Development of a site-directed integration plasmid for heterologous gene expression in Mycoplasma gallisepticum

PLoS One. 2013 Nov 20;8(11):e81481. doi: 10.1371/journal.pone.0081481. eCollection 2013.

Abstract

Deciphering the molecular basis of the interactions between the parasite Mycoplasma gallisepticum and its avian hosts suffers from the lack of genetic tools available for the pathogen. In the absence of well established methods for targeted disruption of relevant M. gallisepticum genes, we started to develop suicide vectors and equipped them with a short fragment of M. gallisepticum origin or replication (oriC MG). We failed to create a disruption vector, although by adding a further short fragment of the M. gallisepticum tufB upstream region we created a "Trojan horse" plasmid. This is fully integrated into the genomic DNA of M. gallisepticum, always at the same site, oriC MG, and is able to carry and express any gene of interest in the genetic background of M. gallisepticum. Successful expression of a heterologous gene was shown with the lacZ gene of E. coli. When used for gene complementation or expression of hybrid genes in M. gallisepticum, a site-specific combined integration/expression vector constitutes an improvement on randomly integrating transposons, which might have unexpected effects on the expression of chromosomal genes.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Base Sequence
  • Gene Expression*
  • Gene Order
  • Gene Targeting
  • Genes, Reporter
  • Genetic Vectors
  • Homologous Recombination*
  • Molecular Sequence Data
  • Mutagenesis, Insertional*
  • Mycoplasma gallisepticum / genetics*
  • Plasmids / genetics*
  • Promoter Regions, Genetic

Grants and funding

This work was supported by grant P20712 from the Austrian Science Fund FWF. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.