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

In Pseudomonas putida P8, three cryptic circular plasmids were detected, i. e. pPP8-1 (2.5 kbp), pPP8-2 (42 kbp) and pPP8-3 (similar to 100 kbp), Cloni ng 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 attribu ted to the latter three ORFs, whereas the predicted ORF A gene product is h omologous to replication proteins known from small multicopy plasmids of Gr am-positive bacteria and single-stranded (ss) phages, genetic elements repl icating 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 p PP8-1. along with a putative single-strand origin, Beyond this, ss replicat ion 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 vec tor was successfully used for the transformation of both Escherichia coli a nd P. putida.