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Conditionally replicating mycobacteriophages: A system for transposon delivery to Mycobacterium tuberculosis

Bardarov, S and Kriakov, J and Carriere, C and Yu, S and Vaamonde, C and Mcadam, RA and Bloom, BR and Hatfull, GF and Jacobs, WR (1997) Conditionally replicating mycobacteriophages: A system for transposon delivery to Mycobacterium tuberculosis. Proceedings of the National Academy of Sciences of the United States of America, 94 (20). 10961 - 10966. ISSN 0027-8424

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Abstract

Transposon mutagenesis provides a direct selection for mutants and is an extremely powerful technique to analyze genetic functions in a variety of prokaryotes. Transposon mutagenesis of Mycobacterium tuberculosis has been limited in part because of the inefficiency of the delivery systems. This report describes the development of conditionally replicating shuttle phasmids from the mycobacteriophages D29 and TM4 that enable efficient delivery of transposons into both fast- and slow-growing mycobacteria. These shuttle phasmids consist of an Escherichia coli cosmid vector containing either a mini-Tn10(kan) or Tn5367 inserted into a nonessential region of the phage genome. Thermosensitive mutations were created in the mycobacteriophage genome that allow replication at 30°C but not at 37°C (TM4) or 38.5°C (D29). Infection of mycobacteria at the nonpermissive temperature results in highly efficient transposon delivery to the entire population of mycobacterial cells. Transposition of mini-Tn10(kan) occurred in a site- specific fashion in M. smegmatis whereas Tn5367 transposed apparently randomly in M. phlei, Bacille Calmette-Guerin (BCG), and M. tuberculosis. Sequence analysis of the M. tuberculosis and BCG chromosomal regions adjacent to Tn5367 insertions, in combination with M. tuberculosis genomic sequence and physical map data, indicates that the transpositions have occurred randomly in diverse genes in every quadrant of the genome. Using this system, it has been readily possible to generate libraries containing thousands of independent mutants of M. phlei, BCG, and M. tuberculosis.

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Details

Item Type: Article
Status: Published
Creators/Authors:
CreatorsEmailPitt UsernameORCID
Bardarov, S
Kriakov, J
Carriere, C
Yu, S
Vaamonde, C
Mcadam, RA
Bloom, BR
Hatfull, GFgfh@pitt.eduGFH
Jacobs, WR
Date: 30 September 1997
Date Type: Publication
Journal or Publication Title: Proceedings of the National Academy of Sciences of the United States of America
Volume: 94
Number: 20
Page Range: 10961 - 10966
DOI or Unique Handle: 10.1073/pnas.94.20.10961
Schools and Programs: Dietrich School of Arts and Sciences > Biological Sciences
Refereed: Yes
ISSN: 0027-8424
MeSH Headings: DNA Transposable Elements; Genetic Vectors; Mycobacteriophages--genetics; Mycobacteriophages--physiology; Mycobacterium--growth & development; Mycobacterium--virology; Species Specificity; Virus Replication--genetics
Other ID: NLM PMC23545, 10.1073/pnas.94.20.10961
PubMed Central ID: PMC23545
PubMed ID: 9380742
Date Deposited: 01 Mar 2013 15:36
Last Modified: 05 Feb 2019 02:55
URI: http://d-scholarship.pitt.edu/id/eprint/17541

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