resistance cassette. (B) Southern blot analysis of WT M. tuberculosis Erdman (lane 1) and the Δ rel Mtb Δ rv1366 strain (lanes 2 to 6) using SmaI-digested genomic DNA and a probe that spans nucleotides 2910191 to 2910844 of the M. tuberculosis genome. SmaI digestion of WT M. tuberculosis yields a band at 2,257 bp. SmaI digestion of the Δ rel Mtb Δ rv1366 strain results in a 4,769-bp band due to the replacement of the rel Mtb gene with a hygromycin resistance cassette. (C and D) M. tuberculosis Δ rel Mtb , Δ rv1366 , and Δ rel Mtb Δ rv1366 strains containing a vector that constitutively expresses Rel Mtb WT or Rv1366 or containing an empty vector (−) integrated at the attB site in the genome were normalized to the same ODλ 600 and either diluted and plated on 7H10 plates (C) or grown in Sauton's media in static, biofilm-forming conditions in a 24-well dish (D). (C) 7H10 plates were incubated for 3 weeks at 37°C. (D) Biofilms were incubated for 4 weeks at 37°C. The parent strain used is labeled above each photo, and the protein expressed from the attB site is labeled below the photo.
Figure Legend Snippet: Rv1366 is not necessary for M. tuberculosis growth or biofilm formation in vitro . (A) Southern blot analysis of WT M. tuberculosis Erdman (lane 1) and the Δ rv1366 strain (lanes 2 to 4) using EcoRI-digested genomic DNA and a probe that spans nucleotides 1537706 to 1538406 of the M. tuberculosis genome. EcoRI digestion of WT M. tuberculosis yields a band at 5,083 bp. EcoRI digestion of Δ rv1366 results in a 2,226-bp band due to the replacement of the rv1366 gene with a hygromycin resistance cassette. (B) Southern blot analysis of WT M. tuberculosis Erdman (lane 1) and the Δ rel Mtb Δ rv1366 strain (lanes 2 to 6) using SmaI-digested genomic DNA and a probe that spans nucleotides 2910191 to 2910844 of the M. tuberculosis genome. SmaI digestion of WT M. tuberculosis yields a band at 2,257 bp. SmaI digestion of the Δ rel Mtb Δ rv1366 strain results in a 4,769-bp band due to the replacement of the rel Mtb gene with a hygromycin resistance cassette. (C and D) M. tuberculosis Δ rel Mtb , Δ rv1366 , and Δ rel Mtb Δ rv1366 strains containing a vector that constitutively expresses Rel Mtb WT or Rv1366 or containing an empty vector (−) integrated at the attB site in the genome were normalized to the same ODλ 600 and either diluted and plated on 7H10 plates (C) or grown in Sauton's media in static, biofilm-forming conditions in a 24-well dish (D). (C) 7H10 plates were incubated for 3 weeks at 37°C. (D) Biofilms were incubated for 4 weeks at 37°C. The parent strain used is labeled above each photo, and the protein expressed from the attB site is labeled below the photo.

Techniques Used: In Vitro, Southern Blot, Plasmid Preparation, Incubation, Labeling

Rel Mtb -mediated (p)ppGpp synthesis is necessary for optimal M. tuberculosis growth and biofilm formation in vitro. M. tuberculosis Δ rel Mtb strains containing a vector that constitutively expresses Rel Mtb WT (WT) or Rel Mtb H344Y (H344Y) or containing an empty vector (−) integrated at the attB site in the genome were normalized to the same ODλ 600 and either diluted and plated on 7H10 plates (A) or grown in Sauton's medium in static, biofilm-forming conditions in a 24-well dish (B). (A) 7H10 plates were incubated for 3 weeks at 37°C. (B) Biofilms were incubated for 4 weeks at 37°C. The parent strain used is labeled above each photo, and the protein expressed from the attB site is labeled below the photo.
Figure Legend Snippet: Rel Mtb -mediated (p)ppGpp synthesis is necessary for optimal M. tuberculosis growth and biofilm formation in vitro. M. tuberculosis Δ rel Mtb strains containing a vector that constitutively expresses Rel Mtb WT (WT) or Rel Mtb H344Y (H344Y) or containing an empty vector (−) integrated at the attB site in the genome were normalized to the same ODλ 600 and either diluted and plated on 7H10 plates (A) or grown in Sauton's medium in static, biofilm-forming conditions in a 24-well dish (B). (A) 7H10 plates were incubated for 3 weeks at 37°C. (B) Biofilms were incubated for 4 weeks at 37°C. The parent strain used is labeled above each photo, and the protein expressed from the attB site is labeled below the photo.

Techniques Used: In Vitro, Plasmid Preparation, Incubation, Labeling

Rel Mtb (p)ppGpp hydrolase activity is essential for acute and chronic M. tuberculosis infection of mice. Bacterial titers in the lungs (A, E, and G) and spleens (B and H) of C57BL/6 mice infected with either control or Tet-Rel Mtb H80A (designated H80A in the figure) M. tuberculosis strains. The parent strain for the control is WT M. tuberculosis Erdman, and the parent strain for Tet-Rel Mtb H80A is M. tuberculosis Δ rel Mtb . Both strains express TetR from a plasmid that confers streptomycin resistance and contain a kanamycin resistance cassette integrated into the attB site that, in the case of Tet-Rel Mtb H80A , carries the rel Mtb H80A allele. The strain symbols in panel A are the same for all panels. (A to C) Mice were given normal mouse chow throughout infection. (E and F) Mice were administered doxycycline-containing mouse chow starting at day 1 postinfection (designated by the arrow). (G to I) Mice were administered doxycycline-containing mouse chow starting at day 63 postinfection (designated by the arrow). (C, F, and I) The ratio of CFU from the lungs or spleens grown on 7H10 plates containing streptomycin and kanamycin compared to 7H10 containing no antibiotics (No AB). ND denotes when no colonies were recovered after plating 5% of the lung homogenate (limit of detection, 20 CFU). Data are means ± SEM of 6 Tet-Rel Mtb H80A strain-infected mice and 3 control strain-infected mice per time point from two replicate experiments. (E, G, and H) The significance of differences were determined by calculating P values by Student's t tests; two asterisks indicate significance with a P value of
Figure Legend Snippet: Rel Mtb (p)ppGpp hydrolase activity is essential for acute and chronic M. tuberculosis infection of mice. Bacterial titers in the lungs (A, E, and G) and spleens (B and H) of C57BL/6 mice infected with either control or Tet-Rel Mtb H80A (designated H80A in the figure) M. tuberculosis strains. The parent strain for the control is WT M. tuberculosis Erdman, and the parent strain for Tet-Rel Mtb H80A is M. tuberculosis Δ rel Mtb . Both strains express TetR from a plasmid that confers streptomycin resistance and contain a kanamycin resistance cassette integrated into the attB site that, in the case of Tet-Rel Mtb H80A , carries the rel Mtb H80A allele. The strain symbols in panel A are the same for all panels. (A to C) Mice were given normal mouse chow throughout infection. (E and F) Mice were administered doxycycline-containing mouse chow starting at day 1 postinfection (designated by the arrow). (G to I) Mice were administered doxycycline-containing mouse chow starting at day 63 postinfection (designated by the arrow). (C, F, and I) The ratio of CFU from the lungs or spleens grown on 7H10 plates containing streptomycin and kanamycin compared to 7H10 containing no antibiotics (No AB). ND denotes when no colonies were recovered after plating 5% of the lung homogenate (limit of detection, 20 CFU). Data are means ± SEM of 6 Tet-Rel Mtb H80A strain-infected mice and 3 control strain-infected mice per time point from two replicate experiments. (E, G, and H) The significance of differences were determined by calculating P values by Student's t tests; two asterisks indicate significance with a P value of

Techniques Used: Activity Assay, Infection, Mouse Assay, Plasmid Preparation

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

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

Journal: Infection and Immunity

doi: 10.1128/IAI.00229-12

Sensitivity of M. tuberculosis strains to oxidative stress. (A and B) Diameters of the zones of inhibition produced in the presence of 500 mM H 2 O 2 (A) or 40 mM menadione (B). KO, knockout. (C) Diameters of the zone of inhibition on 7H10 agar (white columns)
Figure Legend Snippet: Sensitivity of M. tuberculosis strains to oxidative stress. (A and B) Diameters of the zones of inhibition produced in the presence of 500 mM H 2 O 2 (A) or 40 mM menadione (B). KO, knockout. (C) Diameters of the zone of inhibition on 7H10 agar (white columns)

Techniques Used: Inhibition, Produced, Knock-Out

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

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

Journal: Infection and Immunity

doi: 10.1128/IAI.00229-12

Sensitivity of M. tuberculosis strains to oxidative stress. (A and B) Diameters of the zones of inhibition produced in the presence of 500 mM H 2 O 2 (A) or 40 mM menadione (B). KO, knockout. (C) Diameters of the zone of inhibition on 7H10 agar (white columns)
Figure Legend Snippet: Sensitivity of M. tuberculosis strains to oxidative stress. (A and B) Diameters of the zones of inhibition produced in the presence of 500 mM H 2 O 2 (A) or 40 mM menadione (B). KO, knockout. (C) Diameters of the zone of inhibition on 7H10 agar (white columns)

Techniques Used: Inhibition, Produced, Knock-Out

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

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

Journal: Infection and Immunity

doi: 10.1128/IAI.00229-12

Sensitivity of M. tuberculosis strains to oxidative stress. (A and B) Diameters of the zones of inhibition produced in the presence of 500 mM H 2 O 2 (A) or 40 mM menadione (B). KO, knockout. (C) Diameters of the zone of inhibition on 7H10 agar (white columns)
Figure Legend Snippet: Sensitivity of M. tuberculosis strains to oxidative stress. (A and B) Diameters of the zones of inhibition produced in the presence of 500 mM H 2 O 2 (A) or 40 mM menadione (B). KO, knockout. (C) Diameters of the zone of inhibition on 7H10 agar (white columns)

Techniques Used: Inhibition, Produced, Knock-Out

17) Product Images from "The EXIT Strategy: an Approach for Identifying Bacterial Proteins Exported during Host Infection"

Article Title: The EXIT Strategy: an Approach for Identifying Bacterial Proteins Exported during Host Infection

Journal: mBio

doi: 10.1128/mBio.00333-17

Strategy for identification of in vivo induced exported proteins. (a) Identification of in vivo induced exported proteins. Spleens from β-lactam-treated mice infected with the EXIT library were harvested after 2 weeks of infection. Spleen homogenates were plated in parallel on 7H10 agar without β-lactam to recover all clones (red Venn diagram) and on 7H10 agar containing β-lactam to recover clones exporting ‘BlaTEM fusion proteins during in vivo growth and in vitro growth (purple Venn diagram). The population of clones identified only or in significantly greater abundance on media lacking β-lactams represents proteins whose export was induced during infection (blue). (b) Sequenced read count values recovered from agar with or without β-lactam for the 593 EXIT proteins were plotted to compare abundances after β-lactam treatment in vivo , with the abundance after dual β-lactam treatment in vivo and in vitro indicated. The majority of proteins identified as exported in vivo remained highly abundant after additional β-lactam treatment in vitro (black). A total of 38 genes (highlighted in red) were identified as statistically less abundant after in vitro β-lactam selection, representing proteins exported significantly more in vivo than in vitro (see Materials and Methods for details on statistical analysis). (c) In vivo induced exported proteins with roles promoting growth in macrophages ( rv1508 :: tn , rv3707c:tn , rv0559c :: tn , and rv2536 :: tn ). Murine bone marrow-derived macrophages were infected with M. tuberculosis CDC1551 transposon mutants lacking individual in vivo induced exported proteins. At specific times postinfection, macrophage lysates were plated to measure intracellular CFU. The fold change in CFU over the course of the infection is plotted relative to the bacterial burden at day 0 postinfection. Statistical significance was determined by one-way analysis of variance (ANOVA) with multiple comparisons performed by the use of the Holm-Sidak (normal by Shapiro-Wilk) or Student-Newman-Keuls (nonnormal) test (*, P
Figure Legend Snippet: Strategy for identification of in vivo induced exported proteins. (a) Identification of in vivo induced exported proteins. Spleens from β-lactam-treated mice infected with the EXIT library were harvested after 2 weeks of infection. Spleen homogenates were plated in parallel on 7H10 agar without β-lactam to recover all clones (red Venn diagram) and on 7H10 agar containing β-lactam to recover clones exporting ‘BlaTEM fusion proteins during in vivo growth and in vitro growth (purple Venn diagram). The population of clones identified only or in significantly greater abundance on media lacking β-lactams represents proteins whose export was induced during infection (blue). (b) Sequenced read count values recovered from agar with or without β-lactam for the 593 EXIT proteins were plotted to compare abundances after β-lactam treatment in vivo , with the abundance after dual β-lactam treatment in vivo and in vitro indicated. The majority of proteins identified as exported in vivo remained highly abundant after additional β-lactam treatment in vitro (black). A total of 38 genes (highlighted in red) were identified as statistically less abundant after in vitro β-lactam selection, representing proteins exported significantly more in vivo than in vitro (see Materials and Methods for details on statistical analysis). (c) In vivo induced exported proteins with roles promoting growth in macrophages ( rv1508 :: tn , rv3707c:tn , rv0559c :: tn , and rv2536 :: tn ). Murine bone marrow-derived macrophages were infected with M. tuberculosis CDC1551 transposon mutants lacking individual in vivo induced exported proteins. At specific times postinfection, macrophage lysates were plated to measure intracellular CFU. The fold change in CFU over the course of the infection is plotted relative to the bacterial burden at day 0 postinfection. Statistical significance was determined by one-way analysis of variance (ANOVA) with multiple comparisons performed by the use of the Holm-Sidak (normal by Shapiro-Wilk) or Student-Newman-Keuls (nonnormal) test (*, P

