title: Combining the hok/sok, parDE, and pnd postsegregational killer loci to enhance plasmid stability.
abstract: To enhance plasmid segregational stability in bacterial cells, two pairs of independent postsegregational killing loci (genes which induce host killing upon plasmid loss) isolated from plasmids R1, R483, or RP4 (hok+/sok+ pnd+ or hok+/sok+ parDE+) were cloned into a common site of the beta-galactosidase expression vector pMJR1750 (ptac::lacZ+) to form a series of plasmids in which the effect of one or two stability loci on segregational plasmid stability could be discerned. Adding two antisense killer loci (hok+/sok+ pnd+) decreased the specific growth rate by 50% though they were more effective at reducing segregational instability than hok+/sok+ alone. With the ptac promoter induced fully (2.0 mM isopropyl-beta-D-thiogalactopyranoside) and no antibiotic selection pressure, the combination of a proteic killer locus (parDE+) with antisense killer loci (hok+/sok+) had a negligible impact on specific growth rate, maintained high beta-galactosidase expression, and led to a 30 and 190% increase in segregational stability (based on stable generations) as compared to plasmids containing either hok+/sok+ or parDE+ alone, respectively. Use of hok+/sok+ or parDE+ alone with high cloned-gene expression led to ninefold and fourfold increases in the number of stable generations, respectively. Two convenient cloning cassettes have been constructed to facilitate cloning the dual hok+/sok+ parDE+ and hok+/sok+ pnd+ killer systems.
authors.affiliation: Department of Chemical and Biochemical Engineering, University of California, Irvine 92697-2575, USA.
authors.forename: D C
authors.initials: DC
authors.lastname: Pecota
authors.forename: C S
authors.initials: CS
authors.lastname: Kim
authors.forename: K
authors.initials: K
authors.lastname: Wu
authors.forename: K
authors.initials: K
authors.lastname: Gerdes
authors.forename: T K
authors.initials: TK
authors.lastname: Wood
mesh_terms: D016375:Antisense Elements (Genetics) / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics*; D001427:Bacterial Toxins; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D015964:Gene Expression Regulation, Bacterial; D007763:Lac Operon; D009687:Nuclear Proteins / Q000235:genetics; D010641:Phenotype; D010957:Plasmids / Q000235:genetics*; D011401:Promoter Regions, Genetic; D012313:RNA; D012329:RNA, Bacterial; D011995:Recombination, Genetic; D015183:Restriction Mapping; D001616:beta-Galactosidase / Q000378:metabolism
chemical_list: D016375:Antisense Elements (Genetics); D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C087025:Hok protein, E coli; D009687:Nuclear Proteins; C489746:ParD protein, E coli; C489749:ParD protein, Plasmid R1; D012329:RNA, Bacterial; C087026:Sok antisense RNA, E coli; C078613:parD protein, Bacteria; C087023:pndA protein, E coli; D012313:RNA; D001616:beta-Galactosidase