FIG. 3.

Accumulation of cytochrome c₅₅₀ polypeptide under various conditions in degP⁺ and degP backgrounds and in the absence or presence of Ccm. (A) B. japonicum cytochrome c₅₅₀ was expressed in hemA strain S905 or hemA degP strain S906 grown in medium containing either 60 μM (L) or 600 μM (H) ALA. The cells also contained pEC86, which carries the ccm genes. Cell extracts were analyzed by immunoblotting using antibodies against a His₄ tag. Thirty micrograms of protein was loaded per lane. (B) Effect of mutation of the heme-binding site on expression. Cell extracts were prepared from either wild-type strain KS272 (Wt) or degP strain KS474 harboring B. japonicum cytochrome c₅₅₀ constructs which expressed unmutated (Wt) or ⁴¹Cys-to-Ser or ⁴⁵His-to-Leu mutated cytochrome c₅₅₀. The cells also contained pEC86, which carries the ccm genes. Proteins were analyzed as described for panel A. (C) Effect of ccm genes on cytochrome c₅₅₀ expression. B. japonicum cytochrome c₅₅₀ was expressed in either a degP⁺ (Wt) or degP E. coli strain in either the absence or presence of the plasmid pEC86 expressing ccm genes. Proteins were analyzed as described for panels A and B.