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Highly fluorescent GFPm 2+ -based genome integration-proficient promoter probe vector to study Mycobacterium tuberculosis promoters in infected macrophages.

Roy S, Narayana Y, Balaji KN, Ajitkumar P - Microb Biotechnol (2011)

Bottom Line: Study of activity of cloned promoters in slow-growing Mycobacterium tuberculosis during long-term growth conditions in vitro or inside macrophages, requires a genome-integration proficient promoter probe vector, which can be stably maintained even without antibiotics, carrying a substrate-independent, easily scorable and highly sensitive reporter gene.In order to meet this requirement, we constructed pAKMN2, which contains mycobacterial codon-optimized gfp(m) (2+) gene, coding for GFP(m) (2+) of highest fluorescence reported till date, mycobacteriophage L5 attP-int sequence for genome integration, and a multiple cloning site. pAKMN2 showed stable integration and expression of GFP(m) (2+) from M. tuberculosis and M. smegmatis genome.Stable expression from genome-integrated format even without antibiotic, and high sensitivity of detection by flow cytometry and fluorescence imaging, in spite of single copy integration, make pAKMN2 useful for the study of cloned promoters of any mycobacterial species under long-term in vitro growth or stress conditions, or inside macrophages.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore - 560012, Karnataka, India.

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A. Map of the transcription start sites (TSS) of T1–T4 and T6 and the span of their respective promoters, P1–P4, P6 and PQ1K1. B. First main column: Comparison of stability of pAKMN2‐PQ1K1 integrant vector over episomal pMN406‐PQ1K1 in the presence (+) or absence (−) of hygromycin (hyg) selection, in terms of cfu (n = 3). Second main column: GFPm2+ intensities were calculated from flow cytometry results (n = 3) in both M. smegmatis (Ms) and M. tuberculosis (Mt) integrants of pAKMN2‐PQ1K1, in comparison to the respective transformants of episomal pMN406‐PQ1K1 vector. C. Representative histogram of a single data set of M. tuberculosis. Grey shaded histogram: exponential Mt; Green histogram:Mt/pAKMN2‐PQ1K1 (left panel) or Mt/pMN406‐PQ1K1 (right panel) in the absence of hygromycin; Purple: Mt/pAKMN2‐PQ1K1 (left panel) or Mt/pMN406‐PQ1K1 (right panel) in the presence of hygromycin. D. Representative dotplots from single flow cytometric data set of P1–P4 and P6, vector control (pAKMN2, devoid of promoter), and M. tuberculosis/pAKMN2‐PQ1K1 integrants. The mean and standard deviations for GFP intensities driven by the cloned promoters were shown as inset. E. Comparison of % activity of P1‐P4, and P6 obtained from flow cytometry (blue bar) and semi‐quantitative RT‐PCR (red bar) for gfpm2+ in M. tuberculosis/pAKMN2‐Promoter integrants in exponential phase. Only significant differences in activity between promoters were indicated by lines [black, green: significant difference obtained by both RT‐PCR and flow cytometry for P1 (black) and P3 (green); red: differences obtained only by RT‐PCR].
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f2: A. Map of the transcription start sites (TSS) of T1–T4 and T6 and the span of their respective promoters, P1–P4, P6 and PQ1K1. B. First main column: Comparison of stability of pAKMN2‐PQ1K1 integrant vector over episomal pMN406‐PQ1K1 in the presence (+) or absence (−) of hygromycin (hyg) selection, in terms of cfu (n = 3). Second main column: GFPm2+ intensities were calculated from flow cytometry results (n = 3) in both M. smegmatis (Ms) and M. tuberculosis (Mt) integrants of pAKMN2‐PQ1K1, in comparison to the respective transformants of episomal pMN406‐PQ1K1 vector. C. Representative histogram of a single data set of M. tuberculosis. Grey shaded histogram: exponential Mt; Green histogram:Mt/pAKMN2‐PQ1K1 (left panel) or Mt/pMN406‐PQ1K1 (right panel) in the absence of hygromycin; Purple: Mt/pAKMN2‐PQ1K1 (left panel) or Mt/pMN406‐PQ1K1 (right panel) in the presence of hygromycin. D. Representative dotplots from single flow cytometric data set of P1–P4 and P6, vector control (pAKMN2, devoid of promoter), and M. tuberculosis/pAKMN2‐PQ1K1 integrants. The mean and standard deviations for GFP intensities driven by the cloned promoters were shown as inset. E. Comparison of % activity of P1‐P4, and P6 obtained from flow cytometry (blue bar) and semi‐quantitative RT‐PCR (red bar) for gfpm2+ in M. tuberculosis/pAKMN2‐Promoter integrants in exponential phase. Only significant differences in activity between promoters were indicated by lines [black, green: significant difference obtained by both RT‐PCR and flow cytometry for P1 (black) and P3 (green); red: differences obtained only by RT‐PCR].

