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

Pearson, RE and Jurgensen, S and Sarkis, GJ and Hatfull, GF and Jacobs, WR (1996) Construction of D29 shuttle phasmids and luciferase reporter phages for detection of mycobacteria. Gene, 183 (1-2). 129 - 136. ISSN 0378-1119

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Diseases caused by Mycobacterium tuberculosis, M. leprae and M. avium, cause significant morbidity and mortality worldwide. Effective treatments require that the organisms be speciated and that drug susceptibilities for the causative organisms be characterized. Reporter phage technology has been developed as a rapid and convenient method for identifying mycobacterial species and evaluating drug resistance. In this report we describe the construction of luciferase reporter phages from mycobacterio-phage D29 DNA. Shuttle phasmids were first constructed with D29 in order to identify non-essential regions of the D29 genomes and to introduce unique cloning sites within that region. Using this approach, we observed that all of the D29 shuttle phasmids had the cosmid vector localized to one area of the phage genome near one cohesive end. These shuttle phasmids had been constructed with a cosmid that could be readily excised from the D29 genome with different sets of restriction enzymes. Luciferase reporter phages were made by substituting the luciferase cassette for the cosmid vector. Recombinant phages with the luciferase cassette fall into two groups. One group produced light and had the expression cassette oriented with the promoter directing transcription away from the cohesive end. In contrast, the other group had the expression cassette in the opposite orientation and failed to produce light during lytic infection, but did produce light in L5 lysogens which are known to repress D29 promoters. These results suggest that a phage promoter of the D29 phage can occlude the expression of a promoter introduced into this region. D29 luciferase reporter phages are capable of detecting low numbers of L5 lysogens like L5 luciferase phages. However, unlike L5 luciferase phages, D29 luciferase phages can readily infect M. tuberculosis and M. bovis BCG, demonstrating that these phages can be used to evaluate drug susceptibilities of many types of mycobacteria.


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Item Type: Article
Status: Published
CreatorsEmailPitt UsernameORCID
Pearson, RE
Jurgensen, S
Sarkis, GJ
Hatfull, GFgfh@pitt.eduGFH
Jacobs, WR
Date: 1 December 1996
Date Type: Publication
Journal or Publication Title: Gene
Volume: 183
Number: 1-2
Page Range: 129 - 136
DOI or Unique Handle: 10.1016/s0378-1119(96)00530-6
Schools and Programs: Dietrich School of Arts and Sciences > Biological Sciences
Refereed: Yes
ISSN: 0378-1119
MeSH Headings: Cloning, Molecular--methods; Cosmids--genetics; Gene Expression; Genes, Reporter--genetics; Genetic Vectors--genetics; Kinetics; Luciferases--biosynthesis; Luciferases--genetics; Lysogeny; Microbial Sensitivity Tests--methods; Mycobacteriophages--genetics; Mycobacteriophages--growth & development; Mycobacterium--isolation & purification; Mycobacterium--virology; Promoter Regions, Genetic--genetics; Restriction Mapping; Superinfection
PubMed ID: 8996097
Date Deposited: 20 Nov 2015 15:48
Last Modified: 02 Feb 2019 15:57


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