Role of the CspD loop region. (A,B) Point mutations in position 43-44 of CspD. 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. (A) The protein sample was subjected to western blotting using anti-FLAG monoclonal antibody and anti-GroEL polyclonal antibodies. (B) The RNA sample was subjected to northern blotting using probes for baxL-bax 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 sample of CspD was set to 1. (C) Sequence alignment of the loop region in E. coli Csps. Amino acid residues with more than 80% identity across all of the shown Csps are marked in dark blue; those with identities of 60%–80% and 40%–60% are marked in blue and light blue, respectively. The CspD 43-44 positions are highlighted in orange. (D,E) Exchange of two amino acid residues of the CspD loop with those of other E. coli Csps. MG1655 cells harboring the indicated plasmids were analyzed as in A. Statistical significance was calculated using an unpaired two-tailed Student's t-test. (ns) Not significant, (*) P < 0.05, (***) P < 0.001.
