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.

Figure 1

oriC plasmid construction. A 2.1 kbp oriC region of M. hyopneumoniae strain 232 was predicted based on the location of putative DnaA boxes (black arrowheads), the location of dnaA and the presence of short AT-rich regions of 144 bp and 208 bp with an AT content of 0.83 and 0.84 respectively (A). The P97 gene (B) and ldh gene (C) promoter sequences were predicted based on proximity to the ATG start codon (shown in bold) and putative TATA box location (underlined). The oriC sequence was cloned into the NcoI and SpeI restriction sites of pGEM-T to generate plasmid pMHO. The tetM gene with the E. faecalis, spiralin gene, P97 and ldh promoter sequence was cloned into the PstI and SpeI sites to produce plasmids pMHO-1, pMHO-2, pMHO-3 and pMHO-4 respectively (D). M. hyopneumoniae strain 232 was transformed with each plasmid and transformants (pMHO-2 in E) grown in Friis medium with tetracycline selection. After 3 passages, plasmid DNA was extracted from two individual clones (C1 and C2) and analysed by agarose gel electrophoresis along with plasmid (P) DNA control (F).

Figure 2

Minimum oriC determination. The predicted oriC region of M. hyopneumoniae strain 232 is shown with the location of two putative DnaA boxes (black arrowheads) lying within AT-rich intergenic regions (grey boxes). The oriC region from plasmid pMHO (oriC-i) was reduced in a series of steps to produce five further plasmids (pOriC-ii, pOriC-iii, pOriC-iv, pOriC-iii/iv and pOriC-iv/v). M. hyopneumoniae strain 232 was transformed in triplicate with each plasmid and the mean transformation efficiencies (TF) are shown, expressed as the number of transformants per colony forming unit of mycoplasma transformed.

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.