Gene Replacement Systems†

Neil G. Stoker; P. Sander; J. M. Reyrat

doi:10.1128/9781555817657.ch12

Chapter 12 : Gene Replacement Systems†

Authors: Neil G. Stoker¹, P. Sander², J. M. Reyrat³

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Affiliations: 1: Department of Pathology & Infectious Diseases, Royal Veterinary College, Royal College Street, London NW1 0TU, United Kingdom; 2: Institut für Medizinische Mikrobiologie, Universität Zürich, Gloriastr. 30/32, CH-8028 Zürich, Switzerland; 3: Faculté de Médecine Necker-Enfants Malades, INSERM U570, 156 rue de Vaugirard, 75730 Paris Cedex 15, France

Content Type: Monograph

Publication Year: 2005

Category: Bacterial Pathogenesis; Clinical Microbiology

Book DOI: 10.1128/9781555817657

Chapter DOI: 10.1128/9781555817657.ch12

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Abstract:

With the completion of the Mycobacterium tuberculosis genome sequence, the focus of research has turned to functional characterization of genes, and, in particular, identification of virulence factors. The inability to identify double-crossover events was a major impediment in the study of M. tuberculosis pathogenicity. The first mutant of the M. tuberculosis complex made through allelic replacement was a M. bovis BCG mutant, created using the ureC gene as a target. A major factor in the small number of mutants obtained using suicide plasmids was the efficiency of transformation. This difficulty is avoided by using replicating plasmids, although there is the new problem of ensuring plasmid loss. The first counterselectable marker to be described in mycobacteria was streptomycin sensitivity. This system takes advantage of the fact that the S12 ribosomal protein is the target of streptomycin. A major drawback when using replicating plasmids was that it was necessary to isolate plasmid free cells. To induce efficient plasmid loss at will, a thermosensitive replicon was isolated. Marked mutants, in which an antibiotic resistance gene is used to interrupt the gene of interest, are the most straightforward to make. Even when the gene studied is essential, studying the phenotype of a conditional mutant can be useful because it can help to unravel the function of the gene. In conclusion, many genetic tools are now available for studying mycobacteria. It is possible to select directly for allelic exchange in a single-step strategy or to construct an unmarked mutation in a two-step strategy.

Citation: Stoker N, Sander P, Reyrat J. 2005. Gene Replacement Systems†, p 183-190. In Cole S, Eisenach K, McMurray D, Jacobs, Jr. W (ed), Tuberculosis and the Tubercle Bacillus. ASM Press, Washington, DC. doi: 10.1128/9781555817657.ch12

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Bacterial Genetics: 0.5351489
Deletion Mutation: 0.5136325
Periplasmic Space: 0.50516593
Plasma Membrane: 0.50516593
Periplasmic Space: 0.50516593
Plasma Membrane: 0.50516593
Mycobacterium tuberculosis: 0.5039593

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Gene Replacement Systems†

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Figure 1

Two-step strategy for allelic replacement. Positive selection of allelic exchange mutants in a two-step selection strategy, using a counterselectable marker, is shown. CSM, counterselectable marker; SM, selectable marker; wt, wild-type allele; mut, mutated allele. Adapted from reference 54 with permission.

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Figure 1

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