1997
DOI: 10.1006/plas.1997.1286
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Unmarked Gene Integration into the Chromosome ofMycobacterium smegmatisvia Precise Replacement of thepyrFGene

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Cited by 23 publications
(28 citation statements)
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“…However, an efficient methodology to generate defined mutations is urgently needed for the rapid development of the functional genomics of the clinically important pathogenic myco- S. Bardarov and others bacteria in order to understand the genetic basis for their tropism, virulence and persistence in the host. Mutant isolation and gene transfer strategies have been successfully used for the fast-growing mycobacteria such as Mycobacterium smegmatis (Boshoff & Mizrahi, 2000 ;Braunstein et al, 2001 ;Frischkorn et al, 1998 ;Knipfer et al, 1997 ;Pavelka & Jacobs, 1999). However, in the slow-growing mycobacterial species the construction of genetically defined isogenic strains containing single or multiple mutations has been notoriously difficult.…”
Section: Introductionmentioning
confidence: 99%
“…However, an efficient methodology to generate defined mutations is urgently needed for the rapid development of the functional genomics of the clinically important pathogenic myco- S. Bardarov and others bacteria in order to understand the genetic basis for their tropism, virulence and persistence in the host. Mutant isolation and gene transfer strategies have been successfully used for the fast-growing mycobacteria such as Mycobacterium smegmatis (Boshoff & Mizrahi, 2000 ;Braunstein et al, 2001 ;Frischkorn et al, 1998 ;Knipfer et al, 1997 ;Pavelka & Jacobs, 1999). However, in the slow-growing mycobacterial species the construction of genetically defined isogenic strains containing single or multiple mutations has been notoriously difficult.…”
Section: Introductionmentioning
confidence: 99%
“…pyrF-based counterselectable gene knockout systems have successfully been applied to yeast (22), archaea (23), and some bacterial species (24)(25)(26). In these systems, a wild-type strain is often susceptible to 5-FOA and the deletion of pyrF confers resistance to 5-FOA.…”
Section: Resultsmentioning
confidence: 99%
“…5-F-UMP is toxic, and its accumulation often leads to cell death (20,21). Based on this feature, URA3-pyrF has been successfully used as a counterselectable marker for targeted mutagenesis in fungi (22), archaea (23), and bacteria (24)(25)(26). T. denticola has a single copy of the pyrF gene and is susceptible to 5-FOA, which is indicative of its potential to use pyrF as a counterselectable marker in the spirochete (13,27).…”
mentioning
confidence: 99%
“…To increase our knowledge of the genetic basis of virulence and persistence in the host and to develop efficacious potential live vaccines, an efficient method for generating targeted gene knockouts is urgently needed. In contrast to the successful gene disruption in fast-growing mycobacteria, such as M. smegmatis (8,10,26,36), gene disruption in slow-growing mycobacteria has traditionally been inefficient, in part due to the high frequency of illegitimate recombination and the characteristic clumping of cells in culture (1,25,28).…”
mentioning
confidence: 99%