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 (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.
Holtwick
Rita
R
Westfälische Wilhelms-Universität Münster, Institut für Mikrobiologie, Corrensstraße 3, 48149 Münster, Germany1.
von Wallbrunn
Angelika
A
Westfälische Wilhelms-Universität Münster, Institut für Mikrobiologie, Corrensstraße 3, 48149 Münster, Germany1.
Keweloh
Heribert
H
Westfälische Wilhelms-Universität Münster, Institut für Mikrobiologie, Corrensstraße 3, 48149 Münster, Germany1.
Meinhardt
Friedhelm
F
Westfälische Wilhelms-Universität Münster, Institut für Mikrobiologie, Corrensstraße 3, 48149 Münster, Germany1.
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D004261:DNA Replication; D004277:DNA, Single-Stranded / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D016366:Open Reading Frames / Q000235:genetics; D010957:Plasmids / Q000235:genetics*; D016958:Pseudomonas putida / Q000235:genetics*; D011995:Recombination, Genetic; D018741:Replication Origin / Q000235:genetics; D017422:Sequence Analysis, DNA; D014170:Transformation, Genetic
D001426:Bacterial Proteins; D004277:DNA, Single-Stranded; D004247:DNA