Identification of a partition and replication region in theAlcaligenes eutrophus megaplasmid pMOL28
A 4.64 kb region of the 180 kb heavy metal resistance plasmid pMOL28 ofAlcaligenes eutrophus CH34, previously shown to be able to replicate autonomously, was sequenced and analyzed. Three genes involved in plasmid maintenance were identified:parA28 andparB28 are involved in plasmid partitioning and stability, whilerepA28 encodes a protein required for replication. In addition to theparAB28 genes, a third locus,parS28, required in cis for active partitioning was identified. TheparABS28 locus of pMOL28 shows strong similarity in organization to thesop, par andrep regions, respectively, of theEscherichia coli F-factor, theE. coli P1 and P7 prophages, and theAgrobacterium pTiB6S3 and pRiA4b plasmids. The ParAB28 proteins of pMOL28 also show similarity to the proteins encoded by two conserved open reading frames present in the replication regions of thePseudomonas putida andBacillus subtilus chromosomes. The functionality of the pMOL28par region was examined by performing stability and incompatibility tests between pMOL28 and pMOL846 or pMOL850, which contain the 4.64EcoRI replicon fragment of pMOL28, cloned in opposite orientations into pSUP202, which is itself unable to replicate inA. eutrophus. The RepA28 replication protein showed similarity to the RepL protein of P1, which is required for lytic replication of thisE. coli phage. The replication origin of pMOL28,oriV28, seems to be located within therepA28 coding region, and pMOL28 replication may depend on transcriptional activation oforiV28.
Key wordsAlcaligenes eutrophus pMOL28 Plasmid replication Plasmid partition Plasmid stability
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