title: Development of a self-replicating plasmid system for Mycoplasma hyopneumoniae.
abstract: 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.
authors.affiliation: Department of Pathology and Pathogen Biology, The Royal Veterinary College, Hawkshead Lane, North Mymms, Hatfield AL9 7TA, United Kingdom. arycroft@rvc.ac.uk.
authors.forename: Gareth A
authors.initials: GA
authors.lastname: Maglennon
authors.forename: Beth S
authors.initials: BS
authors.lastname: Cook
authors.forename: Dominic
authors.initials: D
authors.lastname: Matthews
authors.forename: Alannah S
authors.initials: AS
authors.lastname: Deeney
authors.forename: Janine T
authors.initials: JT
authors.lastname: Bossé
authors.forename: Paul R
authors.initials: PR
authors.lastname: Langford
authors.forename: Duncan J
authors.initials: DJ
authors.lastname: Maskell
authors.forename: Alexander W
authors.initials: AW
authors.lastname: Tucker
authors.forename: Brendan W
authors.initials: BW
authors.lastname: Wren
authors.forename: Andrew N
authors.initials: AN
authors.lastname: Rycroft
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D045705:Mycoplasma hyopneumoniae / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D051738:Origin Recognition Complex / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids / Q000235:genetics* / Q000378:metabolism; D045729:Pneumonia of Swine, Mycoplasmal / Q000382:microbiology*; D013552:Swine; D013553:Swine Diseases / Q000382:microbiology*
chemical_list: D001426:Bacterial Proteins; C114650:OriC chromosomal replication origin; D051738:Origin Recognition Complex
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