A novel rolling-circle-replicating plasmid from Pseudomonas putida P8: molecular characterization and use as vector The EMBL and GenBank accession number for the nucleotide sequence of pPP8–1 is AJ289784.

Abstract

In Pseudomonas putida P8, three cryptic circular plasmids were detected, i.e. pPP8-1 (2.5 kbp), pPP8-2 (42 kbp) and pPP8-3 (approximately 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.