Figure 6.

In vivo effects of C-box O_L spacer variants on temporal expression of C.PvuII. (A) Diagram of experiment. Four E. coli TOP10’ cultures carrying plasmids with different O_L spacers fused to a cat (chloramphenicol acetyltransferase) promotorless reporter gene were infected with recombinant M13pvuIIwt phage at MOI = 15. We used three spacer variants tested previously in this study: WT-Sym (CAT/CAA; pDK178; closed circles), CAA/CAA (pIM22; open circles) and nonrepressing WT-Sym (box 2B mutated, pWWWR; gray circles) as a control. The first two variants have identical, symmetrized C-boxes sequences, and differ only at a single nt in O_R. A plasmid with no C-box sequence (pKK-238; triangles) was also tested and gave only background levels of cat expression (shown in Figure S6). (B) Growth was monitored at OD_{600 nm} before and after phage addition. (C) CAT production over infection time for two independently performed experiments was measured by sensitive colorimetric assay based on ELISA sandwich method (see ‘Materials and methods’ section). CAT production data were normalized to the value for each strain prior to infection. Curves were fitted and statistical analysis was performed using the SAS package (SAS Institute Inc., Cary, NC). The analysis of covariance indicated significant difference of slopes over time with P < 0.0001. Individual slopes for variants in pairs were also compared having: P = 0.0057 (pDK178 versus pIM22), P = 0.0001 (pDK178 versus pWWWR) and P = 0.0001 (pIM22 versus pWWWR).