Techniques Used: In Vivo, Mouse Assay, Infection, Clone Assay, In Vitro, Selection, Derivative Assay

(a) The ‘BlaTEM reporter. The ‘BlaTEM reporter is compatible with proteins localized to the bacterial cytoplasmic membrane or cell wall or secreted from the bacterial cell. The right panel indicates in-frame fusions to categories of exported proteins that confer β-lactam resistance (red). In-frame fusions to cytoplasmic proteins or the cytoplasmic domain of integral membrane proteins (purple) do not confer β-lactam resistance. (b) EXIT strategy. In step 1, a comprehensive library of 5 × 10 6 plasmids containing fragments of M. tuberculosis (Mtb) genomic DNA fused to the ‘ bla TEM reporter was constructed. The plasmid library was transformed into the ΔblaC β-lactamase-sensitive mutant of M. tuberculosis , and 5 × 10 6 transformants were pooled to generate the EXIT library. In step 2, mice were infected by intravenous injection with the EXIT library and treated with β-lactam antibiotics (oral gavage twice daily) to select for EXIT clones exporting ‘BlaTEM fusion proteins. β-lactam treatment began 1 day after infection and continued to 2 weeks after infection. Mice were sacrificed, and spleens and lungs were harvested and homogenized. In step 3, organ homogenates were plated on 7H10 agar and grown to recover M. tuberculosis clones that survived β-lactam treatment during infection. Plates were scraped, and colonies were pooled separately for lungs and spleens. In step 4, plasmids from the recovered bacteria and the input samples were isolated and the fusion junction was sequenced using next-generation sequencing. Sequencing primers were designed to read out of the ‘ bla TEM reporter and sequence the immediately adjacent M. tuberculosis DNA. Sequences were aligned to the M. tuberculosis genome. Unique sequences were counted to identify the abundance of each fusion junction site within the population. The genes that were most highly abundant after in vivo β-lactam treatment were identified, and the results corresponded to plasmids producing in-frame exported ‘BlaTEM fusion proteins.
Figure Legend Snippet: (a) The ‘BlaTEM reporter. The ‘BlaTEM reporter is compatible with proteins localized to the bacterial cytoplasmic membrane or cell wall or secreted from the bacterial cell. The right panel indicates in-frame fusions to categories of exported proteins that confer β-lactam resistance (red). In-frame fusions to cytoplasmic proteins or the cytoplasmic domain of integral membrane proteins (purple) do not confer β-lactam resistance. (b) EXIT strategy. In step 1, a comprehensive library of 5 × 10 6 plasmids containing fragments of M. tuberculosis (Mtb) genomic DNA fused to the ‘ bla TEM reporter was constructed. The plasmid library was transformed into the ΔblaC β-lactamase-sensitive mutant of M. tuberculosis , and 5 × 10 6 transformants were pooled to generate the EXIT library. In step 2, mice were infected by intravenous injection with the EXIT library and treated with β-lactam antibiotics (oral gavage twice daily) to select for EXIT clones exporting ‘BlaTEM fusion proteins. β-lactam treatment began 1 day after infection and continued to 2 weeks after infection. Mice were sacrificed, and spleens and lungs were harvested and homogenized. In step 3, organ homogenates were plated on 7H10 agar and grown to recover M. tuberculosis clones that survived β-lactam treatment during infection. Plates were scraped, and colonies were pooled separately for lungs and spleens. In step 4, plasmids from the recovered bacteria and the input samples were isolated and the fusion junction was sequenced using next-generation sequencing. Sequencing primers were designed to read out of the ‘ bla TEM reporter and sequence the immediately adjacent M. tuberculosis DNA. Sequences were aligned to the M. tuberculosis genome. Unique sequences were counted to identify the abundance of each fusion junction site within the population. The genes that were most highly abundant after in vivo β-lactam treatment were identified, and the results corresponded to plasmids producing in-frame exported ‘BlaTEM fusion proteins.

Techniques Used: Transmission Electron Microscopy, Construct, Plasmid Preparation, Transformation Assay, Mutagenesis, Mouse Assay, Infection, Injection, Clone Assay, Isolation, Next-Generation Sequencing, Sequencing, In Vivo

18) Product Images from "The EXIT Strategy: an Approach for Identifying Bacterial Proteins Exported during Host Infection"

Article Title: The EXIT Strategy: an Approach for Identifying Bacterial Proteins Exported during Host Infection

Journal: mBio

doi: 10.1128/mBio.00333-17

Strategy for identification of in vivo induced exported proteins. (a) Identification of in vivo induced exported proteins. Spleens from β-lactam-treated mice infected with the EXIT library were harvested after 2 weeks of infection. Spleen homogenates were plated in parallel on 7H10 agar without β-lactam to recover all clones (red Venn diagram) and on 7H10 agar containing β-lactam to recover clones exporting ‘BlaTEM fusion proteins during in vivo growth and in vitro growth (purple Venn diagram). The population of clones identified only or in significantly greater abundance on media lacking β-lactams represents proteins whose export was induced during infection (blue). (b) Sequenced read count values recovered from agar with or without β-lactam for the 593 EXIT proteins were plotted to compare abundances after β-lactam treatment in vivo , with the abundance after dual β-lactam treatment in vivo and in vitro indicated. The majority of proteins identified as exported in vivo remained highly abundant after additional β-lactam treatment in vitro (black). A total of 38 genes (highlighted in red) were identified as statistically less abundant after in vitro β-lactam selection, representing proteins exported significantly more in vivo than in vitro (see Materials and Methods for details on statistical analysis). (c) In vivo induced exported proteins with roles promoting growth in macrophages ( rv1508 :: tn , rv3707c:tn , rv0559c :: tn , and rv2536 :: tn ). Murine bone marrow-derived macrophages were infected with M. tuberculosis CDC1551 transposon mutants lacking individual in vivo induced exported proteins. At specific times postinfection, macrophage lysates were plated to measure intracellular CFU. The fold change in CFU over the course of the infection is plotted relative to the bacterial burden at day 0 postinfection. Statistical significance was determined by one-way analysis of variance (ANOVA) with multiple comparisons performed by the use of the Holm-Sidak (normal by Shapiro-Wilk) or Student-Newman-Keuls (nonnormal) test (*, P
Figure Legend Snippet: Strategy for identification of in vivo induced exported proteins. (a) Identification of in vivo induced exported proteins. Spleens from β-lactam-treated mice infected with the EXIT library were harvested after 2 weeks of infection. Spleen homogenates were plated in parallel on 7H10 agar without β-lactam to recover all clones (red Venn diagram) and on 7H10 agar containing β-lactam to recover clones exporting ‘BlaTEM fusion proteins during in vivo growth and in vitro growth (purple Venn diagram). The population of clones identified only or in significantly greater abundance on media lacking β-lactams represents proteins whose export was induced during infection (blue). (b) Sequenced read count values recovered from agar with or without β-lactam for the 593 EXIT proteins were plotted to compare abundances after β-lactam treatment in vivo , with the abundance after dual β-lactam treatment in vivo and in vitro indicated. The majority of proteins identified as exported in vivo remained highly abundant after additional β-lactam treatment in vitro (black). A total of 38 genes (highlighted in red) were identified as statistically less abundant after in vitro β-lactam selection, representing proteins exported significantly more in vivo than in vitro (see Materials and Methods for details on statistical analysis). (c) In vivo induced exported proteins with roles promoting growth in macrophages ( rv1508 :: tn , rv3707c:tn , rv0559c :: tn , and rv2536 :: tn ). Murine bone marrow-derived macrophages were infected with M. tuberculosis CDC1551 transposon mutants lacking individual in vivo induced exported proteins. At specific times postinfection, macrophage lysates were plated to measure intracellular CFU. The fold change in CFU over the course of the infection is plotted relative to the bacterial burden at day 0 postinfection. Statistical significance was determined by one-way analysis of variance (ANOVA) with multiple comparisons performed by the use of the Holm-Sidak (normal by Shapiro-Wilk) or Student-Newman-Keuls (nonnormal) test (*, P

Techniques Used: In Vivo, Mouse Assay, Infection, Clone Assay, In Vitro, Selection, Derivative Assay

(a) The ‘BlaTEM reporter. The ‘BlaTEM reporter is compatible with proteins localized to the bacterial cytoplasmic membrane or cell wall or secreted from the bacterial cell. The right panel indicates in-frame fusions to categories of exported proteins that confer β-lactam resistance (red). In-frame fusions to cytoplasmic proteins or the cytoplasmic domain of integral membrane proteins (purple) do not confer β-lactam resistance. (b) EXIT strategy. In step 1, a comprehensive library of 5 × 10 6 plasmids containing fragments of M. tuberculosis (Mtb) genomic DNA fused to the ‘ bla TEM reporter was constructed. The plasmid library was transformed into the ΔblaC β-lactamase-sensitive mutant of M. tuberculosis , and 5 × 10 6 transformants were pooled to generate the EXIT library. In step 2, mice were infected by intravenous injection with the EXIT library and treated with β-lactam antibiotics (oral gavage twice daily) to select for EXIT clones exporting ‘BlaTEM fusion proteins. β-lactam treatment began 1 day after infection and continued to 2 weeks after infection. Mice were sacrificed, and spleens and lungs were harvested and homogenized. In step 3, organ homogenates were plated on 7H10 agar and grown to recover M. tuberculosis clones that survived β-lactam treatment during infection. Plates were scraped, and colonies were pooled separately for lungs and spleens. In step 4, plasmids from the recovered bacteria and the input samples were isolated and the fusion junction was sequenced using next-generation sequencing. Sequencing primers were designed to read out of the ‘ bla TEM reporter and sequence the immediately adjacent M. tuberculosis DNA. Sequences were aligned to the M. tuberculosis genome. Unique sequences were counted to identify the abundance of each fusion junction site within the population. The genes that were most highly abundant after in vivo β-lactam treatment were identified, and the results corresponded to plasmids producing in-frame exported ‘BlaTEM fusion proteins.
Figure Legend Snippet: (a) The ‘BlaTEM reporter. The ‘BlaTEM reporter is compatible with proteins localized to the bacterial cytoplasmic membrane or cell wall or secreted from the bacterial cell. The right panel indicates in-frame fusions to categories of exported proteins that confer β-lactam resistance (red). In-frame fusions to cytoplasmic proteins or the cytoplasmic domain of integral membrane proteins (purple) do not confer β-lactam resistance. (b) EXIT strategy. In step 1, a comprehensive library of 5 × 10 6 plasmids containing fragments of M. tuberculosis (Mtb) genomic DNA fused to the ‘ bla TEM reporter was constructed. The plasmid library was transformed into the ΔblaC β-lactamase-sensitive mutant of M. tuberculosis , and 5 × 10 6 transformants were pooled to generate the EXIT library. In step 2, mice were infected by intravenous injection with the EXIT library and treated with β-lactam antibiotics (oral gavage twice daily) to select for EXIT clones exporting ‘BlaTEM fusion proteins. β-lactam treatment began 1 day after infection and continued to 2 weeks after infection. Mice were sacrificed, and spleens and lungs were harvested and homogenized. In step 3, organ homogenates were plated on 7H10 agar and grown to recover M. tuberculosis clones that survived β-lactam treatment during infection. Plates were scraped, and colonies were pooled separately for lungs and spleens. In step 4, plasmids from the recovered bacteria and the input samples were isolated and the fusion junction was sequenced using next-generation sequencing. Sequencing primers were designed to read out of the ‘ bla TEM reporter and sequence the immediately adjacent M. tuberculosis DNA. Sequences were aligned to the M. tuberculosis genome. Unique sequences were counted to identify the abundance of each fusion junction site within the population. The genes that were most highly abundant after in vivo β-lactam treatment were identified, and the results corresponded to plasmids producing in-frame exported ‘BlaTEM fusion proteins.

