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
| Autor: | Angelika von Wallbrunn, Friedhelm Meinhardt, Rita Holtwick, Heribert Keweloh |
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| Rok vydání: | 2001 |
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| Zdroj: | Microbiology. 147:337-344 |
| ISSN: | 1465-2080 1350-0872 |
| DOI: | 10.1099/00221287-147-2-337 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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