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 Nco I and Spe I 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 Pst I and Spe I 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).