Techniques Used: Transmission Electron Microscopy, Construct, Plasmid Preparation, Transformation Assay, Mutagenesis, Mouse Assay, Infection, Injection, Clone Assay, Isolation, Next-Generation Sequencing, Sequencing, In Vivo

19) Product Images from "Interaction of CarD with RNA Polymerase Mediates Mycobacterium tuberculosis Viability, Rifampin Resistance, and Pathogenesis"

Article Title: Interaction of CarD with RNA Polymerase Mediates Mycobacterium tuberculosis Viability, Rifampin Resistance, and Pathogenesis

Journal: Journal of Bacteriology

doi: 10.1128/JB.00879-12

Weakening the interaction between CarD and the RNAP compromises the survival of mycobacteria during oxidative stress. (A and B) Survival of M. smegmatis strains during oxidative stress. The log-phase M. smegmatis Δ carD attB ∷ tet-carD strains expressing CarD WT , CarD R25E , or CarD R47E in LB were treated for 1 h with either 10 mM or 25 mM H 2 O 2 . After treatment, dilutions were plated on LB. Panel A shows one such experiment, and B graphically represents survival as a ratio of CFU in treated cultures to that in untreated cultures. (C) Survival of the M. tuberculosis strains during oxidative stress. The log-phase M. tuberculosis Δ carD attB ∷ tet-carD strains expressing CarD WT or CarD R47E growing in 7H9 broth were treated for 75 h with 25 mM H 2 O 2 . After treatment, dilutions were plated on 7H10 agar, and survival is graphically represented as the ratio of CFU in treated cultures to that in untreated cultures. The graphs in panels B and C show the mean± standard error of the mean (SEM), and each sample is represented by a black circle. The significance levels in panels B and C were determined by calculating P values by Student's t test; an asterisk indicates significance with a P value of
Figure Legend Snippet: Weakening the interaction between CarD and the RNAP compromises the survival of mycobacteria during oxidative stress. (A and B) Survival of M. smegmatis strains during oxidative stress. The log-phase M. smegmatis Δ carD attB ∷ tet-carD strains expressing CarD WT , CarD R25E , or CarD R47E in LB were treated for 1 h with either 10 mM or 25 mM H 2 O 2 . After treatment, dilutions were plated on LB. Panel A shows one such experiment, and B graphically represents survival as a ratio of CFU in treated cultures to that in untreated cultures. (C) Survival of the M. tuberculosis strains during oxidative stress. The log-phase M. tuberculosis Δ carD attB ∷ tet-carD strains expressing CarD WT or CarD R47E growing in 7H9 broth were treated for 75 h with 25 mM H 2 O 2 . After treatment, dilutions were plated on 7H10 agar, and survival is graphically represented as the ratio of CFU in treated cultures to that in untreated cultures. The graphs in panels B and C show the mean± standard error of the mean (SEM), and each sample is represented by a black circle. The significance levels in panels B and C were determined by calculating P values by Student's t test; an asterisk indicates significance with a P value of

Techniques Used: Expressing

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

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

Journal: Infection and Immunity

doi: 10.1128/IAI.00229-12

Sensitivity of M. tuberculosis strains to oxidative stress. (A and B) Diameters of the zones of inhibition produced in the presence of 500 mM H 2 O 2 (A) or 40 mM menadione (B). KO, knockout. (C) Diameters of the zone of inhibition on 7H10 agar (white columns)
Figure Legend Snippet: Sensitivity of M. tuberculosis strains to oxidative stress. (A and B) Diameters of the zones of inhibition produced in the presence of 500 mM H 2 O 2 (A) or 40 mM menadione (B). KO, knockout. (C) Diameters of the zone of inhibition on 7H10 agar (white columns)

Techniques Used: Inhibition, Produced, Knock-Out

21) Product Images from "Essential Roles for Mycobacterium tuberculosis Rel beyond the Production of (p)ppGpp"

Article Title: Essential Roles for Mycobacterium tuberculosis Rel beyond the Production of (p)ppGpp

Journal: Journal of Bacteriology

doi: 10.1128/JB.00759-13

Rel Mtb -mediated (p)ppGpp hydrolysis is required for growth in vitro and maintenance of ATP and GTP levels when (p)ppGpp is being produced. (A and B) An M. tuberculosis Δ rel Mtb strain containing an episomal vector expressing the WT TetR that induces expression of rel Mtb H80A from a cassette integrated at the attB site in the genome in the presence of ATc (Tet-Rel Mtb H80A strain, designated H80A here) and a WT M. tuberculosis strain containing an empty vector in the attB site and transformed with the same TetR-expressing plasmid (control strain). (A) Transcript levels in exponential-growth-phase cultures in liquid 7H9 media. rel Mtb H80A transcripts from the cassette at the attB site were detected in the Tet-Rel Mtb H80A strain, and transcripts from the endogenous rel Mtb gene were detected in the control strain. The first comparison shows the ratio of transcript levels in the presence compared to the absence of ATc in each strain (+ATc/−ATc), and it illustrates the induction of rel Mtb H80A in the Tet-Rel Mtb H80A strain when exposed to ATc and the unresponsiveness of the rel Mtb gene to ATc in the control strain. The second comparison shows the ratio of transcript levels in the Tet-Rel Mtb H80A strain compared to the control strain (H80A/control) under each condition to illustrate the low level of transcription of rel Mtb H80A in the absence of ATc compared to that of induced cultures. (B) M. tuberculosis Tet-Rel Mtb H80A and control strains were diluted and plated on 7H10 plates in the absence or presence of ATc. (C to G) M. smegmatis Δ rel Msm strains containing an episomal vector expressing the WT TetR that turns on expression of rel Mtb WT (Tet-Rel Mtb WT strain; designated the control) or rel Mtb H80A (Tet-Rel Mtb H80A strain; designated H80A here) from cassettes integrated at the attB site in the genome in the presence of ATc. (C and D) M. smegmatis strains were diluted and plated on 7H10 (C) and LB (D) plates in the absence or presence of ATc. (E) Graphic representation of the number of M. smegmatis CFU that grew in the presence compared to the absence of ATc when grown on LB, where all Tet-Rel Mtb H80A strain bacteria that grew in the presence of ATc on LB were suppressors and were no longer responsive to ATc treatment. Data are means ± SEM from 13 replicates. (F) ATP and GTP levels in M. smegmatis strains grown in the absence or presence of ATc for 6 h to an ODλ 600 of ∼0.6 in LB liquid media were measured by LC-MS/MS. The ratio of levels in the Tet-Rel Mtb H80A strain (H80A) to those in the Tet-Rel Mtb WT -expressing strain (control) under the same conditions is graphed. Data are the means ± SEM from 4 to 6 replicates. (E and F) The significance of differences was determined by calculating P values by Student's t tests; one asterisk indicates significance with a P value of
Figure Legend Snippet: Rel Mtb -mediated (p)ppGpp hydrolysis is required for growth in vitro and maintenance of ATP and GTP levels when (p)ppGpp is being produced. (A and B) An M. tuberculosis Δ rel Mtb strain containing an episomal vector expressing the WT TetR that induces expression of rel Mtb H80A from a cassette integrated at the attB site in the genome in the presence of ATc (Tet-Rel Mtb H80A strain, designated H80A here) and a WT M. tuberculosis strain containing an empty vector in the attB site and transformed with the same TetR-expressing plasmid (control strain). (A) Transcript levels in exponential-growth-phase cultures in liquid 7H9 media. rel Mtb H80A transcripts from the cassette at the attB site were detected in the Tet-Rel Mtb H80A strain, and transcripts from the endogenous rel Mtb gene were detected in the control strain. The first comparison shows the ratio of transcript levels in the presence compared to the absence of ATc in each strain (+ATc/−ATc), and it illustrates the induction of rel Mtb H80A in the Tet-Rel Mtb H80A strain when exposed to ATc and the unresponsiveness of the rel Mtb gene to ATc in the control strain. The second comparison shows the ratio of transcript levels in the Tet-Rel Mtb H80A strain compared to the control strain (H80A/control) under each condition to illustrate the low level of transcription of rel Mtb H80A in the absence of ATc compared to that of induced cultures. (B) M. tuberculosis Tet-Rel Mtb H80A and control strains were diluted and plated on 7H10 plates in the absence or presence of ATc. (C to G) M. smegmatis Δ rel Msm strains containing an episomal vector expressing the WT TetR that turns on expression of rel Mtb WT (Tet-Rel Mtb WT strain; designated the control) or rel Mtb H80A (Tet-Rel Mtb H80A strain; designated H80A here) from cassettes integrated at the attB site in the genome in the presence of ATc. (C and D) M. smegmatis strains were diluted and plated on 7H10 (C) and LB (D) plates in the absence or presence of ATc. (E) Graphic representation of the number of M. smegmatis CFU that grew in the presence compared to the absence of ATc when grown on LB, where all Tet-Rel Mtb H80A strain bacteria that grew in the presence of ATc on LB were suppressors and were no longer responsive to ATc treatment. Data are means ± SEM from 13 replicates. (F) ATP and GTP levels in M. smegmatis strains grown in the absence or presence of ATc for 6 h to an ODλ 600 of ∼0.6 in LB liquid media were measured by LC-MS/MS. The ratio of levels in the Tet-Rel Mtb H80A strain (H80A) to those in the Tet-Rel Mtb WT -expressing strain (control) under the same conditions is graphed. Data are the means ± SEM from 4 to 6 replicates. (E and F) The significance of differences was determined by calculating P values by Student's t tests; one asterisk indicates significance with a P value of

Techniques Used: In Vitro, Produced, Plasmid Preparation, Expressing, Transformation Assay, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry

Rv1366 is not necessary for M. tuberculosis growth or biofilm formation in vitro . (A) Southern blot analysis of WT M. tuberculosis Erdman (lane 1) and the Δ rv1366 strain (lanes 2 to 4) using EcoRI-digested genomic DNA and a probe that spans nucleotides 1537706 to 1538406 of the M. tuberculosis genome. EcoRI digestion of WT M. tuberculosis yields a band at 5,083 bp. EcoRI digestion of Δ rv1366 results in a 2,226-bp band due to the replacement of the rv1366 gene with a hygromycin resistance cassette. (B) Southern blot analysis of WT M. tuberculosis Erdman (lane 1) and the Δ rel Mtb Δ rv1366 strain (lanes 2 to 6) using SmaI-digested genomic DNA and a probe that spans nucleotides 2910191 to 2910844 of the M. tuberculosis genome. SmaI digestion of WT M. tuberculosis yields a band at 2,257 bp. SmaI digestion of the Δ rel Mtb Δ rv1366 strain results in a 4,769-bp band due to the replacement of the rel Mtb gene with a hygromycin resistance cassette. (C and D) M. tuberculosis Δ rel Mtb , Δ rv1366 , and Δ rel Mtb Δ rv1366 strains containing a vector that constitutively expresses Rel Mtb WT or Rv1366 or containing an empty vector (−) integrated at the attB site in the genome were normalized to the same ODλ 600 and either diluted and plated on 7H10 plates (C) or grown in Sauton's media in static, biofilm-forming conditions in a 24-well dish (D). (C) 7H10 plates were incubated for 3 weeks at 37°C. (D) Biofilms were incubated for 4 weeks at 37°C. The parent strain used is labeled above each photo, and the protein expressed from the attB site is labeled below the photo.
Figure Legend Snippet: Rv1366 is not necessary for M. tuberculosis growth or biofilm formation in vitro . (A) Southern blot analysis of WT M. tuberculosis Erdman (lane 1) and the Δ rv1366 strain (lanes 2 to 4) using EcoRI-digested genomic DNA and a probe that spans nucleotides 1537706 to 1538406 of the M. tuberculosis genome. EcoRI digestion of WT M. tuberculosis yields a band at 5,083 bp. EcoRI digestion of Δ rv1366 results in a 2,226-bp band due to the replacement of the rv1366 gene with a hygromycin resistance cassette. (B) Southern blot analysis of WT M. tuberculosis Erdman (lane 1) and the Δ rel Mtb Δ rv1366 strain (lanes 2 to 6) using SmaI-digested genomic DNA and a probe that spans nucleotides 2910191 to 2910844 of the M. tuberculosis genome. SmaI digestion of WT M. tuberculosis yields a band at 2,257 bp. SmaI digestion of the Δ rel Mtb Δ rv1366 strain results in a 4,769-bp band due to the replacement of the rel Mtb gene with a hygromycin resistance cassette. (C and D) M. tuberculosis Δ rel Mtb , Δ rv1366 , and Δ rel Mtb Δ rv1366 strains containing a vector that constitutively expresses Rel Mtb WT or Rv1366 or containing an empty vector (−) integrated at the attB site in the genome were normalized to the same ODλ 600 and either diluted and plated on 7H10 plates (C) or grown in Sauton's media in static, biofilm-forming conditions in a 24-well dish (D). (C) 7H10 plates were incubated for 3 weeks at 37°C. (D) Biofilms were incubated for 4 weeks at 37°C. The parent strain used is labeled above each photo, and the protein expressed from the attB site is labeled below the photo.

