Combining the hok/sok, parDE, and pnd postsegregational killer loci to enhance plasmid stability

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 β-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- β-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 β-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.