title: Characterization of pMRI 5.2, a rolling-circle-type plasmid from Lactobacillus plantarum BFE 5092 which harbours two different replication initiation genes.
abstract: Plasmid pMRI 5.2 from Lactobacillus plantarum BFE 5092 was sequenced and analysed. The sequence consists of 5206bp with a mol% G+C content of 35.8%. Nine putative open reading frames were identified. A typical pC194 family double strand origin (dso) and a putative single strand origin (sso) were predicted upstream of a rep gene. This rep gene encoded a replication protein of 314 amino acids exhibiting 98% amino acid sequence identity to the Rep protein of plasmid pLAB1000 from Lactobacillus hilgardii. A mob gene encoding a mobilization protein was also identified and this protein showed high amino acid similarity to Mob proteins from various L. plantarum plasmids. Downstream of the mob gene, a second putative replication region was identified that is similar to the pMV158 family of plasmids. It contains a dso as well as a putative sso, and encodes the 52 amino acid repressor-like protein RepA, the replication initiation protein RepB of 215 amino acids, and the 48 amino acid RepC that is similar to ORFD of the lactococcal plasmid pWVO1. RT-PCR and qRT-PCR expression analyses of the rep and repB genes showed that the repB gene was expressed at a higher level. To confirm that the plasmid replicated by the rolling-circle-type mechanism, the presence of a characteristic single strand intermediate DNA was shown to be produced during replication. Plasmid copy number was ca. 30 per equivalent chromosome copy number based on qRT-PCR analyses. The plasmid also encodes four additional putative proteins of unknown function. The unusual feature of a rolling-circle plasmid having two different plasmid-encoded replication initiation proteins from different replicon families suggests that the genes for these may have originated from different plasmids.
authors.affiliation: Department of Safety and Quality of Fruit and Vegetables, Max Rubner-Institute, Federal Research Institute for Nutrition and Food, Haid-und-Neu-Strasse 9, D-76131 Karlsruhe, Germany.
authors.forename: Gyu-Sung
authors.initials: GS
authors.lastname: Cho
authors.forename: Melanie
authors.initials: M
authors.lastname: Huch
authors.forename: Julius Maina
authors.initials: JM
authors.lastname: Mathara
authors.forename: Marco J
authors.initials: MJ
authors.lastname: van Belkum
authors.forename: Charles M A P
authors.initials: CM
authors.lastname: Franz
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D004270:DNA, Circular / Q000235:genetics*; D004277:DNA, Single-Stranded; D004587:Electrophoresis, Agar Gel; D018628:Gene Dosage / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial / Q000235:genetics*; D048191:Lactiplantibacillus plantarum / Q000235:genetics*; D008969:Molecular Sequence Data; D010802:Phylogeny; D010957:Plasmids / Q000235:genetics*; D016133:Polymerase Chain Reaction; D018741:Replication Origin / Q000235:genetics*; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
chemical_list: D001426:Bacterial Proteins; D004270:DNA, Circular; D004277:DNA, Single-Stranded