Bacillussubtilis holo‐cytochrome c‐550 can be synthesised in aerobic Escherichia coli

Abstract

Bacillus subtilis membrane‐bound holo‐cytochrome c‐550 was found to be expressed from the structural gene cloned on a plasmid vector in aerobically grown Escherichia coli and exhibited normal biochemical properties. This occurs despite the lack of endogenous eytochrome c and suggests that eytochrome c‐heme lyase activity is also present in aerobic E. coli. The membrane topology of B. subtilis eytochrome c‐550 was studied using fusions to alkaline phosphatase (PhoA). The results show that the heme domain (at least when fused to PhoA) can be translocated as apo‐cytochrome and confirm that the N‐terminal part of the cytochrome functions as both export signal and membrane anchor for the C‐tenninal heme domain. A model for the organisation of B. subtilis cytochrome c‐550 in the cytoplasmic membrane is presented.

Bacillus subtilis ResA Is a Thiol-Disulfide Oxidoreductase involved in Cytochrome c Synthesis

“…Horse heart myoglobin (16951.48 Da) was used as a calibrant. Alkaline phosphatase activities of cell lysates of E. coli were measured using p-nitrophenyl phosphate as substrate (36). UVvisible absorbance spectra were recorded on a PerkinElmer 800 spetrophotometer.…”

Bacillus subtilis Contains Two Small c-Type Cytochromes with Homologous Heme Domains but Different Types of Membrane Anchors

“…The N-terminal parts of the two proteins are clearly different. In CccA, the first 32 residues are known to function as a noncleaved signal sequence for membrane insertion and peptide membrane anchor for the cytochrome domain (5). The N-terminal part of CccB also has the features of a signal peptide but contains the bacterial lipoprotein consensus sequence, Leu-Ala-Ala-Cys.…”

“…The latter domain, like that of all bacterial c-type cytochromes, is located on the outer surface of the cytoplasmic membrane (5). At pH 7.0, cytochrome c 550 has a midpoint redox potential of ϩ178 mV (6).…”

A cytochrome c fusion protein domain for convenient detection, quantification, and enhanced production of membrane proteins in Escherichia coli—Expression and characterization of cytochrome‐tagged Complex I subunits

“…Furthermore, large amounts of holo-cytochrome c 550 have previously been produced in E. coli under aerobic or microaerophile conditions. 28 In bacteria, the cytochrome c-type covalent thioether bonds can only form in the oxidizing environment on the periplasmic side of the membrane. Thus, only proteins where the C-terminal end is located in the periplasm can be tagged with holo-cytochrome c. On the other hand, once the heme is incorporated, it will not be lost from the protein during purification or handling and will be retained even under the denaturing conditions of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).…”

“…28,37 To produce the fusion proteins under aerobic conditions and minimize production of apo-cytochrome, the proteins were coexpressed with the ccm proteins responsible for heme insertion and cytochrome c maturation from pEC86 (kindly provided by Linda Thöny-Meyer, see Table I). The plasmid pEC86 expresses the genes in the ccm operon constitutively from the tetracycline promoter.…”