Techniques Used: In Vitro, Southern Blot, Plasmid Preparation, Incubation, Labeling

Rel Mtb -mediated (p)ppGpp synthesis is necessary for optimal M. tuberculosis growth and biofilm formation in vitro. M. tuberculosis Δ rel Mtb strains containing a vector that constitutively expresses Rel Mtb WT (WT) or Rel Mtb H344Y (H344Y) or containing an empty vector (−) integrated at the attB site in the genome were normalized to the same ODλ 600 and either diluted and plated on 7H10 plates (A) or grown in Sauton's medium in static, biofilm-forming conditions in a 24-well dish (B). (A) 7H10 plates were incubated for 3 weeks at 37°C. (B) Biofilms were incubated for 4 weeks at 37°C. The parent strain used is labeled above each photo, and the protein expressed from the attB site is labeled below the photo.
Figure Legend Snippet: Rel Mtb -mediated (p)ppGpp synthesis is necessary for optimal M. tuberculosis growth and biofilm formation in vitro. M. tuberculosis Δ rel Mtb strains containing a vector that constitutively expresses Rel Mtb WT (WT) or Rel Mtb H344Y (H344Y) or containing an empty vector (−) integrated at the attB site in the genome were normalized to the same ODλ 600 and either diluted and plated on 7H10 plates (A) or grown in Sauton's medium in static, biofilm-forming conditions in a 24-well dish (B). (A) 7H10 plates were incubated for 3 weeks at 37°C. (B) Biofilms were incubated for 4 weeks at 37°C. The parent strain used is labeled above each photo, and the protein expressed from the attB site is labeled below the photo.

Techniques Used: In Vitro, Plasmid Preparation, Incubation, Labeling

Rel Mtb (p)ppGpp hydrolase activity is essential for acute and chronic M. tuberculosis infection of mice. Bacterial titers in the lungs (A, E, and G) and spleens (B and H) of C57BL/6 mice infected with either control or Tet-Rel Mtb H80A (designated H80A in the figure) M. tuberculosis strains. The parent strain for the control is WT M. tuberculosis Erdman, and the parent strain for Tet-Rel Mtb H80A is M. tuberculosis Δ rel Mtb . Both strains express TetR from a plasmid that confers streptomycin resistance and contain a kanamycin resistance cassette integrated into the attB site that, in the case of Tet-Rel Mtb H80A , carries the rel Mtb H80A allele. The strain symbols in panel A are the same for all panels. (A to C) Mice were given normal mouse chow throughout infection. (E and F) Mice were administered doxycycline-containing mouse chow starting at day 1 postinfection (designated by the arrow). (G to I) Mice were administered doxycycline-containing mouse chow starting at day 63 postinfection (designated by the arrow). (C, F, and I) The ratio of CFU from the lungs or spleens grown on 7H10 plates containing streptomycin and kanamycin compared to 7H10 containing no antibiotics (No AB). ND denotes when no colonies were recovered after plating 5% of the lung homogenate (limit of detection, 20 CFU). Data are means ± SEM of 6 Tet-Rel Mtb H80A strain-infected mice and 3 control strain-infected mice per time point from two replicate experiments. (E, G, and H) The significance of differences were determined by calculating P values by Student's t tests; two asterisks indicate significance with a P value of
Figure Legend Snippet: Rel Mtb (p)ppGpp hydrolase activity is essential for acute and chronic M. tuberculosis infection of mice. Bacterial titers in the lungs (A, E, and G) and spleens (B and H) of C57BL/6 mice infected with either control or Tet-Rel Mtb H80A (designated H80A in the figure) M. tuberculosis strains. The parent strain for the control is WT M. tuberculosis Erdman, and the parent strain for Tet-Rel Mtb H80A is M. tuberculosis Δ rel Mtb . Both strains express TetR from a plasmid that confers streptomycin resistance and contain a kanamycin resistance cassette integrated into the attB site that, in the case of Tet-Rel Mtb H80A , carries the rel Mtb H80A allele. The strain symbols in panel A are the same for all panels. (A to C) Mice were given normal mouse chow throughout infection. (E and F) Mice were administered doxycycline-containing mouse chow starting at day 1 postinfection (designated by the arrow). (G to I) Mice were administered doxycycline-containing mouse chow starting at day 63 postinfection (designated by the arrow). (C, F, and I) The ratio of CFU from the lungs or spleens grown on 7H10 plates containing streptomycin and kanamycin compared to 7H10 containing no antibiotics (No AB). ND denotes when no colonies were recovered after plating 5% of the lung homogenate (limit of detection, 20 CFU). Data are means ± SEM of 6 Tet-Rel Mtb H80A strain-infected mice and 3 control strain-infected mice per time point from two replicate experiments. (E, G, and H) The significance of differences were determined by calculating P values by Student's t tests; two asterisks indicate significance with a P value of

Techniques Used: Activity Assay, Infection, Mouse Assay, Plasmid Preparation

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

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

Journal: Infection and Immunity

doi: 10.1128/IAI.00229-12

Sensitivity of M. tuberculosis strains to oxidative stress. (A and B) Diameters of the zones of inhibition produced in the presence of 500 mM H 2 O 2 (A) or 40 mM menadione (B). KO, knockout. (C) Diameters of the zone of inhibition on 7H10 agar (white columns)
Figure Legend Snippet: Sensitivity of M. tuberculosis strains to oxidative stress. (A and B) Diameters of the zones of inhibition produced in the presence of 500 mM H 2 O 2 (A) or 40 mM menadione (B). KO, knockout. (C) Diameters of the zone of inhibition on 7H10 agar (white columns)

Techniques Used: Inhibition, Produced, Knock-Out

23) Product Images from "The EXIT Strategy: an Approach for Identifying Bacterial Proteins Exported during Host Infection"

Article Title: The EXIT Strategy: an Approach for Identifying Bacterial Proteins Exported during Host Infection

Journal: mBio

doi: 10.1128/mBio.00333-17

Strategy for identification of in vivo induced exported proteins. (a) Identification of in vivo induced exported proteins. Spleens from β-lactam-treated mice infected with the EXIT library were harvested after 2 weeks of infection. Spleen homogenates were plated in parallel on 7H10 agar without β-lactam to recover all clones (red Venn diagram) and on 7H10 agar containing β-lactam to recover clones exporting ‘BlaTEM fusion proteins during in vivo growth and in vitro growth (purple Venn diagram). The population of clones identified only or in significantly greater abundance on media lacking β-lactams represents proteins whose export was induced during infection (blue). (b) Sequenced read count values recovered from agar with or without β-lactam for the 593 EXIT proteins were plotted to compare abundances after β-lactam treatment in vivo , with the abundance after dual β-lactam treatment in vivo and in vitro indicated. The majority of proteins identified as exported in vivo remained highly abundant after additional β-lactam treatment in vitro (black). A total of 38 genes (highlighted in red) were identified as statistically less abundant after in vitro β-lactam selection, representing proteins exported significantly more in vivo than in vitro (see Materials and Methods for details on statistical analysis). (c) In vivo induced exported proteins with roles promoting growth in macrophages ( rv1508 :: tn , rv3707c:tn , rv0559c :: tn , and rv2536 :: tn ). Murine bone marrow-derived macrophages were infected with M. tuberculosis CDC1551 transposon mutants lacking individual in vivo induced exported proteins. At specific times postinfection, macrophage lysates were plated to measure intracellular CFU. The fold change in CFU over the course of the infection is plotted relative to the bacterial burden at day 0 postinfection. Statistical significance was determined by one-way analysis of variance (ANOVA) with multiple comparisons performed by the use of the Holm-Sidak (normal by Shapiro-Wilk) or Student-Newman-Keuls (nonnormal) test (*, P
Figure Legend Snippet: Strategy for identification of in vivo induced exported proteins. (a) Identification of in vivo induced exported proteins. Spleens from β-lactam-treated mice infected with the EXIT library were harvested after 2 weeks of infection. Spleen homogenates were plated in parallel on 7H10 agar without β-lactam to recover all clones (red Venn diagram) and on 7H10 agar containing β-lactam to recover clones exporting ‘BlaTEM fusion proteins during in vivo growth and in vitro growth (purple Venn diagram). The population of clones identified only or in significantly greater abundance on media lacking β-lactams represents proteins whose export was induced during infection (blue). (b) Sequenced read count values recovered from agar with or without β-lactam for the 593 EXIT proteins were plotted to compare abundances after β-lactam treatment in vivo , with the abundance after dual β-lactam treatment in vivo and in vitro indicated. The majority of proteins identified as exported in vivo remained highly abundant after additional β-lactam treatment in vitro (black). A total of 38 genes (highlighted in red) were identified as statistically less abundant after in vitro β-lactam selection, representing proteins exported significantly more in vivo than in vitro (see Materials and Methods for details on statistical analysis). (c) In vivo induced exported proteins with roles promoting growth in macrophages ( rv1508 :: tn , rv3707c:tn , rv0559c :: tn , and rv2536 :: tn ). Murine bone marrow-derived macrophages were infected with M. tuberculosis CDC1551 transposon mutants lacking individual in vivo induced exported proteins. At specific times postinfection, macrophage lysates were plated to measure intracellular CFU. The fold change in CFU over the course of the infection is plotted relative to the bacterial burden at day 0 postinfection. Statistical significance was determined by one-way analysis of variance (ANOVA) with multiple comparisons performed by the use of the Holm-Sidak (normal by Shapiro-Wilk) or Student-Newman-Keuls (nonnormal) test (*, P

Techniques Used: In Vivo, Mouse Assay, Infection, Clone Assay, In Vitro, Selection, Derivative Assay

(a) The ‘BlaTEM reporter. The ‘BlaTEM reporter is compatible with proteins localized to the bacterial cytoplasmic membrane or cell wall or secreted from the bacterial cell. The right panel indicates in-frame fusions to categories of exported proteins that confer β-lactam resistance (red). In-frame fusions to cytoplasmic proteins or the cytoplasmic domain of integral membrane proteins (purple) do not confer β-lactam resistance. (b) EXIT strategy. In step 1, a comprehensive library of 5 × 10 6 plasmids containing fragments of M. tuberculosis (Mtb) genomic DNA fused to the ‘ bla TEM reporter was constructed. The plasmid library was transformed into the ΔblaC β-lactamase-sensitive mutant of M. tuberculosis , and 5 × 10 6 transformants were pooled to generate the EXIT library. In step 2, mice were infected by intravenous injection with the EXIT library and treated with β-lactam antibiotics (oral gavage twice daily) to select for EXIT clones exporting ‘BlaTEM fusion proteins. β-lactam treatment began 1 day after infection and continued to 2 weeks after infection. Mice were sacrificed, and spleens and lungs were harvested and homogenized. In step 3, organ homogenates were plated on 7H10 agar and grown to recover M. tuberculosis clones that survived β-lactam treatment during infection. Plates were scraped, and colonies were pooled separately for lungs and spleens. In step 4, plasmids from the recovered bacteria and the input samples were isolated and the fusion junction was sequenced using next-generation sequencing. Sequencing primers were designed to read out of the ‘ bla TEM reporter and sequence the immediately adjacent M. tuberculosis DNA. Sequences were aligned to the M. tuberculosis genome. Unique sequences were counted to identify the abundance of each fusion junction site within the population. The genes that were most highly abundant after in vivo β-lactam treatment were identified, and the results corresponded to plasmids producing in-frame exported ‘BlaTEM fusion proteins.
Figure Legend Snippet: (a) The ‘BlaTEM reporter. The ‘BlaTEM reporter is compatible with proteins localized to the bacterial cytoplasmic membrane or cell wall or secreted from the bacterial cell. The right panel indicates in-frame fusions to categories of exported proteins that confer β-lactam resistance (red). In-frame fusions to cytoplasmic proteins or the cytoplasmic domain of integral membrane proteins (purple) do not confer β-lactam resistance. (b) EXIT strategy. In step 1, a comprehensive library of 5 × 10 6 plasmids containing fragments of M. tuberculosis (Mtb) genomic DNA fused to the ‘ bla TEM reporter was constructed. The plasmid library was transformed into the ΔblaC β-lactamase-sensitive mutant of M. tuberculosis , and 5 × 10 6 transformants were pooled to generate the EXIT library. In step 2, mice were infected by intravenous injection with the EXIT library and treated with β-lactam antibiotics (oral gavage twice daily) to select for EXIT clones exporting ‘BlaTEM fusion proteins. β-lactam treatment began 1 day after infection and continued to 2 weeks after infection. Mice were sacrificed, and spleens and lungs were harvested and homogenized. In step 3, organ homogenates were plated on 7H10 agar and grown to recover M. tuberculosis clones that survived β-lactam treatment during infection. Plates were scraped, and colonies were pooled separately for lungs and spleens. In step 4, plasmids from the recovered bacteria and the input samples were isolated and the fusion junction was sequenced using next-generation sequencing. Sequencing primers were designed to read out of the ‘ bla TEM reporter and sequence the immediately adjacent M. tuberculosis DNA. Sequences were aligned to the M. tuberculosis genome. Unique sequences were counted to identify the abundance of each fusion junction site within the population. The genes that were most highly abundant after in vivo β-lactam treatment were identified, and the results corresponded to plasmids producing in-frame exported ‘BlaTEM fusion proteins.

