title: Ability of IncP-9 plasmid pM3 to replicate in Escherichia coli is dependent on both rep and par functions.
abstract: IncP-9 plasmids are common in Pseudomonas species and can be transferred to other Gram-negative eubacteria but tend not to be stably maintained outside their natural host genus. A 1.3 kb ori V-rep fragment from IncP-9 plasmid pM3 was sufficient for autonomous replication in Pseudomonas putida but not in Escherichia coli. Replication of ori V-rep in E. coli was restored when additional rep was provided in trans, suggesting that the replication defect resulted from insufficient rep expression from its natural promoter. A promoter deficiency in E. coli was confirmed by reporter gene assays, transcriptional start point mapping and mutation of the promoter recognition elements. Dissection of the pM3 mini-replicon, pMT2, showed that this replication deficiency in E. coli is suppressed by additional determinants from its par operon: ParB, which can be supplied in trans, and its target, the par operon promoter, required in cis to ori V-rep. We propose that ParB binding to its target either changes plasmid DNA and thus promoter conformation or by spreading or looping contacts RNAP at the rep promoter so that rep expression is sufficient to activate ori V.
authors.affiliation: School of Biosciences, The University of Birmingham, Edgbaston, Birmingham B15 2TT, UK.
authors.forename: Yanina R
authors.initials: YR
authors.lastname: Sevastsyanovich
authors.forename: Marina A
authors.initials: MA
authors.lastname: Titok
authors.forename: Renata
authors.initials: R
authors.lastname: Krasowiak
authors.forename: Lewis E H
authors.initials: LE
authors.lastname: Bingle
authors.forename: Christopher M
authors.initials: CM
authors.lastname: Thomas
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism*; D001483:Base Sequence; D004265:DNA Helicases / Q000378:metabolism*; D019915:DNA Primase; D004261:DNA Replication; D004706:Endodeoxyribonucleases / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D005090:Exodeoxyribonucleases / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids / Q000235:genetics*; D011401:Promoter Regions, Genetic; D018741:Replication Origin; D014158:Transcription, Genetic
chemical_list: D029968:Escherichia coli Proteins; C506744:rep protein, E coli; D019915:DNA Primase; D004706:Endodeoxyribonucleases; D005090:Exodeoxyribonucleases; C121791:dnaG protein, E coli; D000251:Adenosine Triphosphatases; D004265:DNA Helicases