title: Replication system of plasmid pMTH4 of Paracoccus methylutens DM12 contains an enhancer.
abstract: The replication system of plasmid pMTH4 (22 kb) of dichloromethane-degrading Paracoccus methylutens DM12 (Alphaproteobacteria) has been cloned within a mini-replicon pMTH100 (4.7 kb) and preliminarily characterized. Functional analysis, performed with a series of mutated plasmid mini-derivatives, showed that the replicator region consists of three elements: (i) gene repA coding for a replication initiation protein RepA, (ii) origin of replication (oriV), placed in the promoter region of repA and containing a set of imperfect directly repeated sequences (iterons) together with putative DnaA and IHF-binding DNA sequences as well as (iii) an enhancer (0.65 kb) upstream of oriV. We showed that the enhancer was not crucial for plasmid replication, however, it was necessary to assure the proper plasmid copy number. Additionally its presence has increased the strength of a determinant of incompatibility (located within the oriV region) as well as the level of transcription carried from the repA promoter. The enhancer region was shown not to encode any proteins or promoter sequences. We speculate that this region might constitute a site of binding of plasmid or host-encoded proteins that are able to interact with the origin, which positively regulates the initiation of replication.
authors.affiliation: Department of Bacterial Genetics, Institute of Microbiology, Warsaw University, Warsaw, Poland.
authors.forename: Michał
authors.initials: M
authors.lastname: Szymanik
authors.forename: Renata
authors.initials: R
authors.lastname: Welc-Faleciak
authors.forename: Dariusz
authors.initials: D
authors.lastname: Bartosik
authors.forename: Mirosława
authors.initials: M
authors.lastname: Włodarczyk
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D017931:DNA Primers / Q000737:chemistry; D004261:DNA Replication / Q000235:genetics*; D004742:Enhancer Elements, Genetic / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D023061:Gene Order; D008969:Molecular Sequence Data; D016366:Open Reading Frames / Q000235:genetics; D010230:Paracoccus / Q000235:genetics*; D010957:Plasmids / Q000235:genetics*; D016133:Polymerase Chain Reaction; D018741:Replication Origin / Q000235:genetics
chemical_list: D017931:DNA Primers