Stability of r-microbes: stabilization of plasmid vectors by the partitioning function of broad-host-range plasmid RP4.

The genes for biosynthesis of the biodegradable polymer poly-beta-hydroxybutyric acid (PHB) cloned from Alcaligenes eutrophus H16 were used for synthesis of PHB with recombinant Escherichia coli strains. It was recognized that the PHB-biosynthesis genes cause segregational instability to the plasmids used as vectors. Recombinant PHB-plasmids are rapidly lost from host cells and plasmid-free cells occur at high rates, even under conditions of selection for the plasmids. Cloning the partitioning region of plasmid RP4 onto such plasmids resulted in a high degree of stabilization. These par-stabilized recombinant PHB-plasmids could be maintained quite efficiently in batch cultivation experiments in the absence of any selection pressure.

Partitioning of broad-host-range plasmid RP4 is a complex system involving site-specific recombination.

The broad-host-range plasmid RP4 encodes a highly efficient partitioning system (par) that was previously mapped within the 6.2-kb PstI C fragment. The essential functions were assigned to a region of 2.2 kb between fiwA and IS21 (IS8). On the basis of the nucleotide sequence data of the entire par locus and of in vitro and in vivo expression studies, three distinct loci encoding polypeptides of 9, 18, and 24 kDa were identified. Evidence for the expression of another polypeptide was found. A putative divergent promoter was localized in an intergenic region and is suggested to be responsible for transcription of these genes. It was found that the RP4 par region includes a function resolving plasmid dimers. The 24-kDa polypeptide is considered to function as a resolvase, since its predicted amino acid sequence shows homology to sequences of resolvases of the Tn3 family. Furthermore, palindromes present in the intergenic region containing the divergent promoter resemble repeat structures specific for res sites of Tn3-related transposons. However, it was found that dimer resolution itself was not sufficient for stabilization; additional functions, including the other two polypeptides, seemed to play an important role. These results suggested that RP4 contains a complex stabilization system involving resolution of plasmid dimers during cell division, thus ensuring the delivery of at least one copy to each daughter cell.