COMBINING THE HOK SOK, PARDE, AND PND POSTSEGREGATIONAL KILLER LOCI TO ENHANCE PLASMID STABILITY/

To enhance plasmid segregational stability in bacterial cells, two pai rs of independent postsegregational killing loci (genes which induce h ost killing upon plasmid loss) isolated from plasmids R1, R483, or RP4 (hok(+)/sok(+) pnd(+) or hok(+)/sok(+) parDE(+)) were cloned into a c ommon site of the beta-galactosidase expression vector pMJR1750 (ptac: :lacZ(+)) to form a series of plasmids in which the effect of one or t wo stability loci on segregational plasmid stability could be discerne d. Adding two antisense killer loci (hok(+)/sok(+) pnd(+)) decreased t he specific growth rate by 50% though they were more effective at redu cing 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 k iller locus (parDE(+)) with antisense killer loci (hok(+)/sok(+)) had a negligible impact on specific growth rate, maintained high beta-gala ctosidase expression, and led to a 30 and 190% increase in segregation al stability (based on stable generations) as compared to plasmids con taining either hok(+)/sok(+) or parDE(+) alone, respectively, Use of h ok(+)/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 t o facilitate cloning the dual hok(+)/sok(+) parDE(+) and hok(+)/sok(+) pnd(+) killer systems.