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

Rasko, David A.; Esteban, Carlos David; Sperandio, Vanessa

doi:10.1016/j.plasmid.2007.03.002

Cited by 15 publications

(12 citation statements)

References 32 publications

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“…Apart from the aforementioned studies, little research has been done to elucidate F. tularensis promoters. To address this problem, new strategies, including the construction of promoter traps, have been developed to allow the assessment of F. tularensis promoter activity under a variety of conditions (22,32,33).…”

Section: Francisella Tularensis Is Classified By the Centers For Diseasementioning

confidence: 99%

Characterization and Application of a Glucose-Repressible Promoter in Francisella tularensis

Horzempa¹,

Tarwacki²,

Carlson³

et al. 2008

Appl Environ Microbiol

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Francisella tularensis, the causative agent of tularemia, is a category A biodefense agent. The examination of gene function in this organism is limited due to the lack of available controllable promoters. Here, we identify a promoter element of F. tularensis LVS that is repressed by glucose (termed the Francisella glucose-repressible promoter, or FGRp), allowing the management of downstream gene expression. In bacteria cultured in medium lacking glucose, this promoter induced the expression of a red fluorescent protein allele, tdtomato. FGRp activity was used to produce antisense RNA of iglC, an important virulence factor, which severely reduced IglC protein levels. Cultivation in glucose-containing medium restored IglC levels, indicating the usefulness of this promoter for controlling both exogenous and chromosomal gene expression. Moreover, FGRp was shown to be active during the infection of human macrophages by using the fluorescence reporter. In this environment, the FGRp-mediated expression of antisense iglC by F. tularensis LVS resulted in reduced bacterial fitness, demonstrating the applicability of this promoter. An analysis of the genomic sequence indicated that this promoter region controls a gene, FTL_0580, encoding a hypothetical protein. A deletion analysis determined the critical sites essential for FGRp activity to be located within a 44-bp region. This is the first report of a conditional promoter and the use of antisense constructs in F. tularensis, valuable genetic tools for studying gene function both in vitro and in vivo.

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Section: Francisella Tularensis Is Classified By the Centers For Diseasementioning

confidence: 99%

Characterization and Application of a Glucose-Repressible Promoter in Francisella tularensis

Horzempa¹,

Tarwacki²,

Carlson³

et al. 2008

Appl Environ Microbiol

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“…Other promoters include the acpA promoter (34), which was used to drive the expression of green fluorescent protein (GFP) in F. tularensis strain LVS during a murine macrophage infection (27), and the FTN_1451 promoter (11), which was used to express the kanamycin resistance gene in the process of adapting the Targeton system for use in F. tularensis (35,36). Promoter trap studies were previously conducted in F. tularensis LVS, resulting in the identification of several promoters that were active in vivo, but to the best of our knowledge, their identity was not disclosed (18,33). In addition, an LVS promoter trap library constructed and screened in Escherichia coli resulted in the characterization of the FTL_0580 glucose-repressible promoter (15).…”

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confidence: 99%

Identification and Characterization of Novel and Potent Transcription Promoters of Francisella tularensis

Zaide

Grosfeld

Ehrlich

et al. 2011

Appl Environ Microbiol

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Two alternative promoter trap libraries, based on the green fluorescence protein (gfp) reporter and on the chloramphenicol acetyltransferase (cat) cassette, were constructed for isolation of potent Francisella tularensis promoters. Of the 26,000 F. tularensis strain LVS gfp library clones, only 3 exhibited visible fluorescence following UV illumination and all appeared to carry the bacterioferritin promoter (Pbfr). Out of a total of 2,000 chloramphenicol-resistant LVS clones isolated from the cat promoter library, we arbitrarily selected 40 for further analysis. Over 80% of these clones carry unique F. tularensis DNA sequences which appear to drive a wide range of protein expression, as determined by specific chloramphenicol acetyltransferase (CAT) Western dot blot and enzymatic assays. The DNA sequence information for the 33 unique and novel F. tularensis promoters reported here, along with the results of in silico and primer extension analyses, suggest that F. tularensis possesses classical Escherichia coli 70 -related promoter motifs. These motifs include the ؊10 (TATAAT) and ؊35 [TTGA(C/T)A] domains and an AT-rich region upstream from ؊35, reminiscent of but distinct from the E. coli upstream region that is termed the UP element. The most efficient promoter identified (Pbfr) appears to be about 10 times more potent than the F. tularensis groEL promoter and is probably among the strongest promoters in F. tularensis. The battery of promoters identified in this work will be useful, among other things, for genetic manipulation in the background of F. tularensis intended to gain better understanding of the mechanisms involved in pathogenesis and virulence, as well as for vaccine development studies.

