Dataset Information

Conditional mutagenesis using site-specific recombination in Plasmodium berghei.

ABSTRACT: Reverse genetics in Plasmodium, the genus of parasites that cause malaria, still faces major limitations. Only red blood cell stages of this haploid parasite can be transfected. Consequently, the function of many essential genes in these and subsequent stages, including those encoding vaccine candidates, cannot be addressed genetically. Here, we establish conditional mutagenesis in Plasmodium by using site-specific recombination and the Flp/FRT system of yeast. Site-specific recombination is induced after cross-fertilization in the mosquito vector of two clones containing either the target sequence flanked by two FRT sites or the Flp recombinase. Parasites that have undergone recombination are recognized in the cross progeny through the expression of a fluorescence marker. This approach should permit to dissect the function of any essential gene of Plasmodium during the haploid phase of its life, i.e., during infection of salivary glands in the mosquito and infection of both the liver and red blood cells in the mammal.

SUBMITTER: Carvalho TG

PROVIDER: S-EPMC522007 | biostudies-literature | 2004 Oct

REPOSITORIES: biostudies-literature

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Conditional mutagenesis using site-specific recombination in Plasmodium berghei.

Carvalho Teresa Gil TG Thiberge Sabine S Sakamoto Hiroshi H Ménard Robert R

Proceedings of the National Academy of Sciences of the United States of America 20041001 41

Reverse genetics in Plasmodium, the genus of parasites that cause malaria, still faces major limitations. Only red blood cell stages of this haploid parasite can be transfected. Consequently, the function of many essential genes in these and subsequent stages, including those encoding vaccine candidates, cannot be addressed genetically. Here, we establish conditional mutagenesis in Plasmodium by using site-specific recombination and the Flp/FRT system of yeast. Site-specific recombination is ind ...[more]

PMID: 15465918

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Project description:Rhodococcus opacus PD630 is a biotechnologically important bacterium with metabolic capability for bioremediation, metal recovery, and storage of triacylglycerols. Genome editing by homologous recombination in R. opacus is hampered by a very low combined frequency of DNA transfer and recombination. To improve recombination in the species, a conjugative, conditional suicide plasmid based on the replicon derived from the Corynebacterium glutamicum plasmid pGA1 was constructed and evaluated in R. opacus. The replication of this plasmid is controlled by a dual inducible and repressible promoter system originally developed for Mycobacterium spp. Next, we demonstrated that a derivative of this plasmid containing sacB as a counterselection marker and homologous regions of R. opacus could be used for homologous recombination, and that the problem of obtaining recombinants had been solved. Like for other Corynebacteriales, the cell wall of Rhodococcus spp. contains mycolic acids which form a hydrophobic and impermeable outer layer. Mycolic acids are essential for Mycobacterium smegmatis, but not for Corynebacterium glutamicum, and the new vector was used to study if mycolic acid is essential for R. opacus. We found that accD3 that is necessary for mycolic acid synthesis could only be deleted from the chromosome in strains containing a plasmid-encoded copy of accD3. This indicates that mycolic acid is important for R. opacus viability. The conditional suicide vector should be useful for homologous recombination or for delivering gene products like recombinases or Cas proteins and gRNA to Rhodococcus and related genera, while the approach should be applicable for any plasmid needing a plasmid-encoded protein for replication. KEY POINTS: • Improved vector for homologous recombination in R. opacus. • Mycolic acid is important for survival of R. opacus like it is for Mycobacterium. • Similar conditional suicide plasmids may be constructed for other bacteria.

Dataset Information

Conditional mutagenesis using site-specific recombination in Plasmodium berghei.

Publications

Conditional mutagenesis using site-specific recombination in Plasmodium berghei.

Similar Datasets

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