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Efficient point mutagenesis in mycobacteria using single-stranded DNA recombineering: Characterization of antimycobacterial drug targets

Van Kessel, JC and Hatfull, GF (2008) Efficient point mutagenesis in mycobacteria using single-stranded DNA recombineering: Characterization of antimycobacterial drug targets. Molecular Microbiology, 67 (5). 1094 - 1107. ISSN 0950-382X

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Abstract

Construction of genetically isogenic strains of mycobacteria is complicated by poor recombination rates and the lack of generalized transducing phages for Mycobacterium tuberculosis. We report here a powerful method for introducing single point mutations into mycobacterial genomes using oligonucleotide-derived single-stranded DNA recombineering and mycobacteriophage-encoded proteins. Phage Che9c gp61-mediated recombination is sufficiently efficient that single base changes can be introduced without requirement for direct selection, with isogenic mutant strains identified simply by PCR. Efficient recombination requires only short (50 nucleotide) oligonucleotides, but there is an unusually strong strand bias and an oligonucleotide targeting lagging strand DNA synthesis can recombine more than 10 000-fold efficiently than its complementary oligonucleotide. This ssDNA recombineering provides a simple assay for comparing the activities of related phage recombinases, and we find that both Escherichia coli RecET and phage λ Red recombination proteins function inefficiently in mycobacteria, illustrating the utility of developing recombineering in new bacterial systems using host-specific bacteriophage recombinases. ssDNA mycobacterial recombineering provides a simple approach to characterizing antimycobacterial drug targets, and we have constructed and characterized single point mutations that confer resistance to isoniazid, rifampicin, ofloxacin and streptomycin. © 2008 The Authors.

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Details

Item Type: Article
Status: Published
Creators/Authors:
CreatorsEmailPitt UsernameORCID
Van Kessel, JC
Hatfull, GFgfh@pitt.eduGFH
Date: 1 March 2008
Date Type: Publication
Journal or Publication Title: Molecular Microbiology
Volume: 67
Number: 5
Page Range: 1094 - 1107
DOI or Unique Handle: 10.1111/j.1365-2958.2008.06109.x
Schools and Programs: Dietrich School of Arts and Sciences > Biological Sciences
Refereed: Yes
ISSN: 0950-382X
MeSH Headings: Anti-Bacterial Agents--metabolism; Anti-Bacterial Agents--pharmacology; Chromosomes, Bacterial--genetics; DNA, Single-Stranded--genetics; DNA, Single-Stranded--metabolism; Drug Resistance, Bacterial; Drug Resistance, Microbial; Mutagenesis, Site-Directed--methods; Mycobacteriophages--enzymology; Mycobacteriophages--genetics; Mycobacterium--drug effects; Mycobacterium--genetics; Mycobacterium--virology; Oligonucleotides--genetics; Plasmids; Point Mutation; Recombinases--genetics; Recombinases--metabolism; Recombination, Genetic; Reverse Transcriptase Polymerase Chain Reaction; Sequence Homology, Nucleic Acid; Viral Proteins--genetics; Viral Proteins--metabolism
PubMed ID: 18221264
Date Deposited: 14 Jan 2013 16:18
Last Modified: 12 Oct 2017 08:57
URI: http://d-scholarship.pitt.edu/id/eprint/16734

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