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 ␤-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-␤-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 ␤-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.

By complementing cell-free extracts of fseudomonas putida Fl/pSMM020 with purified soluble methane monooxygenase (sMMO) components of Methylosinus trichosporium OB3b, the low cloned-gene sMMO activity in the recombinant strain was found to be due to incomplete activity of the hydroxylase component. To address this incomplete activity, additional sMMO-expressing strains were formed by transferring mmo-containing pSMM020 and pSMM050 into various bacterial species including pseudomonads and a-2 subdivision strains such as methanotrophs, methylotrophs, Agrobacterium tumefaciens A114, and Rhizobium meliloti 102F34 (11 new strains screened); sMMO activity was detected in the last two strains. To increase plasmid segregational stability, the hok/sok locus originally from Escherichia coli plasmid R1 was inserted downstream of the mmo locus of pSMMO2O (resulting in pSMM040) and found to enhance plasmid stability in f . putida F1 and R. meliloti 102F34 (first report of hok/sokin Rhizobium). To further increase sMMO activity, a modified Whittenbury minimal medium was selected from various minimal and complex media based on trichloroethylene (TCE) degradation and growth rates and was improved by removing the sMMO-inhibiting metal ions [Cu(ll), Ni(ll), and Zn(ll)l and chloramphenicol from the medium and by supplementing with an iron source (3.6 pM of ferrous ammonium sulfate). Using chemostat-grown f . putida Fl/pSMM040, it was found that sMMO activity was higher for cells grown at higher dilution rates. These optimization efforts resulted in a twofold increase in the extent of TCE degradation and more consistent sMMO activity.