Mentions: The construction of pAKMN2, from the source vectors, pMN406 (Roy et al., 2004) and pDK20 (DasGupta et al., 1998), through the intermediate episomal pAKMN1, is given in the self‐explanatory Fig. 1. In order to verify the stability of genome‐integrated pAKMN2‐promoter constructs, M. tuberculosis/pAKMN2‐PQ1K1 (Mt) and M. smegmatis/pAKMN2‐PQ1K1 (Ms) integrants, carrying total promoter region, Q1‐K1, of M. tuberculosis cell division gene, ftsZ, MtftsZ (Fig. 2A; Roy and Ajitkumar, 2005), were grown to mid‐log phase without hygromycin and plated on hygromycin‐containing and hygromycin‐free plates. The colony‐forming units (cfu) for both the integrants were comparable in the presence and absence of hygromycin (checked up to 30 and 60 generations for Mt and Ms integrants respectively) (Fig. 2B), with statistically insignificant values (two‐sided P‐values obtained by unpaired t‐test: 0.3078 for Mt and 0.1374 for Ms). On the contrary, statistically significant reduction (two‐sided P‐values: 0.0008 for Mt and 0.0035 for Ms) in cfu was found in the absence of hygromycin for the episomal pMN406‐PQ1K1 transformants of M. tuberculosis and M. smegmatis, grown under same conditions (Fig. 2B).


Highly fluorescent GFPm 2+ -based genome integration-proficient promoter probe vector to study Mycobacterium tuberculosis promoters in infected macrophages.

Roy S, Narayana Y, Balaji KN, Ajitkumar P - Microb Biotechnol (2011)

A. Map of the transcription start sites (TSS) of T1–T4 and T6 and the span of their respective promoters, P1–P4, P6 and PQ1K1. B. First main column: Comparison of stability of pAKMN2‐PQ1K1 integrant vector over episomal pMN406‐PQ1K1 in the presence (+) or absence (−) of hygromycin (hyg) selection, in terms of cfu (n = 3). Second main column: GFPm2+ intensities were calculated from flow cytometry results (n = 3) in both M. smegmatis (Ms) and M. tuberculosis (Mt) integrants of pAKMN2‐PQ1K1, in comparison to the respective transformants of episomal pMN406‐PQ1K1 vector. C. Representative histogram of a single data set of M. tuberculosis. Grey shaded histogram: exponential Mt; Green histogram:Mt/pAKMN2‐PQ1K1 (left panel) or Mt/pMN406‐PQ1K1 (right panel) in the absence of hygromycin; Purple: Mt/pAKMN2‐PQ1K1 (left panel) or Mt/pMN406‐PQ1K1 (right panel) in the presence of hygromycin. D. Representative dotplots from single flow cytometric data set of P1–P4 and P6, vector control (pAKMN2, devoid of promoter), and M. tuberculosis/pAKMN2‐PQ1K1 integrants. The mean and standard deviations for GFP intensities driven by the cloned promoters were shown as inset. E. Comparison of % activity of P1‐P4, and P6 obtained from flow cytometry (blue bar) and semi‐quantitative RT‐PCR (red bar) for gfpm2+ in M. tuberculosis/pAKMN2‐Promoter integrants in exponential phase. Only significant differences in activity between promoters were indicated by lines [black, green: significant difference obtained by both RT‐PCR and flow cytometry for P1 (black) and P3 (green); red: differences obtained only by RT‐PCR].
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Related In: Results  -  Collection

