title: Efficient electrotransformation of corynebacterium diphtheriae with a mini-replicon derived from the Corynebacterium glutamicum plasmid pGA1.
abstract: Efficient transformation of the human pathogen Corynebacterium diphtheriae was achieved with novel cloning vectors consisting of a mini-replicon from the cryptic C. glutamicum plasmid pGA1 as well as of the aph(3')-IIa or tetA(Z ) antibiotic resistance genes. Plasmid-containing transformants of C. diphtheriae were recovered at frequencies ranging from 1.3 x 10(5) to 4.8 x 10(6) colony forming units (cfu)/microg of plasmid DNA. Vector DNA was directly transferred from Escherichia coli into C. diphtheriae with frequencies up to 5.6 x 10(5) cfu/microg of plasmid DNA. On the basis of the pGA1 mini-replicon, an expression vector system was established for C. diphtheriae by means of the P(tac) promoter and the green fluorescent reporter protein. In addition, other commonly used vector systems from C. glutamicum, including the pBL1 and pHM1519 replicons, and the sacB conditionally lethal selection marker from Bacillus subtilis, were shown to be functional in C. diphtheriae. Thus, the ability to apply the standard methods of C. glutamicum recombinant DNA technology will greatly facilitate the functional analysis of the recently completed C. diphtheriae genome sequence.
authors.affiliation: Zentrum für Genomforschung, Universität Bielefeld, Universitätsstrasse 25, D-33615 Bielefeld, Germany. Andreas.Tauch@genetik.unibielefeld.de
authors.forename: Andreas
authors.initials: A
authors.lastname: Tauch
authors.forename: Oliver
authors.initials: O
authors.lastname: Kirchner
authors.forename: Britta
authors.initials: B
authors.lastname: Löffler
authors.forename: Susanne
authors.initials: S
authors.lastname: Götker
authors.forename: Alfred
authors.initials: A
authors.lastname: Pühler
authors.forename: Jörn
authors.initials: J
authors.lastname: Kalinowski
mesh_terms: D001709:Biotechnology; D003353:Corynebacterium diphtheriae / Q000737:chemistry / Q000235:genetics*; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics*; D018274:Electroporation; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics; D005822:Genetic Vectors / Q000737:chemistry; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000737:chemistry; D010957:Plasmids / Q000737:chemistry / Q000235:genetics*; D011454:Prostaglandins A; D014169:Transformation, Bacterial
chemical_list: D004269:DNA, Bacterial; D008164:Luminescent Proteins; D011454:Prostaglandins A; D049452:Green Fluorescent Proteins; C100573:prostaglandin A1