wild type erdman m tuberculosis  (ATCC)

Journal:

Article Title: Glutamine Synthetase GlnA1 Is Essential for Growth of Mycobacterium tuberculosis in Human THP-1 Macrophages and Guinea Pigs

doi: 10.1128/IAI.71.7.3927-3936.2003

Figure Lengend Snippet: Bacterial strains and plasmids

Article Snippet: Hygromycin (50 μg ml −1 ) and/or kanamycin (20 or 50 μg ml −1 ) were included as appropriate. l -Glutamine was sterilized by filtration and added aseptically to broth and agar after autoclaving when required. table ft1 table-wrap mode="anchored" t5 TABLE 1. caption a7 Strain or plasmid Description Reference or source Strains E. coli DH5α Gibco BRL E. coli XL10-Gold Stratagene M. tuberculosis a Wild-type Erdman strain ATCC 35801 M. tuberculosis pNBV1 39 M. tuberculosis glnA1 Insertionally inactivated glnA1 locus; Km r This study M. tuberculosis glnA1 pNBV1 This study M. tuberculosis glnA1 pNBV1- Mtb GS This study M. tuberculosis glnA1 pNBV1- St GS This study Plasmids pUC19 E. coli cloning vector 42 pPR27 Mycobacterial allelic exchange vector; ori (ts) sacB Gm r 31 pEX1 Mycobacterial allelic exchange vector derived from pPR27 and pGFPuv; ori (ts) sacB Hyg r GFP This study pNBV1 E. coli mycobacterial shuttle vector; Hyg r 20 pNBV1- Mtb GS M. tuberculosis glnA1 expression vector 39 pNBV1- St GS S. enterica serovar Typhimurium glnA with M. tuberculosis glnA1 promoter 39 pGFPuv UV-optimized Aequorea victoria GFP gene Clontech Open in a separate window a All M. tuberculosis strains are derived from the Erdman wild-type strain.

Techniques: Plasmid Preparation, Clone Assay, Derivative Assay, Expressing

Journal:

Article Title: Glutamine Synthetase GlnA1 Is Essential for Growth of Mycobacterium tuberculosis in Human THP-1 Macrophages and Guinea Pigs

doi: 10.1128/IAI.71.7.3927-3936.2003

Figure Lengend Snippet: Allelic exchange vector, pEX1, used in the construction of the glnA1 mutant. Unique restriction sites are shown.

Article Snippet: Hygromycin (50 μg ml −1 ) and/or kanamycin (20 or 50 μg ml −1 ) were included as appropriate. l -Glutamine was sterilized by filtration and added aseptically to broth and agar after autoclaving when required. table ft1 table-wrap mode="anchored" t5 TABLE 1. caption a7 Strain or plasmid Description Reference or source Strains E. coli DH5α Gibco BRL E. coli XL10-Gold Stratagene M. tuberculosis a Wild-type Erdman strain ATCC 35801 M. tuberculosis pNBV1 39 M. tuberculosis glnA1 Insertionally inactivated glnA1 locus; Km r This study M. tuberculosis glnA1 pNBV1 This study M. tuberculosis glnA1 pNBV1- Mtb GS This study M. tuberculosis glnA1 pNBV1- St GS This study Plasmids pUC19 E. coli cloning vector 42 pPR27 Mycobacterial allelic exchange vector; ori (ts) sacB Gm r 31 pEX1 Mycobacterial allelic exchange vector derived from pPR27 and pGFPuv; ori (ts) sacB Hyg r GFP This study pNBV1 E. coli mycobacterial shuttle vector; Hyg r 20 pNBV1- Mtb GS M. tuberculosis glnA1 expression vector 39 pNBV1- St GS S. enterica serovar Typhimurium glnA with M. tuberculosis glnA1 promoter 39 pGFPuv UV-optimized Aequorea victoria GFP gene Clontech Open in a separate window a All M. tuberculosis strains are derived from the Erdman wild-type strain.

