Mycoplasma hyopneumoniae is a prevalent swine respiratory pathogen that is a major cause of economic loss to
pig producers. Control is achieved by a combination of antimicrobials, vaccination and management practices, but
current vaccines offer only partial control and there is a need for improved preventative strategies. A major barrier
to advances in understanding the pathogenesis of M. hyopneumoniae and in developing new vaccines is the lack
of tools to genetically manipulate the organism. We describe the development and optimisation of the first
successful plasmid-based system for the genetic manipulation of M. hyopneumoniae. Our artificial plasmids contain
the origin of replication (oriC) of M. hyopneumoniae along with tetM, conferring resistance to tetracycline. With
these plasmids, we have successfully transformed M. hyopneumoniae strain 232 by electroporation, generating
tetracycline resistant organisms. The persistence of extrachromosomal plasmid and maintenance of plasmid DNA
over serial passages shows that these artificial plasmids are capable of self-replication in M. hyopneumoniae. In
addition to demonstrating the amenability of M. hyopneumoniae to genetic manipulation and in optimising the
conditions necessary for successful transformation, we have used this system to determine the minimum functional
oriC of M. hyopneumoniae. In doing so, we have developed a plasmid with a small oriC that is stably maintained
over multiple passages that may be useful in generating targeted gene disruptions. In conclusion, we have
generated a set of plasmids that will be valuable in studies of M. hyopneumoniae pathogenesis and provide a major
step forward in the study of this important swine pathogen.