Construction of a shuttle vector for use in Francisella tularensis

Norqvist, Anders

doi:10.1016/0928-8244(95)00115-8

Cited by 23 publications

(47 citation statements)

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“…This vector, pFNL100, was made by fusing pFNL10 to the E. coli vector pBR328, and encodes resistance to tetracycline and chloramphenicol (Norqvist et al, 1996). A deletion derivative of this vector, pFNL200, which lacks the E. coli origin, was used in the development of the shuttle vector pKK202, which bears the E. coli p15A origin from pACYC184 (Norqvist et al, 1996).…”

Section: Discussionmentioning

confidence: 99%

“…tularensis (Maier et al, 2004(Maier et al, , 2006. Some of the currently available genetic tools encode tetracycline and/or chloramphenicol resistance, which limits their use to non-select agent strains of F. tularensis (Golovliov et al, 2003;Norqvist et al, 1996). Thus, there is a need to test marker genes and develop genetic tools for highly pathogenic strains of subsp.…”

Section: Introductionmentioning

confidence: 99%

“…holarctica (Eigelsbach & Downs, 1961;Golovliov et al, 2003;Lauriano et al, 2003;Nano et al, 2004;Sjostedt et al, 1996). So far, three Escherichia coli-Francisella shuttle plasmids have been described for use in these two subspecies (Maier et al, 2004;Norqvist et al, 1996;Pavlov et al, 1996); allelic exchange methods were developed for novicida and LVS (Golovliov et al, 2003;Lauriano et al, 2003), shuttle transposon mutagenesis has been used for novicida (Cowley et al, 2000), while the EZ : : TN transposome system (Kawula et al, 2004) and a transposon based upon the Himar1 element (Maier et al, 2006) have been used for mutagenesis of LVS. However, few genetic tools have been tested with highly pathogenic, select agent strains of F. tularensis subsp.…”

Section: Introductionmentioning

confidence: 99%

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Genetic tools for highly pathogenic Francisella tularensis subsp. tularensis

et al. 2006

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This paper is the first detailed description of the development and use of new genetic tools specifically for the safe manipulation of highly pathogenic Francisella tularensis subsp. tularensis. Most of these tools are also demonstrated to work with other F. tularensis subspecies. Kanamycin and hygromycin resistance determinants that function as genetic markers in F. tularensis subsp. tularensis strain Schu and sets of episomal shuttle vectors that are either unstable or stably maintained in the absence of selection were developed. In addition, the hyg gene, expressed from the F. tularensis groESL promoter, was successfully used as a marker for transposon mutagenesis. This work also includes the development of sacB-based suicide plasmids expressing kanamycin resistance that can be used for electroporation-mediated allelic exchange of unmarked mutations in Schu and the F. tularensis live vaccine strain (LVS). Using these plasmids, the two predicted b-lactamase genes, blaA and blaB, in Schu and LVS were deleted. Only the DblaB1 mutants had increased susceptibility to ampicillin, and this phenotype was complemented by a plasmid expressing blaB + . The results suggest that the b-lactam antibiotic resistance phenotype of Schu and LVS is likely due to only one of the two b-lactamase genes present and that ampicillin resistance can be used as an additional selectable marker in b-lactamase deletion mutants. The collection of tools presented in this report will be helpful for the genetic analyses of F. tularensis subsp. tularensis pathogenesis.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Genetic tools for highly pathogenic Francisella tularensis subsp. tularensis

et al. 2006

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“…The literature in the last year has contained descriptions of a new transposon mutagenesis system for F. tularensis as well as the description of the plasmids available for the use in F. tularensis (Lovullo et al, 2006), however, both were variations of the existing technologies. To date the known shuttle vectors for use in F. tularensis and Escherichia coli are all based on the F. novicida cryptic plasmid pFNL10 (Lovullo et al, 2006;Maier et al, 2004;Norqvist et al, 1996;Pavlov et al, 1996).…”

Section: Introductionmentioning

confidence: 99%

“…In this work, we describe the construction of two plasmid vectors for use in F. tularensis that are not based on the pFNL10 plasmid and thus can work in concert with these established F. tularensis vectors for complementation and or multiple gene replacements (Lovullo et al, 2006;Maier et al, 2004;Norqvist et al, 1996;Pavlov et al, 1996). We have constructed three novel plasmids named pCU18, pCUG1, and pCUG2.…”

Section: Introductionmentioning

confidence: 99%

Development of novel plasmid vectors and a promoter trap system in Francisella tularensis compatible with the pFLN10 based plasmids

Rasko

Esteban

Sperandio³

2007

Plasmid

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Francisella tularensis is a category A bioterror pathogen which in some cases can cause a severe and fatal human infection. Very few virulence factors are known in this species due to the difficulty in working with it as well as the lack of tools for genetic manipulation. This work describes the construction of a shuttle vector that can replicate in Escherichia coli and F. tularensis as well as two distinct promoter trap constructs based on the shuttle vector backbone. Replication in F. tularensis is based on the promiscuous origin of replication from the Staphylococcus aureus plasmid pC194. We demonstrate the novel plasmids can coexist with established F. tularensis vectors based on the pFNL10 plasmid, the current workhorse of F. tularensis genetics. Our promoter trap can identify promoters that are activated during intracellular growth and survival. These new vectors provide additional tools for the genetic manipulation of F. tularensis.

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Construction of a reporter plasmid for screening in vivo promoter activity in Francisella tularensis

Kuoppa

2001

FEMS Microbiology Letters

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Francisella tularensis is a facultative intracellular bacterium that survives and multiplies inside macrophages. Here we constructed a new promoter probe plasmid denoted pKK214 by introduction of a promoter-less chloramphenicol acetyltransferase (cat) gene into the shuttle vector pKK202. A promoter library was created in F. tularensis strain LVS by cloning random chromosomal DNA fragments into pKK214. Approximately 15% of the recombinant bacteria showed chloramphenicol resistance in vitro. The promoter library was also used to infect macrophages in the presence of chloramphenicol and after two cycles of infection the library contained essentially only chloramphenicol resistance clones which shows that pKK214 can be used to monitor F. tularensis genes that are expressed during infection. ß

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Construction of a shuttle vector for use in Francisella tularensis

Cited by 23 publications

References 1 publication

Genetic tools for highly pathogenic Francisella tularensis subsp. tularensis

Genetic tools for highly pathogenic Francisella tularensis subsp. tularensis

Development of novel plasmid vectors and a promoter trap system in Francisella tularensis compatible with the pFLN10 based plasmids

Construction of a reporter plasmid for screening in vivo promoter activity in Francisella tularensis

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