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“…Recent advances, however, have overcome this limitation with the development of shuttle plasmids (6,40,41,57), transposons (23,33,42), and allelic replacement strategies (27,36). The genes predicted to foster intracellular growth and virulence include the genes in operons encoding iglABCD and pdpABCD proteins present on the Francisella pathogenicity island (FPI), as well as genes involved in oxidative stress or protein turnover, capsule or lipopolysaccharide (LPS) biosynthesis, type IV pilin assembly, iron uptake, outer membrane channels, purine biosynthesis, and regulation through mglAB or pmrA (44,46,50,51,53,56,65,66).…”

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Identification ofFrancisella tularensis Himar1-Based Transposon Mutants Defective for Replication in Macrophages

et al. 2007

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Francisella tularensis, the etiologic agent of tularemia in humans, is a potential biological threat due to its low infectious dose and multiple routes of entry. F. tularensis replicates within several cell types, eventually causing cell death by inducing apoptosis. In this study, a modified Himar1 transposon (HimarFT) was used to mutagenize F. tularensis LVS. Approximately 7,000 Km r clones were screened using J774A.1 macrophages for reduction in cytopathogenicity based on retention of the cell monolayer. A total of 441 candidates with significant host cell retention compared to the parent were identified following screening in a high-throughput format. Retesting at a defined multiplicity of infection followed by in vitro growth analyses resulted in identification of approximately 70 candidates representing 26 unique loci involved in macrophage replication and/or cytotoxicity. Mutants carrying insertions in seven hypothetical genes were screened in a mouse model of infection, and all strains tested appeared to be attenuated, which validated the initial in vitro results obtained with cultured macrophages. Complementation and reverse transcription-PCR experiments suggested that the expression of genes adjacent to the HimarFT insertion may be affected depending on the orientation of the constitutive groEL promoter region used to ensure transcription of the selective marker in the transposon. A hypothetical gene, FTL_0706, postulated to be important for lipopolysaccharide biosynthesis, was confirmed to be a gene involved in O-antigen expression in F. tularensis LVS and Schu S4. These and other studies demonstrate that therapeutic targets, vaccine candidates, or virulence-related genes may be discovered utilizing classical genetic approaches in Francisella.Francisella tularensis is a gram-negative intracellular pathogen and the etiologic agent of human tularemia. The CDC has classified F. tularensis as a category A select agent due to its highly infectious nature and ease of dissemination. Four subspecies of F. tularensis have been recognized, including (i) the virulent type A F. tularensis subsp. tularensis, (ii) the less virulent type B F. tularensis subsp. holarctica, (iii) F. tularensis subsp. mediasiatica, and (iv) F. tularensis subsp. novicida. The F. tularensis LVS (live vaccine strain) is derived from F. tularensis subsp. holarctica and is used as a model system to identify Francisella virulence factors since it is attenuated in humans but virulent in mice (8, 21). The limited genetic variation (2 to 4%) between the subspecies of Francisella suggests that there is potential overlap among genes related to pathogenesis (7,54,59). In fact, F. tularensis LVS and Schu S4 vary in genomic sequence by less than 1% (59). Regardless of the high sequence similarity at the genomic level, genome rearrangement and variation at the functional or regulatory level among the subspecies clearly result in phenotypes that impact virulence and pathogenesis (12,15,29,54,73,74).The life cycle of F. tularensis inside the macrophage ...

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

Cited by 15 publications

References 32 publications

Characterization and Application of a Glucose-Repressible Promoter in Francisella tularensis

Characterization and Application of a Glucose-Repressible Promoter in Francisella tularensis

Identification and Characterization of Novel and Potent Transcription Promoters of Francisella tularensis

Identification ofFrancisella tularensis Himar1-Based Transposon Mutants Defective for Replication in Macrophages

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