Techniques Used: Transmission Electron Microscopy, Construct, Plasmid Preparation, Transformation Assay, Mutagenesis, Mouse Assay, Infection, Injection, Clone Assay, Isolation, Next-Generation Sequencing, Sequencing, In Vivo

24) Product Images from "Essential Roles for Mycobacterium tuberculosis Rel beyond the Production of (p)ppGpp"

Article Title: Essential Roles for Mycobacterium tuberculosis Rel beyond the Production of (p)ppGpp

Journal: Journal of Bacteriology

doi: 10.1128/JB.00759-13

Rel Mtb -mediated (p)ppGpp hydrolysis is required for growth in vitro and maintenance of ATP and GTP levels when (p)ppGpp is being produced. (A and B) An M. tuberculosis Δ rel Mtb strain containing an episomal vector expressing the WT TetR that induces expression of rel Mtb H80A from a cassette integrated at the attB site in the genome in the presence of ATc (Tet-Rel Mtb H80A strain, designated H80A here) and a WT M. tuberculosis strain containing an empty vector in the attB site and transformed with the same TetR-expressing plasmid (control strain). (A) Transcript levels in exponential-growth-phase cultures in liquid 7H9 media. rel Mtb H80A transcripts from the cassette at the attB site were detected in the Tet-Rel Mtb H80A strain, and transcripts from the endogenous rel Mtb gene were detected in the control strain. The first comparison shows the ratio of transcript levels in the presence compared to the absence of ATc in each strain (+ATc/−ATc), and it illustrates the induction of rel Mtb H80A in the Tet-Rel Mtb H80A strain when exposed to ATc and the unresponsiveness of the rel Mtb gene to ATc in the control strain. The second comparison shows the ratio of transcript levels in the Tet-Rel Mtb H80A strain compared to the control strain (H80A/control) under each condition to illustrate the low level of transcription of rel Mtb H80A in the absence of ATc compared to that of induced cultures. (B) M. tuberculosis Tet-Rel Mtb H80A and control strains were diluted and plated on 7H10 plates in the absence or presence of ATc. (C to G) M. smegmatis Δ rel Msm strains containing an episomal vector expressing the WT TetR that turns on expression of rel Mtb WT (Tet-Rel Mtb WT strain; designated the control) or rel Mtb H80A (Tet-Rel Mtb H80A strain; designated H80A here) from cassettes integrated at the attB site in the genome in the presence of ATc. (C and D) M. smegmatis strains were diluted and plated on 7H10 (C) and LB (D) plates in the absence or presence of ATc. (E) Graphic representation of the number of M. smegmatis CFU that grew in the presence compared to the absence of ATc when grown on LB, where all Tet-Rel Mtb H80A strain bacteria that grew in the presence of ATc on LB were suppressors and were no longer responsive to ATc treatment. Data are means ± SEM from 13 replicates. (F) ATP and GTP levels in M. smegmatis strains grown in the absence or presence of ATc for 6 h to an ODλ 600 of ∼0.6 in LB liquid media were measured by LC-MS/MS. The ratio of levels in the Tet-Rel Mtb H80A strain (H80A) to those in the Tet-Rel Mtb WT -expressing strain (control) under the same conditions is graphed. Data are the means ± SEM from 4 to 6 replicates. (E and F) The significance of differences was determined by calculating P values by Student's t tests; one asterisk indicates significance with a P value of
Figure Legend Snippet: Rel Mtb -mediated (p)ppGpp hydrolysis is required for growth in vitro and maintenance of ATP and GTP levels when (p)ppGpp is being produced. (A and B) An M. tuberculosis Δ rel Mtb strain containing an episomal vector expressing the WT TetR that induces expression of rel Mtb H80A from a cassette integrated at the attB site in the genome in the presence of ATc (Tet-Rel Mtb H80A strain, designated H80A here) and a WT M. tuberculosis strain containing an empty vector in the attB site and transformed with the same TetR-expressing plasmid (control strain). (A) Transcript levels in exponential-growth-phase cultures in liquid 7H9 media. rel Mtb H80A transcripts from the cassette at the attB site were detected in the Tet-Rel Mtb H80A strain, and transcripts from the endogenous rel Mtb gene were detected in the control strain. The first comparison shows the ratio of transcript levels in the presence compared to the absence of ATc in each strain (+ATc/−ATc), and it illustrates the induction of rel Mtb H80A in the Tet-Rel Mtb H80A strain when exposed to ATc and the unresponsiveness of the rel Mtb gene to ATc in the control strain. The second comparison shows the ratio of transcript levels in the Tet-Rel Mtb H80A strain compared to the control strain (H80A/control) under each condition to illustrate the low level of transcription of rel Mtb H80A in the absence of ATc compared to that of induced cultures. (B) M. tuberculosis Tet-Rel Mtb H80A and control strains were diluted and plated on 7H10 plates in the absence or presence of ATc. (C to G) M. smegmatis Δ rel Msm strains containing an episomal vector expressing the WT TetR that turns on expression of rel Mtb WT (Tet-Rel Mtb WT strain; designated the control) or rel Mtb H80A (Tet-Rel Mtb H80A strain; designated H80A here) from cassettes integrated at the attB site in the genome in the presence of ATc. (C and D) M. smegmatis strains were diluted and plated on 7H10 (C) and LB (D) plates in the absence or presence of ATc. (E) Graphic representation of the number of M. smegmatis CFU that grew in the presence compared to the absence of ATc when grown on LB, where all Tet-Rel Mtb H80A strain bacteria that grew in the presence of ATc on LB were suppressors and were no longer responsive to ATc treatment. Data are means ± SEM from 13 replicates. (F) ATP and GTP levels in M. smegmatis strains grown in the absence or presence of ATc for 6 h to an ODλ 600 of ∼0.6 in LB liquid media were measured by LC-MS/MS. The ratio of levels in the Tet-Rel Mtb H80A strain (H80A) to those in the Tet-Rel Mtb WT -expressing strain (control) under the same conditions is graphed. Data are the means ± SEM from 4 to 6 replicates. (E and F) The significance of differences was determined by calculating P values by Student's t tests; one asterisk indicates significance with a P value of

Techniques Used: In Vitro, Produced, Plasmid Preparation, Expressing, Transformation Assay, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry

Rv1366 is not necessary for M. tuberculosis growth or biofilm formation in vitro . (A) Southern blot analysis of WT M. tuberculosis Erdman (lane 1) and the Δ rv1366 strain (lanes 2 to 4) using EcoRI-digested genomic DNA and a probe that spans nucleotides 1537706 to 1538406 of the M. tuberculosis genome. EcoRI digestion of WT M. tuberculosis yields a band at 5,083 bp. EcoRI digestion of Δ rv1366 results in a 2,226-bp band due to the replacement of the rv1366 gene with a hygromycin resistance cassette. (B) Southern blot analysis of WT M. tuberculosis Erdman (lane 1) and the Δ rel Mtb Δ rv1366 strain (lanes 2 to 6) using SmaI-digested genomic DNA and a probe that spans nucleotides 2910191 to 2910844 of the M. tuberculosis genome. SmaI digestion of WT M. tuberculosis yields a band at 2,257 bp. SmaI digestion of the Δ rel Mtb Δ rv1366 strain results in a 4,769-bp band due to the replacement of the rel Mtb gene with a hygromycin resistance cassette. (C and D) M. tuberculosis Δ rel Mtb , Δ rv1366 , and Δ rel Mtb Δ rv1366 strains containing a vector that constitutively expresses Rel Mtb WT or Rv1366 or containing an empty vector (−) integrated at the attB site in the genome were normalized to the same ODλ 600 and either diluted and plated on 7H10 plates (C) or grown in Sauton's media in static, biofilm-forming conditions in a 24-well dish (D). (C) 7H10 plates were incubated for 3 weeks at 37°C. (D) Biofilms were incubated for 4 weeks at 37°C. The parent strain used is labeled above each photo, and the protein expressed from the attB site is labeled below the photo.
Figure Legend Snippet: Rv1366 is not necessary for M. tuberculosis growth or biofilm formation in vitro . (A) Southern blot analysis of WT M. tuberculosis Erdman (lane 1) and the Δ rv1366 strain (lanes 2 to 4) using EcoRI-digested genomic DNA and a probe that spans nucleotides 1537706 to 1538406 of the M. tuberculosis genome. EcoRI digestion of WT M. tuberculosis yields a band at 5,083 bp. EcoRI digestion of Δ rv1366 results in a 2,226-bp band due to the replacement of the rv1366 gene with a hygromycin resistance cassette. (B) Southern blot analysis of WT M. tuberculosis Erdman (lane 1) and the Δ rel Mtb Δ rv1366 strain (lanes 2 to 6) using SmaI-digested genomic DNA and a probe that spans nucleotides 2910191 to 2910844 of the M. tuberculosis genome. SmaI digestion of WT M. tuberculosis yields a band at 2,257 bp. SmaI digestion of the Δ rel Mtb Δ rv1366 strain results in a 4,769-bp band due to the replacement of the rel Mtb gene with a hygromycin resistance cassette. (C and D) M. tuberculosis Δ rel Mtb , Δ rv1366 , and Δ rel Mtb Δ rv1366 strains containing a vector that constitutively expresses Rel Mtb WT or Rv1366 or containing an empty vector (−) integrated at the attB site in the genome were normalized to the same ODλ 600 and either diluted and plated on 7H10 plates (C) or grown in Sauton's media in static, biofilm-forming conditions in a 24-well dish (D). (C) 7H10 plates were incubated for 3 weeks at 37°C. (D) Biofilms were incubated for 4 weeks at 37°C. The parent strain used is labeled above each photo, and the protein expressed from the attB site is labeled below the photo.

Techniques Used: In Vitro, Southern Blot, Plasmid Preparation, Incubation, Labeling

Rel Mtb -mediated (p)ppGpp synthesis is necessary for optimal M. tuberculosis growth and biofilm formation in vitro. M. tuberculosis Δ rel Mtb strains containing a vector that constitutively expresses Rel Mtb WT (WT) or Rel Mtb H344Y (H344Y) or containing an empty vector (−) integrated at the attB site in the genome were normalized to the same ODλ 600 and either diluted and plated on 7H10 plates (A) or grown in Sauton's medium in static, biofilm-forming conditions in a 24-well dish (B). (A) 7H10 plates were incubated for 3 weeks at 37°C. (B) Biofilms were incubated for 4 weeks at 37°C. The parent strain used is labeled above each photo, and the protein expressed from the attB site is labeled below the photo.
Figure Legend Snippet: Rel Mtb -mediated (p)ppGpp synthesis is necessary for optimal M. tuberculosis growth and biofilm formation in vitro. M. tuberculosis Δ rel Mtb strains containing a vector that constitutively expresses Rel Mtb WT (WT) or Rel Mtb H344Y (H344Y) or containing an empty vector (−) integrated at the attB site in the genome were normalized to the same ODλ 600 and either diluted and plated on 7H10 plates (A) or grown in Sauton's medium in static, biofilm-forming conditions in a 24-well dish (B). (A) 7H10 plates were incubated for 3 weeks at 37°C. (B) Biofilms were incubated for 4 weeks at 37°C. The parent strain used is labeled above each photo, and the protein expressed from the attB site is labeled below the photo.

