FIGURE 9.

Engineering of the Δp1cat mutant strain. A, possible crossover events between the WT G37 genome and the pΔp1 plasmid. Solid lines, crossover events resulting in the deletion of peptide 1 coding region and in the insertion of cat. Dashed lines indicate a crossover event resulting also in the insertion of cat but not in the deletion of peptide 1. B, screening of pΔp1cat transformant colonies by PCR using genomic DNA as template. The deletion of the region coding for peptide 1 was identified by the presence of a PCR band (∼700 bp) smaller than that of the WT (clones 1, 4, and 9). C, Western blot analysis of G37 WT and Δp1 clones 1, 4, 8, and 9 using a polyclonal antiserum against the M. pneumoniae P41 protein (ortholog of MG491). Clones 1, 4, and 9 show a shift in the MG491 band, consistent with the deletion of the 25 residues of the MG491-Ct_peptide 1. D, Western blot analysis of G37 WT and Δp1clones 1 and 9 using a polyclonal antiserum against MG219. Because no band shift is apparent, polar effects derived from the deletion of peptide 1 were discarded. E, Western blot analysis using anti-P41 antibodies of G37 WT, Δp1c1, and Δp1TC6 (Δp1 clone complemented with a copy of mg491) cells. Δp1TC6 cells present two bands corresponding to MG491 and MG491ΔCt_peptide 1 mutant protein.