Effects of alanine substitutions in the SpoIVFB interdomain linker. (A) Diagram of TM-SpoIVFB (designated 1–288) emphasizing the replacement of residues 201–212 in the linker with 12 Ala residues to create 1–288 A12. See Fig. 1 for additional explanation. (B) Cleavage assays. SpoIVFB 1–288 or 1–288 A12 was coexpressed with Pro-σK(1-127)-His6 (designated Pro-σK) (pZR12) in E. coli, and samples collected at the indicated times after induction were subjected to immunoblot analysis with anti-FLAG (Top) or anti-His (Bottom) antibodies to detect the indicated protein, including the cleavage product. (C) Interaction with Pro-σK. SpoIVFB 1–288 A12 E44Q (pSH27) was expressed with (+) or without (−) Pro-σK in E. coli for 2 h, and samples were subjected to pull-down assays. Input (I), unbound (U), and bound (B) fractions were subjected to immunoblot analysis as in B. (D) Cleavage assays of triple-Ala substitutions. SpoIVFB 1–288 or its derivative with the indicated three residues changed to Ala (pSH08-pSH16) was coexpressed with Pro-σK in E. coli for 2 h, and samples were subjected to immunoblot analysis as in B. (E) Cleavage assays of single-Ala substitutions. As in B, except with 1–288 or its derivative with the indicated residue changed to Ala (pDP74, pDP75, pSH28-pSH54). (F) Interaction with Pro-σK. As in C, except with 1–288 E44Q (pYZ68) or its derivative with the indicated residue(s) changed to Ala (pSH55-pSH58). The range of ratios of bound/unbound (B/U) SpoIVFB is shown at the bottom.