Figure 3

oriC plasmid stability. Individual transformants generated with four plasmids containing various oriC regions were passaged up to 19 times in Friis medium containing tetracycline (A). Genomic DNA was extracted from cultures and analysed by Southern hybridisation using a probe annealing to the bla ampicillin resistance gene. For each plasmid, there was no decline in extrachromosomal DNA over time, and plasmid DNA could still be readily detected after 19 passages. For plasmid pOriC-iv/v, three transformants were passaged up to six times without tetracycline before undergoing selection with tetracycline (B). With each passage without selection, the number of tetracycline-resistant colonies reduced, demonstrating a loss of tetracycline-resistance. Possible integration events were predicted for plasmid pMHO-2 containing oriC-i (C). Three further pMHO-2 transformants were passaged 19 times and total DNA extracts subjected to Southern hybridisation with a tetM DIG-labelled probe, along with plasmid DNA control (P) and untransformed M. hyopneumoniae strain 232 DNA (C) (D). Two clones showed the presence of 7.4 kbp plasmid DNA and also a 4.6 kbp fragment consistent with plasmid integration by homologous recombination.