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A novel rolling-circle-replicating plasmid from Pseudomonas putida P8: molecular characterization and use as vector

Westfälische Wilhelms-Universität Münster, Institut für Mikrobiologie, Corrensstraße 3, 48149 Münster, Germany¹

Author for correspondence: Friedhelm Meinhardt. Tel: +49 251 83 39825. Fax: +49 251 83 38388. e-mail: meinhar{at}uni-muenster.de

In Pseudomonas putida P8, three cryptic circular plasmids were detected, i.e. pPP8-1 (2·5 kbp), pPP8-2 (42 kbp) and pPP8-3 ( 100 kbp). Cloning and complete sequencing of pPP8-1 revealed a 2534 bp element harbouring four open reading frames (ORFs A, B, C and D). No function could be attributed to the latter three ORFs, whereas the predicted ORF A gene product is homologous to replication proteins known from small multicopy plasmids of Gram-positive bacteria and single-stranded (ss) phages, genetic elements replicating via a rolling circle (RC) mechanism involving characteristic ssDNA intermediates. Consistently, a double-strand origin of replication, highly conserved in rolling-circle-replicating (RCR) elements, was identified in pPP8-1, along with a putative single-strand origin. Beyond this, ss replication intermediates were confirmed by Southern analysis and mungbean-nuclease digestion. This being the first element of this type known in pseudomonads, a kanamycin-resistance gene was ligated into pPP8-1 and the resulting vector was successfully used for the transformation of both Escherichia coli and P. putida.

Keywords: extrachromosomal element, cryptic plasmid, cloning, pP81–1, repA

Abbreviations: ds, double-stranded;; DSO, double-strand origin;; IR, imperfect repeat;; Km^R, kanamycin resistance;; RC, rolling circle;; RCR, rolling circle replicating;; S/D, Shine–Dalgarno;; SSB, single-strand DNA-binding protein;; ss, single-stranded;; SSO, single-strand origin.

The EMBL and GenBank accession number for the nucleotide sequence of pPP8–1 is AJ289784.