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

• Published in: Microbiology (Society for General Microbiology) Vol. 147; no. 2; pp. 337 - 344
• Main Authors: Holtwick, Rita, von Wallbrunn, Angelika, Keweloh, Heribert, Meinhardt, Friedhelm
• Format: Journal Article
• Language: English
• Published: Reading Soc General Microbiol 01.02.2001; Society for General Microbiology
• Subjects: Amino Acid Sequence; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Bacteriology; Base Sequence; Biological and medical sciences; Biotechnology; Cloning, Molecular; DNA - genetics; DNA Replication; DNA, Single-Stranded - genetics; Fundamental and applied biological sciences. Psychology; Gene Expression Regulation, Bacterial; Genetic engineering; Genetic technics; Genetic Vectors; Genetics; Methods. Procedures. Technologies; Microbiology; Molecular Sequence Data; Open Reading Frames - genetics; Plasmids - genetics; Pseudomonas putida - genetics; Recombination, Genetic; Replication Origin - genetics; Sequence Analysis, DNA; Transformation, Genetic; Vectors (cloning, transfer, expression). Insertion sequences and transposons; Pseudomonadales; Plasmid; Nucleotide sequence; Bacteria; Pseudomonadaceae; Homology; Replication; Molecular cloning; Pseudomonas putida; Vector; Mechanism; Aminoacid sequence
• ISSN: 1350-0872; 1465-2080
• DOI: 10.1099/00221287-147-2-337

Westfälische Wilhelms-Universität Münster, Institut für Mikrobiologie, Corrensstraße 3, 48149 Münster, Germany 1 Author for correspondence: Friedhelm Meinhardt. Tel: +49 251 83 39825. Fax: +49 251 83 38388. e-mail: meinhar{at}uni-muenster.de 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. Keywords: extrachromosomal element, cryptic plasmid, cloning, pP81–1, repA Abbreviations: ds, double-stranded;; DSO, double-strand origin;; IR, imperfect repeat;; Km R , kanamycin resistance;; RC, rolling circle;; RCR, rolling circle replicating;; S/D, Shine–Dalgarno;; SSB, single-strand DNA-binding protein;; ss, single-stranded;; SSO, single-strand origin. The EMBL and GenBank accession number for the nucleotide sequence of pPP8–1 is AJ289784 .