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.

A replicating plasmid-based vector for GFP expression in Mycoplasma hyopneumoniae

“…The slow growth of this pathogen in liquid medium, difficultly of growth in solid medium, and its resistance to accept foreign genes by standard transformation methods have further complicated the development of genetic tools for functional genomics studies of M. hyopneumoniae 168L. With the exception of two reports by the British authors describing a replicative plasmid and transposon tool for M. hyopneumoniae strain 232 (Maglennon et al, 2013a;Maglennon et al, 2013b), little progress has been made in this area. Therefore, evidence supporting the use of M. hyopneumoniae as a host for heterologous gene expression is important as the complementation of bacterial mutants requires the expression of exogenous genes.…”

“…The origin of replication (oriC) of M. hyopneumoniae was predicted based on previously described methods (Cordova et al, 2002;Maglennon et al, 2013b), as was the p97 gene promoter (Maglennon et al, 2013b). Touchdown PCR was performed with the Ex Taq Polymerase Kit (TaKaRa, Otsu, Japan) to obtain the target genes.…”

“…Electroporation of M. hyopneumoniae was done according to previously published methods with minor modifications (Maglennon et al, 2013b). Briefly, approximately 40 mL cultured M. hyopneumoniae were centrifuged at 12,000 rpm for 20 min at 4°C, and the *Restriction enzyme sites are in bold and underlined.…”

“…More recently, Maglennon et al (2013a;2013b) established methods to transform M. hyopneumoniae strain 232 with oriC-plasmids, and suggested the use of transposon mutagenesis to establish libraries of random mutations in this strain. Therefore, evidence supporting the use of M. hyopneumoniae as a host for heterologous gene expression has become a prerequisite for genetic studies regarding the pathogenicity of this organism, as the complementation of mutants requires the cloning and expression of exogenous genes.…”

Development of oriC -Based Plasmids for Mesoplasma florum

“…Many Mollicutes phylogenetically related to M. florum, including M. mycoides, M. capricolum, and Spiroplasma citri, have been successfully transformed with artificial plasmids containing a chromosomal origin of replication (oriC) (18)(19)(20)(21)(22)(23)(24)(25)(26). oriC-based plasmids have multiple uses, such as expression of exogenous genes, inactivation of target genes by recombination, or complementation of chromosomal mutations.…”