Chimeric variants of CspD and CspA. (A) Sequence alignment of CspA, CspD, and chimeras. For chimeras, the regions originating from CspA and CspD are marked in green and orange, respectively. RNA-binding motifs, RNP1 and RNP2, a loop region, and β-strands are indicated by lines on top of the sequence. (B) The structure of E. coli CspA monomer (Protein Data Bank ID: 1MJC). The junctions in chimeras (jct1 to jct5) are indicated by arrows. (C,D) Expression and properties of chimeras. MG1655 cells harboring the indicated plasmids were grown in LB-ampicillin medium. At an OD600 of 0.3, 0.2 mM IPTG was added to cultures to induce Csps. Incubation was continued for 30 min. (C) The protein sample was subjected to western blotting using anti-FLAG monoclonal antibody and anti-GroEL polyclonal antibodies. (D) The RNA sample was subjected to northern blotting using probes for baxL-bax mRNA, gdx mRNA, or tmRNA. Quantitation data are shown below the northern blot; averages of relative RNA levels were calculated from three independent experiments, with error bars representing the standard deviations. The RNA samples of CspD and CspA were set to 1 for baxL-bax mRNA and gdx mRNA, respectively. Statistical significance was calculated using an unpaired two-tailed Student's t-test. (ns) Not significant, (*) P < 0.05, (***) P < 0.001.