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f2: A. Map of the transcription start sites (TSS) of T1–T4 and T6 and the span of their respective promoters, P1–P4, P6 and PQ1K1. B. First main column: Comparison of stability of pAKMN2‐PQ1K1 integrant vector over episomal pMN406‐PQ1K1 in the presence (+) or absence (−) of hygromycin (hyg) selection, in terms of cfu (n = 3). Second main column: GFPm2+ intensities were calculated from flow cytometry results (n = 3) in both M. smegmatis (Ms) and M. tuberculosis (Mt) integrants of pAKMN2‐PQ1K1, in comparison to the respective transformants of episomal pMN406‐PQ1K1 vector. C. Representative histogram of a single data set of M. tuberculosis. Grey shaded histogram: exponential Mt; Green histogram:Mt/pAKMN2‐PQ1K1 (left panel) or Mt/pMN406‐PQ1K1 (right panel) in the absence of hygromycin; Purple: Mt/pAKMN2‐PQ1K1 (left panel) or Mt/pMN406‐PQ1K1 (right panel) in the presence of hygromycin. D. Representative dotplots from single flow cytometric data set of P1–P4 and P6, vector control (pAKMN2, devoid of promoter), and M. tuberculosis/pAKMN2‐PQ1K1 integrants. The mean and standard deviations for GFP intensities driven by the cloned promoters were shown as inset. E. Comparison of % activity of P1‐P4, and P6 obtained from flow cytometry (blue bar) and semi‐quantitative RT‐PCR (red bar) for gfpm2+ in M. tuberculosis/pAKMN2‐Promoter integrants in exponential phase. Only significant differences in activity between promoters were indicated by lines [black, green: significant difference obtained by both RT‐PCR and flow cytometry for P1 (black) and P3 (green); red: differences obtained only by RT‐PCR].
Mentions: The construction of pAKMN2, from the source vectors, pMN406 (Roy et al., 2004) and pDK20 (DasGupta et al., 1998), through the intermediate episomal pAKMN1, is given in the self‐explanatory Fig. 1. In order to verify the stability of genome‐integrated pAKMN2‐promoter constructs, M. tuberculosis/pAKMN2‐PQ1K1 (Mt) and M. smegmatis/pAKMN2‐PQ1K1 (Ms) integrants, carrying total promoter region, Q1‐K1, of M. tuberculosis cell division gene, ftsZ, MtftsZ (Fig. 2A; Roy and Ajitkumar, 2005), were grown to mid‐log phase without hygromycin and plated on hygromycin‐containing and hygromycin‐free plates. The colony‐forming units (cfu) for both the integrants were comparable in the presence and absence of hygromycin (checked up to 30 and 60 generations for Mt and Ms integrants respectively) (Fig. 2B), with statistically insignificant values (two‐sided P‐values obtained by unpaired t‐test: 0.3078 for Mt and 0.1374 for Ms). On the contrary, statistically significant reduction (two‐sided P‐values: 0.0008 for Mt and 0.0035 for Ms) in cfu was found in the absence of hygromycin for the episomal pMN406‐PQ1K1 transformants of M. tuberculosis and M. smegmatis, grown under same conditions (Fig. 2B).

Bottom Line: Study of activity of cloned promoters in slow-growing Mycobacterium tuberculosis during long-term growth conditions in vitro or inside macrophages, requires a genome-integration proficient promoter probe vector, which can be stably maintained even without antibiotics, carrying a substrate-independent, easily scorable and highly sensitive reporter gene.In order to meet this requirement, we constructed pAKMN2, which contains mycobacterial codon-optimized gfp(m) (2+) gene, coding for GFP(m) (2+) of highest fluorescence reported till date, mycobacteriophage L5 attP-int sequence for genome integration, and a multiple cloning site. pAKMN2 showed stable integration and expression of GFP(m) (2+) from M. tuberculosis and M. smegmatis genome.Stable expression from genome-integrated format even without antibiotic, and high sensitivity of detection by flow cytometry and fluorescence imaging, in spite of single copy integration, make pAKMN2 useful for the study of cloned promoters of any mycobacterial species under long-term in vitro growth or stress conditions, or inside macrophages.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore - 560012, Karnataka, India.

Show MeSH
Related in: MedlinePlus