A conditional suicide system for Saccharomyces cerevisiae relying on the intracellular production of the Serratia marcescens nuclease.
A conditional lethal system for biological containment of genetically modified strains of Saccharomyces cerevisiae is described. This suicide system is based on the intracellular production of the Serratia marcescens nuclease in the yeast cell, aiming at the destruction of the host genetic material. The S. marcescens nuclease, encoded by the nucA gene, is normally secreted by the bacterium into the medium. In the present work, the nucA gene, devoid of its signal peptide coding sequence, was cloned in a yeast expression vector, under control of the glucose-repressed S. cerevisiae alcohol dehydrogenase 2 gene (ADH2) promoter. When transformed into S. cerevisiae, the recombinant plasmid proved to be effective in killing the host cells upon glucose depletion from the medium, and the nuclease activity was found in lysates prepared from the transformants. In addition, the nuclease degrading effect was shown to reach chromosomal DNA in the yeast host. The killing effect of the nucA plasmid was also demonstrated in soil microcosm assays, indicating that whenever the GMM escapes into the environment where glucose is scarce, the nucA gene will be expressed and the resulting nuclease will destroy the genetic material and kill the cells. In contrast to other suicide systems that target the cell envelope, the advantage of the one described here is that it disfavours horizontal gene transfer from recombinant yeast cells to other microorganisms found in the environment.
Balan
Andrea
A
Universidade de São Paulo, Instituto de Ciências Biomédicas, Departamento de Microbiologia, Avenida Professor Lineu Prestes 1374, São Paulo 05508-900, SP, Brasil.
Schenberg
Ana Clara G
AC
D015153:Blotting, Western; D015825:Chromosomes, Fungal / Q000235:genetics; D020552:Comet Assay; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics; D004706:Endodeoxyribonucleases / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D004722:Endoribonucleases / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D041722:Genes, Transgenic, Suicide / Q000235:genetics*; D005947:Glucose / Q000378:metabolism; D016133:Polymerase Chain Reaction; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics / Q000254:growth & development*; D012706:Serratia marcescens / Q000201:enzymology* / Q000235:genetics*; D012988:Soil Microbiology; D014170:Transformation, Genetic
D004271:DNA, Fungal; D004706:Endodeoxyribonucleases; D004722:Endoribonucleases; C021835:Serratia marcescens nuclease; D005947:Glucose