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Specialized transduction: an efficient method for generating marked and unmarked targeted gene disruptions in Mycobacterium tuberculosis, M. bovis BCG and M. smegmatis.

Bardarov, Stoyan and Bardarov, Svetoslav and Pavelka, Martin S and Sambandamurthy, Vasan and Larsen, Michelle and Tufariello, JoAnn and Chan, John and Hatfull, Graham and Jacobs, William R Specialized transduction: an efficient method for generating marked and unmarked targeted gene disruptions in Mycobacterium tuberculosis, M. bovis BCG and M. smegmatis. Microbiology, 148 (Pt 10). 3007 - 3017. ISSN 1350-0872

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Abstract

The authors have developed a simple and highly efficient system for generating allelic exchanges in both fast- and slow-growing mycobacteria. In this procedure a gene of interest, disrupted by a selectable marker, is cloned into a conditionally replicating (temperature-sensitive) shuttle phasmid to generate a specialized transducing mycobacteriophage. The temperature-sensitive mutations in the mycobacteriophage genome permit replication at the permissive temperature of 30 degrees C but prevent replication at the non-permissive temperature of 37 degrees C. Transduction at a non-permissive temperature results in highly efficient delivery of the recombination substrate to virtually all cells in the recipient population. The deletion mutations in the targeted genes are marked with antibiotic-resistance genes that are flanked by gammadelta-res (resolvase recognition target) sites. The transductants which have undergone a homologous recombination event can be conveniently selected on antibiotic-containing media. To demonstrate the utility of this genetic system seven different targeted gene disruptions were generated in three substrains of Mycobacterium bovis BCG, three strains of Mycobacterium tuberculosis, and Mycobacterium smegmatis. Mutants in the lysA, nadBC, panC, panCD, leuCD, Rv3291c and Rv0867c genes or operons were isolated as antibiotic-resistant (and in some cases auxotrophic) transductants. Using a plasmid encoding the gammadelta-resolvase (tnpR), the resistance genes could be removed, generating unmarked deletion mutations. It is concluded from the high frequency of allelic exchange events observed in this study that specialized transduction is a very efficient technique for genetic manipulation of mycobacteria and is a method of choice for constructing isogenic strains of M. tuberculosis, BCG or M. smegmatis which differ by defined mutations.


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Details

Item Type: Article
Status: Published
Creators/Authors:
CreatorsEmailPitt UsernameORCID
Bardarov, Stoyan
Bardarov, Svetoslav
Pavelka, Martin S
Sambandamurthy, Vasan
Larsen, Michelle
Tufariello, JoAnn
Chan, John
Hatfull, Grahamgfh@pitt.eduGFH
Jacobs, William R
Journal or Publication Title: Microbiology
Volume: 148
Number: Pt 10
Page Range: 3007 - 3017
DOI or Unique Handle: 10.1099/00221287-148-10-3007
Schools and Programs: Dietrich School of Arts and Sciences > Biological Sciences
Refereed: Yes
Uncontrolled Keywords: Alleles, Bacterial Proteins, Bacteriological Techniques, Cosmids, Gene Deletion, Genetic Markers, Mycobacteriophages, Mycobacterium, Mycobacterium bovis, Mycobacterium smegmatis, Mycobacterium tuberculosis, Plasmids, Recombination, Genetic, Transduction, Genetic
ISSN: 1350-0872
Funders: NIAID NIH HHS (AI26170), NIAID NIH HHS (AI28927), NIAID NIH HHS (AI46690), NIAID NIH HHS (AI49375), NIGMS NIH HHS (GMG2410)
MeSH Headings: Alleles; Bacterial Proteins--genetics; Bacteriological Techniques; Cosmids--genetics; Gene Deletion; Genetic Markers; Mycobacteriophages--genetics; Mycobacterium--genetics; Mycobacterium bovis--genetics; Mycobacterium smegmatis--genetics; Mycobacterium tuberculosis--genetics; Plasmids; Recombination, Genetic; Transduction, Genetic--methods
PubMed ID: 12368434
Date Deposited: 12 Nov 2012 15:53
Last Modified: 25 Feb 2018 03:55
URI: http://d-scholarship.pitt.edu/id/eprint/16124

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