Genetic Organization of the Francisella Plasmid pFNL10

Pomerantsev, Andrei P.; Golovliov, Igor; Ohara, Yoshikazu; Мокриевич, А. Н.; Obuchi, Masatsugu; Norqvist, T. Anders; Kuoppa, Kerstin; Павлов, В. М.

doi:10.1006/plas.2001.1548

Cited by 37 publications

(69 citation statements)

References 26 publications

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“…PCR reactions with various thermostable polymerases were done according to the manufacturer's recommendations. All plasmids used in this study (Table 1) were from our laboratory's collection, except the cryptic Francisella plasmid pFNL10 (Pomerantsev et al, 2001a), which was a generous gift from Fran Nano of the University of Victoria, BC, Canada, and the shuttle vector pFNLTP1 (Maier et al, 2004), which was kindly provided by Tom Zahrt of the Medical College of Wisconsin. Plasmids were prepared from E. coli and F. tularensis strains using either a standard alkaline lysis protocol or with Qiagen columns.…”

Section: Methodsmentioning

confidence: 99%

“…1) (Pomerantsev et al, 2001a) that includes the origin of replication, the repA and ORF2 genes and their promoter region, but no other intact ORFs from pFNL10.…”

Section: First-generation Shuttle Vectorsmentioning

confidence: 99%

“…To this end, we cloned the ORF4-ORF5 region of pFNL10, encoding a putative 'addiction system' (Pomerantsev et al, 2001a), into pMP527 to produce pMP607 (Fig. 1).…”

Section: Plasmid Maintenance and Second-generation Shuttle Vectorsmentioning

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: Methodsmentioning

confidence: 99%

“…1) (Pomerantsev et al, 2001a) that includes the origin of replication, the repA and ORF2 genes and their promoter region, but no other intact ORFs from pFNL10.…”

Section: First-generation Shuttle Vectorsmentioning

confidence: 99%

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Genetic tools for highly pathogenic Francisella tularensis subsp. tularensis

et al. 2006

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“…In constructing these plasmid vectors, we took advantage of the promiscuous nature of the pC194 origin of replication (Horinouchi and Weisblum, 1982) to overcome the problem of the pFNL10 based plasmid vectors that are commonly used in F. tularensis work (Pavlov et al, 1996;Pomerantsev et al, 2001aPomerantsev et al, , 2001b. The pC194 origin can replicate within F. tularensis LVS.…”

Section: Resultsmentioning

confidence: 99%

“…These plasmids have an advantage over existing shuttle or promoter trap vectors in that they are not based on the pFNL10 plasmid and thus can be used in tandem with those vectors that have been described in the literature and are truly the gold standard for F. tularensis vectors (Pavlov et al, 1996;Pomerantsev et al, 2001aPomerantsev et al, , 2001b. Our promoter trap strategy allows the identification of promoters that can be activated by defined stimuli or during intracellular growth.…”

Section: Resultsmentioning

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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Limited Genomes and Gene Transfer in the Evolution of Intracellular Parasitism and Symbiosis

Somboonna

Dean

2009

Intracellular Niches of Microbes

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Genetic Organization of the Francisella Plasmid pFNL10

Cited by 37 publications

References 26 publications

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

Limited Genomes and Gene Transfer in the Evolution of Intracellular Parasitism and Symbiosis

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