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Recombineering in Mycobacterium tuberculosis

van Kessel, JC and Hatfull, GF (2007) Recombineering in Mycobacterium tuberculosis. Nature Methods, 4 (2). 147 - 152. ISSN 1548-7091

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Abstract

Genetic dissection of M. tuberculosis is complicated by its slow growth and its high rate of illegitimate recombination relative to homologous DNA exchange. We report here the development of a facile allelic exchange system by identification and expression of mycobacteriophage-encoded recombination proteins, adapting a strategy developed previously for recombineering in Escherichia coli. Identifiable recombination proteins are rare in mycobacteriophages, and only 1 of 30 genomically characterized mycobacteriophages (Che9c) encodes homologs of both RecE and RecT. Expression and biochemical characterization show that Che9c gp60 and gp61 encode exonuclease and DNA-binding activities, respectively, and expression of these proteins substantially elevates recombination facilitating allelic exchange in both M. smegmatis and M. tuberculosis. Mycobacterial recombineering thus provides a simple approach for the construction of gene replacement mutants in both slow- and fast-growing mycobacteria.


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Details

Item Type: Article
Status: Published
Creators/Authors:
CreatorsEmailPitt UsernameORCID
van Kessel, JC
Hatfull, GFgfh@pitt.eduGFH
Date: 1 February 2007
Date Type: Publication
Journal or Publication Title: Nature Methods
Volume: 4
Number: 2
Page Range: 147 - 152
DOI or Unique Handle: 10.1038/nmeth996
Schools and Programs: Dietrich School of Arts and Sciences > Biological Sciences
Refereed: Yes
ISSN: 1548-7091
MeSH Headings: DNA-Binding Proteins--genetics; Exonucleases--genetics; Gene Targeting--methods; Mycobacteriophages--chemistry; Mycobacteriophages--genetics; Mycobacterium smegmatis--genetics; Mycobacterium smegmatis--growth & development; Mycobacterium tuberculosis--genetics; Mycobacterium tuberculosis--growth & development; Mycobacterium tuberculosis--virology; Recombination, Genetic; Transformation, Genetic; Viral Proteins--genetics
PubMed ID: 17179933
Date Deposited: 14 Jan 2013 16:19
Last Modified: 02 Feb 2019 15:57
URI: http://d-scholarship.pitt.edu/id/eprint/16733

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