Control of DegP-dependent degradation of c-type cytochromes by heme and the cytochrome c maturation system in Escherichia coli

Abstract

c-Type cytochromes are located partially or completely in the periplasm of gram-negative bacteria, and the heme prosthetic group is covalently bound to the protein. The cytochrome c maturation (Ccm) multiprotein system is required for transport of heme to the periplasm and its covalent linkage to the peptide. Other cytochromes and hemoglobins contain a noncovalently bound heme and do not require accessory proteins for assembly. Here we show that Bradyrhizobium japonicum cytochrome c550 polypeptide accumulation in Escherichia coli was heme dependent, with very low levels found in heme-deficient cells. However, apoproteins of the periplasmic E. coli cytochrome b562 or the cytosolic Vitreoscilla hemoglobin (Vhb) accumulated independently of the heme status. Mutation of the heme-binding cysteines of cytochrome c550 or the absence of Ccm also resulted in a low apoprotein level. These levels were restored in a degP mutant strain, showing that apocytochrome c550 is degraded by the periplasmic protease DegP. Introduction of the cytochrome c heme-binding motif CXXCH into cytochrome b562 (c-b562) resulted in a c-type cytochrome covalently bound to heme in a Ccm-dependent manner. This variant polypeptide was stable in heme-deficient cells but was degraded by DegP in the absence of Ccm. Furthermore, a Vhb variant containing a periplasmic signal peptide and a CXXCH motif did not form a c-type cytochrome, but accumulation was Ccm dependent nonetheless. The data show that the cytochrome c heme-binding motif is an instability element and that stabilization by Ccm does not require ligation of the heme moiety to the protein.

FIG. 1.

Heme-dependent expression of heme protein polypeptides. B. japonicum cytochrome c₅₅₀, E. coli cytochrome b₅₆₂, and Vhb were expressed from a plasmid as His-tagged proteins in E. coli hemA strain S905 grown in medium containing either 60 μM (L) or 600 μM (H) ALA. Cells that expressed B. japonicum cytochrome c₅₅₀ also harbored the ccm genes on pEC86. Cell extracts were analyzed by immunoblotting using antibodies against the His tag. Thirty micrograms of protein was loaded per lane.

FIG. 2.

Absorption spectra of heme proteins in heme-sufficient or heme-deficient cells. B. japonicum cytochrome c₅₅₀ (A), E. coli cytochrome b₅₆₂ (B), and Vhb (C) were overexpressed in E. coli hemA strain S905 grown in medium containing either 60 μM (low) or 600 μM (high) ALA. Cells that expressed B. japonicum cytochrome c₅₅₀ also harbored the ccm genes on pEC86. The soluble cell fraction containing cytochrome c₅₅₀ or cytochrome b₅₆₂ was reduced with sodium dithionite (A and B). The dithionite-reduced spectrum of the pyridine hemochromagen of Vhb was taken (C).

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.

FIG. 4.

Effects of Ccm on E. coli cytochrome b₅₆₂ and c-b₅₆₂ absorption spectra and heme staining assay. E. coli cytochrome b₅₆₂ (A) or a derivative containing heme-liganding cysteines (c-b₅₆₂) (B) was expressed in the parent strain KS272 in either the presence or absence of the plasmid pEC86 expressing the E. coli ccm genes. Absorption spectra for the soluble cell fraction (12 mg/ml) were determined for the 500- to 600-nm region as described in the text. (C) Cell fractions were separated by SDS-polyacrylamide gel electrophoresis and stained for covalently bound heme as described in Material and Methods. Thirty micrograms of protein was loaded per lane.

FIG. 5.

Expression of E. coli cytochrome b₅₆₂ and c-b₅₆₂ polypeptides under various conditions. (A) Effects of heme and DegP on cytochrome b₅₆₂ and c-b₅₆₂ polypeptide accumulation. E. coli cytochrome b₅₆₂ or c-b₅₆₂ was expressed in either 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 the His tag. Thirty micrograms of protein was loaded per lane. (B) Effects of Ccm and DegP on cytochrome b₅₆₂ and c-b₅₆₂ polypeptide accumulation. E. coli cytochrome b₅₆₂ or c-b₅₆₂ was expressed in degP⁺ (Wt) strain KS272 or degP strain KS474 in either the absence or presence of the plasmid pEC86 containing the E. coli ccm genes. Proteins were analyzed as described for panel A.

FIG. 6.

Effects of heme and Ccm on expression of Vhb derivatives. (A) Vhb constructs. SPVhb was generated by fusion of the 30-amino-acid N-terminal signal sequence of B. japonicum c₅₅₀ to Vhb as described in Materials and Methods. Lys³-to-Ala and Ala²⁷-to-Glu mutations represent the periplasmic targeting and leader peptide cleavage-site mutations of SPVhb, respectively. c′SPVhb was generated by the addition of heme-binding cysteines Cys¹²⁴ and Cys¹²⁷ to SPVhb. (B) Wild-type or mutant derivatives of Vhb were expressed in hemA strain S905 grown in medium containing either 60 μM (L) or 600 μM (H) ALA. c′SPVhb, which contains heme-liganding cysteines, was grown in either the absence of presence of the plasmid pEC86 expressing the E. coli ccm genes. Cell extracts were analyzed by immunoblotting using antibodies against the His tag. Thirty micrograms of protein was loaded per lane.