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 × 10^5 to 4.8 × 10^6 colony forming units (cfu)/μg of plasmid DNA. Vector DNA was directly transferred from Escherichia coli into C. diphtheriae with frequencies up to 5.6 × 10^5 cfu/μg 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.