Unmarked Gene Integration into the Chromosome ofMycobacterium smegmatisvia Precise Replacement of thepyrFGene

Knipfer, Nancianne; Seth, Anjali; Shrader, Thomas E.

doi:10.1006/plas.1997.1286

Cited by 23 publications

(28 citation statements)

References 18 publications

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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%

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

et al. 2002

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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 temperaturesensitive mutations in the mycobacteriophage genome permit replication at the permissive temperature of 30 SC but prevent replication at the nonpermissive temperature of 37 SC. 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 γδ-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 γδ-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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Section: Introductionmentioning

confidence: 99%

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

et al. 2002

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“…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).…”

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pyrF as a Counterselectable Marker for Unmarked Genetic Manipulations in Treponema denticola

Kurniyati

2016

Appl Environ Microbiol

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The pathophysiology of Treponema denticola, an oral pathogen associated with both periodontal and endodontic infections, is poorly understood due to its fastidious growth and recalcitrance to genetic manipulations. Counterselectable markers are instrumental in constructing clean and unmarked mutations in bacteria. Here, we demonstrate that pyrF, a gene encoding orotidine-5=-monophosphate decarboxylase, can be used as a counterselectable marker in T. denticola to construct marker-free mutants. T. denticola is susceptible to 5-fluoroorotic acid (5-FOA). To establish a pyrF-based counterselectable knockout system in T. denticola, the pyrF gene was deleted. The deletion conferred resistance to 5-FOA in T. denticola. Next, a single-crossover mutant was constructed by reintroducing pyrF along with a gentamicin resistance gene (aacC1) back into the chromosome of the pyrF mutant at the locus of choice. In this study, we chose flgE, a flagellar hook gene that is located within a large polycistronic motility gene operon, as our target gene. out was free of selection markers (i.e., pyrF and aacC1). Compared to previously constructed flgE mutants that contain an antibiotic selection marker, the deletion of flgE in FlgE out has no polar effect on its downstream gene expression. The system developed here will provide us with a new tool for investigating the genetics and pathogenicity of T. denticola. Treponema denticola is an obligatory anaerobic and highly motile bacterium that is associated with human periodontitis (for a review, see references 1-3), which is a chronic inflammatory disease that damages the supporting connective tissues around the teeth and ultimately leads to tooth loss. The use of targeted mutagenesis followed by phenotypic characterizations in vitro and in vivo is a routine method for identifying bacterial virulence factors and elucidating their roles in bacterial pathogenicity. Due to its fastidious growth requirements, only a few virulence factors have been characterized in T. denticola (for a review, see references 4 and 5). During the last decade, although tremendous efforts have been devoted to the identification of genetic tools, only a few have been developed for the genetic manipulation of T. denticola (6-10). Even now, genetic manipulation (e.g., gene deletion) of T. denticola is still inefficient and cumbersome (7,11). In addition, all of the current genetic tools for T. denticola are built upon positive selection by inserting an antibiotic resistance marker into a targeted gene on the chromosome (6,8,9,12). The drawback of this method is that the insertion of an antibiotic resistance cassette often impairs downstream gene expression, particularly for a gene within a large polycistronic gene cluster. To precisely interpret the function of a targeted gene, the cognate mutant has to be genetically complemented by reintroducing the targeted gene either back into the chromosome or to the cells through a shuttle vector. However, T. denticola is not amenable to the introduction of exogenous genetic el...

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“…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).…”

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confidence: 99%

Demonstration of Allelic Exchange in the Slow-Growing Bacterium Mycobacterium avium subsp. paratuberculosis , and Generation of Mutants with Deletions at the pknG, relA , and lsr2 Loci

Park

Dahl

Bannantine

et al. 2008

Appl Environ Microbiol

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Mycobacterium avium subsp. paratuberculosis is the causative pathogen of Johne's disease, a chronic inflammatory wasting disease in ruminants. This disease has been difficult to control because of the lack of an effective vaccine. To address this need, we adapted a specialized transduction system originally developed for M. tuberculosis and modified it to improve the efficiency of allelic exchange in order to generate site-directed mutations in preselected M. avium subsp. paratuberculosis genes. With our novel optimized method, the allelic exchange frequency was 78 to 100% and the transduction frequency was 1.1 ؋ 10 ؊7 to 2.9 ؋ 10 ؊7 . Three genes were selected for mutagenesis: pknG and relA, which are genes that are known to be important virulence factors in M. tuberculosis and M. bovis, and lsr2, a gene regulating lipid biosynthesis and antibiotic resistance. Mutants were successfully generated with a virulent strain of M. avium subsp. paratuberculosis (M. avium subsp. paratuberculosis K10) and with a recombinant K10 strain expressing the green fluorescent protein gene, gfp. The improved efficiency of disruption of selected genes in M. avium subsp. paratuberculosis should accelerate development of additional mutants for vaccine testing and functional studies.Johne's disease (paratuberculosis) is a chronic wasting disease of the intestine of ruminants caused by Mycobacterium avium subsp. paratuberculosis. It causes significant economic loss to animal producers, especially in the dairy industry, due to an increase in forage consumption, decreased milk production, and early culling due to poor health of affected animals (6,24,31). This disease has been difficult to control because of the lack of sensitive specific diagnostic assays and the lack of an efficacious vaccine. Available diagnostic assays, such as M. avium subsp. paratuberculosis antigen enzyme-linked immunosorbent assays and the gamma interferon assay, vary in the capacity to detect infected animals in the early stages of the disease (29). This is a major problem since infected animals begin to shed M. avium subsp. paratuberculosis in their feces early in the course of the disease before clinical signs appear. Available vaccines have been shown to reduce the severity of pathology but not to stop the shedding of bacteria (20). Consequently, there is a continuing need to develop both a better diagnostic assay and a better vaccine that, at a minimum, stops shedding of bacteria during the productive life of dairy cattle.An important prerequisite before strategies can be developed to control this disease is a better understanding of the molecular mechanisms of M. avium subsp. paratuberculosis pathogenesis. 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-gro...

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Unmarked Gene Integration into the Chromosome ofMycobacterium smegmatisvia Precise Replacement of thepyrFGene

Cited by 23 publications

References 18 publications

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

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

pyrF as a Counterselectable Marker for Unmarked Genetic Manipulations in Treponema denticola

Demonstration of Allelic Exchange in the Slow-Growing Bacterium Mycobacterium avium subsp. paratuberculosis , and Generation of Mutants with Deletions at the pknG, relA , and lsr2 Loci

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