Fig. 1.

Schematic representation of the triparental mating and genomic integration process. A helper strain (E. coli Stbl3) is used to provide the genes necessary for conjugation in trans on the helper plasmid pRK2013. Once the helper plasmid is transferred through the conjugation bridge, the donor strain with the plasmid of interest can transfer its cargo to the recipient Clostridium strain via conjugation. The mechanism of plasmid transfer between Gram-negative and Gram-positive bacteria is currently unknown and is indicated by a dotted arrow. Genomic integration is induced by the addition of 2% xylose to the medium. Plasmid loss is promoted by repeated cultivation in YTF medium with selection on d-cycloserine and clarithromycin only, omitting thiamphenicol