Overproduction of theBradyrhizobium japonicum c-Type Cytochrome Subunits of thecbb3Oxidase inEscherichia coli

Abstract We report on a system to improve expression of maturec-type cytochromes inEscherichia coli.It is based on the use of plasmid pEC86 that expresses theE. colicytochromecmaturation genesccmABCDEFGHconstitutively, whereby the production of both endogenous and foreignc-type cytochromes was increased substantially. The periplasmic soluble domains of thec-type cytochrome subunits FixO and FixP of theBradyrhizobium japonicum cbb3oxidase could be expressed inE. colionly when pEC86 was provided in adegP-deficient strain. This shows that a stimulation of heme attachment by the Ccm maturase system combined with the diminished proteolytic activity in the periplasm can increasec-type cytochrome yields.

[1]  H. Hennecke,et al.  Prototype of a heme chaperone essential for cytochrome c maturation. , 1998, Science.

[2]  J. Beckwith,et al.  Characterization of degP, a gene required for proteolysis in the cell envelope and essential for growth of Escherichia coli at high temperature , 1989, Journal of bacteriology.

[3]  H. Hennecke,et al.  Cytochrome c biogenesis in bacteria: a possible pathway begins to emerge , 1994, Molecular microbiology.

[4]  H. Hennecke,et al.  A high-affinity cbb3-type cytochrome oxidase terminates the symbiosis-specific respiratory chain of Bradyrhizobium japonicum , 1996, Journal of bacteriology.

[5]  Linda Thony-Meyer,et al.  Biogenesis of respiratory cytochromes in bacteria. , 1997 .

[6]  R. Poole,et al.  10 The Analysis of Cytochromes , 1985 .

[7]  R. A. Fabianek,et al.  The Active-Site Cysteines of the Periplasmic Thioredoxin-Like Protein CcmG of Escherichia coli Are Important but Not Essential for Cytochrome c Maturation In Vivo , 1998, Journal of bacteriology.

[8]  V. Méjean,et al.  A reassessment of the range of c-type cytochromes synthesized by Escherichia coli K-12. , 1994, FEMS microbiology letters.

[9]  George Georgiou,et al.  Construction and Characterization of a Set of E. coli Strains Deficient in All Known Loci Affecting the Proteolytic Stability of Secreted Recombinant Proteins , 1994, Bio/Technology.

[10]  R. E. Tully,et al.  Bradyrhizobium japonicum cytochrome c550 is required for nitrate respiration but not for symbiotic nitrogen fixation , 1995, Journal of bacteriology.

[11]  W. Reznikoff,et al.  Identification of the regulatory sequence of anaerobically expressed locus aeg-46.5 , 1993, Journal of bacteriology.

[12]  M. D. Page,et al.  Contrasting routes of c-type cytochrome assembly in mitochondria, chloroplasts and bacteria. , 1998, Trends in biochemical sciences.

[13]  L. Hederstedt,et al.  Bacillus subtilis holo‐cytochrome c‐550 can be synthesised in aerobic Escherichia coli , 1990, FEBS letters.

[14]  H. Hennecke,et al.  Requirements for maturation of Bradyrhizobium japonicum cytochrome c550 in Escherichia coli. , 1996, European journal of biochemistry.

[15]  G. Thomas,et al.  Escherichia coli K‐12 genes essential for the synthesis of c‐type cytochromes and a third nitrate reductase located in the periplasm , 1996, Molecular microbiology.

[16]  H. Hennecke,et al.  Assembly and Function of the Cytochrome cbb Oxidase Subunits in Bradyrhizobium japonicum(*) , 1996, The Journal of Biological Chemistry.

[17]  B. Goldman,et al.  Mmicular mechanisms of cytochrome c biogenesis: three distinct systems , 1998, Molecular microbiology.

[18]  P. Künzler,et al.  Translocation to the periplasm and signal sequence cleavage of preapocytochrome c depend on sec and lep, but not on the ccm gene products. , 1997, European journal of biochemistry.

[19]  H. Hennecke,et al.  Escherichia coli genes required for cytochrome c maturation , 1995, Journal of bacteriology.