Schematic diagram for construction of the isogenic strains DP-L1510 and DP-L1535, in which readthrough transcription of prfA was blocked.
A. Chromosomal integration of pDP1498 by homologous recombination between the cloned Pstl (P)- and EcoRI (E)-generated fragment present on the plasmid and the homologous chromosomal sequence. The designated cross-over points are arbitrary. P1 and P2 indicate the two promoter elements immediately upstream of prfA while PplcA indicates the single plcA promoter (Mengaud et al., 1989; 1991; N. Freitag unpublished).
B. The resulting integration structure on the chromosome of both mutant strains. The integration strains were selected for, and maintained by, growth at a non-permissive temperature for plasmid replication in the presence of chloramphenicol. The plasmid was shown by Southern blot analysis to have integrated multiple times in a head-to-head configuration (data not shown).