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Characterization of the replicator region of megaplasmid pTAV3 of Paracoccus versutus and search for plasmid-encoded traits

Department of Bacterial Genetics, Institute of Microbiology, Warsaw University, Miecznikowa 1, 02-096 Warsaw, Poland¹

Author for correspondence: Dariusz Bartosik. Tel: +48 22 554 13 44. Fax: +48 22 554 14 04. e-mail: bartosik{at}biol.uw.edu.pl

The replicon of the pTAV3 megaplasmid (approx. 400 kb) of Paracoccus versutus has been localized to a 4·3 kb EcoRI restriction fragment and its entire nucleotide sequence determined. The G+C content of the entire sequence is 66 mol%, which is within the range (62–66 mol%) previously determined for P. versutus total DNA. ORF1 encodes a replication initiation protein Rep (47·2 kDa), which shares substantial similarity with putative proteins of the Coxiella burnetii plasmids QpH1 and QpDV, and the replication protein of Pseudomonas syringae plasmid pPS10. ORF2, located in the opposite transcriptional orientation to ORF1, encodes a putative protein that shares similarity to a subfamily of ATPases involved in plasmid partitioning. The highest similarity was observed with homologous proteins (RepA) encoded by the repABC family of replicons found in several plasmids of Agrobacterium, Rhizobium and Paracoccus spp. The predicted product of ORF3 was similar to AcoR, Nif and NtrC transcriptional activators. A strong incompatibility determinant (inc) was localized between ORF1 (rep) and ORF2 (parA). The origin of replication of pTAV400 contains a short A+T-rich region and several imperfect palindromic sequences. Curing experiments demonstrated that the megaplasmid bears genes required for growth in minimal media and can therefore be referred to as a mini-chromosome. Megaplasmids pTAV3 of P. versutus UW1 and pKLW2 of Paracoccus pantotrophus DSM 11073 were found to carry closely related, incompatible replicons. It has been shown that plasmid pORI6 (containing oriV of pTAV3 cloned into plasmid pABW1, which does not replicate in Paracoccus spp.) can be trans activated not only by pTAV3, but also by pKLW2. Using pORI6, it was demonstrated that replication systems related to pTAV3 are also present in the replicons of Paracoccus alcaliphilus JCM 7364, Paracoccus thiocyanatus IAM 12816 and Paracoccus methylutens DM 12.

Keywords: Paracoccus versutus, pTAV3 megaplasmid, mini-replicon

Abbreviations: DR, direct repeat; IR, inverted repeat; Km, kanamycin; MCS, multiple cloning site; Rif, rifampicin; Tc, tetracycline

The GenBank accession number for the sequence reported in this paper is AF390867.

^a Present address: Department of Microbial Biochemistry, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawinskiego 5a, 02-106 Warsaw, Poland.

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