Techniques Used: In Vitro, Plasmid Preparation, Incubation, Labeling

Rel Mtb (p)ppGpp hydrolase activity is essential for acute and chronic M. tuberculosis infection of mice. Bacterial titers in the lungs (A, E, and G) and spleens (B and H) of C57BL/6 mice infected with either control or Tet-Rel Mtb H80A (designated H80A in the figure) M. tuberculosis strains. The parent strain for the control is WT M. tuberculosis Erdman, and the parent strain for Tet-Rel Mtb H80A is M. tuberculosis Δ rel Mtb . Both strains express TetR from a plasmid that confers streptomycin resistance and contain a kanamycin resistance cassette integrated into the attB site that, in the case of Tet-Rel Mtb H80A , carries the rel Mtb H80A allele. The strain symbols in panel A are the same for all panels. (A to C) Mice were given normal mouse chow throughout infection. (E and F) Mice were administered doxycycline-containing mouse chow starting at day 1 postinfection (designated by the arrow). (G to I) Mice were administered doxycycline-containing mouse chow starting at day 63 postinfection (designated by the arrow). (C, F, and I) The ratio of CFU from the lungs or spleens grown on 7H10 plates containing streptomycin and kanamycin compared to 7H10 containing no antibiotics (No AB). ND denotes when no colonies were recovered after plating 5% of the lung homogenate (limit of detection, 20 CFU). Data are means ± SEM of 6 Tet-Rel Mtb H80A strain-infected mice and 3 control strain-infected mice per time point from two replicate experiments. (E, G, and H) The significance of differences were determined by calculating P values by Student's t tests; two asterisks indicate significance with a P value of
Figure Legend Snippet: Rel Mtb (p)ppGpp hydrolase activity is essential for acute and chronic M. tuberculosis infection of mice. Bacterial titers in the lungs (A, E, and G) and spleens (B and H) of C57BL/6 mice infected with either control or Tet-Rel Mtb H80A (designated H80A in the figure) M. tuberculosis strains. The parent strain for the control is WT M. tuberculosis Erdman, and the parent strain for Tet-Rel Mtb H80A is M. tuberculosis Δ rel Mtb . Both strains express TetR from a plasmid that confers streptomycin resistance and contain a kanamycin resistance cassette integrated into the attB site that, in the case of Tet-Rel Mtb H80A , carries the rel Mtb H80A allele. The strain symbols in panel A are the same for all panels. (A to C) Mice were given normal mouse chow throughout infection. (E and F) Mice were administered doxycycline-containing mouse chow starting at day 1 postinfection (designated by the arrow). (G to I) Mice were administered doxycycline-containing mouse chow starting at day 63 postinfection (designated by the arrow). (C, F, and I) The ratio of CFU from the lungs or spleens grown on 7H10 plates containing streptomycin and kanamycin compared to 7H10 containing no antibiotics (No AB). ND denotes when no colonies were recovered after plating 5% of the lung homogenate (limit of detection, 20 CFU). Data are means ± SEM of 6 Tet-Rel Mtb H80A strain-infected mice and 3 control strain-infected mice per time point from two replicate experiments. (E, G, and H) The significance of differences were determined by calculating P values by Student's t tests; two asterisks indicate significance with a P value of

Techniques Used: Activity Assay, Infection, Mouse Assay, Plasmid Preparation

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

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

Journal: Infection and Immunity

doi: 10.1128/IAI.00229-12

Sensitivity of M. tuberculosis strains to oxidative stress. (A and B) Diameters of the zones of inhibition produced in the presence of 500 mM H 2 O 2 (A) or 40 mM menadione (B). KO, knockout. (C) Diameters of the zone of inhibition on 7H10 agar (white columns)
Figure Legend Snippet: Sensitivity of M. tuberculosis strains to oxidative stress. (A and B) Diameters of the zones of inhibition produced in the presence of 500 mM H 2 O 2 (A) or 40 mM menadione (B). KO, knockout. (C) Diameters of the zone of inhibition on 7H10 agar (white columns)

Techniques Used: Inhibition, Produced, Knock-Out

Related Articles

Mutagenesis:

Article Title: A Ferritin Mutant of Mycobacterium tuberculosis Is Highly Susceptible to Killing by Antibiotics and Is Unable To Establish a Chronic Infection in Mice
Article Snippet: .. The resulting complementing plasmid, pSM852, was electroporated into bfrB mutant strain ST214, and transformants were selected on 7H10 agar containing Strp and Spec. ..

Isolation:

Article Title: Real-Time Investigation of Tuberculosis Transmission: Developing the Respiratory Aerosol Sampling Chamber (RASC)
Article Snippet: .. For determination of CFU in controlled aerosol release experiments, bacilli were propagated in liquid 7H9 medium (Difco) supplemented with 0.05% Tween-80, 0.2% glycerol and albumin/NaCl/ glucose (ADC) complex and/or isolated on solid 7H10 agar (Difco) supplemented with 0.2% glycerol and oleic acid/albumin/dextrose/catalase (OADC) complex. .. Owing to the selectivity provided by the aph cassette on pMSP12GFP, all media contained kanamycin (Sigma) at a final concentration of 20 μg/mL.

other:

Article Title: A Ferritin Mutant of Mycobacterium tuberculosis Is Highly Susceptible to Killing by Antibiotics and Is Unable To Establish a Chronic Infection in Mice
Article Snippet: Briefly, M. tuberculosis strains were grown to the logarithmic phase (optical density at 595 nm [OD595 ] of 0.5) in 7H9 medium, and approximately 3 × 107 bacteria were plated onto 7H10 agar or 7H10 agar plus 5 μM the iron chelator 2′,2-dypyridyl (DPI) and spread evenly.

Article Title: ideR, an Essential Gene in Mycobacterium tuberculosis: Role of IdeR in Iron-Dependent Gene Expression, Iron Metabolism, and Oxidative Stress Response †
Article Snippet: M. tuberculosis strain H37Rv (American Type Culture Collection) was maintained in Middlebrook 7H9 broth or on 7H10 agar (Difco) supplemented with 0.2% glycerol, 0.05% Tween 80, and 10% albumin-dextrose-NaCl complex (ADN) ( ).

Article Title: The EXIT Strategy: an Approach for Identifying Bacterial Proteins Exported during Host Infection
Article Snippet: M. tuberculosis strains were grown with Middlebrook 7H9 broth or 7H10 agar (Difco) supplemented with 1× albumin dextrose saline (ADS), 0.5% glycerol, and 0.05% Tween 80 (7AGT) ( ).

Article Title: Essential Roles for Mycobacterium tuberculosis Rel beyond the Production of (p)ppGpp
Article Snippet: Bacterial burden was determined by plating serial dilutions of lung and spleen homogenates onto 7H10 agar plates in the absence or presence of kanamycin and streptomycin.

Derivative Assay:

Article Title: Essential Roles for Mycobacterium tuberculosis Rel beyond the Production of (p)ppGpp
Article Snippet: .. All M. tuberculosis strains were derived from Erdman and were grown planktonically at 37°C in 7H9 (broth) or 7H10 (agar) (Difco) medium supplemented with 10% oleic acid-albumin-dextrose-catalase (OADC), 0.5% glycerol, and 0.05% Tween 80 (broth). ..

Plasmid Preparation:

Article Title: A Ferritin Mutant of Mycobacterium tuberculosis Is Highly Susceptible to Killing by Antibiotics and Is Unable To Establish a Chronic Infection in Mice
Article Snippet: .. The resulting complementing plasmid, pSM852, was electroporated into bfrB mutant strain ST214, and transformants were selected on 7H10 agar containing Strp and Spec. ..

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    Rel Mtb -mediated (p)ppGpp hydrolysis is required for growth in vitro and maintenance of ATP and GTP levels when (p)ppGpp is being produced. (A and B) An M. tuberculosis Δ rel Mtb strain containing an episomal vector expressing the WT TetR that induces expression of rel Mtb H80A from a cassette integrated at the attB site in the genome in the presence of ATc (Tet-Rel Mtb H80A strain, designated H80A here) and a WT M. tuberculosis strain containing an empty vector in the attB site and transformed with the same TetR-expressing plasmid (control strain). (A) Transcript levels in exponential-growth-phase cultures in liquid 7H9 media. rel Mtb H80A transcripts from the cassette at the attB site were detected in the Tet-Rel Mtb H80A strain, and transcripts from the endogenous rel Mtb gene were detected in the control strain. The first comparison shows the ratio of transcript levels in the presence compared to the absence of ATc in each strain (+ATc/−ATc), and it illustrates the induction of rel Mtb H80A in the Tet-Rel Mtb H80A strain when exposed to ATc and the unresponsiveness of the rel Mtb gene to ATc in the control strain. The second comparison shows the ratio of transcript levels in the Tet-Rel Mtb H80A strain compared to the control strain (H80A/control) under each condition to illustrate the low level of transcription of rel Mtb H80A in the absence of ATc compared to that of induced cultures. (B) M. tuberculosis Tet-Rel Mtb H80A and control strains were diluted and plated on <t>7H10</t> plates in the absence or presence of ATc. (C to G) M. smegmatis Δ rel Msm strains containing an episomal vector expressing the WT TetR that turns on expression of rel Mtb WT (Tet-Rel Mtb WT strain; designated the control) or rel Mtb H80A (Tet-Rel Mtb H80A strain; designated H80A here) from cassettes integrated at the attB site in the genome in the presence of ATc. (C and D) M. smegmatis strains were diluted and plated on 7H10 (C) and LB (D) plates in the absence or presence of ATc. (E) Graphic representation of the number of M. smegmatis CFU that grew in the presence compared to the absence of ATc when grown on LB, where all Tet-Rel Mtb H80A strain bacteria that grew in the presence of ATc on LB were suppressors and were no longer responsive to ATc treatment. Data are means ± SEM from 13 replicates. (F) ATP and GTP levels in M. smegmatis strains grown in the absence or presence of ATc for 6 h to an ODλ 600 of ∼0.6 in LB liquid media were measured by LC-MS/MS. The ratio of levels in the Tet-Rel Mtb H80A strain (H80A) to those in the Tet-Rel Mtb WT -expressing strain (control) under the same conditions is graphed. Data are the means ± SEM from 4 to 6 replicates. (E and F) The significance of differences was determined by calculating P values by Student's t tests; one asterisk indicates significance with a P value of
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    Rel Mtb -mediated (p)ppGpp hydrolysis is required for growth in vitro and maintenance of ATP and GTP levels when (p)ppGpp is being produced. (A and B) An M. tuberculosis Δ rel Mtb strain containing an episomal vector expressing the WT TetR that induces expression of rel Mtb H80A from a cassette integrated at the attB site in the genome in the presence of ATc (Tet-Rel Mtb H80A strain, designated H80A here) and a WT M. tuberculosis strain containing an empty vector in the attB site and transformed with the same TetR-expressing plasmid (control strain). (A) Transcript levels in exponential-growth-phase cultures in liquid 7H9 media. rel Mtb H80A transcripts from the cassette at the attB site were detected in the Tet-Rel Mtb H80A strain, and transcripts from the endogenous rel Mtb gene were detected in the control strain. The first comparison shows the ratio of transcript levels in the presence compared to the absence of ATc in each strain (+ATc/−ATc), and it illustrates the induction of rel Mtb H80A in the Tet-Rel Mtb H80A strain when exposed to ATc and the unresponsiveness of the rel Mtb gene to ATc in the control strain. The second comparison shows the ratio of transcript levels in the Tet-Rel Mtb H80A strain compared to the control strain (H80A/control) under each condition to illustrate the low level of transcription of rel Mtb H80A in the absence of ATc compared to that of induced cultures. (B) M. tuberculosis Tet-Rel Mtb H80A and control strains were diluted and plated on 7H10 plates in the absence or presence of ATc. (C to G) M. smegmatis Δ rel Msm strains containing an episomal vector expressing the WT TetR that turns on expression of rel Mtb WT (Tet-Rel Mtb WT strain; designated the control) or rel Mtb H80A (Tet-Rel Mtb H80A strain; designated H80A here) from cassettes integrated at the attB site in the genome in the presence of ATc. (C and D) M. smegmatis strains were diluted and plated on 7H10 (C) and LB (D) plates in the absence or presence of ATc. (E) Graphic representation of the number of M. smegmatis CFU that grew in the presence compared to the absence of ATc when grown on LB, where all Tet-Rel Mtb H80A strain bacteria that grew in the presence of ATc on LB were suppressors and were no longer responsive to ATc treatment. Data are means ± SEM from 13 replicates. (F) ATP and GTP levels in M. smegmatis strains grown in the absence or presence of ATc for 6 h to an ODλ 600 of ∼0.6 in LB liquid media were measured by LC-MS/MS. The ratio of levels in the Tet-Rel Mtb H80A strain (H80A) to those in the Tet-Rel Mtb WT -expressing strain (control) under the same conditions is graphed. Data are the means ± SEM from 4 to 6 replicates. (E and F) The significance of differences was determined by calculating P values by Student's t tests; one asterisk indicates significance with a P value of

