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: |
Creators | Email | Pitt Username | ORCID  |
---|
Van Kessel, JC | | | | Hatfull, GF | gfh@pitt.edu | GFH | |
|
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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