Techniques: Plasmid Preparation, Mutagenesis

Journal:

Article Title: Glutamine Synthetase GlnA1 Is Essential for Growth of Mycobacterium tuberculosis in Human THP-1 Macrophages and Guinea Pigs

doi: 10.1128/IAI.71.7.3927-3936.2003

Figure Lengend Snippet: Construction of the M. tuberculosis glnA1 mutant. (A) Maps of the wild-type glnA1 locus and the disrupted allele, which contains a Kmr cassette (aphA-2) inserted into the unique EcoNI site in the middle of the glnA1 coding region. Only a small portion of the 3′ end of glnE is present on the SmaI fragment. (B) Genomic DNA from the M. tuberculosis wild-type strain and the glnA1 mutant was digested to completion with SmaI and probed with a 1.8-kb fragment containing glnA1 (hatched bar in panel A). M, molecular mass markers in kilobases; wt, wild-type.

Article Snippet: Hygromycin (50 μg ml −1 ) and/or kanamycin (20 or 50 μg ml −1 ) were included as appropriate. l -Glutamine was sterilized by filtration and added aseptically to broth and agar after autoclaving when required. table ft1 table-wrap mode="anchored" t5 TABLE 1. caption a7 Strain or plasmid Description Reference or source Strains E. coli DH5α Gibco BRL E. coli XL10-Gold Stratagene M. tuberculosis a Wild-type Erdman strain ATCC 35801 M. tuberculosis pNBV1 39 M. tuberculosis glnA1 Insertionally inactivated glnA1 locus; Km r This study M. tuberculosis glnA1 pNBV1 This study M. tuberculosis glnA1 pNBV1- Mtb GS This study M. tuberculosis glnA1 pNBV1- St GS This study Plasmids pUC19 E. coli cloning vector 42 pPR27 Mycobacterial allelic exchange vector; ori (ts) sacB Gm r 31 pEX1 Mycobacterial allelic exchange vector derived from pPR27 and pGFPuv; ori (ts) sacB Hyg r GFP This study pNBV1 E. coli mycobacterial shuttle vector; Hyg r 20 pNBV1- Mtb GS M. tuberculosis glnA1 expression vector 39 pNBV1- St GS S. enterica serovar Typhimurium glnA with M. tuberculosis glnA1 promoter 39 pGFPuv UV-optimized Aequorea victoria GFP gene Clontech Open in a separate window a All M. tuberculosis strains are derived from the Erdman wild-type strain.

Techniques: Mutagenesis

Journal:

Article Title: Glutamine Synthetase GlnA1 Is Essential for Growth of Mycobacterium tuberculosis in Human THP-1 Macrophages and Guinea Pigs

doi: 10.1128/IAI.71.7.3927-3936.2003

Figure Lengend Snippet: Intracellular growth of M. tuberculosis wild-type, glnA1, and complemented strains in human THP-1 macrophages. The tissue culture medium included the standard amount of l-glutamine (2 mM) (A) or 0.2 mM l-glutamine (B). When THP-1 cells were cultured in the presence of 2 mM l-glutamine, the glnA1 mutant multiplied intracellularly but at a reduced rate compared with the wild-type strain. When THP-1 cells were cultured in the presence of only 0.2 mM l-glutamine, the strain did not multiply and slowly died (∼0.5 log reduction in 6 days). The wild-type strain and both complemented strains grew normally in the presence of 0.2 mM l-glutamine. Data are the means ± standard errors for three wells per time point. In many instances, the error bars are smaller than the symbols. For all measurements, the standard error was <2% of the mean. The experiment was repeated once with similar results. Mtb, M. tuberculosis.

