RT Journal Article SR Electronic(1) A1 Holtwick, Rita A1 von Wallbrunn, Angelika A1 Keweloh, Heribert A1 Meinhardt, FriedhelmYR 2001 T1 A novel rolling-circle-replicating plasmid from Pseudomonas putida P8: molecular characterization and use as vectorThe EMBL and GenBank accession number for the nucleotide sequence of pPP8–1 is AJ289784. JF Microbiology, VO 147 IS 2 SP 337 OP 344 DO https://doi.org/10.1099/00221287-147-2-337 PB Microbiology Society, SN 1465-2080, AB 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. , UL https://www.microbiologyresearch.org/content/journal/micro/10.1099/00221287-147-2-337