    Journal: Journal of Bacteriology

    Article Title: Essential Roles for Mycobacterium tuberculosis Rel beyond the Production of (p)ppGpp

    doi: 10.1128/JB.00759-13

    Figure Lengend Snippet: Rel Mtb -mediated (p)ppGpp hydrolysis is required for growth in vitro and maintenance of ATP and GTP levels when (p)ppGpp is being produced. (A and B) An M. tuberculosis Δ rel Mtb strain containing an episomal vector expressing the WT TetR that induces expression of rel Mtb H80A from a cassette integrated at the attB site in the genome in the presence of ATc (Tet-Rel Mtb H80A strain, designated H80A here) and a WT M. tuberculosis strain containing an empty vector in the attB site and transformed with the same TetR-expressing plasmid (control strain). (A) Transcript levels in exponential-growth-phase cultures in liquid 7H9 media. rel Mtb H80A transcripts from the cassette at the attB site were detected in the Tet-Rel Mtb H80A strain, and transcripts from the endogenous rel Mtb gene were detected in the control strain. The first comparison shows the ratio of transcript levels in the presence compared to the absence of ATc in each strain (+ATc/−ATc), and it illustrates the induction of rel Mtb H80A in the Tet-Rel Mtb H80A strain when exposed to ATc and the unresponsiveness of the rel Mtb gene to ATc in the control strain. The second comparison shows the ratio of transcript levels in the Tet-Rel Mtb H80A strain compared to the control strain (H80A/control) under each condition to illustrate the low level of transcription of rel Mtb H80A in the absence of ATc compared to that of induced cultures. (B) M. tuberculosis Tet-Rel Mtb H80A and control strains were diluted and plated on 7H10 plates in the absence or presence of ATc. (C to G) M. smegmatis Δ rel Msm strains containing an episomal vector expressing the WT TetR that turns on expression of rel Mtb WT (Tet-Rel Mtb WT strain; designated the control) or rel Mtb H80A (Tet-Rel Mtb H80A strain; designated H80A here) from cassettes integrated at the attB site in the genome in the presence of ATc. (C and D) M. smegmatis strains were diluted and plated on 7H10 (C) and LB (D) plates in the absence or presence of ATc. (E) Graphic representation of the number of M. smegmatis CFU that grew in the presence compared to the absence of ATc when grown on LB, where all Tet-Rel Mtb H80A strain bacteria that grew in the presence of ATc on LB were suppressors and were no longer responsive to ATc treatment. Data are means ± SEM from 13 replicates. (F) ATP and GTP levels in M. smegmatis strains grown in the absence or presence of ATc for 6 h to an ODλ 600 of ∼0.6 in LB liquid media were measured by LC-MS/MS. The ratio of levels in the Tet-Rel Mtb H80A strain (H80A) to those in the Tet-Rel Mtb WT -expressing strain (control) under the same conditions is graphed. Data are the means ± SEM from 4 to 6 replicates. (E and F) The significance of differences was determined by calculating P values by Student's t tests; one asterisk indicates significance with a P value of

    Article Snippet: Bacterial burden was determined by plating serial dilutions of lung and spleen homogenates onto 7H10 agar plates in the absence or presence of kanamycin and streptomycin.

    Techniques: In Vitro, Produced, Plasmid Preparation, Expressing, Transformation Assay, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry

    Rv1366 is not necessary for M. tuberculosis growth or biofilm formation in vitro . (A) Southern blot analysis of WT M. tuberculosis Erdman (lane 1) and the Δ rv1366 strain (lanes 2 to 4) using EcoRI-digested genomic DNA and a probe that spans nucleotides 1537706 to 1538406 of the M. tuberculosis genome. EcoRI digestion of WT M. tuberculosis yields a band at 5,083 bp. EcoRI digestion of Δ rv1366 results in a 2,226-bp band due to the replacement of the rv1366 gene with a hygromycin resistance cassette. (B) Southern blot analysis of WT M. tuberculosis Erdman (lane 1) and the Δ rel Mtb Δ rv1366 strain (lanes 2 to 6) using SmaI-digested genomic DNA and a probe that spans nucleotides 2910191 to 2910844 of the M. tuberculosis genome. SmaI digestion of WT M. tuberculosis yields a band at 2,257 bp. SmaI digestion of the Δ rel Mtb Δ rv1366 strain results in a 4,769-bp band due to the replacement of the rel Mtb gene with a hygromycin resistance cassette. (C and D) M. tuberculosis Δ rel Mtb , Δ rv1366 , and Δ rel Mtb Δ rv1366 strains containing a vector that constitutively expresses Rel Mtb WT or Rv1366 or containing an empty vector (−) integrated at the attB site in the genome were normalized to the same ODλ 600 and either diluted and plated on 7H10 plates (C) or grown in Sauton's media in static, biofilm-forming conditions in a 24-well dish (D). (C) 7H10 plates were incubated for 3 weeks at 37°C. (D) Biofilms were incubated for 4 weeks at 37°C. The parent strain used is labeled above each photo, and the protein expressed from the attB site is labeled below the photo.

    Journal: Journal of Bacteriology

    Article Title: Essential Roles for Mycobacterium tuberculosis Rel beyond the Production of (p)ppGpp

    doi: 10.1128/JB.00759-13

    Figure Lengend Snippet: Rv1366 is not necessary for M. tuberculosis growth or biofilm formation in vitro . (A) Southern blot analysis of WT M. tuberculosis Erdman (lane 1) and the Δ rv1366 strain (lanes 2 to 4) using EcoRI-digested genomic DNA and a probe that spans nucleotides 1537706 to 1538406 of the M. tuberculosis genome. EcoRI digestion of WT M. tuberculosis yields a band at 5,083 bp. EcoRI digestion of Δ rv1366 results in a 2,226-bp band due to the replacement of the rv1366 gene with a hygromycin resistance cassette. (B) Southern blot analysis of WT M. tuberculosis Erdman (lane 1) and the Δ rel Mtb Δ rv1366 strain (lanes 2 to 6) using SmaI-digested genomic DNA and a probe that spans nucleotides 2910191 to 2910844 of the M. tuberculosis genome. SmaI digestion of WT M. tuberculosis yields a band at 2,257 bp. SmaI digestion of the Δ rel Mtb Δ rv1366 strain results in a 4,769-bp band due to the replacement of the rel Mtb gene with a hygromycin resistance cassette. (C and D) M. tuberculosis Δ rel Mtb , Δ rv1366 , and Δ rel Mtb Δ rv1366 strains containing a vector that constitutively expresses Rel Mtb WT or Rv1366 or containing an empty vector (−) integrated at the attB site in the genome were normalized to the same ODλ 600 and either diluted and plated on 7H10 plates (C) or grown in Sauton's media in static, biofilm-forming conditions in a 24-well dish (D). (C) 7H10 plates were incubated for 3 weeks at 37°C. (D) Biofilms were incubated for 4 weeks at 37°C. The parent strain used is labeled above each photo, and the protein expressed from the attB site is labeled below the photo.

    Article Snippet: Bacterial burden was determined by plating serial dilutions of lung and spleen homogenates onto 7H10 agar plates in the absence or presence of kanamycin and streptomycin.

    Techniques: In Vitro, Southern Blot, Plasmid Preparation, Incubation, Labeling

    Rel Mtb -mediated (p)ppGpp synthesis is necessary for optimal M. tuberculosis growth and biofilm formation in vitro. M. tuberculosis Δ rel Mtb strains containing a vector that constitutively expresses Rel Mtb WT (WT) or Rel Mtb H344Y (H344Y) or containing an empty vector (−) integrated at the attB site in the genome were normalized to the same ODλ 600 and either diluted and plated on 7H10 plates (A) or grown in Sauton's medium in static, biofilm-forming conditions in a 24-well dish (B). (A) 7H10 plates were incubated for 3 weeks at 37°C. (B) Biofilms were incubated for 4 weeks at 37°C. The parent strain used is labeled above each photo, and the protein expressed from the attB site is labeled below the photo.

    Journal: Journal of Bacteriology

    Article Title: Essential Roles for Mycobacterium tuberculosis Rel beyond the Production of (p)ppGpp

    doi: 10.1128/JB.00759-13

    Figure Lengend Snippet: Rel Mtb -mediated (p)ppGpp synthesis is necessary for optimal M. tuberculosis growth and biofilm formation in vitro. M. tuberculosis Δ rel Mtb strains containing a vector that constitutively expresses Rel Mtb WT (WT) or Rel Mtb H344Y (H344Y) or containing an empty vector (−) integrated at the attB site in the genome were normalized to the same ODλ 600 and either diluted and plated on 7H10 plates (A) or grown in Sauton's medium in static, biofilm-forming conditions in a 24-well dish (B). (A) 7H10 plates were incubated for 3 weeks at 37°C. (B) Biofilms were incubated for 4 weeks at 37°C. The parent strain used is labeled above each photo, and the protein expressed from the attB site is labeled below the photo.

    Article Snippet: Bacterial burden was determined by plating serial dilutions of lung and spleen homogenates onto 7H10 agar plates in the absence or presence of kanamycin and streptomycin.

    Techniques: In Vitro, Plasmid Preparation, Incubation, Labeling

    Rel Mtb (p)ppGpp hydrolase activity is essential for acute and chronic M. tuberculosis infection of mice. Bacterial titers in the lungs (A, E, and G) and spleens (B and H) of C57BL/6 mice infected with either control or Tet-Rel Mtb H80A (designated H80A in the figure) M. tuberculosis strains. The parent strain for the control is WT M. tuberculosis Erdman, and the parent strain for Tet-Rel Mtb H80A is M. tuberculosis Δ rel Mtb . Both strains express TetR from a plasmid that confers streptomycin resistance and contain a kanamycin resistance cassette integrated into the attB site that, in the case of Tet-Rel Mtb H80A , carries the rel Mtb H80A allele. The strain symbols in panel A are the same for all panels. (A to C) Mice were given normal mouse chow throughout infection. (E and F) Mice were administered doxycycline-containing mouse chow starting at day 1 postinfection (designated by the arrow). (G to I) Mice were administered doxycycline-containing mouse chow starting at day 63 postinfection (designated by the arrow). (C, F, and I) The ratio of CFU from the lungs or spleens grown on 7H10 plates containing streptomycin and kanamycin compared to 7H10 containing no antibiotics (No AB). ND denotes when no colonies were recovered after plating 5% of the lung homogenate (limit of detection, 20 CFU). Data are means ± SEM of 6 Tet-Rel Mtb H80A strain-infected mice and 3 control strain-infected mice per time point from two replicate experiments. (E, G, and H) The significance of differences were determined by calculating P values by Student's t tests; two asterisks indicate significance with a P value of

    Journal: Journal of Bacteriology

    Article Title: Essential Roles for Mycobacterium tuberculosis Rel beyond the Production of (p)ppGpp

    doi: 10.1128/JB.00759-13

    Figure Lengend Snippet: Rel Mtb (p)ppGpp hydrolase activity is essential for acute and chronic M. tuberculosis infection of mice. Bacterial titers in the lungs (A, E, and G) and spleens (B and H) of C57BL/6 mice infected with either control or Tet-Rel Mtb H80A (designated H80A in the figure) M. tuberculosis strains. The parent strain for the control is WT M. tuberculosis Erdman, and the parent strain for Tet-Rel Mtb H80A is M. tuberculosis Δ rel Mtb . Both strains express TetR from a plasmid that confers streptomycin resistance and contain a kanamycin resistance cassette integrated into the attB site that, in the case of Tet-Rel Mtb H80A , carries the rel Mtb H80A allele. The strain symbols in panel A are the same for all panels. (A to C) Mice were given normal mouse chow throughout infection. (E and F) Mice were administered doxycycline-containing mouse chow starting at day 1 postinfection (designated by the arrow). (G to I) Mice were administered doxycycline-containing mouse chow starting at day 63 postinfection (designated by the arrow). (C, F, and I) The ratio of CFU from the lungs or spleens grown on 7H10 plates containing streptomycin and kanamycin compared to 7H10 containing no antibiotics (No AB). ND denotes when no colonies were recovered after plating 5% of the lung homogenate (limit of detection, 20 CFU). Data are means ± SEM of 6 Tet-Rel Mtb H80A strain-infected mice and 3 control strain-infected mice per time point from two replicate experiments. (E, G, and H) The significance of differences were determined by calculating P values by Student's t tests; two asterisks indicate significance with a P value of

    Article Snippet: Bacterial burden was determined by plating serial dilutions of lung and spleen homogenates onto 7H10 agar plates in the absence or presence of kanamycin and streptomycin.