Article Snippet: Hygromycin (50 μg ml −1 ) and/or kanamycin (20 or 50 μg ml −1 ) were included as appropriate. l -Glutamine was sterilized by filtration and added aseptically to broth and agar after autoclaving when required. table ft1 table-wrap mode="anchored" t5 TABLE 1. caption a7 Strain or plasmid Description Reference or source Strains E. coli DH5α Gibco BRL E. coli XL10-Gold Stratagene M. tuberculosis a Wild-type Erdman strain ATCC 35801 M. tuberculosis pNBV1 39 M. tuberculosis glnA1 Insertionally inactivated glnA1 locus; Km r This study M. tuberculosis glnA1 pNBV1 This study M. tuberculosis glnA1 pNBV1- Mtb GS This study M. tuberculosis glnA1 pNBV1- St GS This study Plasmids pUC19 E. coli cloning vector 42 pPR27 Mycobacterial allelic exchange vector; ori (ts) sacB Gm r 31 pEX1 Mycobacterial allelic exchange vector derived from pPR27 and pGFPuv; ori (ts) sacB Hyg r GFP This study pNBV1 E. coli mycobacterial shuttle vector; Hyg r 20 pNBV1- Mtb GS M. tuberculosis glnA1 expression vector 39 pNBV1- St GS S. enterica serovar Typhimurium glnA with M. tuberculosis glnA1 promoter 39 pGFPuv UV-optimized Aequorea victoria GFP gene Clontech Open in a separate window a All M. tuberculosis strains are derived from the Erdman wild-type strain.

Techniques: Cell Culture, Mutagenesis

Journal:

Article Title: Glutamine Synthetase GlnA1 Is Essential for Growth of Mycobacterium tuberculosis in Human THP-1 Macrophages and Guinea Pigs

doi: 10.1128/IAI.71.7.3927-3936.2003

Figure Lengend Snippet: Glutamine requirement of the M. tuberculosis glnA1 mutant during intracellular growth in human THP-1 macrophages. The concentration of l-glutamine in the tissue culture medium was varied from 0.2 to 10 mM. At the highest concentration, the mutant grew at a rate similar to that of the wild-type strain. Data are the means ± standard errors for three wells per time point. In many instances, the error bars are smaller than the symbols. For all measurements, the standard error was <2% of the mean. The experiment was repeated once with similar results. Mtb, M. tuberculosis.

Article Snippet: Hygromycin (50 μg ml −1 ) and/or kanamycin (20 or 50 μg ml −1 ) were included as appropriate. l -Glutamine was sterilized by filtration and added aseptically to broth and agar after autoclaving when required. table ft1 table-wrap mode="anchored" t5 TABLE 1. caption a7 Strain or plasmid Description Reference or source Strains E. coli DH5α Gibco BRL E. coli XL10-Gold Stratagene M. tuberculosis a Wild-type Erdman strain ATCC 35801 M. tuberculosis pNBV1 39 M. tuberculosis glnA1 Insertionally inactivated glnA1 locus; Km r This study M. tuberculosis glnA1 pNBV1 This study M. tuberculosis glnA1 pNBV1- Mtb GS This study M. tuberculosis glnA1 pNBV1- St GS This study Plasmids pUC19 E. coli cloning vector 42 pPR27 Mycobacterial allelic exchange vector; ori (ts) sacB Gm r 31 pEX1 Mycobacterial allelic exchange vector derived from pPR27 and pGFPuv; ori (ts) sacB Hyg r GFP This study pNBV1 E. coli mycobacterial shuttle vector; Hyg r 20 pNBV1- Mtb GS M. tuberculosis glnA1 expression vector 39 pNBV1- St GS S. enterica serovar Typhimurium glnA with M. tuberculosis glnA1 promoter 39 pGFPuv UV-optimized Aequorea victoria GFP gene Clontech Open in a separate window a All M. tuberculosis strains are derived from the Erdman wild-type strain.

