Construction of D29 shuttle phasmids and luciferase reporter phages for detection of mycobacteria

Pearson, Robert E.; Jurgensen, Stewart; Sarkis, Gary J.; Hatfull, Graham F.; Jacobs, William R.

doi:10.1016/s0378-1119(96)00530-6

Cited by 60 publications

(59 citation statements)

References 16 publications

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“…8 Using these reporter phages to deliver the firefly luciferase (Fflux) gene into mycobacteria it has been possible to develop highly sensitive tests for detection of drug resistance in clinical isolates of M. tuberculosis and M. avium and diagnosis of M. tuberculosis. [8][9][10][11] The luciferase reporter phage assay was highly specific but required 10 4 organisms per ml to give a detectable level of light output expressed as relative light units (RLU) in a luminometer. 12 Luciferase reporter phages developed from temperate phage L5 showed increased light output from M. smegmatis compared to lytic phage constructs 13 suggesting that a similar construct from temperate phage infecting M. tuberculosis would bring about a sustained light output leading to better sensitivity of LRP assay.…”

Section: Introductionmentioning

confidence: 99%

Characterization of temperate phage Che12 and construction of a new tool for diagnosis of tuberculosis

et al. 2008

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SUMMARYA temperate phage, Che12, able to infect Mycobacterium tuberculosis, was isolated from soil samples taken from tuberculosis sanatorium area in Chennai, India. The plaque morphology of this phage showed varying grades of turbidity on lawns of M. tuberculosis. The temperate nature of Che12 was established by super infection immunity. Phage integration into the host genomic DNA was confirmed by Southern hybridization using Che12 DNA as a probe. PCR amplification and sequencing of a part of the integrated phage genome in a M. tuberculosis lysogen also confirmed the temperate nature of Che12. The morphology of the phage particles was observed by electron microscopy, revealing similarities to other mycobacteriophages like L5, D29 and TM4. A luciferase reporter phage, phAETRC16, was constructed by cloning firefly luciferase gene into Che12. Infection of viable M. tuberculosis cells by phAETRC16 resulted in expression of luciferase leading to sustained light output. Che12, a true temperate phage infecting M. tuberculosis, is thus ideally suited for developing a diagnostic tool facilitating rapid diagnosis of M. tuberculosis.

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Section: Introductionmentioning

confidence: 99%

Characterization of temperate phage Che12 and construction of a new tool for diagnosis of tuberculosis

et al. 2008

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“…9). The high Fflux activity in this experiment suggests that the hsp60 promoter, which directs transcription of Fflux on phBD8 (Pearson et al, 1996), may be an excellent choice for a promoter to replace the fortuitous promoter found on phGS15. Such a phage would combine the necessity of high light production with expression of gp71 to inhibit the phage from undergoing the lytic cycle.…”

Section: Expression Of Fflux Is Strong From Phbd8 Lysogensmentioning

confidence: 83%

“…The genomic deletion in phGS18 could have resulted in the loss of a gene that encodes a phage particle component essential for adsorption to BCG but not to M. smegmatis. The impact of this deletion suggests that the previously identified 'non-essential' region shared by L5 and D29 (Sarkis et al, 1995;Pearson et al, 1996) may be dispensable for infection of M. smegmatis but is essential for infection of slow-growing strains. Further support for the role of this region in infection of slow-growing strains is that a host range mutant of L1 was also uncovered.…”

Section: Discussionmentioning

confidence: 96%

“…Mycobacteriophages L5, D29, TM4, and L1 (Froman et al, 1954;Doke, 1960;Timme and Brennan, 1984;Hatfull and Jacobs, 1994) and luciferase reporter phages phGS15, phGS18, phBD8, and phAE40 Sarkis et al, 1995;Pearson et al, 1996) have all been described previously. All phages were propagated by the plate lysate method from Tryptone Soy Agar as described previously .…”

Section: Methodsmentioning

confidence: 99%

“…Phage phBD8, a luciferase reporter phage derived from D29, gave sharp peaks of luciferase activity that reached a maximum at 3 h (Pearson et al, 1996 and Fig. 7B).…”

Section: Fig 5 Super-infection Immunity In Bcg-cmentioning

confidence: 99%

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Mycobacteriophage L5 infection of Mycobacterium bovis BCG: implications for phage genetics in the slow‐growing mycobacteria

Fullner

Hatfull

1997

Molecular Microbiology

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SummaryMycobacteriophage L5 is a well-characterized temperate phage that forms stable lysogens in Mycobacterium smegmatis. The host range of L5 is, however, unclear because previous reports suggested that it does not infect slow-growing mycobacteria such as Mycobacterium tuberculosis and bacille CalmetteGué rin (BCG). Moreover, luciferase reporter phage derivatives of L5 failed to produce light from BCG, suggesting that infection is blocked at or before the stage of DNA injection. In this study, we demonstrate that L5 infection of slow growing mycobacteria specifically requires a high concentration of Ca 2þ , conditions that differs from those required for infection of M. smegmatis by L5 and for infection of BCG by the closely related phage D29. In addition, we show that there are specific genetic determinants of L5 that confer the ability to infect slow growing mycobacteria, without altering infection of M. smegmatis. These observations extend the use of phage L5 for the diagnosis and analysis of tuberculosis and other mycobacterial diseases.

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Luciferase Reporter Bacteriophages

Hagens

Loessner

2007

Handbook of Biosensors and Biochips

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Reporter bacteriophages harness the biological specificity of the interaction between bacterial cells and their bacteriophages. The use of phages that have been genetically engineered to carry luciferase genes which provide an easily quantifiable signal upon infection represents a highly specific means for the detection of viable bacteria. Luciferases of different origin can be used, in conjunction with phages infecting a variety of different pathogenic microorganisms. This chapter aims to describe the biology behind the approach and summarizes the current standing with respect to the various luciferase reporter phages and their applications.

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Construction of D29 shuttle phasmids and luciferase reporter phages for detection of mycobacteria

Cited by 60 publications

References 16 publications

Characterization of temperate phage Che12 and construction of a new tool for diagnosis of tuberculosis

Characterization of temperate phage Che12 and construction of a new tool for diagnosis of tuberculosis

Mycobacteriophage L5 infection of Mycobacterium bovis BCG: implications for phage genetics in the slow‐growing mycobacteria

Luciferase Reporter Bacteriophages

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