    Techniques: Activity Assay, Infection, Mouse Assay, Plasmid Preparation

    Course of M. tuberculosis aerosol challenge in the lungs of memory and naive mice. Memory immune (●) and naive (□) mice were infected with ∼60 M. tuberculosis bacilli via aerosol. The numbers of viable bacilli were determined by plating serial dilutions of lung homogenates onto 7H10 plates and counting the colonies after 3 weeks at 37°C. Each time point represents four mice, and the experiment was repeated once. The standard error bars are too small to see on this graph. ∗, P ≤ 0.001, comparing naive and memory mice at each time point.

    Journal: Infection and Immunity

    Article Title: CD8+ T Cells Participate in the Memory Immune Response to Mycobacterium tuberculosis

    doi: 10.1128/IAI.69.7.4320-4328.2001

    Figure Lengend Snippet: Course of M. tuberculosis aerosol challenge in the lungs of memory and naive mice. Memory immune (●) and naive (□) mice were infected with ∼60 M. tuberculosis bacilli via aerosol. The numbers of viable bacilli were determined by plating serial dilutions of lung homogenates onto 7H10 plates and counting the colonies after 3 weeks at 37°C. Each time point represents four mice, and the experiment was repeated once. The standard error bars are too small to see on this graph. ∗, P ≤ 0.001, comparing naive and memory mice at each time point.

    Article Snippet: Mice were infected intravenously (i.v.) via tail vein with 2 × 105 live bacilli in 100 μl or by aerosol with approximately 100 live bacilli as determined by viable counts on 7H10 agar plates (Difco Laboratories, Detroit, Mich.).

    Techniques: Mouse Assay, Infection

    Strategy for identification of in vivo induced exported proteins. (a) Identification of in vivo induced exported proteins. Spleens from β-lactam-treated mice infected with the EXIT library were harvested after 2 weeks of infection. Spleen homogenates were plated in parallel on 7H10 agar without β-lactam to recover all clones (red Venn diagram) and on 7H10 agar containing β-lactam to recover clones exporting ‘BlaTEM fusion proteins during in vivo growth and in vitro growth (purple Venn diagram). The population of clones identified only or in significantly greater abundance on media lacking β-lactams represents proteins whose export was induced during infection (blue). (b) Sequenced read count values recovered from agar with or without β-lactam for the 593 EXIT proteins were plotted to compare abundances after β-lactam treatment in vivo , with the abundance after dual β-lactam treatment in vivo and in vitro indicated. The majority of proteins identified as exported in vivo remained highly abundant after additional β-lactam treatment in vitro (black). A total of 38 genes (highlighted in red) were identified as statistically less abundant after in vitro β-lactam selection, representing proteins exported significantly more in vivo than in vitro (see Materials and Methods for details on statistical analysis). (c) In vivo induced exported proteins with roles promoting growth in macrophages ( rv1508 :: tn , rv3707c:tn , rv0559c :: tn , and rv2536 :: tn ). Murine bone marrow-derived macrophages were infected with M. tuberculosis CDC1551 transposon mutants lacking individual in vivo induced exported proteins. At specific times postinfection, macrophage lysates were plated to measure intracellular CFU. The fold change in CFU over the course of the infection is plotted relative to the bacterial burden at day 0 postinfection. Statistical significance was determined by one-way analysis of variance (ANOVA) with multiple comparisons performed by the use of the Holm-Sidak (normal by Shapiro-Wilk) or Student-Newman-Keuls (nonnormal) test (*, P

    Journal: mBio

    Article Title: The EXIT Strategy: an Approach for Identifying Bacterial Proteins Exported during Host Infection

    doi: 10.1128/mBio.00333-17

    Figure Lengend Snippet: Strategy for identification of in vivo induced exported proteins. (a) Identification of in vivo induced exported proteins. Spleens from β-lactam-treated mice infected with the EXIT library were harvested after 2 weeks of infection. Spleen homogenates were plated in parallel on 7H10 agar without β-lactam to recover all clones (red Venn diagram) and on 7H10 agar containing β-lactam to recover clones exporting ‘BlaTEM fusion proteins during in vivo growth and in vitro growth (purple Venn diagram). The population of clones identified only or in significantly greater abundance on media lacking β-lactams represents proteins whose export was induced during infection (blue). (b) Sequenced read count values recovered from agar with or without β-lactam for the 593 EXIT proteins were plotted to compare abundances after β-lactam treatment in vivo , with the abundance after dual β-lactam treatment in vivo and in vitro indicated. The majority of proteins identified as exported in vivo remained highly abundant after additional β-lactam treatment in vitro (black). A total of 38 genes (highlighted in red) were identified as statistically less abundant after in vitro β-lactam selection, representing proteins exported significantly more in vivo than in vitro (see Materials and Methods for details on statistical analysis). (c) In vivo induced exported proteins with roles promoting growth in macrophages ( rv1508 :: tn , rv3707c:tn , rv0559c :: tn , and rv2536 :: tn ). Murine bone marrow-derived macrophages were infected with M. tuberculosis CDC1551 transposon mutants lacking individual in vivo induced exported proteins. At specific times postinfection, macrophage lysates were plated to measure intracellular CFU. The fold change in CFU over the course of the infection is plotted relative to the bacterial burden at day 0 postinfection. Statistical significance was determined by one-way analysis of variance (ANOVA) with multiple comparisons performed by the use of the Holm-Sidak (normal by Shapiro-Wilk) or Student-Newman-Keuls (nonnormal) test (*, P

    Article Snippet: M. tuberculosis strains were grown with Middlebrook 7H9 broth or 7H10 agar (Difco) supplemented with 1× albumin dextrose saline (ADS), 0.5% glycerol, and 0.05% Tween 80 (7AGT) ( ).

    Techniques: In Vivo, Mouse Assay, Infection, Clone Assay, In Vitro, Selection, Derivative Assay

    (a) The ‘BlaTEM reporter. The ‘BlaTEM reporter is compatible with proteins localized to the bacterial cytoplasmic membrane or cell wall or secreted from the bacterial cell. The right panel indicates in-frame fusions to categories of exported proteins that confer β-lactam resistance (red). In-frame fusions to cytoplasmic proteins or the cytoplasmic domain of integral membrane proteins (purple) do not confer β-lactam resistance. (b) EXIT strategy. In step 1, a comprehensive library of 5 × 10 6 plasmids containing fragments of M. tuberculosis (Mtb) genomic DNA fused to the ‘ bla TEM reporter was constructed. The plasmid library was transformed into the ΔblaC β-lactamase-sensitive mutant of M. tuberculosis , and 5 × 10 6 transformants were pooled to generate the EXIT library. In step 2, mice were infected by intravenous injection with the EXIT library and treated with β-lactam antibiotics (oral gavage twice daily) to select for EXIT clones exporting ‘BlaTEM fusion proteins. β-lactam treatment began 1 day after infection and continued to 2 weeks after infection. Mice were sacrificed, and spleens and lungs were harvested and homogenized. In step 3, organ homogenates were plated on 7H10 agar and grown to recover M. tuberculosis clones that survived β-lactam treatment during infection. Plates were scraped, and colonies were pooled separately for lungs and spleens. In step 4, plasmids from the recovered bacteria and the input samples were isolated and the fusion junction was sequenced using next-generation sequencing. Sequencing primers were designed to read out of the ‘ bla TEM reporter and sequence the immediately adjacent M. tuberculosis DNA. Sequences were aligned to the M. tuberculosis genome. Unique sequences were counted to identify the abundance of each fusion junction site within the population. The genes that were most highly abundant after in vivo β-lactam treatment were identified, and the results corresponded to plasmids producing in-frame exported ‘BlaTEM fusion proteins.

    Journal: mBio

    Article Title: The EXIT Strategy: an Approach for Identifying Bacterial Proteins Exported during Host Infection

    doi: 10.1128/mBio.00333-17

    Figure Lengend Snippet: (a) The ‘BlaTEM reporter. The ‘BlaTEM reporter is compatible with proteins localized to the bacterial cytoplasmic membrane or cell wall or secreted from the bacterial cell. The right panel indicates in-frame fusions to categories of exported proteins that confer β-lactam resistance (red). In-frame fusions to cytoplasmic proteins or the cytoplasmic domain of integral membrane proteins (purple) do not confer β-lactam resistance. (b) EXIT strategy. In step 1, a comprehensive library of 5 × 10 6 plasmids containing fragments of M. tuberculosis (Mtb) genomic DNA fused to the ‘ bla TEM reporter was constructed. The plasmid library was transformed into the ΔblaC β-lactamase-sensitive mutant of M. tuberculosis , and 5 × 10 6 transformants were pooled to generate the EXIT library. In step 2, mice were infected by intravenous injection with the EXIT library and treated with β-lactam antibiotics (oral gavage twice daily) to select for EXIT clones exporting ‘BlaTEM fusion proteins. β-lactam treatment began 1 day after infection and continued to 2 weeks after infection. Mice were sacrificed, and spleens and lungs were harvested and homogenized. In step 3, organ homogenates were plated on 7H10 agar and grown to recover M. tuberculosis clones that survived β-lactam treatment during infection. Plates were scraped, and colonies were pooled separately for lungs and spleens. In step 4, plasmids from the recovered bacteria and the input samples were isolated and the fusion junction was sequenced using next-generation sequencing. Sequencing primers were designed to read out of the ‘ bla TEM reporter and sequence the immediately adjacent M. tuberculosis DNA. Sequences were aligned to the M. tuberculosis genome. Unique sequences were counted to identify the abundance of each fusion junction site within the population. The genes that were most highly abundant after in vivo β-lactam treatment were identified, and the results corresponded to plasmids producing in-frame exported ‘BlaTEM fusion proteins.

    Article Snippet: M. tuberculosis strains were grown with Middlebrook 7H9 broth or 7H10 agar (Difco) supplemented with 1× albumin dextrose saline (ADS), 0.5% glycerol, and 0.05% Tween 80 (7AGT) ( ).

    Techniques: Transmission Electron Microscopy, Construct, Plasmid Preparation, Transformation Assay, Mutagenesis, Mouse Assay, Infection, Injection, Clone Assay, Isolation, Next-Generation Sequencing, Sequencing, In Vivo

    Sensitivity to oxidative stress. Shown are the diameters of the zones of growth inhibition produced in the presence of 600 mM hydrogen peroxide (A) and 5 mM plumbagin (B). Wild-type H37Rv, ST22 ( ideR :: aph ), and ST52 ( ideR complemented) were grown in 7H9 medium and plated in 7H10 medium. The values represent means plus standard deviations (the experiments were performed in triplicate).

    Journal: Infection and Immunity

    Article Title: ideR, an Essential Gene in Mycobacterium tuberculosis: Role of IdeR in Iron-Dependent Gene Expression, Iron Metabolism, and Oxidative Stress Response †

    doi: 10.1128/IAI.70.7.3371-3381.2002

    Figure Lengend Snippet: Sensitivity to oxidative stress. Shown are the diameters of the zones of growth inhibition produced in the presence of 600 mM hydrogen peroxide (A) and 5 mM plumbagin (B). Wild-type H37Rv, ST22 ( ideR :: aph ), and ST52 ( ideR complemented) were grown in 7H9 medium and plated in 7H10 medium. The values represent means plus standard deviations (the experiments were performed in triplicate).

    Article Snippet: M. tuberculosis strain H37Rv (American Type Culture Collection) was maintained in Middlebrook 7H9 broth or on 7H10 agar (Difco) supplemented with 0.2% glycerol, 0.05% Tween 80, and 10% albumin-dextrose-NaCl complex (ADN) ( ).

    Techniques: Inhibition, Produced