Techniques: Mutagenesis, Concentration Assay

Journal:

Article Title: Glutamine Synthetase GlnA1 Is Essential for Growth of Mycobacterium tuberculosis in Human THP-1 Macrophages and Guinea Pigs

doi: 10.1128/IAI.71.7.3927-3936.2003

Figure Lengend Snippet: Glutamine requirement of the M. tuberculosis glnA1 mutant in broth culture. Cultures of the glnA1 strain (A and B) and the wild-type strain (C) were inoculated into 7H9-OADC-TW containing various concentrations of l-glutamine (indicated next to the corresponding lines on the graphs) to an initial calculated A550 of 0.0001. Growth was monitored by assaying absorbance (A and C) and CFU (B). Data are the means ± standard errors for duplicate or triplicate cultures. In many instances, the error bars are smaller than the symbols. For all measurements, the standard error was <14% of the mean. The limits of detection were 0.02 absorbance units (A and C) and 1.22 log CFU/ml (16 CFU/ml) (B), as indicated by the dashed lines (measurements below the detection limit were scored as equal to the detection limit). The experiment was repeated once with similar results (only the 0 and 0.2 mM l-glutamine cultures were plated for CFU in the second experiment). Growth of the wild-type strain was unaffected by the l-glutamine concentration. Mtb, M. tuberculosis.

Article Snippet: Hygromycin (50 μg ml −1 ) and/or kanamycin (20 or 50 μg ml −1 ) were included as appropriate. l -Glutamine was sterilized by filtration and added aseptically to broth and agar after autoclaving when required. table ft1 table-wrap mode="anchored" t5 TABLE 1. caption a7 Strain or plasmid Description Reference or source Strains E. coli DH5α Gibco BRL E. coli XL10-Gold Stratagene M. tuberculosis a Wild-type Erdman strain ATCC 35801 M. tuberculosis pNBV1 39 M. tuberculosis glnA1 Insertionally inactivated glnA1 locus; Km r This study M. tuberculosis glnA1 pNBV1 This study M. tuberculosis glnA1 pNBV1- Mtb GS This study M. tuberculosis glnA1 pNBV1- St GS This study Plasmids pUC19 E. coli cloning vector 42 pPR27 Mycobacterial allelic exchange vector; ori (ts) sacB Gm r 31 pEX1 Mycobacterial allelic exchange vector derived from pPR27 and pGFPuv; ori (ts) sacB Hyg r GFP This study pNBV1 E. coli mycobacterial shuttle vector; Hyg r 20 pNBV1- Mtb GS M. tuberculosis glnA1 expression vector 39 pNBV1- St GS S. enterica serovar Typhimurium glnA with M. tuberculosis glnA1 promoter 39 pGFPuv UV-optimized Aequorea victoria GFP gene Clontech Open in a separate window a All M. tuberculosis strains are derived from the Erdman wild-type strain.

Techniques: Mutagenesis, Concentration Assay

Journal:

Article Title: Glutamine Synthetase GlnA1 Is Essential for Growth of Mycobacterium tuberculosis in Human THP-1 Macrophages and Guinea Pigs

doi: 10.1128/IAI.71.7.3927-3936.2003

Figure Lengend Snippet: Growth rates of the M. tuberculosis glnA1 mutant in broth culture and in human THP-1 macrophages

Article Snippet: Hygromycin (50 μg ml −1 ) and/or kanamycin (20 or 50 μg ml −1 ) were included as appropriate. l -Glutamine was sterilized by filtration and added aseptically to broth and agar after autoclaving when required. table ft1 table-wrap mode="anchored" t5 TABLE 1. caption a7 Strain or plasmid Description Reference or source Strains E. coli DH5α Gibco BRL E. coli XL10-Gold Stratagene M. tuberculosis a Wild-type Erdman strain ATCC 35801 M. tuberculosis pNBV1 39 M. tuberculosis glnA1 Insertionally inactivated glnA1 locus; Km r This study M. tuberculosis glnA1 pNBV1 This study M. tuberculosis glnA1 pNBV1- Mtb GS This study M. tuberculosis glnA1 pNBV1- St GS This study Plasmids pUC19 E. coli cloning vector 42 pPR27 Mycobacterial allelic exchange vector; ori (ts) sacB Gm r 31 pEX1 Mycobacterial allelic exchange vector derived from pPR27 and pGFPuv; ori (ts) sacB Hyg r GFP This study pNBV1 E. coli mycobacterial shuttle vector; Hyg r 20 pNBV1- Mtb GS M. tuberculosis glnA1 expression vector 39 pNBV1- St GS S. enterica serovar Typhimurium glnA with M. tuberculosis glnA1 promoter 39 pGFPuv UV-optimized Aequorea victoria GFP gene Clontech Open in a separate window a All M. tuberculosis strains are derived from the Erdman wild-type strain.

Techniques: Mutagenesis

Journal:

Article Title: Glutamine Synthetase GlnA1 Is Essential for Growth of Mycobacterium tuberculosis in Human THP-1 Macrophages and Guinea Pigs

doi: 10.1128/IAI.71.7.3927-3936.2003

Figure Lengend Snippet: GS expression in M. tuberculosis wild-type, glnA1, and complemented strains. (A) SDS-PAGE analysis of cell lysates (∼18 μg of total protein per lane). (B) Immunoblot analysis of the cell lysates (∼18 μg of total protein per lane). Blots were probed with polyclonal rabbit anti-M. tuberculosis GS and, as a control, rabbit anti-M. tuberculosis superoxide dismutase antibody. The GS band (arrow) present in the wild-type lysates is absent in the M. tuberculosis glnA1 pNBV1 and M. tuberculosis glnA1 pNBV1-StGS lysates. GS is overexpressed in the M. tuberculosis glnA1 pNBV1-MtbGS strain due to its expression from a multicopy plasmid. The M. tuberculosis GS polyclonal antibodies used for immunodetection of GS were not capable of detecting <1 μg of purified S. enterica serovar Typhimurium GS (data not shown); therefore, the absence of a band for S. enterica serovar Typhimurium GS shows only that expression is <1 μg. +l-Gln, cultures grown in the presence of 20 mM l-glutamine; M, molecular mass markers in kilodaltons; Mtb, M. tuberculosis.

Article Snippet: Hygromycin (50 μg ml −1 ) and/or kanamycin (20 or 50 μg ml −1 ) were included as appropriate. l -Glutamine was sterilized by filtration and added aseptically to broth and agar after autoclaving when required. table ft1 table-wrap mode="anchored" t5 TABLE 1. caption a7 Strain or plasmid Description Reference or source Strains E. coli DH5α Gibco BRL E. coli XL10-Gold Stratagene M. tuberculosis a Wild-type Erdman strain ATCC 35801 M. tuberculosis pNBV1 39 M. tuberculosis glnA1 Insertionally inactivated glnA1 locus; Km r This study M. tuberculosis glnA1 pNBV1 This study M. tuberculosis glnA1 pNBV1- Mtb GS This study M. tuberculosis glnA1 pNBV1- St GS This study Plasmids pUC19 E. coli cloning vector 42 pPR27 Mycobacterial allelic exchange vector; ori (ts) sacB Gm r 31 pEX1 Mycobacterial allelic exchange vector derived from pPR27 and pGFPuv; ori (ts) sacB Hyg r GFP This study pNBV1 E. coli mycobacterial shuttle vector; Hyg r 20 pNBV1- Mtb GS M. tuberculosis glnA1 expression vector 39 pNBV1- St GS S. enterica serovar Typhimurium glnA with M. tuberculosis glnA1 promoter 39 pGFPuv UV-optimized Aequorea victoria GFP gene Clontech Open in a separate window a All M. tuberculosis strains are derived from the Erdman wild-type strain.

Techniques: Expressing, SDS Page, Western Blot, Plasmid Preparation, Immunodetection, Purification

Journal:

Article Title: Glutamine Synthetase GlnA1 Is Essential for Growth of Mycobacterium tuberculosis in Human THP-1 Macrophages and Guinea Pigs

doi: 10.1128/IAI.71.7.3927-3936.2003

Figure Lengend Snippet: Expression of glutamine synthetase

Article Snippet: Hygromycin (50 μg ml −1 ) and/or kanamycin (20 or 50 μg ml −1 ) were included as appropriate. l -Glutamine was sterilized by filtration and added aseptically to broth and agar after autoclaving when required. table ft1 table-wrap mode="anchored" t5 TABLE 1. caption a7 Strain or plasmid Description Reference or source Strains E. coli DH5α Gibco BRL E. coli XL10-Gold Stratagene M. tuberculosis a Wild-type Erdman strain ATCC 35801 M. tuberculosis pNBV1 39 M. tuberculosis glnA1 Insertionally inactivated glnA1 locus; Km r This study M. tuberculosis glnA1 pNBV1 This study M. tuberculosis glnA1 pNBV1- Mtb GS This study M. tuberculosis glnA1 pNBV1- St GS This study Plasmids pUC19 E. coli cloning vector 42 pPR27 Mycobacterial allelic exchange vector; ori (ts) sacB Gm r 31 pEX1 Mycobacterial allelic exchange vector derived from pPR27 and pGFPuv; ori (ts) sacB Hyg r GFP This study pNBV1 E. coli mycobacterial shuttle vector; Hyg r 20 pNBV1- Mtb GS M. tuberculosis glnA1 expression vector 39 pNBV1- St GS S. enterica serovar Typhimurium glnA with M. tuberculosis glnA1 promoter 39 pGFPuv UV-optimized Aequorea victoria GFP gene Clontech Open in a separate window a All M. tuberculosis strains are derived from the Erdman wild-type strain.

Techniques: Expressing, Activity Assay

Journal:

Article Title: Glutamine Synthetase GlnA1 Is Essential for Growth of Mycobacterium tuberculosis in Human THP-1 Macrophages and Guinea Pigs

doi: 10.1128/IAI.71.7.3927-3936.2003

Figure Lengend Snippet: The M. tuberculosis glnA1 mutant is avirulent in guinea pigs. Guinea pigs were infected by aerosol with M. tuberculosis strains as indicated, and 10 weeks later, bacterial load in the right lung (A) and spleen (B) was quantified. Guinea pigs were infected with the M. tuberculosis glnA1 mutant at the standard dose (1×) used for the other strains and at 10× and 100× the standard dose. Data are the means ± standard errors for all animals in a group (n = 5). No CFU were detected in plated aliquots of the lung and spleen for any of the M. tuberculosis glnA1-infected animals, and all of these organs were scored as 2.1 log CFU for statistical purposes (2.1 log CFU/organ, the limit of detection, is indicated by the dashed lines). Mtb, M. tuberculosis.

Article Snippet: Hygromycin (50 μg ml −1 ) and/or kanamycin (20 or 50 μg ml −1 ) were included as appropriate. l -Glutamine was sterilized by filtration and added aseptically to broth and agar after autoclaving when required. table ft1 table-wrap mode="anchored" t5 TABLE 1. caption a7 Strain or plasmid Description Reference or source Strains E. coli DH5α Gibco BRL E. coli XL10-Gold Stratagene M. tuberculosis a Wild-type Erdman strain ATCC 35801 M. tuberculosis pNBV1 39 M. tuberculosis glnA1 Insertionally inactivated glnA1 locus; Km r This study M. tuberculosis glnA1 pNBV1 This study M. tuberculosis glnA1 pNBV1- Mtb GS This study M. tuberculosis glnA1 pNBV1- St GS This study Plasmids pUC19 E. coli cloning vector 42 pPR27 Mycobacterial allelic exchange vector; ori (ts) sacB Gm r 31 pEX1 Mycobacterial allelic exchange vector derived from pPR27 and pGFPuv; ori (ts) sacB Hyg r GFP This study pNBV1 E. coli mycobacterial shuttle vector; Hyg r 20 pNBV1- Mtb GS M. tuberculosis glnA1 expression vector 39 pNBV1- St GS S. enterica serovar Typhimurium glnA with M. tuberculosis glnA1 promoter 39 pGFPuv UV-optimized Aequorea victoria GFP gene Clontech Open in a separate window a All M. tuberculosis strains are derived from the Erdman wild-type strain.

Techniques: Mutagenesis, Infection